Second Edition

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Second Edition

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 Bibliotf!ca I C A P

Second Edition

Dwight C. Hirsh
N. James Maclachlan
Richard L. Walker

Blackwell
Publishing
Dwight C. Hirsh, DVM, PhD, is cmeritus professor,
Department of Patl1ology, Microl.Jiulogy a11d
Im mu nology, l Jnivcrsity of California-Davis.
N. Jan1es MacLachla11, DVM. PhD, is protcssor,
Dcpart1ncnt of Pathology, Microbiology and
l1nmunology, Universíty of California-Davis.
Richard L. Walker, DVM, Pl1D, MPVM, is profe:;sor of
clinical cliagnostic bacteriology, California Animal Health
and Food Safcty Laboratory, Univcrsity of Califorrúa-
Davis.
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J\11 rights reserved Second edition, ©7Jl04 Bl;ir.kwell Publishing

Blackwell Publishing Profession¡ll Library (>f Congress Cataloging-in-PubJicatio11 LJata

2121 Slatc Ave11ue, An1es, Iowa 50014, USJ\ Vctcrinary microbiology / [edited byj Dwight C. Hirsh,
N. James MacLachlan, llichar<.l L. Walker.-2n<.l e<.J.
Ordcrs: 1-800-862-6657 p.; cm.
Office: 1-515-292-0140 lncludes bibliograpllical refe1 en ces a11d index.
fax: 1 515 292-3348 ISBN 0-8138-0379-9 (;ilk. paper)
web s1te: www.blackwellprofesslonal.com l. Yelerü1ary microbiology.
lDNLM: l. Animal Population Groups-microbiology.
Blackwell Publislung Ltd 2. Veterinary Medicine. QW 70 VS86 2001] J. Hirsh,
9600 í.;irsington Road, Oxford OX4 2DOJ UK Dwight C. 11. MacLachlan, N1gel James. fII. Walker,
Tel.: +44 (0)1865 776868 Richard L., DVM.

Blackwcll Publishing J\sia Sl'780.2.V48 2004

s:io Swanston Street, Carlton, Victoria 3053, Australia 636.089' 69041-dc22
Tel.: +61 (0)3 8359 1011 2004008413

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Dedications

To Lucy, Dw1ght, and Ellzabeth for years of patience and understanding; and to the memory of my brother, Harry.
-DH1ight C. Hirsh

'lo Kcn and Marion for lighting the fire of in quisitivcncss.

-N. [ames MacLachlan

To Dee Dee and Mary for thPir ~11pport, 11nderstandi.ng, and focus on what is really important.
- Richard L. Walker
Contents

Contributors iX 19 Moraxella 119

DwTr.l-IT C. HrRSH A>'\JD ERNST L. B1RERSTEJN
Preface Xi 20 Pseudornonas 122
DV\llGHT c. HtRSH
21 Taylorella equígenitalis 12 5
PAR'r l IN'fROl)UCfION 1
F.RNST L. BJBERSTEIN Ai'\ID DWIGHT c . HIRSH
1 Parasitism and Pathogeni(_ity 3 22 Spiral-Curved Organisms l: Borrelía 128
ERNST L. lliBERSTEIN R.ANCE B. LEFF..BVKE
2 lmmune Responses to Infectious Agents 6 2~ Spiral-C:urved Organisms TI: Rrachyspira
LAUREL]. GERSHWJN (Serpulina) 131
3 Laboratory Diagnosis 15 DvVIGHT C . H IRSH
Dw1c1 1T C. HrRs11, YUAN CmJNc. Zr.E, ANO 24 Spiral -Curved Organisms 111: Campylobacter,
ANTHONY E. CASTRO Arcobacter, Lawsonia 134
4 Antimicrobial C hen1 o therapy 26 DWlGAT c. HIHSH
]OHN F. PR1:sc<J1T 25 Spiral-Curved Organisms TV: Helicobacter-
5 Antimicrobial Drugs: A Str<itegy for R<ition<il TJse The Spiral Shaped Microorganisms o f the
and Ll1e Ran1iflcalio11s of Misuse 44 Gastroi.ntestinal Tract and Livcr 141
DwtGHT C. H1RSH JAMES G. Fox
6 Vaccincs 48 26 Spiral ~Curved ()rganisms V: Leptospira 118
N . ]AMES MACLi\CHLAN AND DWIGHT C. I-IIRSH RANCE B. LEFEBVRE
27 Staphylococcus 153
Ü\.Y ICiHT C. H lR.'iH ANU ERNST L. BIBERSTEIN
PART II BACTERIA A.NO FUNGl 55
28 Streptococcus and Enterococcus 159
7 Family Enterubacleríac:eae 57 DWlGHT C. H TRSH AND ERNST L. BlllF.RSTETN
DvVTGHT e:. HrRSH 29 Arcanohacterium 168
8 E11terobact:eriaceae: Escherichía 61 Ü\"/JGHT C. H lRSH AND ERNST L. BIBERSTEIN
DWIGHT C. HIRSH 30 Bacillus 170
9 Enterobacteriaceae: Salmonella 69 Ü\.YIGHT c . HIRSH AND ERNST L . BlllERSTEIN
DWIGHT C. HIR~H 31 Corynebacterium 175
10 Enterobacteriaceae: Ye.rsinia 75 DvVIG HT C. H 1RSH AND ERNST L . RTUERSTEJN
DW!GHT C . HIRSH 32 Erysipelothrix 181
11 Enterobacteriaceae: Shigella 81 RJCHARO L. WALKER
ÜWJGHT C . lllRSH 33 Listeria 185
12 Pasteurellaceae: Pasteurella, Mannhein1ia 84 R ICllA RI) L. WA l. KBH
DWTGTTT c . HrnsH AND ERNST L. BIBERSTEIN 34 Rhodococcus 190
13 Pasteurellaceae: Actinoba.cillus 91 D\\TIGHT C . I-l!RSH AND ERNST L. BIBP.RSTEIN
DWIGHT e . HIPSH AND IlP.NST L. BlBEPSrEIN 35 Non- Spore-Forming Clbligat e Anaerohes 193
14 Pasteurellaceae: Haen1ophilus a11d Histophilus 95 DWIGHT C. H!RSH
Dw1GHT C. H IRSH AND EHNST L. BIBERSTEIN 36 Clostridiu1n 198
15 Bordetella 100 DWIGHT C. HJRSH AND ERNST L. IllllERSTE!N
lYWIGHT C. H IRSH ANO ERNST L. Hl.BERSTl'.fN 37 filamentou s Bacteria : Actinorrzyces, Nocardia,
16 Brucella 105 Dermatophilus, and Strcptobacillus 215
RlCHAilD L . WALKER ERNST L. B IBERSTElN i\ND ÜWIGHT C . H!RSH
17 Burkholderia mallei and Burkholderia :38 A!fycobacterium 223
p::;eutlornu lid 113 DWKiHT C. HlRSli AND ERNST L. BIBERSTEIN
DWTGHT C. HTRSH AND ERNST L. BIDERSTEIN 39 C;h/an1ydíaceae 2~.'i
18 Francisclla tularensis 117 DWIGHT C. TlJRSii AND ERNS'l' L . DIJJERSTEIN
IJWTGHT C. H TRSH AND ERNST L . .BIBEHSTEIN

Vll
viii Contents

40 Mollicutes 240 58 Tognviridae ;in<I Plaviviridae 351

RrCHARU L. WALKLJ{ N. ]AMF.5 MAcLACHLAN ANO ]F.FFRF.Y L. STOTr
41 Rir.kett.~iae: Rickettsia, Coxiella, and Orientia 250 59 Orthomyxoviridac and Bunyaviridae :~61
JANET E. FoLtY, ERNST L. nrnr.RsTe::1N, AND Au:x A. J\ROJ\NS AND N. JAMES MAcLACHLl\N
DWJGHT c. HIRSH 60 Paramyxoviridae, Fifoviridae, and Bornaviridae 369
42 .Hhrlichiac: Ehrlíchia and Neorickettsia 253 YVAN C HUNG Z F.F. ANO N . ]AMES MACLAC:HLAN
JANtT E. fOLEY, ERNST L. BJBERSTEIN1 ANO 61 Rhabdoviridae 377
DwJGHT C. HmsH Y UAN CHIJN(; 7FF AND N. ]i\MFS MACLA.CHLAN
43 Anaplasmataceae 257 62 Coronaviridae and Arteriviridae 383
}ANn F.. Frn.FY A1'TI F.RNST I .. B1BF.RSTEIN UDE."Jl .B. R. BALJ\SURIYA A:--10 I EFFREY L. STOTI
44 Darro11e/laccae 260 63 Rcoviridac 398
BRUNO B. CHOMEL ANO füCKI~. w. KAsTEN N. JAMf.S MACLAC:Hl.AN ANI) JtHIU.Y L. StUTI
45 Ycasts Cryptococcus, Malassezia, and Candida 265 64 Birnaviridae 407
DWIGHT C . HIRSH ANO ERNST L. BIBERSTEII\ N. ] ANt.tS MAcL ACHLAN ANU ]EFFRF.Y L. STurr
16 Dermatophytes 273 65 Retroviridae 409
El!.NST L. BmF.RSTEIN ANU Dw1c;111· C. HtK.~H RICHARD M . D oNOVAN 1\Nt) Í'REDERICK]. fuu.F.R
47 Agents of Subcutaneou s Myro<;p<; 279 66 Transmissible Spongiform Encephalopathies 427
DWIGHT C. HIRSH ANO ERNST L. BIBER~TCIN BRADD c.
BAIU\ t\J'fü YUAN CHUNG ZEE
48 Agents of Systemic Mycoses 285
DwtGHT C . HJRSll ANO ER~ST L. B 11mnSTEIN
PARTIV CLINICAL APPLI CATIONS 435

67 Circulatory System and Lymphoid T issues 4~7

PART l1l VJRUSF.S 299
RICHARD L. WAt.Kf.K
49 Pathogenesis of Viral Diseases 301 68 Digestive System anrl A<;<;oci;ited Organs 446
YUAN CHUNG ZH AND N.) AMES MACLACHLAt-. RICHARD L. WALKER
50 Parvoviridae and Circovirirlae ~0.5 69 lntegumentary System 45!:)
YUAN CttUNG Z F.F. AND N . JAM t::S Mi\CLACHLAN RICHARD L. WALKl\R
.'il Asfarviridae and Iridoviridae 312 70 Musculoskeletal System 468
N. )AMES MACLAC:HLAN ANL> J.i.;FFREY L . STOTr RtCHARU L. WALKER
52 Papillomaviridae and Polyomaviridae 315 7l Ner vous System 474
YuAN C11UNG ZEI'. AND N. j AMJ;S MACLACHLAN RICHARD L. WALKER
53 Adenoviridae 317 72 Oculdr lnf~ctiurn, 482
Y UAN CHUNG z.u ANO N . J A\AJ:<; MACl.AC:Hl.AN RICHARD L. WALI<.ER
54 Hetpesviridae 320 73 Respiratory Systcm 487
ALEX A . ARDANS RlCHARD L. WALKER
55 Poxviridac 333 74 U rogenital System 496
N. jAMFS MACLACHLAN ANO JEFFRF.Y L. $TOTI RICHARD L. WALKLR
56 Picornaviridae 339
N . JAMES MACLACHLAN, YU/\N C llUNG ZEE, Al'\D Index 505
JEHREY L. Sron
57 Culidviridu~ 346
YtJAN CHUNG ZF.F. ANO N.J AMES MACLACHLAN
Con tribu tors

Alex A. Artl<tn:>, DVM, MS janet E. Foley, DVM, PhD

Protcssor, Department of Medicine and Epidemiology; Assistant Professor, Department of Medicine anrl
Director, California Animal Health and Food Safety Epidemiology
Laboratory Systern Scl1ool of Vele1ina1y Mcuil.:ir1e
School of Veterinary Medicine University oí California
University of California Davis, California
Davis, California
James G. Fox, DVM
Udeni B. R. Balasuriya, BVSc, PhD ProfC'c;c;or anrl Ilirector, Division of Comparative Medicine
Associate Research Molecular Virologist Massachusctts lnstitutc ofTcchnology
Dcpartn1ent of Pathology, Microbiology and Cambridge, Massachusetts
Immunology
School of Veterinary tvledicine Frederickj. Fuller, PhD
Univcrsity of California Professor, Depa1l111enl of Povulaliu11 Health and
Dav1S, c:aiifornia PathobioJogy
College of Veterinary Medicine
Bradd C. Barr, DVM, Pl1D North Carolina State Univcrsily
Professo r, Departme11t of Pathology, Microbiology, and Raleigh, North Carolina
Tm munology;
California Animal Hcal th and Pood Safcty Lnboratory LnurelJ. Gershwin, DVM, PhD
SchooI of Veterinary Medicine Professor, [ )epartment of Pathology, ~1icrobiology, and
University of California Immunology;
Davis, CA 95616 Chief, Clinical Immunology, Virology, and Microbiology
Service
Ernst L. Biberstein, DVM, PhD Veterinary Medica! Teaching Hospital
Professor Emeritus, Department of Pathology, School of Veterinary Medicine
Microbiology, and In1111unology Universily ofCali(urnia
School of Veterinary Medicine Davis, California
lJnivcrsity of California
Dovi:i, California D"vight C. Ili.l'.sh, DVM, I'hD
Professor Emeritus, Departmcnt of Pathology,
Anthony E. Castro, DVM, PhD Microbiology, and Immunology
Department of Veterinary Scicnccs School ofVeterinary Medicine
Pennsylvania State U11iversity U1 1iver:.ity uf Califurnia
University l'ark, l'ennsylvania Davis, California

Bruno B. Chomel, DVM, PhD Rickie W. Kaste11, Plill

Associate Professor, Dcpartment of Population Health lJepartment ot Population Health and Reproduction
and Reproduction School of Veterinary Medicine
School of Veterinary Medicine Univcrsity of California
U rli ver:.i l y uf CCtlifur1lia Davis, California
Davis, California
Rance B. LeFebvre, PhD
Richard M. Donovan, PhD Prufessor, Department of Pathology, Microbiology, and
V iral and R1ckettsial Visease Laboratory Tmmunology
í:!'lliforni;i Oep;irtment of Health Services School of Veterinary Medicine
Richmond, California University of California
Davis, California

.IX
Preface
Thls book is intended for vcterlnary students, to accom-
pany and supplPmPnt thPir first courses in pathogenic
bacteriology-mycology and virology (Parts l tl1rough III),
as well as subsequent courses dealing with infectious dis-
cascs (Part IV). The focus includes pathogcnic mcchanis1ns
and proccsses in infectious diseases; methods of diagnosis;
and principies of resistance, prevention, and therapy. A
worki11g k11uwl1::<.lge uf general mlcroblology is assumed.
Beyond serving as a rPso11ríP for students, the book is
also meant to serve as a convenient reference for vete1li1a1-
ians and veterinary scientists whose main line of activity
and expertise is outside the areas of microbiology.
The book is divided into four sections. l'art l deats with
the general characteristics of the host-parasite relation-
shlp, laboratory diagnosis of condltlons Involving an in-
fectious etiology, antimicrobial treatment, and prevention
of iufecLious disea:;1::.
Parts n (bacteria anrl fungi) anrl 111 (vin JSPS) present the
infectious agents that affcct thc veterinary species. TJ1c
chapters deating with the bacteria! agents are grouped
mainly by morphology, and their gram stainingcharacter-
lsttcs. ·rhe fungal agents are S?;rouped mainly by morpho-
logic characteristics (yeast, mold). ·rhe viruses are grouped
a long taxonomic grounds.
Part TV deals with the intectious agcnts in the context
of the host. This section is organized by organ system.
Each urgan system is dtscussed flrst as a microbial habitat,
followerl hy disc11ssion of tbose infecti01.1s agents that
mainly affect that particular systen1.
'fhe content and organization of this book varíes some-
what from the last cdition of Veterinary Micro/Jiology
(1999). Most notable is the organization of the infectious
agents discussed in Part 11, the bacteria and fungí. Thcsc
agcots are now dlscussed w!thout regard to major organ
system, huta long morP traditional grounds (i.e., morphol-
ogy and gram reaction in the case of U1e bacteria! ageut:s,
and yeast vs. mold for the fungal agents). A major changc
in the content of this cdition is thc addition of Part TV,
which deals with the infectious agents along more clini-
catl y useful lincs. In addition, we havc climinatcd rcfcr-
ences fron1 the end of each chapter. Aside from a space-
saving device, allnost universal access to the Internet and
vi1 Lual librarie:; cuak<:: :;uch references redundant.
We gratefully acknowlcdge Natalie Karst, whose help is
much apprcciatcd. Spccial thanks go to Dede Andersen
and Tad Ringo of Htackwell Publishing, who have been un-
believably patient and extrcmcly hclpful in gctting our cf-
fort to press.
•
XI
Introduction
 - ... Parasitisni and
Pathogenicity
ERNST L. B IBERSTEIN
Veterlnary m icrot>iology deals wlth mlcroblal agents af-
fecting animals. Such agpnts arP ch;:iractPrizPd according
to their ecologic arrangements: parasites live in pern1anent
association with , and at the expense of, animal hosts;
saprophytes normally inhabit the inanimatc cnvironmcnt.
Parasites that cause their host no discernible harm are
called co1nmensals. The term symbiosis usually refers to re-
ci prucall y !Jeneficial asso<:iatlons of organisms. ·rhls
arrangement is also callPci n1utuali.~1n .
Pathogenic organisms are parasites or saprophytes that
cause disease. The process by which they establish them-
selves in a host in.dividua] is infection, but infcction nccd
not be followed by clinical illness. The term virulence is
somctimes used to inean pathogenicity but sometimes to
express degrees of pat hogenlclty.
Sorne Attributes of Host-Parasite Relationships
Many pathogenic microorganisms are host-specilic in that
they parasitize only one ora few animal species. 1'he cause
of equine strangles, Streptococcus equi subspecies equi, is es-
scntially limited to horses. Others- certain Salnione/la
tyµc::., íor cxarnple-liavt.: a bruaú liust rangc. Tltc l>asís for
this difference in host specificity is incompletely under-
stood, bu t it may in part be related to the need for specific
attachmcnt ctevices between host (receptors) an d parasite
(adhesins).
Sorne agents in.f ect sevcral host species but with varying
effects. The plague bacillus Yersinia pestis behaves as a com-
111e11sal parasile i11111a11y, bul l>y 110 111ca11s all, s1nall rodent
species but causes fatal disease in rats and humans. Evol11-
tionary pressure may have produced sorne of these diffcr-
ences, but not others: C'occidioides in1mitis, a saprophytic
funi:,'"Us requiring no living host, infects cattle and dogs
w1th equal ease; yet it produces no clinical signs in cattle
bt1t frequently causes progrcssive fatal d isease in dogs.
Poten ti al path ogens vary 1n thel r effects on different tis-
sues in thc san1c h ost. 'l'hP P.sr:hr•richin coli that is com:men-
sal in thc intcstine can cause severe disease in t11e urü1ary
tract and peritoneal cavity.
Sorne microorganisms that are commensal in one ha-
bitar may turn pathogenic in a habitat that is patholo,gí-
cally altered or otherwise compromised. 'fhus, oral strepto-
cocci, which occasionally c1llcr the !Jloollstream, may
colonize a damaged heart valve and initiate b acteria! en-
docarditis. In the absence of such a lesion, however, they
would be clearecl u11eve1 1Lfully via the ruacrop hage system.
Similarly, the frequent entranc.e of i ntPstin;i 1 h;ictPria int(l
vascular channels normally leads to their disposal by hu-
moral and cellular defense mechanisms. In immuno-
incompetent hosts, however, such cntrancc may lcad to
fatal sepncemia.
'fransfer to a new host or tissue, or a change in host re-
sislance, are con1rno11 ways lltat cou1Iuer1sal parasites are
convcrted into active pathogens. Com1nensalism is thP sta-
blc form of parasitic existence. Tt ensures survival of the
microorga11ism, which active disease would jeopardize by
killing the h ost or evoking an active im1nunc response.
Either effect deprives the agent of its habitat. Evolution-
ary selective pressure therefore tends to eliminate bost-
l-'ara:.ile relatio11slilp~ tl1at threaten the survival of either
partner. It does so hy allowing milciPr str;iins of thP
-
pathogen, "vhich permit Iongcr surviva1 of the host and
thereby facilitate thcir own dissemination, to replace the
more lethal ones. It also favors a rcsistant host population
by screening out highly susceptible stock. ·1he trend is thus
toward commensalism. Most agents causing serious dis-
ease have alternative modes of survival as com mensa1s in
ti~~11Ps o r hosts not subjcct to disease (e.g., plague) or in the
inanimate environment (e.g., coccidioidon1ycosis) . Olhers
cause cl1ronic infections lasting months or years (tubercu-
losis, syphilis), during which thcir disscmination to othcr
hosts ensures t heir survival.
Criteri a of Pathogenicity-Koch's Postulates
The presence of a microorganism in diseased individuals
does not prove its pathogenlc s1gnificance. To demonstrate
thP ca11s;:il role of an agent in a disease, the following qual-
ificatio11s or "postulales" for 1nulaLed by Ruuert Kuclt
(1843-1910) should hP s;:iti~fiPrl:
l. The suspected agent is present in all cases of the
<.liscase.
2 . The agent is isolated from such disease anci rropa-
gatcd scrially in pure culture, apart from its natural
host.
3. Upon introduction into an experimental host, thc
isolate produces the original disease.
3
4 PART 1 Introtluction
4. ·rhe agent can be reisolated from this experimental
infection.
Thcsc postulutcs are idcals that are not satisfied in ali
cases ot infectious diseases. 'Ihe prcsence of sorne rnicroor-
ganisms cannot be demonstrated at the time of disease, es-
pccially in affected tissues (e.g., tetanus, botulism). Others
lose virulence rapidly after isolation (e.e., T.f'ptnspira spp.),
wllilc slill ull 1e1 s, Lhough i11dispcnsable for pathogenesis,
require undetermined accessory factors (e.g., Pasteurella-
related pneumonias). For sorne human viral pathogens
(e.g., Cytomegalovirus), no experimental host is known,
and sorne agents (e.g., Mycobacterium leprae) have not been
grown apart from thcir natural hosts.
Elements in the Producti on of an lnfectious
Disease
Effectiv<-' rransmission through indirect contact occurs by
ingestion; inhalation; o r mucosa], cutancous, or wound
contamination. Airborne infection takes place largely via
droplct nuclci, which are 0.1 to Smm in diameter. Particles
ot this size stay suspended in ai r and can be inhaled. Larger
particles settle out but can be resuspended in dust, which
may also harl)Or infcctlous agents fruu1 11011re::;piralury
sources (e.g., skin sq11;imps, fpcf's, saliva). Arthropods may
se1 ve as 111ecl1anical carriers of pathogens (e.g., Shigella,
Dermatophilus) or play an indispensable part in the lite cy-
cles of diseasc-producing agc11ts (plague, eh rlichioses, viral
encephalitides).
Attachment to host surfaces requires interaction be-
tween the agent's adhesins, whlch are usually proteir1s,
and the host's receptors, whicl1 are most oftpn carhohy-
c..lrale residues. Exan1plcs of bacteria! adhesins are fimbria!
proteins (Escherichia co/i, Salmonella spp.), P-1 protein of
Mycoplas1na pncumoniac, and afimbrial surface proteins
(sorne streptococci). Examples of host receptor substances
include fibronectin for sorne streptococci and staphylo-
COCCI, 1nannose for many E. l ·uli strairis, C111d sialic acid for
M '. pneumoniaf..
Attachment is inhibitcd by normal commensal flora
that occupy or block available receptor sltcs and discour-
age colonization by excrcting toxic metabolites, bacteri
ocins and microcins. ·¡ his "colon1zat1on resistance" is an
important defense mechanism and may be assisted by mu-
cosa! antibody and other antlbactcrlal sutJs'tances (dc-
fensins, lysozyme, lactoferrin, organic ilcids).
Pc11clralio11 of hosl surfaces is a variable requircmcnt
among pathogens. Some agents, having rcached a primary
target cell population, pcnctratc no farthe r (e.g., entero
toxigenic E. co/i). Others traverse surface membranes after
inducing cytoskeletal rearrangements, resulting in "ruf-
fles" that entrap adhered bacteria or passage betwcc11 cp-
ithelial cclls (e.g., Salmonella, Yersinia) . Tnhaled facultative
i11tracellulC1r pa1 asi Les like Mycobacteriu111 tuberculosis are
takcn up by pulmonary macrophages, in wl1ich they may
multiply and travcl via lymphatics to lymph nodes and
other tissues. l'ercu taneous penetrat1on occurs mostly
through injuries, including arthropod bites.
Dissemination takes place by cxtenslon, aitled perhaps
by such bacrerlal enzymes as collagenase and hyaluru-
nidase, which are produced by .m.any pathogPns. Mic:ro-
urga11is1ns are also spread via lyinph and blood vcssels,
the bronchial tree, bile ducts, nerve trunks, and mobile
phagocytcs.
c;rowth in or on host tissue is a prerequisite of patho-
genesis for ali pathogenic organisms, except the few that
produce toxins In foodstuffs prior to lnges'tion. In urc..lcr Lu
multiply to pathogenic levels, thPy m11st hP. ahle to neu-
tralize liusL defense efforts. Rclevant adaptations of various
bacteria include firn1 attachment to prevent mechanical
removai; repulsion or nonattraction of phagocytes; and in-
terference with phagocytic funct1on by capsules and cell
walls, by leukotoxic activity, or by prevention of phago-
cytic digestion. Sorne bacteria dlgest or tlivert autibuc..lie:-.
and deplete complement. Sorne dcstroy the vascular sup-
ply to tissuc, sltulling out dcfcnsive resourccs and sus-
pP.nding antimicrobial activity in the affccted area.
With host defcnscs ncutralizcd, microbial growth can
proceed it nutritional supplies are adequate and the pH,
temperaturc, and oxidation-rcduction potcntial (Eh) are
appropriate. Iron is often a llmltlng nutrie11t. Microbial
ability to appropriate iron from iron-binding host proteins
(transferrin, lactoferri11) is a faclor in virulence. Gastric
acidity acco11nt.~ for the resistance of the stornach to n1ost
pathogenic bacteria, alth ough exprcssion of altcrnativc
sigma factors when bacteria are in stationary phase rcsults
in an RNA polymerase that transcribes genes whosc prod-
ucts help the pathogcn resistan acidic environmen1 (c.g.,
Salmonella, E. colí) . The hlgh body temperature of birds
may cxplain thelr resistance to sorne discases (e.g., an-
thrax, histoplas1nosis), while low F.h rcquirements ac-
cuu11l for lhe 1eslriclio11 of an<terobic growth to devitalized
(Le., nonoxygenated) tissues or tissues i11 which sirnulta-
neous aerobic growth has lowcrcd the Eh.
Pathogenic Action
Microbial disease manifests itself either as dircct damagc
to host structurcs and functions by cxotox.in.s or viruses, or
as damagc duc to host reactions such as those triggercd by
e11dotoxin or immU11e responses.
Direct Damage
Exotoxins are bacteria! proteins, which are often frccly cx-
creted into the environment. The differences between en-
dotoxins and cxotoxins are shown in "fable 1.1.
'lwo types of exotox.ins exist. One acts cxtracellularly or
on cell membranes, attacking interccllular substanccs or
cell surfaccs by enzyrnatic or c..lclerge11l-like 1nccha11isms.
It includcs, for ex;implP., bacteria! hemolysins, leuko-
cidi11s, collagenases, and hyaluronidases, which may play
an ancillary role in infections.
·rhc othcr type of exotoxin consists of proteins or poly-
peptides that en ter cells an d enzymatically disn1pt cellular
processes. 'I'hesc usually consist of an A fragment , \"1hich
has enzymatlc activity, ar1d a B frag1 ne11L, wh.ich is respon-
sible for binding the toxin to its target cell. Exotoxins are
e11coded chromosomally, on plasmids, or on bactcrio-

Table 1.1. Exo- and Endotoxins Compared
Exotoxins
Oftcn spontaneously diffusible
t'rotems or po1ypept111es
Produced by gram-positive and gram--negative bacteria
P1udule a )inyle, pharrnacologically specific effect
Each is distinct in structure and reactivity a<cording to
its bacteria! species of origin
Lethal in minute amounts (mice : nanograms)
Labile to heat, chemicals, storage
Convertible to toxoids (= nontoxic, immunog-enic toxin-
u~r ÍV<SlÍV~)); ~lid l i!lllilUXÍll prodUttiOll
phages. 'fh e function the toxins serve th<> hac:tr.riri is not
known.
Viruscs produce injury by destroying the cells in which
thcy replica te or by nltcring ccll function, appearance, and
growth c11aractcr1st1cs.
Endotoxins are lipopolysaccharides, whích are part of
Lhe g1an1-negali vt: ct:ll wall. Tlit:y cu11:si:st uf puly:;accharide
snrfacr ch;iins, which m;iy be viruJence factors and so-
matic (0) antigens¡ a core polysaccharide; and lipid A,
where the to.xicity resides.
Lipopolysaccharides bind to lipopolysaccharidc-
binding prote1 n (a serum protcin), which in turn transfers
it to the blood phasc of CD14. The CD14-LPS complex
blnds to Toll-llke receptor protelns on the surface of
macrophagP cPll<; triggering the release of proinflamma-
tory cytokines. These substances eUcit n1anifestations of
endotoxemia, including fever, headache, hypotension,
lcukopcnia, thrombocytopcnia, intravascular coagula-
tion, intlammation, endothelial dan1age, hemorrhage,
fluid extravasation, and circulatory collapse. Many of
thcsc rcsult from l) activation of thc complement cascade
and 2) production of arachido11ic acid metabolites: prosta-
gl<111di11:-, lt:ukuldeut::-, a11d LlinJ111buxa11e~. T11t: piler1u1u-
f'na proctuc:f'cl hy P.nclotoxins closrly rrsrmhlr risprcts of
gram-ncgativc septicemias, but most of them can also be
duplicatcd by pcptidoglycans of gram-positive bacteria.
Chaprer 1 Parasitism and Pathogenicity 5
Cell-bound as part of ccll wall
t1popo1ysaccnande (llprd A1s toxic component)
limíted to gram-negative bacteria
Produce a range of efferu, largely due to host-
derived mediators
Ali similar in structurc and effect regardless of
bacteria! species of origin
lethal in largcr amounts (micc ª micrograms)
Very stable to heat, chem1cals, storage
Not convertible to toxoids
lmmune-Mediated Damage
Tissue damage due to immune reactions is considered
elsewhere (see Chapter 2). Co1nplement-mcdiated re-
sponses (such as inflammation) and rcaction5 rct.cmbling
lmmedlatc-type allergic phenomena can occur in response
to rnrlotoxins or to peptídoglycan without preceding
sen si liL.ali011.
Specific imrnune responses partic:ipatf' in thr patho-
genesis of many infections, particularly chronic !n"anulo-
matous intections such as tuberculosis. Lesions a:'e dueto
cell-mediated hypersensitivi.ty, which is established in the
~arly weeks of lnfection. Cell-mediated responses intensify
inflammatory responses and tissue destruction upon sub-
.sequcnl encounle1s wilh Lhe ageul ur ils vrulcili tl1ruugh
thc relcase of effector substances from T lymphocytes (e.g.,
cytokincs, pcrforins).
lmmune mechanisms apparently contribute to ane-
mias seen in anaplasmosis, and the haemotrophic my-
coplasmas. ·rhe antibody response to hemoparasitism does
not distinguish between the parasite and the host erythro-
cyte. Both are removed by phagocytosis.
 lllllllune Responses to
Infectious A gen ts
LAUREL] . GERSIIWIN
The imrnune response to infectious agcnts is comprised of
a series of i11nal.c and acquired mcchanisms that work to-
gether tO prevent lnfectl011, if possible, auu to COJllrol Ll lt::
pathogeoic effects of organ ism~ that arC' ahlc to evade the
ilútiaJ i11nale in11nu11e dcfenses. In recent ,vears t here have
hc>cn significant advances in undcrstanding several
heretofore unappreciated innatc dcfcnscs. Direction of im
mune responses tor appropriate anti-pathogen responses
by immunon1odulatory cytoki nes and appropriate m echa-
nisms of antigen presentation are now wcll defined. This
chapter emphasizes the current knowlcdge defining how
the irnrnUI1e :,y:,t¡;111 cu~Lon1i2es Lhc response depending
11pon thP typc of pathogen encountcred.
lnnate lmmunity
Anatomic Features, Physiological Processes, and Normal Flora
'Irauitio11ally, i111 1'1lt:: i111111Lu1ily has bccn con:;idered to be
composed of anatomic features. physiological functions ,
microbiological barricrs (thc normal flora), fluid phase
components, and ceUular constituents. t-or examplc, thc
mucosa covered nasal turbinates form an initial "sticky"
barrier to remove largcr particles from the inhalcd alr.
Smaller particlcs that gain access to the respiratory trllc-t
encou11ter t ltt cilia lt::d t::pill1elil1111 and 111ucus blanket that
pff0ctively produces a mucociliary apparatus; this works to
rcmovc inhalcd n1atcrial from the lower respiratory tract.
Within the gastro1ntcst111al tract, peristalsis and the vomit
rcflex have similar effects on removal of undesired mate-
rial from the body. The flushing actlon of the bla<.luer i:,
important in prcvcnting urine stasis, which prf'<lisposps to
IJacterial iufcct io11.
'fhe nor111al flora plays a11 iJ11portan t role in preventíon
of colonization by more viru1ent organisms. These bacte-
ria and fungí establ1sh a unique relationship with t he host,
a relationship that begins as the microbiologically sterilc
fctus begtns its journey <Iown the blrth canal. Acquisitiuu
of bacteria and fungi begins immediatPly, with infPction
(colonizatiuu) uf ali t::Xposed su1faccs, including rnucosal
s11rfacP.~ (alimen tary canal, upper respiratory tract, and
distal gcnitourinnr y truct), "'ith microorgonisms from the
birth canal and from tl1e 1nother's immediate environ-
ment. The association of rnicrobe with thc host is not hap-
hazard but rather is an association that depends upon 1)
6
receptors (usually 1n the for111 uf <.:'11 bol1ydratcs that are
part of glycoprotrins nn the surface of t11e host cell) and
adl1esins on the n1icrobe cell surfacc, 2) thc chcmicals in
the imrnediate environment ot thc microbe- l1ost interac-
tion, in part duc to products sccreted by competing mi-
croorganisms (e.g., microcins, bacteriocins, and volatilc
fatty acids) and in part dueto products secreted by thP host
(e.g., acid envlronmcnt of the stu111'1cl 1, defe11sü1s sccreted
by Paneth cells, the contents of hilC' in the uppcr small in-
tcstil1t, ur Ll 11:: 1..onle11L of sebun1 on thc ~kin), and 3) thc
availahility of nutrient substances.
The establishment of thc normal flora is a dynam ic one,
with replacement at various exposed Iocations with ml-
crobes more capable of living at a particular site (nichf')
than the ones preceding. In auditio11, lltt in1n1une systcn1
appears to play some role, sinrP it has heen shown that
111e1ubers of Lhe norn1al flora are very poorly immu-
nogenir in thf' host fron1 which thc microbes are isolatcd.
This suggests that in1mune responses to microbcs attcmpt-
ing to colonizc a particular locatlon (niche) will rcsult 1n
thc blockage of association between adhesin (microbe)
with receptor (host). Jf a microbc cannot associatc, then lt
'"'ill be replaced with one that "vil!. 'fhis occurs 11ntil ll
strain uf microlJ~ i~ e1H.:ou11Ltred Lhal is n1ore sin1ilar to thc
host than it.~ prP<l0ccssor, which is subsequently "acceptcd"
as part of the normal flora of that particular animal. Thc re
sult ís an ecosystem composed ot numerous species of bac-
teria and fungí that are associated '"'ith an abundance of
ruches, each of \\ºhlch is oclupieu wil11 i:1 particular species
of mícrobe most s11itP<l to liYP at that location. TI1is "occu-
JJl1tio11" no'.:>Ull:> i11 a barrier to colonization (infection) by
microhes that are not members of the normal flora, thus
the term colo11iz(,tion resistancc.
Cellular Responses in lnnate Defensc
The innate phl1:>C uf the iu1n1unc response has bccn de-
scribed as a "speed bump" for pathogens. ·rhere are several
ce11 l) pes Lhal participate in innatt: defense. Thc phago-
cytic cell is of particular importance in detensc against bac-
teria! pathogcns. Thcrc are primarily two types of phago-
cytes in 1nammals, the polymorphonuclear Jeukocyte
(11eutrophil) and the macrophag.: Whilf-> thP ni>ntrophil'~
1nain role i:, reuLoval uf foreign n1aterial a11d t hc killing
and sub~P<¡11Pnt rP1noyaJ ofbactcria, the macrophage plays
<t tlual 1ole by acling asan antigcn presenting ccll for initi-
 Chapter 2 ln1111une Responses Lo Infeclious AgenLs 7

atio.n of an acquired immune response_ A third type of cell
that is important in the innate response against viruses is
lhe natural killer cell.
The Neutrophil. Thc polymorphonuclcar lcukocytc, ncu-
trophíl, is a bone-marrow- derived end-cell that normally
comprises 30% to 70% percent of the total leukocytes in
the peripheral blooct of various species. The neutrophil is a
gr::inn l{)r'ytir lPn k{)rytP ::inri rr)nt::iin <: two typP<: ()f gr;.in1.1 les·
primary or azurophilic granules and secondary or specific
gra11ules_ Neutrophils spend 011ly about 12 hours in circu-
lation, then go into the tissucs where they survive for an
additionaJ two to three days. Within the bone marrow
there is a large storage cornpartment for neutr.ophils. A
bacteria! infection within the lJody causes a rapid mobl-
lization of this pool and thP. nP.utrophils ac.c.11m11l<itf' <it thf'
site of the infectious process. They are attracted by the
chemotactic factors, C3a and CSa, which are generated
subsequent to activation of the complement system. The
process of neutrophil accumulation begins by adhcrence of
the circulating neutropl1ils to the vascular endotheliurn
(111argi11atio11), exlruvusuLiun i11to ti:;:;ue space:;, and chemo-
taxis of the cells toward the focus of injury. Tnvacling mi-
croorganisn1s are ingested by neutrophils in a process
called phagocytosís (Figs 2.1 and 2.2).
Phagocytosis of bacteria by neutrophils involves severa!
steps. First, initial recognition and binding occur. This
process is made more efficie11t by the presence of opsonins
con:>i:>tir1g of irnrr1unoglobulin and/or complement con1-
ponents. Opsoni7.<ition co<its t hf' s11rf<ice of a partic.le, neu-
tralizing the net negative charges, which n1ight otherwise
cause the neutrophil and bacteria! cell to repel each other.
In addition, on thc ccll mcmbranc of thc ncutrophil, rc-
ceptors are present for antibody (Fe receptors) and for com-
pleme11t (CR). These receptors facilitate firm attachment
of the opsonized bacterium to the neutrophil. Next,
pseudopoclia for111 arou11d tht: orgaui:>ru a11d ther1 fu:;e to
form a phagocytíc vacuo/e containing the organism. Sorne
organisms are more rcadily cngulfcd than others. f.or ex-
ample, the presence of a polysaccharide capsule causes an
organism to be resistant to phagocytosis. J\fter engulf-
1nent, lysosomal granules fuse with the phagosome mem-
br.;.ine to fo(tll the phngol)lSOsome. 'l'he eventual eliminati()n
of the engulfed organisn1 occurs witl1in tl1is slruclure.
Bacterial killing is accomplished by a series of metabolic
and enzymatic events. Metabolic activity increases within
a neutrophil during phagocytosis. Oxygen consumption
increases and light energy is emitted (chcmilumincs
cence). ·rhis metabolic or respiratory burst involves oxida-
tion of glncose by tl1e hexosemonophosphate shunt. Bac-
tericida! p roducts are ge11erated. Superoxide radical:> are
produced and converted to H 2 0 2 by superoxide disrr1utase.
Hydrogen pcroxidc is toxic for bacteria that lack catalase.
The enzyme myeloperoxidase, present in azuropl1il gran-
ules, catalyzes the oxidation of halide ions to hypohalite,
which Is also toXic to microorganisms. Thus the myeloper-
oxici<ise-hydroge11-peroxide-halide system is efficient in
bacteria! killing. Susceptible orga11is111s are killed vviLhi11
rninutes. lnside the prilnary granules of neutrophils, en-
zymes rclcascd during dcgranulation act on protcins,
liptds, carbohydrates, and nucleic acicts to degrade the
killed bacteria! cells. Some of these enzymes are collage-
nase, elastase, acid. phosphatase, phospholipase, lyso-
zyme, hyaluronidase, acid ribonuclease, and deox yribonu-
clease. Lysoz;yrne can cleave glycosyl bouds i11 lhe bacLerial
ceJJ wall, making the cell susceptible to lysis. Also, lyso-
somcs contain cationic pcptidcs (dcfensins) that form
lethal pores in bacteria as well as fungal cell walls.

F 1G U R E 2 . 1 . NP.11trophil response toan infP.dious agP.nt: A. Neutrophils arP. prP.sP.nt in thP.

circulation. 8. Neutrophils express adhesin molecules (CD18) and adhere to the endothelia/ ce/Is in the
blood vessel. This process is cal/ed margination. c. Neutrophils pass rhrough rhe endorhelial ce/Is by
díapedesís. D. Neutrophí/s, now extravascu/ar, respond and move along a chemotactic gradient.

A B e D
8 PART 1 I11troduclio11

F 1G U RE 2. 2. The process of phagocytosis: A. A bacterium is opsonized by antibody. The antibody

binds to ;:in Fe receptor on a phagocyte. B. The phagocyte begins to engulf the attached bacterium.
C. The phagosome containing the bacterium fuses with lysosorr1e~ ;,, !11e µhdyutyte tytoplasn1 to forn1
a phagolysosome. D. The bacterium is killed and digested. E. The bacteria/ breakdown products are
eliminated from the ce//. Some parts of the bacterium wi/I remain on macrophage membrane associated
MHC lid}} /1 10 be used in antigen presentation to T ce/Is.

•
- A B e

o E

Macrophage. The macrophage is a mononuclear cell de-
rived from the bone marrow. For severa! days after release
from the bon e marrow, it circ11latPs ;:is a hlood rnonocyte hc-
fore going into the tissues, where it becomes a functional
macrophage. Free macrophages are prcsent in many parts
of the body and are named accordi ngly, for cxa1nplc, alve-
olar macropl1ages (lung) and peritoneal macrophages.
Fixcd macrophages line sinus cavities that filter blood.
These lnclude the Kupffer cells (Uver), Langerhans cell~
(skin), histiocytes (connective tissue), mesa ngial cells (kicl-
11ey~), a11d sinus-lining cells of the spleen., lymph n odes,
and bone marrow. Sorne of thesc macrophages are impor-
tant in antigen processing for induction of an immune re-
sponse (described later in this chapter).
The macrophage differs from the neutrophil in that it
has a longer life span in tlssue and can reuse phagolyso-
somes. 111 additio11, macroph;iee<> stim11 l;itprl hy rytokines
(e.g., i11lerfero11) or n1icrobial products (e.g., lipopoly-
saccharide) result in activation of n itric oxide synthasc
that catalyzcs thc production of nitric oxide (NO) from
l-arg1n1ne. NV 1s toxic to many bacteria, espec1a11y those
residing 1N"ithin macrophages (e.g., Salmonella, Listeria).
The macropl1agt: is silnilar to llie 111:ulrupl1il it1 ll1al loxic
oxygen metabolites are generated for h;ictpri;il killing and
Lhe lysosomes contain potent hydrolytic enzymes and
cationic peptides (defensins) WhiJe the ne11trophil rP-
:.µ0111.l:. lo a sli111ulus rapidly, lile n1acrophage is n ot p res-
ent until later in an infectious proccss, often after 8 to 12
hours. In sorne instar1ccs, ncutrophils may eliminate an
organism before macropl1agcs arrive in any great number.
When tissue destruction has occurred as a result of an in-
fla1nmatory response, macrophagcs are attracted to tht:
area by products from dying neutrophil" and hac.teria.
·rhey pl1agocytose the debris and remove it. In sorne in-
stances, macrophages may engulf particulate material that
they are unablc to digcst. Whcn this occurs, the macro
phage may migrate to a mucosa! surface such as the respi-
ratory or gastrointestinal tract for elirnination from thc
body.
Sorne microorganisms !lrP morP e.asi ly killed by phago-
cylcs than others. Bacteria that have polysaccharide
capsules are less readily engulfed than unencapsula ted
countcrparts. ()psonizing antib<>dy or complement corn-
ponents must be present to coat such organisrns before
phagocytes can engulf them (as described above) Sorne rni-
croorganls1n:; are rea<.llly !Jlld!)Ul.ylu;)eu, 1.Jut <Ht: able Lo
grow intracellularly. For example, microorganisms such as
Listeria 111011ocytogenes and Mycobactcrium tuberculosis are
not killed after phagocytosis by macrophages. Organisms
may produce factors that inhibit phagolysosome fusion or
 Chupler 2
even escape the phagolysosome into the cytoplasm, thus
preventing exposure to enzymatic degradation. These or-
ganis1ns rcquirc n1acrophagc activalion by i11lerferon ·y, a
product of both NK ce lis and activated T helper 1 cells.
Natural Killer (NK) Cells. Natural killer cells are a central and
important component of the innate immune response to
vlruses and sorne bacteria. ·rhe NK ceu is a distinct lineage
of lymphocyte that, unlike the T and B lymphocyte, does
not have a specific receptor for antigen-i.e., tl1ey do nol
rearrange genes encoding membrane receptors. Yet, this
cell type is able to recognize and target cells for destruc-
tion. NK cells comprise trom .)-:lUo/o ot Jymphocytes in the
peripheral blood and more than 90o/o of lym phocytes in
the placenta. 'fhelr functlon lncludes cell mediated immu-
nity, :inti horly-rlPpPn<lf'n t <'Plh1 l;ir cytotoxir.ity, produc-
tion of interferon yearly in lnfection, and secretio11 of a va-
riety of other cytokincs. 1n the ad u lt m am mal NK cells
develop within the bone marrow from hcmatopoictic ccll
precursors. NK cells are responsive to several cytokines and
they secrete cytokines, including interferon y, and tumor
necrosis factor a. (TNFa.). The NK cell response is coordi-
n;itpcl ;inri mnrl11l1itPrl hy rytnkinP( th;¡t inr.lude interfer-
ons Cl and ~ and lntcrleukins 2, 12, 15, and 18. Recently a
variety of reccptors havc been identified on NK cells. These
receptors allow thc NK ccll to cithcr kill a targct ccll, orto
turn off the potent1al k1ll1ng response. Inhibitory recep-
tors expressed on NK cells includc the killer immunoglob-
ulin-llke receptor (KIR). This receptor binds to MHC class I
moleculcs on body cclls and facilitates an inhibitory re-
sponse lh1ough aclivalion of inuacellula1 Lyrosi11e- ln1=>ell
inhibitory motifs. This function prevents the powerful
killillg capacity of thc NK cell from acting on normal body
cells. ln contrast, when a cell Jacks MHC class 1, the NK cell
recognizes the absence and begins the process of killing
that target ceu.
The NK cell is particularly effective in eliminating
tumor cells and cells infectecl wlth sorne viruses. A number
of vin1sf'~ (:inrl ~nml' ti1mor rf'lls) rlown-regulate cellulilr
MHC class l 1nolecule synLhesis. 'l'hese vi1uses effeclively
evade the acquircd T cytotoxic cell (CD8) response (de-
tailcd latcr in thi:; chaptcr) by rcmoving th c 'lvfHC molc -
cule tt1at presents antigen to cytotoxic T cells. However,
the NK cell targets these cells that display the "missing
self" appearance. NK cells are important in eli1nination of
hPrpPs vir11.~f'S . For l'x:implf', in h11m:in hPings thatJackNK
cells, the diseases varicella zoster and cytomegalovirus in-
fection (both herpes viruses) are often fatal, whereas the
normal individual is ablc to succcssfully recover from
tnese 1nfect1ons. 1 he mecnan1sm by whicn tne NK cell
kills the target involves secretion of perforin molecules
llii:tt C:1rc étlJh: tu puke hules in the cell membrane, permlt-
ting caustic granzymf'~ f'ntry tn thf' rytnsol.
The carly production of interferon y by NK cells is 11elp-
ful in injtiating a T helpcr 1 type response {described later
in this chapter) that activates macrophagcs for more cffi-
Cient killing of sorne bacteria. Other killing mechanisms
includc antibody-dependent cellular cytotoxicity (ADCC).
NK cell:; lli=>pléty Lltt: ccll 11u.:1n1Jrane receptor CD16, a low-
affinity IgG receptor. Ry 11sing this rf'<'Pptor to billd IgG,
the NI< cell is able to participate in ADCC, ki1Ji11g cells ll1al
Iuuuune Responses to lnfectious Agents 9
are recognized by the attached immunoglobulin. In th1s
way the NK cell collaborates "l>Vith the acquired immune
sysle1u lu eli111i11ale i11fcctcu ct:ll=>.
Gamma-Delta T Cells. T cells that display membrane recep-
tors called yo chains (gamma-delta T cells) constitute a
small percentage of the circulating pool of lymphocytcs ill
nonruminant species. In ruminants, however, gamma-
delta T cells may rcpresent up to 30% of the circulating
Jyo1phocyle poul. I11 111ust spccics, garnrna-tlelta 'f cells are
pre.sP.nt in thP l:imin:i propria underlying mucosa! epithe-
lia, strategically beneficia! sites for cells il1volved in host
detense.
Functional studies in mouse models and also data from
human bcings has demonstrated a role for these cells in
defense against mycobacterial pat hogens. The role of
gan11na-deJ La T ct:lls i11 i1111ate defense again st l\1ycobacte-
riurn tuberculosis appears to he most import :int f''1rly in the ·.
infcction. Activation of these cells by M. tuberculosis anti-
gens is dcpendent on accessory cells, such as alveolar
macrophagcs, which providc co-stimulatory molecules.
The l1gands on the rnycobacterium that stunulate the
gamma-delta T cclls have recently been shown to be small
phosphale-cvr 1lai11i11g u1ulecules. The major effector func-
tions of gamma-delta T lymphocytes in <leff'nsf' against M .
tuberculosis is in cytokine secretjon and in cytotoxic effec-
tor cell tunctjon. These cells produce interferon y, 'l'Nfa, r
anda smaU amount of IL 2.
Gamma-delta l ' ce lis ha ve a role 1n limiting viral replica-
tion in sorne viral diseases. In mouse models of influenza
vi rLI:> iufc<.:tlun these ce lis accumulate in the lung presum-
ahly to resolvf' thf' pnf'11monir prorf'~. Tn both mouse
n1odels and in human patients the role of gamn1a-delta T
cells in resolution of herpes simplex-1 infection has been
demonstrated.
Toll-like Receptors. ·10!1-like receptors (TLR) are a primitive
mechanlsm of lnnate immunity. They are defined as
pattern-recognition receptors and are p resent on a variety
uf i111111u11e a11tl utlier cclls. Originally described in Droso-
phila, these molecules and thei r signaling p:ithw:iys :irf'
important for dctcction of invud in g puth.o gens in fruit
flJes, mammals, and ptants. ·rhere are 10 k.11own ·rLR jn
mammals. Jlach 'fLR recognizes a specific compon en t of a
pathogen. for exampte, TLRl , 2, and 6 recognize various
inicrobial cornpon ents. TLR2 recognizes lipoproteins,
lipoleichoic acid fio1n gra111-pu::.ilive lJacteriC1, and lipoara-
hinoman nan from myroh:irtPria . TLR3 recognizes double-
stranded RNA, and is therefore in1portant in viral recog-
nition. ·rtl<4 has a role in transducing the signals of
lipopolysaccharide (LPS) from gram negative bacteria.
·rLR5 ts act1vated by flagellin, its spectfic ligand. Thus.
TLRS is important in the response to flagellated bacteria,
bul nol tu Ll 1v:.e ll 1at C1re not. When the baso lateral surface
of the intestinal epíthelium is e.xposerl to fl:igellin an in-
flammatory response occurs. This is the site of expression
ot the ·rLRS. ·rLR7 is activated by certain synthetic com-
pounds that have anti viral activity. TLR9 rccognizcs bac-
teria! CpG motifs in DNA. These motifs have recently been
identificd as important immune modulators (stirnulating
a T Hl res¡.¡uu~c).
1O PART 1 Tntroduction
RPrognition of viruses by TT.R has hcen reported for
sorne viruses. 'fL1~4 binds to onc of thc major surface pro-
teins on respiratory syncytial virus (l~V), an important
pathogen of human children and bovine calves. In an ex-
perin1ental mouse model with mutated 1'LR4, RSV was
found to be cleared less efficiently than from mice that
have normal TLR4. Otl1er vJruses, such a:> rnuuse II1a1u-
mary tumor virus, have been ~hown to hincl 1'I.R4 to envc-
lupe p1olelt1s. 111 addition, signaling through ·rLRJ and
·rLR7 has been shown to induce synthesis of alpha and
beta intcrfcrons.
Other studies with ·rLH. mutant mice have demon-
stratcd the importance of thcsc receptors in resistance to
bacteria! lnfection. TLR4-mutant mlce are higlrly suscepti-
ble to infection with the gram-ncgative bacteria, Salmn-
nellu lyphirnuriurr1 . Whereas, 1llicc dcfective in TLR2 are
highly sensitive to infection with g ram-positive bacteria,
Streptococcus pneun1oniae.
Acquired lmmunity
Generation of the lmmune Response
The initial step rcquired for initiation of an acquired
immune response 1s presentation of an antigen to T lym-
phocytes.
Antigen Presentation. There are severa! cell types that are c.a-
pal>lt:: uf a11tige11 presenlalion: de11dritic cell, macrophage,
.B lymphocyte, anrl speriali7.Pd c.el ls in the skin called
Langerllan's ce/Is. Of these cell typcs, the dendritic cell is
considered to be a "professional antigen presenting cell"
antl as such is most efficient at antigen presentation. Tt is
currently bel1eved that the dendritic cell involved in pri-
mary antigen presentation is rcsponsible for determining
what type of iuuuw1e re::.)JoH::.e i::. geue1 aled in response Lo
the pathogen . Suhsets of dcndritíc cells are responsive to
different cytokines¡ thcy in turn produce cytokines that
influence the 'l' Cell response towards a humoral ('fH2) Of a
ccllu lar Cftn rcspo11se). For example, production of lnter-
Jeukln 12 (IL-12) by c.1endritic cells is required for initita-
tion of a Tu 1 response in T lymphocytes. Binding of 'l'LH.
by dendritic cells ar1tl )Jhagucyte::. U1dl pi esenl a11Ligc11 ir1-
fl11PnrP~ the type of response that is induced, so that it
is appropriate for thc type of pathogen that is invading
the host.
The proccss of antigen presentation occurs either by
the extraceUular (phagocytic) pathway, or by the endo-
cytic (cytosolic) pathway. Jnactivatcd and killed organ-
isms, once digestetl a11J )Jro<:e::.:.eú iu a pl1agoson1c, are
ho11nd to (major histocompatibility) MHC class II mole-
cules and are brougl1t to thc ccll surf<icc for presentation to
CD4 ·r (helper) lymphocytes.
Recognition of the aotigen/MHC class 11 complex by a 'l'
cell wíth the same MHC: class ll ls refcrred to as MHC-
reslrictio11 and is a characteristic of the arquirt>rl immune
response. Productiun uf JL-2 l>y ll1e T <:ell occurs afler bind-
ing with the antigenic. peptide and co-stimulatory mole-
cules. Interleukin-2 is a T ccll growth factor that facilitates
clonal expansion of the participating 1· cell. ·rhese ·r cells,
which are phenotypically c:D4 and functionally called
hclpcr T cclls, produce additional cytokines to influence the
dcvclop n1ent of B ceus, which are spccific for the antigen.
Under the influence of ·r ccll-produced IL-4, B cells dc-
velop and rnature i11tu pla::.111<1 cells secreling antibodies.
1-IPlpPr '(' rells procltJC.P rred()minantly IL-4cr11elper 2 cells
v11'u2), whicl1 facilita le production of IgG 1 and lgE.
Another subset of T helper cells responds to presented
antigen by making other cytokincs. l 'hese cytokines are
important in act:ivation of macropbages for killing fa cu l ra-
tive intracellular bacteria and for supporting otl1er cell-
mediated responses. ·r helper type 1 cells produce i11Le1-
feron y and IL-2, ü1 addition to 11,.. 12. As noted above, the
i11ili<tl p1oduclio11 of IL- 12 by the dendritic cell or NK cell
can prejudice the T 11clper cell respon se towards 1'111 • 'fhis
response may be initiatcd through 'fLR binding to the dcn-
dritic cell.
J\ntigen presentation for intracellular pathogens fol-
lows the endocytic pathway through tl1e cylu:.ol. When a
virus is replicating in thP rytnsol, v iral proteins are proc-
c:.::.eJ ar1d u11iLed with MJ-IC class l mole<.."Ules. 1'hesc pep-
tide-MHC complexes are then sent to the surtace ot thc
cell where they are bound by the CD8 cytotoxic T cells.
Effcctor Function. 1'he ultimate result of antigen presenta-
tlon to CD4 T lympllocyLcs Is the development of an im-
mune response. As stated above this response is mPdiaterl
by the type of cytukir1e e11v i1 onn1enl ll1at has been cre-
ated. If the T H 2 cytokínes are present, and for most patho-
gcns there are usually sorne, thcrc will be a humoral (anti
body) response. When the pathogen has skewed thc T
hclpcr ccll response towards Ttti cytokine production (this
occurs with facultativc rntracellular bacteria a11d viral
pathogens), cytokines are produced to actívate 1nacro-
phages and CD8 ·r cells to become mure effective killer:..
Humoral lmmunity (The Antibody Response). The initial introduc-
tion of antigen to a host followed by presentation of anti-
gcnic peptides to C~D4 'l' cclls rcsults in stimulation of lhcse
cells to become ·r helper typc 2 cells secret:ing cytokines
that assist B cells in diffcrentiatiing into cells that become
antibody produclng plas1na cells. ProJuctiou of lnlerleukin
4 (ll.-4) by thPSf' T 112 rell~ results in expansion of B cell
clones specific to the dífferent epitopes on thc antigen. The
J3 cells also recognize antigenic epitopes on the microbc
and in additio11 develop binding of co-stimulatory mole-
cules on the T cell. Then under the influence of ·r cell cy-
tokincs, these B cells differentiatc into antibody-produring
plasma cells (Fig 2.3).
The fir.~t antihorly to he produced is lgM and it appears
in the circulation 7 to 10 days aftcr initiation of the im-
mune response. Next, TgG appcars but does not rise to very
high titcrs in this primary immune response. Subsequent
c11counters ;vith antigen rcsult In a secuuuary ur <111a111nes-
tic response. In the secondary r<'~ponse, the kinetics of an-
tiliuJy appearar1ce lt1 Lhe circulation are more rapid and
the quantity of antibody produced is greater. Most impor-
tantly, the isotype that predominates in the sccondary re
sponse is predomjnantly lgG. ·r11e longer half-Jife of IgG fa-
cilita tes the maintenance of an antibody titer higher for a
Jonger duration of time. Often evaluation of the immunc
 Cltapter 2 J1nrnuue Res¡;o11ses tu h1fectiuu~ Agent~ 11

F1G U RE 2 . 3 . lnteraction of antigen-presenting ce//, T helper ce//, and 8 ce// in the

production of antibody.

+ Antigen

•
l'W'J . .

~º'\ ¿_ _ TL-4, TL-5

\( )) ....__, ' ti·o1n 1'h2 cell

L>evelop1nent of
Expru1sion ofB plasma cells
ce 11 elon!".'> mak.ing
antibodies
Antigen-presenting
Th2 cell cell

response (lgG versus IgM) to a disease agent can yield im- have capsules are particularly resistant to engulfment hy
portant information as to the chronicity of the exposurc. phagocytcs unlcss thcy have opsonins present.
It is a well-accept ed diagnostic procedure to obtain acute Generally, the IgE response is limited to parasitic infec-
and convalescent serum samples to be evaluated for anti- tions and hypersensitivity reactions to various cnviron-
lJody tlter an<l tsotype. Cienerally, when a cllsease agent is mental allergens, such as pollens and grasses. Occas1onally,
rPsponsible for clinical sign.s two to three weeks after the IgE is elicited in response to vaccination against sorne bac-
initial appearance of sign.s, the titer will l1ave h1creased by lerial a11d viral palhngeu::;. Whe11 tlll::; occur~, very ~eriuus
at least fourfold if the agent was in volved in the infectious aclvP.rsP. rP.sponsPs, s11ch ;:is ;:in;:iphylactic shock, can result.
process. In an initial exposure to a disease agent, IgM is th.e Often tl1ere is a hereditary predisposition toward IgE pro-
predominant isotype, while a second or tertiary exposure duction and these individuals are at increased risk of hav-
(or vaccination) will elicit mostly JgG. ing such a vaccine reaction. For immunity to parasite infec-
tions, IgE may assist in the phenomenon of "self cure," in
Effector Functions of Antibody. Antibodies can neutralize which large numbers of nematodes are purged from the ¡,rut
virus, bacteria, and soluble toxins. Sorne isotypes of anti- by mast cell mediator-induced smooth muscle contrac-
hndy (TgG, TeM) Ciln lysf' tilT8Pt CPlls ;:iftf'r ;¡('tiv;iting thP tion AltPrn;itivPly, ~nmP i nfP~t;itinns ilrP. contr.olled by :.:in-
complement cascade. Antibodies can facilitate phagocyto- tibody dependent cellular cytotoxicity (ADCC), in which
sis by acting as opsonins to help phagocytes adhere to mi- IgE binds to eosinophils by low-affinity Fe receptors and fa-
ccobes. The antibody response tbat is important in defense cilita tes release of major basic protein and othcr caustic cn-
again.st bacteria! disease depends on the pathogenicmech- zymes on the parasite surface.
anisms involved, the site of th.e infectious process, and the
isoLype of Lhe a11Libody elic:iled (Tal>le 2.1). Ir1 a <.lisease
caused by an extracellular toxin, such as tetanus, antitoxin Table 2.1 . Effector Functions of Antibody
antibodies are important to neutralize and bind tl1e toxin
before it can biI1d to cellular sites and initiate clinical Sife of Actione
signs. Th·is mechanism is ilnportant in diseases such as
tetanus, anthrax, and botulism- all roxin-mediated dis- lgM lntravascular Complement fixatíon,
eases. In some instances, when a nonimmune host is at agglutiaation
risk of developing a Loxin -n1edi.aLed disease, iuuue<.liate a<.1- lgG lntravascular CómP.lei;ner1t fixation,
ministration of antitoxin (a solution containing antihocl- neutralization
ies to the toxin) is required to prevent the disease. In order Ti,,ue 'fli:lt't!' Opsonization, ADCCº
to eliminate intectious agents, antibodies serve as opso- JgA Secretions: respiratory, , Neutralízation,on
nins as well as initiating the complernent cascade (activa- GI, salivary mucosa! surfaces
tion through the classical pathway). Opsonins lead to in- lgE 5ubcutaneous Mast-Gell sensitization,
creased uptake by phagocytic cells, whereas complement ~ubm.uc0sal ADCC
activation leads to il1ilialion of iufla1111uatio11 an<.l genera-
tion of compounds that are detrimental to thP. infP.<:tions ªAntíhorly-ilepP.n8eot r.ellular ~t~dty
agcnt (e.g., 1ne1nbrane attack complex), as bacteria that __. . .
 •
12 PART I Introduction
lgA is a ver y desiratJle response to infectious agents that
affect mucosal surfaces. Since secretory IgA (STgA) is pro-
tected by a secretory component from digestion in the gut
t>: proteolytic e11zymes, it is the most efficient antibody to
be active in the cnvironment of thc gastrointestinal
lumea. Ihere lt ca11 neutralize viruses and bacteria to pre-
wnt tbeir respective attachn1ent to cellular receptors.
.:>Unllar!y, SlgA ls effect:lve wltl1l11 the secretlor1s of tt1e re::;-
piratory tract. Refore a virus ora hac.terinm can infecta cell
it must first bind t o a ccll surface protein that acts as a re-
cep:or for the infectious agent. Thus. binding of the infec-
~o:lS agent by antibody can inhibit bü1ding to thc rect~p -
:, a.J.d thus lower the intectivity ot the agent. For
_11a:n¡J'.e. influenza virus exprcsses a hernagglutiJ1in that
biI1ds :o certain glycoproteins expressed on epithelial cells
~r.. ~he respiratory tract. Bindi11g of a11tibodies to thc he-
::nagglutiniI1 preveuts e11try uf the virus iutu tl1e:,t: <.:ell:,,
and di<>ea<;e is preventcd.
Antibodies are 1nost effective against viruses that un-
dergo a vire1nic phase, when there are numerous virus
particles in the extracellular environment. Viruses such
as intluenza virus, for example, are r1eutralized by antibod-
ies specific for the major surface antigens (hernagglutinin
and neuran1inidase). Other viruses- herpes virus, for
exainple-r.emain very closely assoriatt>ci with ct>lls ;inci clo
n ot present n1uch opportu11ity for antibody-111ediated inac-
tivation. ·rhe IgA isotype is especially effective on mucosa!
su1·faccs and fu11ctions to ncutralizc virui;cs bcfore entry
into the body. Secretory IgA is an extremely effective de-
fense against respiratory and gastrointestinal viruses, as well
as viruses tl1at cause systemic disease but enter vía the oral
route. Virus neutralization occurs because the antibody
binds to surface detern1inants of the vlrus a.n d prevenls ll1e
virus from binding to the cellular receptors to vvhich it n1ust
attach in ordcr to initiatc thc infcctious proccss.
The relat1ve 1mportance of ant1body and ce11-mearatec1
inunune respo11sc depends u pon the pathogenesis of the
<.lisease. Fur t'.Xdru¡;le, l.Jacteria tl1at produce potent exotox-
i ns, su ch as Clostridiurn tetani, require a11tibodies to neu-
tral izc the toxiJ1. Heavily e.n capsulaled bacteria, sucl1 as
.Klebsiella pneurnonía, require opsonizing antibodies for ef-
fcctivc i;cmoval of thc bacteria and ulti1natc killing by
phagocytes. ln contrast, bacteria that are capable of living
within pl1agocytcs, such as Listería rnonocytogenes, are not
effectively killecl by an antibody, and requlre the ·r Hl
respo11s<~ Jor cffective eli1nination. Sin1ilarly, viral infec-
Lio11s th.at produce viren1ia, sucl1 as il1fluenz:a, are l1<111dled
well by an appropriate a11Ubody response; compared witb
herpes viruses that are cell-associated and require a cell
mediated imn1une response for effective control.
Cell·Mediated lmmunity. Cell-medlated ilnmune responses
mediated by T cells involve two different mech.anisms: ma-
cropl1agc activation ar1d cytotoxic T cell activity (Table 2.2).
Killing of Facultative lntracel/ular Bacteria by Activated Macrophages.
A variety of bacteria have the ability to prevent phago-
son1e-lysome fusion. Bacteria that fall into this category
include Brucella, !v'lycobacteriun-1, Listeria, Saln1onella, Rho-
dococcus equi, and others. Wl1en tl1is occurs, tl1e macro-
phage is oflen killed by Ll1e bacteria, ratl1er Ll1a11 tl1e ex-
Table 2.2. Effector Funaions of Cell-mediated lmmune
Responses
Medianism
CDS MHC class 1with pept1de Perforin, granzymes, fas-fas
li9and, TNFa
M:icroph:igc with intr3ccllul3r lntcrfcron y mcdiatcd
organtsms activaJion of macrophage
NK CPll fell~ l~ckin!J MHCI PP.rforin-mP.rfüitP.ci
pected opposite response. In the presence of CD4 ·r lyn1-
phocytes that make the so called ·r helper cytokines, thc
1nacrophage is armed and able to circumvent the bacteria
and to kili. The cytokines induce lysosomal fusion and in-
crease macrophage !Jacteriocitlal activity. Macroµhagt:::. are
ac.tivated fol lovvi ng the production of interferon y by these
'fHl cells. This subset of T helper cells is stimulated subse-
q uent to the production of IL-12 by intected macrophages
an<l NK cells. Th1.1S, the "arn1ing" of inacrophages results
in destruction of the infectious agent that tl1e macrophage
prcv.iously had been unable to destroy (Fig 2.4).
Killíng of Vir11s-lnfected Ce/Is by Cytotoxic TCe/Is. Pathogt>ns, s11rh
as viruses, ll1at live a11d grow inside of cells are best con-
tained by elirnination of the cell in which they are repro-
ducing. Viru:; protcin.:; nJ.ad<: in th<: cyto::;ol are ablc to ar.no
ciate 'INith surface molecules of the cell for presentation to
1' cells. Cytotoxic T cells (CD8) recogn.ize an antige.n.ic pep-
tide that is l1eld in the groove forrned by the chains of the
MHC class I molecule on tl1e cell surface. All nucleated
ce.lls llave MHC cla~s I 011 t.l1eir surfa(.:e a11<.l are LJ terefure
ahle to hind and present antigen from inside the cell in
thi:; manner. Recognition of the combination of MTIC
cJass l ana antigenic determinant by cytotoxic J' cells (by
;vay of aspecific 'f cell receptor) results in the release of pe:-
forins that make small pores in the cell membrane. 'fhis a!-
lows destructive enzyn1es called granzyrnes to enter the C) -
tuplC;1su1. In C;1d<.li.tiu11, tuu1ur uec.rusis fdctor u. ls ¡;rutl ut=~­
Death of the cell is facil itated hy the interaction of Fa<;-Fas
ligand system, stin1ulating apoptosis. One CD8 T cell car:
repeatedly kili infected target cells, programn1ing one x
d.ie, and the11 movi.ng on to the next ce11 to kili it (Fig? .5
The cyrotox.ic T ceu is an efficient way to decrease \42.
progeny in an infected host.
Effector Ce/Is Can Use Antibody to Bind Target Ce/Is. Antibod' ct...=...
pendent cellular cytotoxicity (Ai)CC) occurs wl1en ar;u
body binds to a cell that has receptors for thé Fe portlO!'
im·n 1unoglobuJin G or E. The Fe receptor for t he ga:::=~
chain is CD21 and the luw affinity lgE rt:(.:e¡.>tur i$ CD2.3
'l'ht>Sf> molecnles are prt>sent on several types of eifectr.c
cells, includir1g neutrophils, 1nacrophages, NI< cells are
eosinophils. 1·11e attachment of antibody to a cell tha: p..--e-
viously had no receptor for antigen renders it and:c~
specific and capable of binding antigen. Besides ~1~ >:c-ll3,.
eosinophils and macrophages can also beco me in\·o,-ec o
ADCC. ADCC is an effective u1ethu<.l uf killiu~ ~ ·
ferteci with microorganisms (viral, hactPrial, or ;'m:=
well as parasites. In the case of parasites, the eosir ::- - ,....._
 0~814
Chupler 2 !II1111uuc Responses tu lnfectious Agents 13

F 1G U RE 2 . 4 . Destrudion of an intracel/ular infectious ae1ent by activated macrophages

stimulated by T helper ce/Is.

o
o
lFN y

• •
Macrophage infected CD4+ Tcell Activatcd n1acrophagc has fusion of
" 'ith facultative activated by ly::.osuu1c::. w itJ1 pliugoso111t: unJ
intraeellular bacteria;
bacteria! kills and digcsts intracellular
phago-lysoson1e fusion is
inhibited and killing
anti gen bacteria nftcr contact wi th y
docs not occur lntcrfcron fro1n ( ;fJ4 1 ht:lpt:r ct:ll, ,.
type l

F IGUR E 2.5. DP'1"r11rtinn nf ;¡ vir11s-infected ce// by CD8 T ce//. u

:r: ,...
CDS T Cell
programs First cell uies and T u~c
viru s- ccll movcs on to
p rogram the neX"t
infected cell
iufecled cell to die
<i -
to die
:J ..J
-
m
• • -
m
.::::. ...
i!lllli

leases granules contalnlng major basic protein rendering
the cuticle of the parasite permeable.
the 1n VIVO sk1n tcsting for cell-mediated responses has
been of even greater use. For determination of recent infec-
tiun status, serum samples are obtained during acute ill-
;incl ;ig¡¡in ?. to 3 ~vccks later. 'fhese acute and con..,:a-
np<;<;

Evaluation of lmmune Responses to lnfectious
Agents
The use of serological assays to evaluate exposure to or in-
fection wilh baclclial and vi1a1 paLhugens has ueen a
mainstay for control of infectious diseases. Tn addition, for
so1nc infcctions, such as those with mycobacterial species,
lescent samples are thcn assayed for anLibody liler u:.i11g
one of the methods describcd in Table 2.3. When the titer
has increascd nt lcast fourfold (two clilutions), seroconYer-
s1on has occurred and the disease agent tor ,,·ruch the
titers are specífic is confirmed as having stimulated a re
cent response. Table 2 .3 lists the common types of immun-
ocli;ignostic tests and sorne examples of disease for \,;hicb
the method has been/is being used.
14 PARr l Jntroduction

Ta b 1e 2. 3. Assay Methods for lnfectious Diseases

Type of Assay Application/Example

Precipitin in gel
. lomplement fixation
Agglutination
lndirea lmmunofluores<.ente
Direct lmmunofluorescence
Virus neutra!ization
ELISA
Coggin's test for antib-Ody to equine infectious anemiavirus (EIA}
Antibody titer to Bruce/la abortus
Antibody titer to harleria/SalmonPl/a
Anti!Jody ti ter to canine distemper virus (CDV)
Antigen detection in cells: CDV, feline leukemia virus (FeLV)
fuoctional titer of antibody thut prcvcnts virill infcction; cquinc influenza,
bovine respirato¡y syncytial virus
Detection of antigen (FeLV, parvovirus} or antibody ag11in~ viral, harterial,
fungal, and protozoan antigen)

Antibody-Based Serology
The current trt:!11u fvr i111111u11odiag11osis utilizes the solid
phasi>-hinding assays, such as thc e nzyme-linked ü11mu-
nosorbent assay (ELISA). ·rhcsc assays are generally more
sensitivc than assays based on precipitin formation or
complcmcnt fixation. Depending on the disease to be di-
agnosed, ELISA can be designed to detect antigen (as in fe-
line leukemia virus and canine parvovirus infections) or
antibody. E.LISA has the atlvantagc tl1at ll 1e fo1 rnal is easily
adaptable to eithPr ;i q11ick (positive or negative) read-out
oc Cl titcr dctcrmina tio n.
Cell-Mediated lrnrnunity-Based Diagnostics
For those pathogens that induce a strong ·r helper 1 type
immune response wlth production of associatt:!d cytvki11e:;
(e.g., interferon y), an intraclermal skin test with antigen
frou1 tl11;: µaLl 1vge11 can often be u~ed to de1nonstrate expo-
surc or infection. Reccntly, in vitro correlates have been
u:;c<l for :iomc di:;cn:;c:;. -,.,1ycobactcriu1n bovis infcction can
be diagnosed with intraderrnal infection of tuberculin.
WithiÓ 48 to 72 hours of antigen injection, crythema and
tnduration at thc site are apparent in infecteu ¡;atienls.
Infection with Mycobacteriurn avium subspPciPs jJseudotu-
berculosis (f uhne:. tli:.case ageul) ca11 be detected by in vitro
incubation of patiPnt lymphocytes with antigen and sub-
sequent analysis of interferon "f levels iJ1 thc culture super
natant. lntection by a<1ditional organ1sms tnat are an1ong
the group callcd facultative intracellular pathogens can be
detected using similar testing strategies.

Laboratory Diagnosis
DWJGHT C. HIRSH
ANTHONY E.
Bacteria and Fungí
A key decision made early in the diagnostic workup is
vvhether the patient's condition has an infcctious ctiology.
This decision is important because drugs used to t reat con-
ditions witl1 noninfectious etiologies- corticosteroids, for
exan1ple- are ofter1 contrainc.licated for treat ment of con-
ditions with an infe.ctious one, for t.vhirh antibiot i.cs are
appropriate.
One of the first majo r goals of tl1e microbiology labora-
tory is to isolate clinically significant microorganisms
from an affected site and, if more than one type of mi-
croorganism is present, to isolate them in approximately
the sarne ratio as occurs in vivo. Whether an isolate is
"clinically si.gnt ficant" or not depends upon the circum-
stances of i50Jation. For ex.a n1ple, the isola lion of large
numbers of a particular microorganis1n from a normally
stcrilc sitc in thc prcscncc of an inflammatory cytology
wou ld be int erpretect as significant .
Attention must be given to the si te cultured as well as to
t he method of obtai11ing the sample for culture. The deter-
n1ínation of significance is rn ade a great deal easier if the
:;arnple i:; ul>taiueu fruu1 a r1ur1nally :;terile site . Obtaining
a sample from the.alimenta ry canal, anci expe.c:ting mPan-
in gful answers, may be unrealistic unless one is looking for
the presence or a bsence of a particular microorganism, for
examplc, Salmonella oc Campylobacter.
Sample Collection
Care mu st be given to how the sample is c:olle.c:te.cl; if not,
interpretation of results niay be difficult. Most infectious
processes arise subsequent to t he contaminatio11 of a com-
p romised sur.tace or site by microorganisms t h at are also a
part of the flora occurring on a contiguous mucosal sur-
face. In other words, microorganisms isolated from an af-
fectec.l site are o ften similar (if not ldentlcal) to those foun d
as part of thf' normri 1flora of the patient.
Transport of Samples
1·11e soo11er the sample is processed in the microbiology
laboratory, the better. Realistically, the time bet ween sam-
ple colleclion and p rocessiug 1uay range from minutes ro
hours. Sample drying (al! m icroorganisms) ancl Pxposure
to a noxio us atmosphere (oxygen for obligate anaerubes)
BtbJioteca I C A P
YUAN CHUNG ZEE
C ASTRO
are the 1na jor dangers in not analyzi11g samples prornptly.
For t his reason, it is important that the sample be kept
rnoist (for a sy.ringe fu ll of exudare, this is obviously not
an important consideration) and, if conditions warrant
(see below), a ir excluded. Molst11ess is inai11lai11eu l>y plac-
ing the sample in a transport (holding) medium com-
poscd of a balanccd salt solutlon usually in a gelled ma-
t nx. Because t11is medium does not contain any nutrient
material, m icroorganisms in the sam ple m ultiply poorly if
at ali (and thereby relative numbers and ra rios are pre-
serve.o) h11t rf'm ain viable for a time, at least for overn ight
(exactly how long depe11ds u pon ll1e n1icroorga11 iSHL
involved- beta hemolytic Strcptococcus, for exan1p le, does
not survivc as long as Eschcrichia coli) . Swabs should al-
ways be placed in transpo.rt mectium, regardless of the
tirne elapsed b etween processing and collection. Fluids
that may contain anaerobic bacteria (e.g., exudate from
ciraining tracts, peritoneal and pleural effusions, abscess
n1aterial) sl1ould be cultured in1n1edialely. If lllis 111aLerlal
is contained in a syringe, then the air should be expelled
a11d a stcrile stopper placed over the needle. If a swab is
used to collect the sample, it should be p laced in an anaer-
obic tra11sport medium. If a syri nge full of san1ple ca11not
be processed immediately, t he syringe should be e1nptjed
into an anaerobic transport rnedi um and held at room
Len1pe1alure. Siu1ilarly p laceu svval>:; are treated ir1 the
same 1nanner. Do not refrigerate sampl <-~'> suspecteci of
containing anaerobes because sorne species do not toler-
ate reduced t emperatures.
Demonstration of an lnfectious Agent
·rhe preser1ce of an infectious agent is acco1nplished by ex-
am.i nation of stained sm ears made fron1 a portion of thc
clinical sample, culture techniques, molecular/in1muno-
logical methods, ora con1bination of t h ese methods.
Direct Smears. Information obtain ed from examination of a
~lai11 eLI ::.111ear ls Vdlud!Jle !Jecause tt m aybe t he first indica-
tion (and sometimes the on ly onP) that anin fectious agent
is present. ;\!so, '"'hat is seen (shape, staining charactecis-
tics) will help guide the choice of therapy 24 hours before
culture results are available. At lcast 104 microorganisms/
ml or gram of n1aterial must be p resent in o rder to be read-
ily detect ed microscopically.
A:s is tlie case witt1 a sample obtalned from a normally
15
16 PART 1 lnl1oduclion
sterile site, the presence of bacteria ln bladder urlne is a slg-
nificant finding. Howcver, lnterprctation of the results of
¡¡11;1ly:.i:. uf uri11c :.Cl111µlc:. u1Jlai11etl IJy ¡;al11eler or by
"catch" is difficult hecause of the confounding presence of
flora flushed from the distal urethra . Finding bacteria by
direct smear in concentrated (the preferred) or unconcen-
trated u rine obtained by percutaneous aspiration of blad
der unne 1s a s1s.rn1ficant f1nding. Demonstration of l bac-
teria/oíl field in a drop of unconcentrated urine (which
has been allowec.I to dry and theu stair1eu) represer1ts <11.Juul
1os to 106 h;:irtPri;:i/ml of 11rinf'.
Two types of stain3 are available, thc grarn :;tain and
Romanovsky-typc stains such as Wright's or viemsa. Each
type of stain has advantages and disadvantages. The gram
stain is useful in that the shapc a11d thc gran1-staining
characteristics of the agent are seen. Tl1e disadvantage of
the gran1 staln IS that the cellular contcnt of the sample is
not readily discerned On t h e other hand, a Rnm;inovsky-
lype stain gives the observer a feel ing'for the cellular na-
ture of the san1plc and whether or not there is an infec-
tious agent prcscnt. Cytologic cvaluation of the sa1nple is
very important in assessing the significance of the n1i-
croorganism sccn and subscquently grown.
Culture Techniques. Media are inoculated with a portian af
the specirne11. l110<..:ulaliuu :.l1uulu IJe ve1(01u1ed ll1 a se111i-
quantitativc fashion (especially samples of bladder urine
obtained by catheter or catch).
Determination of the relative numbers ot microorgan-
isms in a sample greatly helps interpretation of signifi-
cance. Colonies of m1croorganísms gro>vlng on ali four
quadrants of a petri plate indicate that there are largc
number:. uf rni<..:ruurga11.l:.111:> ill tl1c :;au1µlc. If a sa111µle
yiPl<IP<I onP or two coloniPs growing on the plate, the sig-
nificancc of thcsc calonics and th us the question as to the
infectious etiology of the condition would be in doubt.
"Enrichment" prior to plating of a sa1nple obtained from a
normally sterile site should never be done beca use one mi-
croorganism can grow to numbers that equal 1nany thou-
sa11ds in a very short period o í l i111c. Obviously n1ore cTe-
<lence wi 11 hf' givPn to a prnc:ess from wh ich thousands of
microorganisn1s were i:;olated than to a sample from
which one was isolated. An important exception to this
general "rule" is whether the presence or absence of a par-
ticular microorganism is s1gnifica11t, e.g., Salmonelfa in a
fecal specimen. from a clinical pcrspective, the use of en-
richrnent !Jrotli:., utllt:r tl1a11 fur ll tt: uele1111 i11aLion of Lhe
prPsPncP or ;:ihsPnC<' of a particular species of bacterium, is
n1ore troublc than it is worth. Too often, eruichment cul-
ture results lead to the unnecessary workup and treatment
of a contaminating microorganism.
Determination of significance is aided by the cytology
of the sample obtained from thc affected site. Isalatian
(clemonstratiou) uf 11uu1cruu:> 1uil.:ruuq~a11is1ns f1on1 a nor-
m;:illy stPrilP sitf' without thc presente of inflammatory
cells should be viewed with suspicion. The one exception
to this rule is cryptococcal infection wherein the samplc
may contain a large number of yeast cells but vcry few in-
flamn1atory cells (thc cryptococcal capsule is immunosup-
pressive). The isolation or demonstration of a "significant
number" of micruurgani:.ru:. fru111 a 11ur111ally :>lerile sile
without cvidcnce of an infta1nmatory response can be ex-
plained by conlaminated collection devices; contamina-
liou uf lite ¡;ull1::clio11 device f1on1 a co11Liguous, i1or111ally
nonsterile si te; contamination of the mcdium inoculation
devicc in the microbiology laboratory; or contamination
of the medium bctorc lnoculation. Collection devices
(e.g., catheters) sterilized by liquid disinfectants quite
often becomc co11tamina1cd by microorganisms able to
live in such fluids (Pse11don1onas is notorious far this).
Plille:. 111ay lJe :>llt:aked iu any fasl1ion as lo11g as individ-
ual colonies are produced after incubation. Assessing rela-
tivc numbcrs is vcry subjcctivc, und cvcry laboratory has its
own way of doing this. Rclative numbers of microorganisms
may be reported by noting how much growth occurs on
the surface of thc platc. Obviously, gro~vth of one colony
(the offspring of 011c bactcdum) vs. growth af colonies over
the whole pl<1te wuulu !Je viewed differe11Uy will11cspect to
clinic.al sign ificance. Determination of tl1e actual numbers
of bacteria present is only important whcn analyzing urine
obtained by "catch" or cathetcr because of the problem of
contamination of the sample by bacteria in tl1e distal ure-
thra. In this instance, disposable calibrated loops contain-
ing 0.001 or 0.01 mi of urine are used to inoculate appropri-
ate media (blooc.l/MacCunkey, fur excuuµle) . Greater ll1an
ios bacteria/mi of 11rinP ohtainPrl hy catheter or "catch" is
co11sidcred significant (i.e., the bacteria are more likely to be
coming from the bladder rather than the distal urethra).
Acrobic Bacteria. l"hc standard med.ium inoculated for
the isolation of facultative lllicroorganisms is a blood agar
plate. Many laboratories include a MacConkey agar plate
as well (or as a "split" plate with blood agar on balf ancl
MacConkey agar on the other half). MacConkey agar is
u:.eful ue¡;au:.t: e11L1::1ic n1ic1oorga11is111s (n1eu1bcrs of the
Family Enterobacteriaceae, e.g., Eschericllia coli, Klebsiella,
Enterobciclcr) grovv very well, as does the noncntcric T'scudo-
1nonas. Most othcr noncntcri.c gram-negative rocts and ali
gram-positíve microorgan ísn1s do not grow well on this
n1cdium. Borcletel/a g rows as tiny pinpoint colonies after 24
hours; b11t aftPr 48 ho11rs of incuhation, the colonies will
he quite large. A:;sessing the growth on MacConkey agar
will help greatly in d etermining the prescnce or absence of
enteric organisms, the group of bacteria n1ost difficult to
dcal with therapeutical ly.
Anaerobic Bacteria. Anaerobic bacteria grow on blood
agar that is spectally prepared by ridding t he metliu111 a:s
much as possible of oxygen a nd its proc1ucts. 'f'he plates
con1e fron1 the n1anufacturer in sealed pouches designcd
to exclude air. After anacrobic plates are inoculated, they
should be placed in a container of flowing oxygen free
Cü 2 or placed directly into an anaerob1c environment.
(Note that anaerobic blood plates that have been removed
from thcir pouchcs should be stured in fluvviI~ uxyge11-
free C02 orinan anaerobic PnvironmPnt.)
Whcn to inocula te n1edia for anaerobic lncubation de-
pends upon the source of the sample. Processing samples
for anacrobcs is tim<.'-cOnsuming and expensive. The most
common si tes or conditions that contain anaerobic bacte-
ria are draining tracts¡ abscesses; pleural, pericardial, and
peritoneal effuslons; pyometra; u~tt:u111yelili:.; aud lungs.
Anaerobic culture of sites that contain a population of
a11aerobic bacteria as part of the normal flora is wasteful

.,...:...: a, d istal urethra, oral cav1t y). An excep-
=-:;:;.:c --e cr.lhl re of cl11oclenal aspirates for assessment
~-_,.• O""<'..-gro"l-,·th. In this instance, the relative num-
'::!:~a:re ··;hat are sought (overgrowth is usually con-
u.:::==.;::::. --::-se~r ~,·ben the total number of bacteria, anacr-
- ~-"bes, exceeds 108 /ml of co11tents). Anaerobic
: ¡:!:e urinary tract is not routinely pe.rformed be-
~:s:-~::: _ecovery of these rnicroorganisms from this site
t:e::e.:nelY ;a re.
.: :c...z ..11munologic Methods. Sometimes it is ilnportant to
dctcrrninc thc prcscncc or absence of a particular microor-
ganísm as quickly as possible so that appropriate measures
can be ta.k en to deal '"'ith the problem. This is especially
true vvhen infectious agents are suspected that pose a
threat to other anima Is, inch1cling h1 1m;:in c·;:irp givers (e.g .1
Saln1011ella, Leptospira). Likewise, son1e infectiou s age11ts
take so long to grow in culture tl1at formulation of a ra-
tional therapeutic strategy is difficult (e.g., sorne funga l
agents, Mycobacteriu1n). Still oth ers are hard to detect be-
cause they are difficult to culture (e.g.1 Leptospira, rick-
t::tt~iae) or 11ave r1ut been cultured in artificial media (e.g.,
C/ostrídium piliforrnis, T,aivsonia intrar.t?ll11/aris) . Tn these il1-
stances, various tech.n iqucs are available.
Immunologically based tecl1niques make use of anti-
bodies specific for t he n1icroorganisrn in question. ·rhese
antibodies are usually imrnobilizect on a soUd support and
are used to trap t he agent. The presence of the t rapped
agent is then detected wJ.th speclflc antibody that has been
labeled in sorne way (usually with a color reagent). Sorne
kits 111aking use o( l11is app1oach are co1nn1ercially avail-
able (e.g., Salrnonella).
Molecular tcchniqucs utilizing DNA probes specific for
a segment oí UNA tl1at is u11ique to the microorganism ir1
q uestion, or the polymerase chain reaction (PCR) using
specific DNA primers, have been designed for a num ber of
agents.
Virus
General Considerations
The diagnosi.s of viral diseases traditiunally has been both
tedious and tüne-consurning, but i11creasingly is expedited
by modern technolohries such as the polymerase chain re-
action. Prompt and accurate diagnosis of viral diseases is
therefore essential for an effective course of disease preven-
tiou a11tl <.:011trul.
Pn1pf'r methods of collecting and processing clinical
specin1ens anda con1plete history are vital lo ll1e successful
isolation of viru ses. 1'issues that are extensively autolyzed
or poorly stored usually do not yield infectious virus be-
cause of the suscept ibility of most viruses to detrimental
en,rironmental conditions. Viral isolation and/or identifi-
catiou shuultl l.Jc atternpted in the followin g conditlons:
l. During outbreaks of vesicular disease in livestock
(e.g., fout-and-mouth d isease in cattle, pigs, sl1eep,
or goats).
2. During outhreaks of clise;:is~ in J;:irgr :1nimal popula-
tiC>ns such as feedlots, poultry houses, or catteries
Chapter3 Laboratorv
-
Dia0"11osis
b
17
where rnany animals are at risk and prompt, accu-
rate cl iagnosis is critic;:il to t he instit11tion of control
methods (such as vaccir1ation).
3. In instances of potentially zoonotic d iseases like ra-
bies, West Nile fever, cquine cncephalomyelitis, par·
ticularly when human exposure has ocurred.
4. In deter1nining the et iology of new d isease, or in
defintng uncharacterlzed aspccts of eXlsting ones.
Tissues for virus isolation should be collected from re-
ccntly dcad animals whenever possible (Tables 3.1 and
3.2). Collection of appropriate specimens during the acute
phase of the disease, and in.clusion of additional submis-
sions from similarly affected animals, enhances the like-
lihood of isolating viruses. Thc following factors should
l.Je cu11~iuered in selecting clin ical specimens: 1) type of
d isease (e.g., respiratory- l11ng or trachi>a, or vc~slcular­
vesicle or skin biopsy), 2) tl1e age and species ofhost, 3) the
nature of the Iesions in affected animals, and 4) the size of
carcasses (able to be shipped on ice?). 'fhe following is a
systematic approach for the rapid laboratory diagnosis of a
viral-caused disease in animals:
l. Exa1u i11atioo (gro~~ a11u histologic) of the dlseased
animal/tissues as a p resumpti ve diagnosis for ;:i vir;:il
etiology.
2. Measurement of the developrnent of viral-specific
antibodies (acute and convalescent sera) during
clinical disease.
3. Imm unohistochemical st ain.ing of tissue sections
with virus-specific antibodies to detect JodJv.idual
vi ral anti gens in the tiss11e_
4. Ex.amination of feces, plasma, o r serun1 by in1-
n1unoassays that detect specific viral antigens (e.g.,
rotavirus in feces, fcline leukemia viru s in serurn,
bovine respiratory syncytial virus in lung).
5. Exarnination of positive- or negative-stained speci-
mens by electron microscopy to identify the rnor-
phology of virus. 'l'his d iagnostic procedure is lim-
ited by the co11ce11tratlon of viral parlicles required
fordetection (>105/ml).
6. Isolation or am p lification of infcctious virus in cell
cultures and identification of the virus propagated
from clinical su bmission.
Many viral diseases do not kili the host, but the host
may serve as a reservoir of virus and disseminate the virus
to other co11tact a11in1als. Serological assays cae1 ~0111eti rnes
be used to determine which animals carry specific viruses
and which animals are suscept ible to infection.
lt is to be emphasized that merely isolating a virus from
an animal does not necessarily implicate that virus as thc
causative agent o.t any <lísease t hat is occurr1ng- 1n tnat an-
imal. It is very important to confirm that the isolated virus
produces a sirnilar cli~t::ast:: in the sau1e u r relatec.I species,
which may even involve the inoculation of susceptible or
nonim1.uune a11i1nals. When two or more viruses are iso-
Jated from a specimen, a clear interpretation of the role of
each isolate in the disease process is alsC> necessary. Finally,
it must be remernbered that vaccine stratns of viruses can
also be reisolated from vaccinated anilnals, and can b€
confused witl1 true field strains.
18 PAKr 1 Introuuctio11

Table3.1. ~uggested ~pecimens from Mammalian Species for Virus tsolation and ldentificatlon

Type of lllness Common Name or Associated Other lnfections Oinical Specimens to Collect Diagnostic klentification
or lnfection Virus Tem

Kespiratory Adenovirus (bovine, porcine, Nasal and ocular secretions, feces, lu119, VI (CPE), HA, CF, FA, VN
canine) brain, tonsil
lnfectious canine heµalilb Spleen, liver, lymph nodes, kidney, blood VI (CrE), HA, FA, VN
(adenovirus)
Bovíne viral díarrheü (muc<>s<>I Genital, abortions, enterk Nasal secretions, oral Jesions, lung, splei!n, VI (CPE and virus interference},
disease) (pestivirus) blood, mesenteric iymph nodes, intestinal FA, VN
mucosa, vaginal secretions, fetal tissues.
uncloned blood
lniectious bovine rhinotracheitis Central nervn11~ ~Pm Nasal and ocular secretions. lung. tracheal VI (CPE). FA. VN
(her ¡.¡e)vir U)) (CNS), yt11ilal, swob, tr0<heol >C9ment, broin, voginol
abortíons secretions, serum, aborted fetus, liver.
spleen, kidney
Feline rhinotracheitis (herpesvirus) Nasal and pharyngeal secretions, VI (CPE and inclusions), FA
conjunctival membranes, livP.r, lung,
1pleen, kiú11ey, )dlivary gland, brain
Equine rhinopneumoniti~ Genital. abortions Placenta, fetus, lung. nasal secretions. FA, VI (ECE and CPE}, VN
(herpesvlrus) lymph nodes
tnfb1P.n2a (f>llnine. porcine) Nasal and ocular secretions, lung, tracheal VI (ECE), HA. Hl
(orthomyxovirus) swab
Parainfluenza (bovíne. equine. Nasal and ocular secretions, lung, tracheal VI (tCE), HA. HI, VN
porcine, ovine, canine) swab
(paramyxovirus)
Bovine respiratory syncytial Trachea. lung, n¡¡s;if iecrPtinns, dnttf'd hlood VI (CPE). fA ELISA
virus (pneumovirus)
Bovine herpesviru~ 4 (Mnv;1r, Abortíons (?) Trachea. lung, nasal secretíons. fetus. clotted VI (CPE), FA, V~
DN599) blood
Reovirus (bovine, equine. canine. Feces, intestinal mucosa, nasal and VI, HA, HI
feline} pharyngeal secretions
African horse sickness Whole blood in anticoagulant. lesion VI (CPE and mice), VN
(orbiviru')ª material. naql ~ml ph;,ryn(JP~I <Pl'rPtionc
Malignant catarrhal feve~ W11ule !Jlood in anticoagulant, lymph nodcs, VI (CPE}, CUSA, rA, VN, EM
(herpesvirus) spleen. lung
Pseudorabies• {herpesvirus) CNS, genital, abortions Nesal secretions, tonsil, lung, brüin (midbrain, VI (CPE and rabbits), VN,
pons, medulla), spinal cord (sheep and EUSA, FA
canle), spleen (swine), vaginal secretion,
serum
Canine herpesvirus Kidney, liver, lung, spleen, nasal. VI (CPE and inclusions), FA, VN
oropharyngeal, añd vayi11dl >t!l.I dions
Por(ine inrlucinn horly rhinitis Turbinate, nasal mucosa EM, VI (CPE), FA, VN
(lytumegalovirus)
Equine rhinovirus Nasal secretions, teces VI (CPE}, VN
Maedi-V15na, ovine progressive CNS CSF, whole blood, salivary glands, lung, VI (CPE and sheep}, VN, Cf
pneumonia (retrovirus, mediastinaf lymph nodes, choroid plexus,
lentivirus} spleen
Bovine rhinovirus Nasal secretion~ VI (CPE}, VN
Rift valley fPv<>rª (hovinP, ovine) Whole blood in anticoagulant. fetus. liver. Vl (CPE and rnice), VN, CF, FA
(pi 1lebovirus) spleen, kidney, brain
Enteric B.ovine enterovirus Feces, oropharyngeal swab VI (CPt)1 VN
Transmissible gastroenteritis Feces, nasal secretions, jejunum, ileum VI (newborn pig~). FA. fM
(coronavirus)
Neonatal diarrheas
1. Rotaviruses Fece), ~mdll inlestine VI (CP( witn trypsin), ELISA,
FA, EM
2. l'a1voviruses Abortion Fcc~,intestinal mucor..o, rcgion;il lymph VI (CPE}, fA. EM, HA, HI, VN
nodes, bram, heart
 Chapter 3 Laboratory Diagn.osis 19

T a b 1e 3. 1 . Continued

T~p_e -of Jllness Comllion Name or Associated óther lnfettions €1inical Specimens to CoJlect Diagnost{c ldentífic.ation
of lnfcdion Virus Tes:t:S

VI (ll'~ witfi trypsin), fA, tM

0

3. Coronaviruses feces, small intestine

Picornavirus. ~Jv1ffll (enteroviru5)
f'olioencephalitis (Teschen,
Talfan) (enterovirus)
Rínderpest• (mO:rbiHivirus)
Peste des petits ruminantsª
(morbilfivírus)
C.entral nP.rvnus Rahi1>' (lyssavirus)
system (CNS)
Equine encephalomyelitis
{VEE,ª EEE, WEE)b (alphavirus)
LotJpíng ill encephalomyelitisá
Feces, intestine, brain, tonsil. fiver VI (CPE), VN, EM
CNS BraiA, intestine, feces VI. (CPf),. VN
Blood in anticoag.ulant, spleen, mesenteric VI{CPE ·and cattle), AGID,
lvmph nodes Cf, VN
Blood in anticoagulant, spleen, mesenteric VI {CPE and goats), VN, Cf,
lympll nodes AGIO
Brain, salivary gtand VI (mice <md indusions), FA, VN
Whole bíood, brain. cerebrospinal fluid, VI (ECE and micel, HA; HI,
nasal and pharyngeal secretions, pancreas vN, cr
~

/li.r..
(flavivirus) Whole bfood, brain, cerebrospina1fluid VI (ECE and CPf), FAr VN, HI
'
Hemagglutinating Brain, sP,irial .eord, tonsH, blood VI (l'PE), HA, HAO, VN, FA
encephalomyelitis virus
(coronavirus)
Caprine arthritis encephalitis Arthritis B[óod, spinaf cord VI (f PE), Afilo, Fl.ISA r
(retrovirus, peAtivirus) ~
-
Japanese Bencephalitisª Brain, CSF VI (ECE and mice). lgM, VN, CF. e
(flavivirus) HI, FA, ELISA
Berna disease (unclassífied} l!rain, spínal cord
r
Vl(ECE and rabbits), FA,.Cf "
Prion diseases" Brajn VI (mice and sheep), MI{?)
Mucous membranes Poxviruses Lesion scrapings, lesions, vesicul.ar fluitls, VI (ECE, CPE,.and rabbits), HA,
and skin 1. swiñe pox (suipoxvirus) crusts. liver. spleen Hi. VN, FA, FM
2. vaccinia (orthopoxvirus)
3. cowpox (ortliojlo:XVirus)
4. sheep and goat.poxª
(capripoxvirus)
•
Foot-and-mouthª disease E.nteric lesion material, tonsil, vesicular fluid, hoof VI (CPE and neonatal mke),
(Aphthovirus) lesions, esophageal-pharyngeal (ep) CF, VN, FA, AGID, ELISA
flaid,s, ali tissues
Bovine ma111millitís (herpesvirus) lesion scrapings, lesions, teat swab, fluid VI (CPE), VN
exudates from lesion
Vcsicul¡ir stomatitisª VcsiCular fluid, epltheliaf (OVering of lesions, VI {CPE), VN, CF
(ves1culov1rus) whole blood, regional lymph nodes,
tongueswab
V.esicular exanthema of swineº Vesicuíar fluid, epith,elía! covering of foot VI (CPE), Cf, VN, AGID
(t!alicivirus) lesíon, tonsil lymph node, serum, nr;il ;ind
nasal leslons
5111/ine vesicular diseaseª Vesicular fluid. epithelial Eovering of lesion. VI (CPE). VN, FA, AGIO
(cntcrovirus) oral or nasal !esions
Papillomaviruses Neoplasia Lesion ma.teri.ál, warts, skin scraping EM, cell transformation, PA
Contagious ecthyma ORF Scabs, lesions on lip VI (fCE and CPE); VN, !\(;ID,
(parapoxvirus) fA,.EM
Bovine papular storhatitis Les.ion hiop~. 'ff~rin9 m11nle, mouth, teats VI (ECE and t':PE), EM
(parapoxvirús)
Genital and/or Enteroviruses CN5, respiratory Vaginal secretions, serum from dam or s-0w, VI (CPE). VN
ab.ortions nasal swab, tonsil, btaín (swine), feces
(cattle and swine)
Parvovirus (swine) Vaginal secretions, serum from dam or sow, VI (CP.E), FA, HA, HI
lung (mummified fetus)
20 PAKr l lntro<..luction

Table 3 . 1 . Continued

Type of lllrress Common Name :or Asso<latéd Ot!Jer lnfections Clinícal Specim!!lls to <oltect Diagnostic ldentíficat.ion
or Jnfection Virus Tests

Bluetongue, ~plzooirc
liemorrhagit disease of deer,
lbaraki íorbiviFu.S)
Equine viral arteritis (Pestivirus)
Border disease (hairy shaker)
(pestivirús)
Alabaneª {Bunyavin1s}
Hemorrliage Ho9 choleraª (pestivirus)
syadrcome (vtremia)
Equine iM.fectious. anen,:iia
(retrovjrus, PeAtivirus}
Atrican swine feVer1 (lridovirus).
Narrobi sneep dísease•
(Nairovirosl
Ríft v<!lley fever" (phlebovirus)
.......,.., .
..~.......asta Retrovirus (bovine, fetirie)
(ontovirinae)
Hemorrh?glC syndrome;
(virertlia), respiratory
· Whole blood, nasal and pharyn9ea~
CNS
lmmunodéficiency,
leo1<emia, anemia
serum from dam, fetal·heari, hepar1n1zed VI (CPE and E·CE)c Cf, AGID, FA,
blood, spleen, hone marrow, lympn nooes, VN, EM
lung,.~emen
VI (€f'El. GF, AGIO, FA
~ecretiops; placenta, fetus; spleen, nostril,
lymph nodes, conjunctíval sac, semen
Brain, spleen, bi·ood, bone marrow VI (CPE and interference),
FA, VN
Plac~nta, 'fetal muscle, Aerve tissues VI íCi'E and svtl<ling min~),
'
.. FA, Vft HI, HA
Tonsíl. spleen, líver. brain, lymph nodes VI (pigs), FA, VN
Who.le blood, spleen, lymph nodes VI (CPE and horses), FA, VN, CF,
EUSA, AGIO
Btood in anticoagofant, spleen, liver, tonsil, VI (CPE and pigs). HAO, HA, Cf,
iy·mph nodes FA, RIA (r;idioimmum'las.lay),
EllS.I\, JtOP (im111unoelectro-
osmophoresís)
S¡¡leen; blood (plasma), mesenteriC'lymph VI (intracerebral suckling mice),
nodes FA
Fetus, J)looa io antkoagtilant; livcr, splccn, VI (CPE and suc~ling )'lamsters
kidÍley, braln, serum or mice), VN, Cf, A(jJD, ,HI, ~A
Lymph·nodes•. metastatie;growths, b.lood in VI, reverse traoscriptase, Ef\111
antiEoagulant serum ELISA, fA, Western
i mmunoblot

AGIO=·aga>gel immunodiffuSion, Cf = Eomplement tixation, EOFAl = complement.fixatioll for avían Jeukosis, CPE= cyto11athic efféct. ECE = embryonating chicken eggs, EM = électron microscopy,
FA = imm11nofloorescenr.e. HA =hem,ag9l11:tinin. HAO = hema~orption. RI = hemagglutination inhioitiori, RJA = radioiinmunoassaY., VI =virus isolation,VN =J1irus neutrali:ration, MI = molewlar/
irnmunofogic.
'lleportable ;'1!$eaie or a foreign animal dísease in l!níted States.
~VEE =Venezuelan equíne enc~flalomyelltis, EEE= eastem equine encephalomyel.itis, WEE =western equtne encépl\alomyelitis.

lsolation of Virus from Clinical Specimens

Cultivation in Tissue Culture. Viruses are isolaled fro111 cli11ical
specimens hy inoculating susce.ptihlP primary or contin11-
ous cell cultures derived fro m the host or related species,
embryonated eggs. o r laboratory animals. Specime11s sub-
mitted for viral isolation should be placed in virus trans-
port media (e.g., a balancect salt solution containing an-
tibiotics) in sealed containers for safet y in handling. They
shoulcl tJe clearly itlent ifiecl l>y appropriate labeling, an<..l
s11hm itte.cl on ícP (4ºC:) or froze.n (- 20º\.)_
Specimens should be collected from live animals during
acute pl1ases of tl1e disease. Dependi11g on the specific dis-
ease process, excretions or secretions, swabs of body orí-
fices or liquids (lymph o r blood), and tissue conected by
biopsy are all suitable specimens for viral isolation. ln the
laboratory, tissue specimens are processe<..l as a 10% or ZOo/o
(w/v) homogf'n<ltf' in ¡¡ h;:il;:inc.Pd s;:ilt snh1tion ;:ilong with
antibiotics. lleavily contaminated specimens may b e fil-
tered to remove other microorganisms. Virus isolation
should be conducted in cell cultures free of contaminating
agents such as noncytopathic bovine viral diarrhea virus
or mycoplasma.
To isolate virus, tissue homogenates are p laced onto cel-
lular monol.ayers and absorbed 1 hour or longer at 3.SºC to
37ºC, and tl1e inoculun1 is left 011 or renioved and fresh
media added. Ino(u!ated and uninfected cell cultures are
observed for 7 to 10 days. Viral cytopathic effect (CPE) in
cells is usually evident betvvee11 24 and 72 11ours tor most
cytopathic viruses (Fig 3.1 ). However, for most clinical ma-
terial containing low concentrations of virus, several (>3)
blind cell passages are recommended.
Afler a virus has been den1onstrated at lin1iting dilution
to replicate in a cell by CPE or other parameters. infectious
virus is released from cells by th..rcc c::yclcs of hcczc- t haw or
sonication, tol1owed by centrifugation anct storage to
maintain maximum infectivity. Eacl1 virus isolated should
be itlentifie<..l as to .s pecies of origiu, 111orpl 1ulogic Lype, pas-
sage leve!, and host cell used for propagation.
Embryonated Eggs. A n1nnher of mammalian viruses, and
many avían viral pathogcn::; cun be isolutcd in cmbryonat-
ing chicl<en eggs (E(:E). A key to successful viral isolation
in ECE is the route of inoculation (Fig 3.2). Candled ECE
that die within 24 hours after inoculation are considered
traumatic deaths. Subsequent deatbs of inoculated ECE
are placed at 4QC for ~everal l1our:s (to avoid he111orrhages)
 Chapter 3 Laboratory Diagnosis 21

Table 3.2. Suggested Specimens trom Avian Species tor Virus lsolat ion and ldent itication

Typ_e 9f fllness Cq11mi9n Name or Assoóated Clinical Specimens to Collect

Respiratory Newcastle disease CNS Tracheal or cloaca! swabs, luffq, spleen, ijver; VI (ECE, CPE), VN, HA, Hí
WNDª (paranwxovirus) kidney, bone marrow
Avlan. Influenza• (fowt plague Diop In egg production, n achea, lung, aif sac, sinus exudate, liver, V~ (tlt), HA, HI, Alli'; VN
virus) (ortfiomyxovirus) enteríc spteen, blnod, croara! ~;ih
lnfectious bro11chitis (corol"lavirus) Ne¡ih~osis, drop in egg lun_g,.trachea¡ ttacheal swabs VI (ECE tr.id1eal rí119 lullur'e~),
production VN, HA HI, EM
Herpesvirus of
1. psittacines (Pacheco's Disease) liver¡ spleen, lntestine VI (ECE, tíirds), VN, FA, EM
2. cranes
3. pigeons
4. owls
s, fakons
Laryngotfacheitis (herpesvirus) Trach~a or tracheal'E!XUdate, lung VliECE, CPE), AGPr VN, FA
J\vian adenovirus Eggclrop syndrome, Trachca, lung. air sacs, intestine, feces VI (ECE, CPE), AGP, VN, FA
hepatitis, enteritis
f'r\terk (pronavira! enteritis of turkeys lotestine, bursa of Fabr(dus, ceca VI (ETE), FA, V.N
Reovirus lntestine, teces EM, VI
Central nervous Avían encephalomyelitis Brain VI (Chkks. ECE), VN; FA
system (enterovirus)
Alphavirus infections (eastern Serum, _b.rain, heart, spteen. liver Vl (ECE, mlce, CPE). VN, CF, HI
and western equine
eneephalitis'Viruses)
Turkey meningoencepñalitis Brain, spleen, serum VI (EE:E, mice, CPE), VN, Hr
(flavivirus)
Mutous membranes Avipoxvirus Nodular skin resiol'is, scab Vl ·(ECE-, CPE), AGP, HA, VN, FA,
~flJ.I ~i'-i11 l. Pi9~011 pox immunoperoxídase
2. Canary pox
3. fowl pox
4. TurJ<ey_ pox
Hemorrh¡¡gic syndrome Vi'ral arthritis- (reovirus) Synovial fluid from ti&iotarsal or VI (CPE, ECE), J\GP, VN, FA
{viremia) tibiofemoral joints, spleen swab
Dt1ck plague (duele virus Liver, spleen, blood VI (CPE, EDE), VN
enterttts) (herpesvirns) .
Hemorrttagic. enteritis of tutlieys Jnte~inal rontP.nts, splP.P.O VI (CPF, poulfs), A'GP, EM
or marble s¡¡leen of pheasants
(adenovir.us, t~pe2J
TurJ(ey viral hepatitis Liver Vl·(fCE)
(unclassified)
fnfectious bursal disease lmmunosuppression Spleen, bursa VI (ECE), VN, AGP
(Birnavirus)
Fledgling disease (papovavirus) Bone marrow, kidney, heart, spleen VI (CPE), VN, E.M, EA
Chicleen anemia agent lmmunosuppression, Spleen, bursa, thymus, 'blood VI (chicks), EM, VN, ELISA
(parvovirus) pancytopenia
Neoplasia leukosis and sarcomas (rettovirus, W.líolc J:> lood, plasm<i, ~loacal 5wab5, RT, VI (CPE, cell ttaosform,ati.on,
oncovirinae) rne<omum, albJJmr.n, embryos, tumors ECE), ELISA, FA; COFAL
RetiEuloP.ni:fotheliosis lmm.unosuppressiori, Spleen, tumor tissue, beparinizeil biood VI (Cl1E, ETE), FA, R-T; AGP, VN
(1el1 oviru~, ontuvitin'éle) lymphoma
Mare!<'s disease (herpesvirus) Blood, tumor, liídney, spleen, feather~ VI (<;PE, !iCE), AGP. VN, FA

=
AGJD =agar gel fmmuoodiffusioQ, AGP agar gel ~recipitin, Cf = ~oniplement fixatioQ, COF.Al =cpmpJement-fixation for avian leukosis, CPE = c:ytopathic effect. ECf =embryooating chicl:en eggs,
EOE = ernbryonating dQd( eggs, EM =electron mía:oscopy, EJE= embryonating turke.}'l!ggs. FA =,immuoofluor.<rPnre,. HA: hom•gglotinin: HAD :hell)adsorption, HI ~hefnaggltitination inhibi-
tion, RIA= tadloJmmunoassay, RT ~ reve,se tra~ciiptase, V1" virus isolatíon, VI>!= viws n•4.traliz,ati 0n, WND = velogeniGviscerotropic f>!cwcastlc diocosc.
22 PART I Introduction

F 1G U RE 3 . 1 . Cytopathic effect (syncyti;i/) nn hnvinP fpt;¡/ kídney ce/Is by the herpes virus of
malignant catarrhal fever (X200).

·,. •
•
• •
.. ~ .
,.~
•
..
•
~~
V, •

·~
• \ l
••
{"
'

..
"• • ,...
...
..•
.~ ••
•

'
•

•
•

• •• •
•

' •
•

.
.i
·~.
.
.)
. .
"'
*'
~ ~·\

'.,/.
•

•
"'. •
• ~~ ,. •
41 ,,., • · • "

F 1 G U RE 3. 2 . The chicken embryo (10 to 12 days olú) <Jnú ruule~ uf ínutuldliu11 lu reach the
various ce// types (as indicated). For chorioallantoic membrane inoculation, a hole is first drilled through
the egg shel/ and shell membrane; the sheU over the air sac is then perforated, causing air to enter
between tf1e sf1el/ rnernbr<Jne cnú l/1e tl1uríuallanloic 111e1nbrane, creating an artificial air sac, where the
sample is deposited. The samp/e comes in contact with the chorionic epithelium. Yolk sac inoculation is
usual/y carried out in younger (6-day-old) embryos, in which the ynlk sac is larger. (RPprnd11rP.d with
pertnission from Davis BD et al. Microbiology. 2nd ed. llagerstown, MD: Harper and Row, 1977.)
Chorioallantoic membranc inoculation

Amniotic cavity
Shell membrane

Shell yA!rsac
Amniot ic inoculation
\
Albumin

\ \ Yolk sac inoculation

IJ_~l~~::::::::::::=C::~----
Yolk soc
Allantoic inoculation

Allantoic
cavity Chorioallantoic membrane

prior to collection ot tluids or visual examination ot the
embryos and egg membranes. Embryos that are stunted,
dcformed, edematous, or hemorrhagic, and membranes
that contain lesions (Le., pocks) should be homogenized
in a slcrilc bala11ccd salinc as a 10% (w/v) suspei1sion aud
repassaged in ECE or cell cultures. To avoid toxicity, a dilu-
tion of thc inoculum is done.
Animal lnoculation. The inoculation of susceptible labora-
rory anlmals remalns a useful procedure for the identifica-
tion proccdure for the idcntification of sorne viral patho-
gens, particularly thosc Lhal are llighly fasLidious and
difficult to propagate in other systems.
ldentification of Viruses or Viral Antigens in Clinical Specimens
Electron Microscopy. Electron mlcroscopy (EM) can be used
to rapidly identify the morphology and size of any virus
prese11t in a speci111en 01 isolaLed in cell cullurt:. uf ECE.
Tentative diagnosis of viral diseases can be made hy EM on
thin sections of affcctcd tissucs and ccll-frcc homogcnatcs
of clinlcal speclmens. !"he use of t::M for diagnosis is lim-
ited, however, because thc mcthod is not very sensitive
(> 105 virus partlc.:les/ml are requlred to see a single viral
partic.lP on ;.i 200 mPsh gri<I), :inri viruses from different
specics have similar morphology and size.
lmmune Electron Microscopy. Immunc clcctron microscopy
(IEM) enhances detection of viruses in tissues, cells, or
fecal specimens by reacting specific immune sera with
Chapter 3 Laboratory Diagnosis 23
virus. In IEM, specific antibody to a virus, prcferably poly-
clonal, is mixcd 'vith virus for 1 hour to produce antigen-
antibody complexes. ·rhese immune complexes are cen-
trifuged at 1000 X g onto Formvar-coated grids, then
:.lai111:Ll wil11 4% PTA, ¡.¡H 7.0 and examined by EM. T he re-
action of viral fluids with <;pPcific ;.in1tP or conv;.ilP<:Cf'nt
serum as vie"ved by EM determines if a virus is associated
with a specific diseasc. This procedure has been used suc-
ccssfully for viruses associated with infectious diarrhca.
lmmunofluorescence. lmmunofluorescence is a visible fluo-
re:;c1:11ct: acce11Luatct.1 uy ultraviulet light as a specific anti-
body covalently ho11n<l to ;.i f111orochromf' (e.g., fluores-
cein isothiocyanatc, rhodamine) t hat con1bines '"'ith a
fixed antigen. This technique provides a sensitive and
rapid method for detecting and identif·ying spccific viruscs
in eíther tissues or cell cultures (Fig 3.3). Tmmunofluore-
scence is detectable by either a d irect or indirect proce
dure. ·rtie c.llrect 1m mu nofluorescence test employs a virus-
spP.c.ific ;.intiho<ly l:ihPlrrl with f111()rP~rPin t h ¡¡t combü1es
with a specific viral antigcn Jocated in cells or tissues. Thc
indirect test requires the use of a fluorescein-labeled anti-
serum to a virus specific immunoglobulin.
Immunohistochemical staining uses tl1e same ap-
proach, except that thc virus-specific antibody is either di-
1eclly 01 iudi11:clly lauele<l \Vith ar1 enzyme. The presence
of the enzyme is determincd hy a<ldition of its s11h.-,;tr;ite,
and the reaction detectcd by a color change. The advan-
tage ot immunohistochemical staining over immunofluo-
rescence is that a fluorescent microscopc is not rcquircd,
F 1G U R E 3 . 3 _ Cytoplasmic immunofluorescence in fetal
bovine lung ce/Is produced by BVD virus (X200). (Reproduu=ú wiú1
perm1ss1on from Castro AE. Bov Pract 1984;19:61.)
24 PAR!' I Introducllo11
and the use of an enzyme-enhancement step greatly in-
creases the sensitivity of the procedure.
Nudeic Acid Hybridization. Molecular hybridization tech-
niques have lcd to the synthetic production of viral DNA
probes that are highly specific for individual víruses. These
probcs are labclcd with various dctcction systcms that
allow idcntitication ot the presence of individual viruses,
citl1cr in tissucs or in extracts of them.
Polymerase Chain Reaction. The relatively recent develop-
rne11l of lile polyrne1ase cl1aü11eaclio11 (PCR) has revolu-
tionized the rapid diagnosis of many viral diseases. The
importance of the procedure lies ü1 its ability to amplify
small amounts of viral DNA or lZNA even trom contami-
nat:ed specimens, and on its ability to be conductcd on a
large seale so that vast numbers of san1ples can sin1ultane-
ously be evaluated. Furthermore, technical developments
such as real-ti111e PCR <tlluw ll11:: 4uaJ1Lilalion of len1plale
th;:it is prcsc nt in a sample, which is reflectíve of the viral
load. Thc PCR assay is based on the cyclic synthesis of a
DNA segmcnt limited by two specífic oligonucleotides
that are uscd as prirners to specifically amplify portions of
the viral genomc. I'ropcrly run, the PCR assay is both sen-
sitive and specific, although thc identification ofviral nu-
clelc acld does not prove that lnfectious virus was present,
so PCR-posiliv<' s;implc>s ohPn shoulci hP suhjerted to tra-
ditional virus isolation procedures.
Enzyme-Linked lmmunosorbent Assay. 'rhc cnzymc-linked im-
m unosorbent assay (ELl:SA) is a rapid, highly sensitive im-
munoassay adapted to measure viral antigen or antibody
(see Chapter 2). ELISJ\s have been developed for numerous
avían viral pathoge11s (e g , avían laryngotracheitis virus,
avian cnccphalitis, Newcastle disease virus, infectious
bronchitis virus, and reovirus), and increasingly are being
dcvclopcd to dctcct viruscs that infcct othcr spccics of do-
mestic animals.
Serologic Detection of Viruses
Mos l viruscs u.:;ually c lic it an in1n1une response in the
host; thus the detection of a humoral (antibody) or cellu-
lar response is often used to determine prior infection of
an animal with a viral pathogen. Serologic assays measure
humoral immunity in animals, and assays for measuring
cellular immunlty to vlruses are used lnfrequcntly in vet-
erinary diagnostic medicine.
Vifuses have cerlain anligens that are type- or group-
spccific and that in part determine the serologic assay
uscd. Scrologic diagnosis of virus infcctions typicully rc-
quires the collection of paired samples: an acute (at or
prior to the onset of clinical signs) and a convalescent
serum (1 O to 28 days later). A fourfold or greater rise in an-
tibody titer (the reciproca! of the serum dilution) inciir;itPs
recen l or ongoing viral ilúeclion. Antibody levels in single
serum samples are more difficult to interpret, although the
presencc of antíbody is indicativc of prior cxposurc (or rc-
sulting trom the passive transfer of maternal antibodies .in
young animals) which is especially important in chronic
discases with a carrler state like bovine leukemia virus In
cattle, equine infectious anemia, and equlne arteritis Virus
infection of stallions.
Serology can help to rapldly establish a diagnosis when
vir;il isol;ition prnc1>f111r1>s <lrP nPgativP. St>rology c:an also
be used to definitively rule out the presence of specific
viruses in a given discase outbreak, whereas a negative
viral isolation cannot.
Serum Virus Neutralization (SN) Test. Most viruses produce a vis-
lble cytopathlc effect (CPE) in cell cultures. CPE is usetl tu
determ íne thP prPsPncP of prot1>ctive or virus-neutralizing
antibodies in a serum. 'fo quantify the amount of neutral-
izing antibody, serum fron1 an animal is serially diluted by
twofold dilutions and mixed with a known amount of
virus (generally !>O to 300 infect1ous doses of virus-·rcrD50)
for 1 hour at 37ºC prior to inoculation of a volume of the
mixture !nto antmals, embryonatlng chicken eggs (ECE) ur
cell cultures. Tbe SN test is vPry spt>cific ;:inrl highly sensi-
live, but lt is titu.e-consun1ing and expc11sive. The SN can
be used to confirm recent infection of animals it paired
sera are cvaluatcd.
Hemagglutination lnhibition (HI) Test. Viruses that possess a
hcmagglutlnln protein (IiA) will agglutinate erythro-
cytes, a fact that has been used to quantify the amount
(titratt:) uf lht:~e viru;)e:> U1al élfe present in a san1ple.
The HI test can he used to identify or type a virus through
the inhibition of hemagglutination by virus-spccific an-
tiserum .
Hemadsorption-lnhlbition (HAD-1) Test. The HAD-1 test is based
on the ability of certain virus-infected cells (monolayers)
to attract speclflc erythrocytes to their surface. 111e prc:;-
ence of hPm;ici~orhin g PrythrocytP ch1st1>rs on a c:ellular
monolayer indicates that viral protein (hemagglutinin)
has accumulated on the surface of the cell membrane. The
hcmadsorption phcnomcnon can be inhibited by pre
treatment of virus-infected cells for 30 minutes, usually at
ambient temperature with twofold dilutions of antisera
followcd by thc addltlon of O.Q5<)'ó to 0.5% erytl1rocytes.
Antibody (Ab) c;in be quantifipfl hy comp;:iring the oh-
served "vashed virus-infected cell monolayers that contain
adhered clumped erythrocytes on the surface of the cell
(Ab negative) with the cell monolayers that contain free-
floating erythrocytes (Ab positive).
Complement Fixation. Compleu1e11t fixalio11 (CF) lesls en1-
ploy thP c;:isc;:icie of compleme nt in reactions of viral anti-
gens that fue complement-usually guinea pig-whcn
combíncd to antibody. Although CF has been used in early
test tube assays to detect virus (e.g., leukemia viruses),
virus-infected cells, or virus-specific antibody, the com-
plexity of the assay and the ti me required have led to its re-
placement by slmpler prucctlur~.
lmmunodiffusion. The immunodiffusion (ID) proccdurc is
routinely used as a diagnostic tool to monitor the spread of
specific viral pathogens in various animal diseases (e.g.,
bluetongue, equíne infcctious anemia, bovíne leukosis,
caprine arthritis, encephalitis, infectious bursal disease).
The basis of the te~1: is thc al.JiliLy uf cerlain soluble viral
 Chapter J Laboratory Diagnosis 25

;:intigPns to d iffuse in a semisolid medium (agar) with the

formation of a precipitin line with specific antisera.
lnununoelectroosmophoresis combines the ditfusion
p rocedure with the principle of movem.e11t of charged pro-
tein molecules in an electrical field. Since most viral anti-
gens assl.nne an electrically negative charge, applicatio.n of
an electrical current u1uve~ tl1e viral µarticle Lu l11e <iuuue.
Following Plec:trical migration, the antigen can he eluci-
dated by the use of a positive antiserum that migrates to-
ward the cathode. 1'his technique has been used to detect
antigcns of African swinc fcvcr virus.
F 1G U RE 3. 4 . Western immunoblot of bluetongue virus
prolein> u>iny >e1 u111 obldined before and after natural infection
with BTV serotype 17. Virus proteins identified are given in the right
margin; LMW represents three low molecular weight virus proteins
that have not been previously defined; X represents an additiona/
noncharacterized virus protein; P designates specific BTV protein; NS
is the nonstructural virus protein. 1 anPs A and R rPpresent prP.- anrl
postinfection serum, respective/y. Jmmune complexes were detected
in lane B using a biorin-avidin-enzyme probe, e.g., biotin-tabeled
rabbit antisheep /gG in association with peroxidase-/abe/ed avidin.
(Reproduccd with pcrmission from MA Adkison <ind JL Stott.)

Radioimmunoassay. 'fl1e radioim1nunoassay (RJA) is an ex- B

quisitely sensitlvc method for quantifying antigens oran-
tibodies when onP C'omponf'nt is r;:idio-J;:¡hf'lf'd . Altho11gh
RIA has an advantage for detecting minute amounts of an-
tibodies, the need for a scintillatio11 counter to measure ra- r
dioactivity and ve.ry pure reagents limits use of this assay r
to appropriately equipped diagnostic laboratories.

Enzyme-Linked lnununosorbent Assay (ELISA). ELISA~ are highly

spf'cific ;:in<l sensitivf' imm11no;:issays in which the spf'ci-
ficity of the react.ion can be enhanccd by increasing the
leve! ot puriíicatio11 ot the antigen or antibody employed.
= :;;t -
-
P2
p3
The ELISA can detect nanogran1 levels of IgG-, IgM-, and
IgA-type antibodies. ELISAs can be made quantitative if
appropriate standard curves are developed. Numerous t
co111111ercially available assays for avían and n1an1n1alian ~
viruses provicle qualitative information on antibody to
various viruses, whereas other.'\ detect the viruses them- ~ ...J
~

selves in clinical specimens. The blocking ELISA evaluates

thc ability of a test serum to displace the binding of a virus
protein-specific antibody to its antigen.
m
- NS2 e""'
Western lmmunoblot Assay. The Western irnn1unoblot assay J-Pe
can detect antibodies to a full range of vir;:i 1 protf'ins ;is rf'- ~
vealed on a slrip oI nitrocellulose paper as discrete bands - .. - - P7
by electrophoresis. When a serun1 sample is applied to the
nitrocellulose strip, antibodics from animals infcctcd with
- X
a specihc virus bind to the specific viral proteins at the ap-
propriate positions. 'fhese bands become dark and distinct
t.vllen tlle n itrocellulose paper is treated with a reagent (Fig
3.4). Because it provides a ful l viral antibody profile of the
serurr1 sarnple, tllis test is the u1osl sµec.ific viral diagnosuc
test c11rrently availahle.
-- LMW
 Antirnicrobial
Chelllotherapy
]OHN
Antimitrohial cirugs exploi t differences in structure or bio-
chemical functio11 between host and parasite. Modcrn
chemotherapy is traced to the work of Paul Ehrlich, who
devotcd his lifc to discovcring agcnts that possessed selec-
tive toxicity. ·rhe first clinically successful broad-spectrum
antibacterial drugs were the sulfonamides, developed in
1935 as a result of Ehrlich's work wlth synthetic úyes. It
was, however, the discovery of pcnicillin by Fleming ano
its úevclu¡.n11c11L 1Jy Chai11 a11d florcy in World Wax 11 that
IPci to the subsequent discovery of turther antibiotics,
chcmical substances produced by rnicroorganisms that at
low concentrations inhibit or kili other microorganisms.
The chemical 1nodification of many of the drugs discov-
ered early in the antibiotic rcvotutlon has led to the devel-
opment of new and powerful antimicrobial drugs with
p1 UJ!C1 lil:::) Ui:>li1!1..l f LVI 11 ll 11;:i 1 p<tt<:u l:>. Anlibiolic:; and
their derivatives have more importance as antimicrobial
agcnts than do thc fcwcr synthctic antibactcrial drugs. By
contrast, antiviral drugs are ali chenucally synthesized.
The term antimicrobial will be uscd throughout to indude
both antibiotics and syntheric ant1mlcrobial drugs.
lmportant milcstones in the development of antimicro-
IJial <.lrug~ are ~l1uw11 i11 Figure 4.1. The LherapeuUc use of
;intin1itrobial drugs in veterinary medicine has followcd
thcir use in human medicine because of the high cost of
thei r <levelopment.
Classification of Antimicrobial Drugs
J\ntimicrobial drugs can be classified in a number of ways:
1. Spectrurn of activity against class of microorganism.
Penicillins are narrow spectrum because they inhibtt
only bacteria; sulfonamides, trimethoprim, and lin-
cosamides are broader bccausc chey inhibit both
bacteria and protozoa. Polycnes only inhihit fungi.
2. Anli/Jucleria.l activity. Son1e antibiotics are narrow-
spectrum in that they inhibit only gram-positive
(bacitracin, vancomycin) or mainly gram-ncgativc
bacteria (polymyxin), whereas broad-spectrum
drugs such as tetracyclines inhibit both gran1-
positive and gram-negartve bacteria. Other drugs
such as penicillin G or Iincosamides arP most active
agai11~L grain-posilive baclcria but will inhibit sorne
gram-negatives.
26
F. PRESCOTI
3. Bacteriostatic or bactericida/. 'fhis distinction is an ap-
proxi1nation that depends on drug concentrations
and tlle organism invoJvcd. For example, penicillin
is bactericida! at high concentrations and bacterio-
static at lower ones. The <.li~tiIH.:tiuu uelween baclc-
ricidal and bacterioSti'ltit is critica! in certain cir-
cwnsta11ces, such as the treatment of meningitis or
of septicemia in neutropenic paticnts.
4. Phannacodynamic activity. Antibactcrial action is
concentration- or timc-dependent (see below under
dosing considerations).
S. Mechanism o( action. Llke pharmacodynamlc activ-
ity, this is dependcnt on the drug class, and is cii~­
cusse<.l below. ·rhis is pruuably t11e n1ost useful oftl1e
classifications, sintP i t cir.1 r.nn i nes the previous four
classification approachc:s.
Mechanism of Action of Antimicrobial Drugs
The rnarked structural and biochcmical diffcrcnccs bc-
twecn eukaryotic and prokaryotic cells give greater oppor-
tunity for selective toxicity of antibacterial drugs com-
pared to antifungal drugs becausc fungí, like mammallan
cells, are eukaryo tic. Dcveloping selectively toxic antiv\ral
<.lrugs is particularly difficulL llccause viral replication de-
pr.nds largely on the metabolic pathways of the host cell.
This chapter mainly discusscs antibactcrial drugs.
·rhe mechanisms ot action ot antibacterial drugs fall
into four catcgorics: 1) inhibition of cell wall synthesis,
2) damagc to cell membrane functton, 3) inhibition of 11u-
cleic acid synthesis or function, and 4) inhihition of pro-
tci11 :.y11Llic~is (Fíg 4.2).
lnhibition of Cell Wall Synthesis
Antihiotics that interfere with cell wall synthesis inc.lude
pcnicill in~ and cephalosporins (bcta.-Jactam nntibiotics),
cycloserine, bacitracin, and vancomycin. ·rhe bacteria! celJ
wall is a thick envelope that gives shape to the cell. This
tough wall outside the cell membrane ls a major differeucl!
between bacteria and mammalian cells. Tn gram-positivc
1.Jactl::ria i Lcou:si:-ls laigely of a t h ick !ayer of peptidoglycan,
which gives the cell rigidity and maintains a high internal
osmotic pressure of about 20 atmospheres. In gram
negative bacteria this !ayer is thinner and the 1nternal
 (;hapter 4 Antimicrobial Chemotl1erapy 27

FIGURE 4.1. Milestones in antimicrobial therapy

Human lnfectious Oisease Antibacterial Agents

8 Penicillin dto;covered

1930
2
4
6
Serious infections - first suttonamide released
respond to sulfonamides ~8

Morey demonstrates '40

penic.illin's effectiveness
z
4 Strcptomycin, fir3t aminoglycoside
Chloramphenicol
6
8 Chlo~tetracycline. first tetracycline

'50
z Erythromycin. first macrolide

Peniciltin.. resistant infections :..- 4

become clinically sioníficant
6 Vancomycin

8
'60 . Mern1c1111n. pen1c1111nase-res1stant penicillin

z
Gentamicín, anüpseudomonal aminoglycosíde
4
Ampicillin
6
Cephalothin. first cephalosporin
Gentamicin-resistant Pseudomonas - - -.... 8
ano melhiC1llin·resistant staphylococcal Am11<acin, ammog1ycos1oe tor
'70
------
infcction~ bec.ome clinicalfy $ignificant 9entamici n-r~si stant strains

Cephalexin
Beginning in earty 1970s, increasing Z
trena ol nosocu111icd i11ret.:tiu11:> üue to
opportunistic pothogens 4 Carbenicillin. first amipseudomonal Betalactam
Amp1cillin-resistant infections _..-__.;y Cefoxitin. expanded-spectrum
6
beconle r1equeot
/ cepna1ospor;n
8

__
Cefaclor, oral cephalosporin
with improved activity
'80
...... Cetotaxime, antipseudomonal cephalosporin
AIDS-related ba.cte1ial i11ftt.:lio11:> ---~
z Mox:il3ct3m, first oxa-BMinhibitor
4 ~~ Clavulanic acid-amoxycillin,
:--...::---.... broad Beta·lactamase 1nn1011or
6 ....... lmipenem cilastatin, first thienamyr.in
Melhicillin·resistant
staphylococcal infections ~
""-•go
8
- Norfloxacin, ncwcr quinolonc-5 for urinary tract in!cctions
ALheu11a111, li1s1 111onobactar11

~~ lmproved macrotides
Vancomycin-resistant Newer quinolones tor systemic use
enterococcl " - 2
~......
M1dtic1r110 rP.!=::iSif::tnce --........_
Myco/Jacterium tuberculosis ......._ ...... 4
..... Oral, e><tended-spectrum
Penicillin-resistant - 6 .....- ceohalosooñns
Streprococcus pneumoniae -.
8 - drug& fara11livi1at
Er r~c..:Live
HlV infection
lncreasino resL<lanr.o nf --->-2000
community-acquired infections: ,_ . Oxazolidinones
·resistance crisis" 2
Reduction antimicrobial ---~
use. bener prescribing guides 4
. Broader·spectrum
ftuoroquinolones

osn1otic pressure correspondingly lower. Peptidoglycan tide bridge from one tetrapeptide to another, so that the
cor1sists of a polysaccharide chain made up of a repeating disaccharide backbone is cross-linked both ;vithin and be-
disaccl1aride backbone of alternating N-acetylglucosa- tween layers. ·rhe cross-linkage between transpeptides
mine ¡:..¡· acetylmuramic acid in beta 1,1 linkage, n tetra g ives the cell wall rcmarkablc strength. Sc..-e ral cn zyme!;
peptide attached to the N-acetylmuramic acid, and a pep- are involved in transpeptidation reactions.
28 PAKT 1 Introduct1on

FIGURE 4.2. Mechanisms of aetion of antibacteria/ drugs.

NITROIMIOAZOLES,
NITROFUR/\NS

~~~ GELL WALL ' -

' - '---. BETA-LACTAM
CELL MEMBRANE. '---. "'-- ANTIBIOTICS,
SULFONAMIDES. PURINE -------.. "'-- "--.. ---GLYCOPEPTIOES,
TRIMETHOPRIM _ .,_,. SYNTHESIS "'-- "--..•~ BACJTRACIN ,
"--.. srncrTOGílAMIHS

DNA
()
C-..- FLUOROOUINOLONES
J NOVOBIOCIN
''V'"-

,_.._,,-. RIFAMPIN
RIBOSOME
• ~
\ ___)
\ MESSENGER )
( NEW
PROTEIN
RNA

IRANSfER
(
ANA
1 •
-
( sos '
AMINO ACJDS
'
~:
\ TETRACYCLINES,
l
-
----- ·~
,, AMl/NOGI vr.osmFS

. '"'--~
OXAZOLIDINONES __.-
1JNr.OSAMJnFS• •/
" ....,...
:
••
'
CHLORAMPHENJCOL
MACROUOES

·rhe ettect of beta-!actam antibiotics (penieillins and
cephalosporins) is to prevent the final cross-linking in the
ce11 wa!l, lnhlblt!ng divlslor1 and creatlng weak points.
Among the targets of these drues ;.irt> pt>nicillin -hincling
protcins (PBPs), of which there are three to eight in bacte-
ria; many of these PBPs are transpeptidase enzymes. They
are rcsponsiblc for thc formation and rc1nodeling of the
cell wall during gro'vth and division. Dúfercnt PBPs have
different affinities for drugs, explaining the variation in
the spectrum of acrton of dlffcrcn t beta-lactarn antibiotics.
L1egradative rnechanisms are also involvPc1 in <Pll wall pro-
duclion. The:;e are carried out by autolysins, and son1e
penicillins act partly by decreasing normal inhibition of
Lhc autolysins.
'!'he action of beta-Jactam antiblol1cs is thus to b lock
pcplidoglycan synthesis, which scvcrcly weakens the cell
wall, an<.l to prornott:' t11e actiu11 uf ll te aulo lysi11s, "vhich
l y~i> tht> CPll. ílPta-lactams are active only against actively
g1owing cell:;. The greater activity of sorne beta-lactams
against gram-positive bacteria is thc rcsult of the greater
quantity of pcpti<loglycan and highcr osmotic pressure in
gram-positive bacteria, the impermeabt11ty of sorne gram-
ncgat1ve bacteria because of their lipopolysaccbaric.te and
lipid exterior, and the presence of beta-lactamase enzymes
lr1 rnany grarn-11egativc orga11i;:.111~. TJ1e re111a1kable acliv-
ity agai nst gram-negatives of sorne of the newest peni-
cillins and cephalosporins is the result not just of thcir im-
proved ability to enter gram-ncgativc cells and bind I'BPs,
but to their ability to resista variety of beta-lactamasc en-
zymes found in the periplasmlc space of gram-11egarive
bacteria. More recently, beta-lactamase-inhibiting clr11gs
witli uo i11tri 11:.ic a11 LilJacle1 ial aclivily, su ch as clavulanic
acid and sulbactam, have been combined witl1 amoxicillin
or ticarcillin to expand thc spcctrum of activity of th ese
latter compou11ds by neutralizing enzyn1es that might
otherwise degrade then1.
1.lacitracin and vancon1yc1n tnhlblt the early stages in
peptidoglycan synthesis. 1'hi>y arP active only against
gran1-positive bacteria.
Penicillins. Sir Alexandcr f.lcming's observation that
colonies ot staphy!ococci lysed on a plate that had become
contaminated ~''ith a Penicillurn fu 11gus was the discovery
that led to the development of antiblotlcs. In 1940, Chain,

Florey, and tl1eir associates succeeded in producing thera-
peutic quantities of perlicillin from Penicilliu111 notaturn.
.Almost a decade Iater, penicillin G becan1e widely avail-
nblc for clinicnl use. In thc ycnrs that followcd, this antibi-
Otic was found to have certain limitations: its relative in-
stability to stomach acid, its susceptibility to inactivation
by penicillinase, and lts relatlve lnactivlty agalnst most
gram-ncgative bacteria . Tsolat.ion of the active moiety, 6-
a111inopenicillanic acid, in the penicillin molecule has re-
sulted in the design and development of se1nisynthetic
penicillins that overcome so1ne of these limitations.
·rne deve1op1nent ot the cep11aJosporin tamily, which
shares with penicillin the beta-lactam ring, has led to a re-
markable array of drugs with in1proved ability to penetrate
rliffP.rt>nt gram-nP.gativt> ha<.tt>ria 1 spP.cit>s anrl to rt>sist ht>til-
lactamase enzymes. ln recent years, other naturally occur-
ring beta-lactam antibiotics have been describcd that lack
the bicyclic ring of the classical beta lactam penicillins and
cephalosporins. Many oftl1ese new drugs have potent anti-
bacterial activity and are highly resistant to beta-lactamase
el tly1ne:;.
C.l"in ically i n1 portant pen icill íns c.an hP. cliviclt>cl i nto six
groups:
l. Beuzyl pe1licilli11s a11d it:; lu11g-acti11g fur1ns. Injec-
tahle penicillins, highest activity against gram-
positive organisms, but susceptible to acid hydrolysis
a11d beta-lactamase inactivatio11 (e.g., penicillin G).
2. Orally absorbed penicillins, spectrum similar to
benzyl penicillins (e.g., pen.icillin V).
3. Antistaphylococcal isoxazolyl pe11icillins. Relatively
reslstant to staphylococcal beta-lacta.n1ases (e.g.,
cloxacillin, methicillin) .
4. Exlended-spectrun1 penicilli11s: An1inopenicillins
(P..g., <imoxic.illin, ampicillin).
5. Antipseudomonal penicillins: Carboxy- and ureido-
penicillins (e.g., carbenicillin, piperacillin, ticarcillin).
6. Bcta-lacta1nnsc rcsistant pcnicillins: 1'crnocillin.
Antitnicrobial Activity. Penicillin G is the most active of
the penicillins against gram-positive aerobic bacteria sucl1
as non beta lactamase- producing coagulase-positive sta-
phylococci, beta-he1nolytic streptococci, Bacillus anthracis
and other gram-positlve rods, corynebacteria, Er)lsipelo-
Lhrix., Lisl1::riu, auu agaiu:;L 1uust a11aerolJes. Tt i.s u1uderately
active against the more fastidious gra1n-negative aerohes
such as Tlae1nophilus, Pasteurella, and s<nne Actinobacillus,
but it is inactive against members of the family Enterobac-
tcriaccac, Bordetclla, and J>scudomonas. 1'hc pcnicillinasc-
resistant isoxazolyl penicillins (oxacillin, cloxacillin me-
thicillin, and nafcillin) are resistant to coagulase-positive
sLaphylococcal penicilli11ase, bul are less aclive lli.au peui-
cillin G against other penicillin-sensitive gram-positive
bacteria. Most gram-negative bacteria are resistant to then1.
Ampicillin and amoxicillin are slightly less active than
pcnicillin G against gram positivc and anacrobic bacteria
and are also inactivated by pe11icilllnase produced by
coagulase-positive staphylococci . 'fhey have considerably
greale1 aclivily agail1st gra111-11egaLive !Jacteria. Tli.ey are ir1-
effective against ]Jseudomonas aeruginosa. C~arhenicillin ancl
ticarcillin resemble ampicillin in spectrum of activity with
the notable difference of having activity against P aerugi-
Chapter 4 Antirnicrobial Che1notherapy 29
nosa. Temocillin is hi.gl1ly resistant to beta-lacta.mases in-
cluding extended-spectru1n cephalosporinases, and has
broad-activity against members of the family Enterobac-
tcriaccae, including othcrwisc rcsistant isolatcs. Myco-
plasma and mycobacteria are resistant to penicil lins.
Resistance. ln gram-positive bacteria (particularly
coagulase-posltlve staphylococci), resistance is mainly
througb production of extracellular beta-lactamase (peni-
cillinase) enzyn1es that break tl1e beta-lactan1 ring oí 111osl
penicillins. Resistance in gram-11egative bacteria result~, in
part, fron1 a wide v ariety of beta-lactamase enzyrr1es and
also trorn 1ow bacteria! permeability or lack ot penicillin-
binding protein receptors. Most or ali gram-negative bacte-
rJa express low levels of species-specific chron1oson1a1Jy
mt>cliatt>cl bet;i-J;ictan1ase enzymes >-11ithin the periplasmic
space, and these sometin1es co11tribute to resisla11ce.
Plasmid-mediated beta-lactamase production is wide-
spread among common gram-ncgntivc bacteria. Thc cn-
zy n1es are constitutively expressed and cause high-level re-
sistan ce. The majority are penicillinases rather than
cephalosporinases. The most widespread are ·rEM-type
ht>t:i-lact:imases, which readily hydrolyze penicillin G and
ampicillil1 rather than n1ethicillin, cloxacillil1, or carbeni-
cillin. The less widespread ()XA-type beta-lactamases hy-
drolyze isoxazolyl pcnicillir1s (oxncillin, cloxacillin, and
related compounds). SHV-type beta-lactamases are founcl
particularly in Klebsiella pneumoniae but may be found in
other members of the family Enterobacteriaceae.
A rnajor recent advance has been the discovery of broad-
spectrun1 inhibitors ofbcta-lacta111ase (e.g., clavula11icacid,
sulbacta1u). These drugs have weak antibacterial activity
but show cxtraordinnry syncrgis1n whcn ad1ninistcrcd
with penicillin G, ampicillin, a1noxicillin or ticarcUlin be-
cause they irreversibly bind the beta lactamase enzymes of
resistant bacteria.
Absorption, Dístríbution, and Excretion. The pe11icillins
are organic acids that are generally available as the sodium
or potassium salt of the free acid. Apart from the isoxazolyl
penicillir1s and penicilliI1 V, acid hydrolysis lin1its the sys-
temic availability of most penicillins from oral prepara-
tions. Both ampicillin and amoxicillin are relatively stable
in acid.
The penicillins are predominantly ionized in the blood
pla~u1a, 11ave relatively ~rnall apparent vulu1nes of distritJu-
tion, and have short half-livP.s (0.:S to 1.2 ho11rs) in :ill
species of domestic animals. After absorption, penicillins
are widely distributed in body fl.uids. Beca use of their high
dcgrcc of ionization and low solubility in lipid, they attain
only low intracellular cqncentrations and do not penetra te
1.rell into transcellular fluids. 'fhe relatively poor di.ffusi.biJ -
ity uf penicillins across cell membranes is reflected in their
rnilk-to-plasma conct>ntration r;i tios (0.3) . The rel<itively
low Lissue levels auained nLay, 1101-vever, lJe clirlically effec-
tive beca use of the high .sensitivity of susceptible bacteria
to pcn.icillins and thcir bactericida! action.. Ampicillin an d
an1oxicillin1 in additlo11to11aving a wider spectrun1 of an-
timicrobial activity, pe11etrate cellular parriers more read-
ily than penicillin G. Their somewhat lor1ger half-lives
might ht> attri buted to enterohepatic circulation. Penetra-
tion to cerebrospinal fluid (CSF) is usually poot bul is e11 -
hanced by int1ammat.ion. In addition, active removal of
30 .PART l lntroduction
penicillin from CSF is diminishcd by inflammation. The
pcnicillins are eJiminated almost entirely by renal excre
tion, which results in very high Ievels in the urine. The
renal excretion mechanisms include glomerular filtration
and mainly proximal tubular secretion.
Adverse Effects. Penicillins are remarkably free of toxic
cffcc..:I~, 1::vc11 al doses grossly in excess of 1l1ose recon1-
me n<l<'d. The major adverse effect is acute anaphylaxis;
mildcr hypersensitivity reactions (urticaria, fever, an-
gioncurotic edema) are more common. A11 penicillins are
cross sensitizin g and cross rcacting. Anaphylactic reac-
tions are less common after oral penicíllin administration
than after parenteral administration. Many of the acute
toxlcl ttes repurted iI1 a11ilual;) a rt: ll 1t: lux.ic effecls of lhe
potassium or proc;iinf' with wh ic:h the penicillin is com-
bincd. The use of penicillin in guinea pigs invariably
causes fata l Clostridium difticile colitis, and use ot ampi-
cillin in rabbits causes fatal C. spiroforrne colitis.
Cephalosporins. Cephalospori ns are natural or semisyn-
thetic products of thc fungí Cephalspurium spp.; tl1t: relalt:d
cephamycins are derived from thP i!C'tinomycetes type of
uaLlt:1ia. 1'11e i1ucleus of thc scmisynthetic cephalospor-
ins, 7-aminocephalosporanic acid, bears a clase structural
rcsemblance to that of tl1e pcnicillins, which accounts for
a common mechanism ot action and other properties
shared by these two classes of drugs. Th ey are bactericidal.
Like thc pcnicillins, cephalosportns have short half-lives,
and most are excreted unchanged in the urine. Attach-
II1e11t uf varivus R groups lo lile cephalospora11ic acid i1u-
c:l<'us has resulted in compounds with low toxicity and
high therapeutic activity. 'i'hough notan ideal description,
the classification of cephalosporins as belonging to gener-
ations relates to their increasíng spectrum of activity
against gram-negative bacteria because of improved pene-
tration of cells and their progressive resistance to the beta-
1<11.lct111d:>t::S of g1a111-negalive bacteria.
Antimicrobial Activíty. The first-generation cephalo-
sporins (e.g ., cephalothin, ccphalcxin, ccphaloridinc,
and cefadroxil) have a similar spcctrum ot activity to
ampicillin, with the notable difference that beta-
lactamasc-produci11g staphylococcl are susceptible. They
are active against a variety of gram-positive bacteria such
as <.:vag ula~e-pvsi li ve staphylococci, r11a11y slrcptococci
(cxcept enterococci), corynebacteria, and gram-positive
anacrobes (Clostridiu1n). Among gram-ncgativc bacteria,
Haen1ophilus and Pasteurel/a are susceptible, as are sorne
Escherichia coli, Klebsiella, J>roteus, and Salmonella.
Enterobaccer and P. aen1ginosa are reslstant. Many anaero-
bic bacteria, except members of the Bacteroides fragilis
gruu¡.¡, are ~u;)ct:plible . The second-ge11eration cephalo-
sporins (e.g .. cefamandole, cefoxitin, and cefuroxime)
ha ve i ncreased resistan ce to g;am -ncgativc bcta-
lactamases and thus broader activity against gram-
ncga l ive bacteria as well as against bacteria susceptible to
the first-generation drugs. Thcy are active against so1ne
strains of Enterobacter and against cephalothin-resistant E.
l·uJi, Klebsiellu, a11tl Prvleu.\. Su111t: B. (iugilis ace susceptible.
f.ik<' the first-generation cephalosporins, these drugs are
not active against P. aen,ginosa or Serratia. Thc third-
gencration cephalosporins (e.g., cetotaxime, moxaJac-
tam. and cefoperazone) are characterized by reduced ac-
tivity against gram positive bacteria, modest activity
against ]"J. aeruginosa, and rcmarkable activity against
m embers of the family Enterobacteriaceae. So1ne third-
generatlon cephalosporins (e.g., ceftal'..idiu1e) are higl!ly
active against .P. aer11ginnsa at thE> Pxpense of activity
against me1nbers ofthe family Enterobacteriaceae. fourth-
generation cephalosporins (e.g., cefepime, cefpirome)
havc vcry broad-spcctrum activity and are stable to hy
drolysis by many beta-Jactamases.
Resistance. Methicillin-resistant coagulase-positive sta-
phylococd are resistant to ali generatiuns uf 1.:epl1aluspvr-
ins. Plasmid-mediated rf'si~tan cP to first-, second-, and
lhi rd-generation drugs has been dcscribed in gram-
negative bacteria. Emergence of rcsistance in Enterobacter,
Serratia, and P. aeruginosa during treatment \vith third
generat1on úrugs resuits from derepress1on or inaucible,
chromosomal beta-lactamase enzymes, which in turn re-
sults in broa<.l-spectrurn resi:;ta11c..:t: lo bela-laclan1 a11tibi-
otics. In addition, plasm írl-mE><liiltP<I resistance to third-
generalio11 cepl1alosporins is increasmgly reported. Tt can
involve either TEM- or SHV-beta-lactamases or other beta-
Iactamase families including CTX-Ml, which hydrolyses
cefotaxime. These beta-lactamases are inhibited by clavu-
Ianic acid. More recently, broad-spectrum cephalospori-
nases (cepham ycinases), CMY-2 beta-lactamases, have
been recognized on plasmids of E. coli and Salmonella;
these are not inhi!Jited l.Jy c..:lavulauic acid.
A hsnrpti.nn, Distrihutinn, and F.x.cretion. Cephalosporins
are water-soluble drugs. Of thc first-generation cephalo-
sporins, cephalexin, and cephadroxil are relatively acid-
stablc and sufficicntly wcll absorb ed from the intestine to
be administered orally to dogs and cats, but not to herbi-
vores. Other first-generation cephalosporins must be ad-
ministered parenterally although they are often painful un
intramuscular ínjection :.in<I irrit;iting on intr;ivenous in-
jection. Second- and third-generation cephalosporins are
someti n1es available for oral use and could be give11 to dogs
and cats by this routc rathcr than parenterally. Following
absorption from injection si tes, cephalosporins are widely
distributed into tissue a11d body flui ds. Third-generation
cephalosporins penetrate cerebrospinal t1uid ruudcraL~ly
well, and because of thPir high ;ictivity against gram-
negative bacteria have particular potential application in
the treatment of meningitis.
Adversc Effccts. Ccphalosporins are relatively nontoxic
antibiotics in humans. Allergic reactions occur in 5% to
10% of human patients who are hypersensitive to peni-
clllln. Jntravenous and intran1u:;c..:uli:ir iujeclions of son1c
drngs ilrf' ;in irrit;int.
Other Beta-Lactam Antibiotics. The last 20 years have seen the
discovcry of other naturally occurring beta-lactam antibi-
otics. These include the cephamyci.ns, clavulanic acld,
thienamycin, the monobactams (such as aztreonam), the
carbapenems (such as irnipe11e111), Lllt: PS-con1pou11ds, a11d
the c;irpf'timycins- ;ill compounds with the basic beta-
lactam ring but v.'ithout the bicyclic ring structurc of thc
classical beta-lactams. All are highly resistant to beta-
Iactamases, and many possess potcnt antibacterial propcr-
ties or are used m combination with earlier beta-lactams

(ampicillin, amoxicillin, ticarcillin) for tht>ir pott>nt ht>ta-
lactan1ase inhibitory effects (clavulanic acid, sulbactam,
tazobactam). Carbapene1ns (biapenem, imipenem-
cilastatin, meropencm) havc cxcept.ional activity against
clinically important aerobic and anaerobic bacteria, with
the greatest activity of ali anti.m icrobials against gram-
negative bacteria.
Damage to Cell Membrane Function
Antibiotics that damage ccU mcmbrane function include
the polymyx.lns, the polyenes lampnoter1c1n, nystat1n),
the imidazoles (tniconazole, ketoconazole, itraconazole,
fluconazole, clotrtmazole), and monensin. 'I"he cell mern-
hrilnt> liPs hPnf>ilth the c:ell \"lall, enc:losing the cytoplasm .
lt controls the passage of materia]s into or out of the cell.
lf its function is damaged, cellular contents (proteins, nu-
cleotides, ions) can leak from the cell and result in cell
damage and deatl1.
Polymyxins. Tl1t: strui.:turt: oí ll1e polyinyxillS is such LltaL
they have well-clefined separate hydrophilic and hydro-
phobic sectors. Polyn1yxins act by binding to n1embrane
phospholipids, which results in strltctural disorganization,
pcrmcability damagc, a11d ccll lysis. The polymyxins are sc-
Ject1ve1y tox1c to gran1-negat1ve bacteria because of the
presence of certain phospholipids in the cell membrax1e
and because the outer surface uf tlu: uuter 1uet111Jra11e u[
grarn-negative b;ictPria consists rna in ly of 1ipopolysac-
ch.aride. Parenteral use is assocíated wíth nephrotoxíc, neu-
rotoxic, and neurornuscular blocking effects. 1'he major
clinical applications are lirnítcd to thc oral trcatrnent of
gram-negative bacteria! infections.
Polyenes. The polyenes are selectively active against fun6'1
since t11ey only affect m.embranes containíng ergosteroL
Polyenes inllilJit tl11:: furruation of 1nen1brane lipids, forn1-
ing porPs through which the vital contents of the cyto-
plasn1 are lost. See the section on antifungal therapy,
below:
lmidazoles. lmidazoles interfere with the biosynthesis of
sterols and bind cell 1nelnbrane phospholipids to cause
leakage of cell i.:outeuts. They are aclive againsl Lhe fungal
cPll mt>mhr;:ine. See the section on antifungal t herapy,
below.
lnhibition of Nucleic Acid Function
Examples of drugs that inhibit nucleic acíd function are
nitroimidazoles, nltrofurans, nalldiXlc acld, the fluoro-
q11inolon<->c; (ri rroflox;iri n, rl;inoflox;irin, rliflnx;icin, Pn-
roflOX3CÍil, orbifloxacin, sarafloxacin), novobiocin, ri-
fampin, sulfonamides, trimethoprim, and 5-flucytosine.
Because the mechanisms of nucleic acid synthesis, replica7
tion, and transcription are similar in ali cells, drugs affect-
ing nucleic acid functío11 have peor selective toxicity. Most
act IJy IJiudiug tu DNA tu inlrilJit it:; replication or tran-
scription. Drugs with greater st>lectivt> toxicity ilrt> the sul-
fonamides and trimethoprim, which inhibit the syntl1esis
offolic. acid.
Chapter 4 Antimicrobial Chemotherapy 31
Nitroimidazoles. Nitroimiclazoles, such ;is mf>troni<l;izolt>
and din1etridazole, possess antiprotozoal and antibacterial
properties. Activity within bacterial cells is due to the
unidentified, rcduccd products of thc drug, which are only
seen in anaerobes or microaerophiles. Nitroimidazoles
cause extensive DNA strand breakage either by inlúbiting
the DNA repair enzyme, UN ase 1, or by forming complexes
1Aríth the nucleotide bases th.at the enzyme <loes not recog-
nl<:t:. Nitrui111ida<:oles are bacLericidal to anaerobic g1an1-
negative and many gram-positive bacteria and are active
against protozoa such as 1ritricho1nonas fetus, Ciardia larn-
/Jlia, and Hzsto1nonas 1neleagnct1s. c11ro1nosoma1 resistance
may cause slíght increases in n1inimum inhibitory con-
centrations (MIC) but, as is tl1e case for 11itrofura11s,
plasn1id-encoded resistance is rilre. Nitroimidazoles ;ire
generally well absorbed after oral administration, but par-
enteral injection is highly irritating. 'f'hey are \.Vell distrib-
utcd throughout body tíssucs and fluids, including brain
and cerebrospinal fluid . Excretion is throu~h the urine.
The most serious potential hazard is tl1e controversia) re-
port of carcinogenicity in laboratory animals. For this rea-
son., these drugs are not used in food animals.
Nitrofurans. Like the nitroimidazoles, the nitrofurans are
antiprotozoal but have wider antibacterial activity; they
are 1nost active unáer anaerobic co.náltions. Atter entry
into the cell, bacteria) nitroreductases produce unchaTac-
terized unstab le reduction products, which differ \.Vith
eacl1 type of nitrofuran . These products cause StTancl
breakage 111 bacteria! DNA.. The nitrofurans are synthetic 5-
nitrofuraldehyde derivatives with broad antimicrobial ac-
tivity. Toxicity and low tissue conccntrations límit thcir
use to the local treatment of infections and to the treat-
ment of urinary tract infections.
Fluoroquinolones. Fh1oroq11in olonPs (c:iprofl oxac:i n, clano-
floxaci11, difloxacin, enrofloxacin, orbifloxacin, sarat1oxa-
cin) are active against gram-negative bacteria. ·rhey cause
selcctive inhibition of bacteria! DNA synthcsis by inhibit-
ing UNA gyrase (topoisomerase JI) and DNA topo1so-
merase IV. DNA gyrase is in.volved in packing (supercoil-
ing) DNA into bacteria! cells whereas topoisomerase IV is
involved in relaxing supercoiled DNA . FluoroquinolonP<;
are baclericidal drugs. Nalidixic acid (a quinolone rarely
used because of toxicity) is most active again~1: gram-
negative bacteria except 1~ aeruginosa, but the newer flu.o-
roquinolone derivatives are broader spectrum and active
against sorne gram-positive bacteria, includü1g mycobac-
teria. /\ctivity against Mycoplasma and rickettsia is also an
important attribute of the newer fluoroquinolones . l' he
fluoroqu1nolo11es are rapidly absorbed after oral ad1ninis-
tr;ition ;inrl h;ivp h;ilf-l iv<->c; v;irying f rom 4 to 17. honr~
·rhey are \.Videly distributed in tissues, and inay be conce11-
trated, for example in. the prostate. Penetration into cere-
brospinal fluid is about half that of serum, whicb makcs
these drugs useful to treat n1eningitis. ·rhey are being in-
troduced rapidly into veterinary use, particularly for use
against gram-11egative bacteria and Mycoplasma. One
drawback is the fairly rapid development of chromosoma-
lly n1edia Led resistan ce, wlricl1 in Curnpylubacter jejuni and
P. aeruginosa can produce high-lcvel resistanc:e ;:iftt>r singlP
32 PART 1 Introduction
nuclcotidc mutations but is more gradual in other bacte-
ria, usually as thc rcsult of cumulative rather than individ-
ual nucleotide mutations. Resistance can also result from
dccrcascd pcrmcability of thc ccll wall as wcll as from ac-
quisition or enhanced activíty of an efflux pump that ac-
tively transports fluoroquinolones from the cell.
Rifampin. Rifampin, which has particular activity against
gra111-¡Ju:>i livt: l.laLlt:lid a11d 1nycobacleria, 11as ren1arkablc
selectivity of inhibition of bacteria! DNA-depe11dent ri-
bonucleic acid (RNA) polymerase. llifan1pin prcvcnts inlti-
ation of transcription. Resistancc deveJops rap idly as the
resul t of chromosomal mutatio11 1 so that this drug is rarely
used on its owr1 but rather is uscd in com b ination with
other antimicrobial drugs.
Sulfonamides and Trimethoprim. S1JI fonamidcs are synthetic
dr ug::; w ith broad antibactcrial an d an tip rotozoal proper-
ties. ·rhey intertere with tJ1c biosynthesis o t tolic acid an d
prevent thc formation of purine nucleotides. Sulfona-
mides are funct lona! analo¡.,rues of para-amin obenzoic acid
and compete '"'ith it for the same enzyme, tetrahydropter-
oate synthetasc, formlng nonfunctiona.1 folie aci<l aua-
Iogues and inhibiting bacteri;il growth. SPIPctivP toxic:ity
of sulfonan1ides occurs beca use man1maJian cells have lost
their ability to synthcsi7.C folie acid but rather absorb it
from thc inlcstinc, whcrcas bacteria must synthcsizc it. In
the bacteria! cell, pretormed folie acid is progressively ex-
l1austed by severa! bacteria! divisions.
Othcr drugs affect folle add synthesis by int erfering
witl1 the enzyme dihydrofolate reductase. One example is
tri111elliupri111, wltiLh i:. :.1:l1:1:lively loxic lo bacteria ralher
than to mammalian cells because of greater affinity for the
bact eria! enzyme. 'fhe enzyme inhibits the convcrsion of
dihydrotolate to tetrahydrololatc, produci ng wit h su lton-
amidcs a scqucntial blockade of folie acid synthesis.
Sulfonamides. The sulfonamides con stitute a series of weak
urgallic acids Lhal e11Le1 n1osl Lissues and body Iluids. 1'11e
degree of io nization ancl lipicl solubility of the large n un1-
ber of individual sulfona1nides influenccs absorptlon, de-
term ines capacity to pc11ctrate cell membranes, and can
affect thc ratc of climination . Sulfonamides exert a bac-
tcriostaric effect agalnst both gram-positive and gra1n-
negative bacteria and can also inhibit other m icro organ -
i:;u1.s (:.u1ue ¡JI ulu1..ua). They are available il1 a wide variet)•
of preparations for either oral or parenteral use. Thcy have
lnrgcly bccn abandoncd bccnusc of widcsprcad rcsistan cc,
ditficulties in administrat1on 1 and the existence of better
alternatives. Ccrtain individual sulfonamides are com-
bined "''lth trlmcthoprlm In fixed ratio (5:1) cornl.JiI1otiu11
preparations that have the advantage of both synergistic
cu1<.l l.léiclt:riLi<.lal t:fft:Ll:>.
Individual sulfonamides are derivatives of sulfanil-
amidc, which contains the structura1 prercquisitcs for an-
tibacterial a<.'1:ivity. 1·he various derivatives ditter in physic-
ochemical and pharmacokinetic properties and in degree
of antimicrobial activity. The sodium salts of sulfonam ldcs
are readily soluble in water, a11d parenteral p reparations
are available for intravc11uus au1niuJ:>LraLio11. Cerlain sul-
fon ilmiclP molPc11IPs ilrf' <IPsigned for Jow solubility (e.g.,
phth;ilyls11lf;ithi;i7.olc-') so th;it thf'y v.'111 hP slaK
sorbed; these are intended far use in the treatme::~
teric infections.
Antin1icrobial Activity. Sulfonamides are broad-spe._-'"'---"--
a11ttm1crob1al drugs. They are active against aerot~ ~ _
positivc cocci and so me rods and sorne gram-n ega::r· =,,._
tcrla, includlng members of the family Enteroba-:.-c-::-f.:a::r::::!
Many anacrobcs are sPn<>itivP.
Resistance. Resistance to sulfonamides in pa~'! -=--
and nonpathogenic bacteria isolated from animals is
sprcad. This situation rcflects their exten ::;ive use in :..:.._•:=?:::::
and veterinary med1ctnc for many years. Sulfona.rr:-d_
sis tan ce may occur as a resul t of m utation causing o< ""--~-:=-
ductlon of para-amlnobenzoic aci<.l (PABA) or a:> o It'SU:1
a structural chane<~ in th e cli hyclrofolic: ;i c:i <l-synth~
en zyn1e wilh a lowcred affinity far sulfon amides. ~ 1,.._.,._
often, sulfonam ide resistan ce is plasmid-mediated.
Absorption, Distribul'ion, and Ex crction. Most sclf
am1C1es a re rap1a1y absorbed trom th e gastroinresrina . - ..... ~­
an d distributed widcly to ali tissues and body fluids
cluding :>yr1uvial auu 1.:c.::reui osp ina! fluid. 'fl1ey aie bo~­
to p lasma proteins to a variable extent. ln actctition - , ....___
fc.::11::11ces a1nong sulC011an1idcs in extent of binding, th~=­
variation among species in binding of individual si.± -
ami des. E.xtcnsive (80%) protein blnding serves to incc
half-life. They enter cerebrospinal tluid well.
Sulfonamides are eliminated by a com bination o f ::-.:n-
excretion and biotransformation processes in the 'i\
This combination of elimi11ation processes contributes
thc spccles varlatlon in tl1e 11alf-life uf iI1uiviuual :...tJ.~.t=:-
amides. While a l;irgP nnmhPr of sulfon;imide prE>pa;a-
is availablc for use in veterinary medicine, many oi 7 3!5
are different dosage forms of sulfamethazine. This St._,,._,_
amidc is most widcly uscd in thc food-producing an.:':l.E
anct can atta in effective plasma concentrations (""•th!:: = u
· _
range 50 to 150 µg/ml) \vh cn ad n1i n istered either o ra:·-
parentcrally. Duc ro thei r a lkalin ity, rr1ost paren:c..
preparations should on ly be ;idm inistPrP<I hy intra,-.,,..,-
iI1jcction. Prolongeu-release oral dosage forms of sulfa.~-­
h azi ne are available .
Advcrsc Ef(ccts . 'fhc sulfonamid es can produce a widc-
riety of sicte effccts, sorne of which may have an alle::.: .
basís w h crcas others are du e to direct toxicity. The m : =
com mon a<lverse effc.::ct:; are uriuary lracl dislurl.Ja1........
(cryst;il lt1ri;i, hPmat11ri;;i, o r even ohstruction) and hema~
poietic disorders (thrombocytopenia and leukopení_
Sorne ad verse effects are associated with particular sul:" -
amidc5. Sulfodiozinc and i;ulfasalazine given for 1011g p.<=-
ods to dogs to control chronlc hemorrhagic colitis h ~
caused keratoconjunctivitis sicca .
Trimethoprim-Sulfonamide Combinations. 1rimethoprim is co::::-
bined with a varicty of sulfonarnides in a fixcd ratio. 7'-
combination produces a bactericida! ettect against a \\iC~
rangc of bacteria, with sorne irnportant cxceptions, ar. _
also inhtb1ts ccrta1n other microorganisms. Veterina.•
prcparations contain trimethoprim combined with sul~­
diazlne or sulfauoxi11c i11 llit: 1:5 1alio. Oll1e.c anlibacter
diaminopyrimidines comhinPcl with sulfonamicles for De'
il1 anin1als include baquiloprim a11d orn1etoprim.
Antirnicrobial Activi ly. ·rrimethoprim-su lfonam ide coc -

"')n~ h;.ive a generally broad spectrum and usually
· ~ricidal action against n1any gram-positive and gram-
__::ve aerobic bacteria, including members of the family
c.;;...:- bactcriaccac. ·rhe combination is active against a largc
?Ort.Jon of anaerobic bacteria, at teast under in vitro
-ditions. A1ycoplasma ar1d J>. ueruginosa are resistant.
·-nerglsm occurs when the mlcroorgan1sms are sensi-
--,..,. "') hoth dn1gs in the combination. When bacteria are
=;:.,:.·:int to sulfonamides, synergism n1ay still he obtained
up to 40% of cases, even when bacteria are only moder-
susceptible to trimethoprim. Becausc of diffcrcnccs
,...,,_ "een the trimethoprim and sulfonamidc in distribu-
n pattern and processes of elimination, the concentra-
......,.•;. ratlos of the two drugs wlll dlffer conslderably in tis-
-.........:. a nci 11rint> frnm tht> ratio in thi> pl11sm;.i. This variation
not important since the synergistic interaction occur~
"'a wide range of conccntration ratios of the two drugs.
Resistance. Resistance to sulfona1nides is due to struc-
·al alteration in the dihydrofolic acid synthesizing en-
-me (dihydropteroate synthetase), whereas resistance to
.....1ellluprirr1 usually results frorn plasmid-encoded syn-
• oCStS of a resistan t ciihyci rofolatf' ri>rl11rtil~f' Pnryme Rae-
- 31 resistance to the combination has progressively de-
ped with use of these preparations in animals.
,bsorption, Distribution, and Elimination. Trimethoprim
- :ipid-soluble organic base that is approx1mately 60o/o
..;nd to plasma proteins and 60ºAi ionized in the plasma.
.:. cun1l>ination of physicochem!cal properties enables
""" ci n1g to distribute widely, to penetra te cellular barriers
nonionic diffusio11, aJ1d to atlain effeclivc co11ce11lra-
t.. ns in most body fluids and tissucs, including brain and
<.... rcbrospinal fluid . Hcpati.c .m ctabolism is thc principal
¡: ¡ocess tor el imination ot trimethopri m. ·rhe halt-lite and
-:-action of the dose that is excreted unchanged in the
L:ine vary widely among different species. The drug is
ell absorbed following oral administration in dogs, cats,
~~llJ horses or from injet:tion sltes in these and other
;w'C'i f'~.
.J.dverse Effects. Serious sidc effccts are uncommon;
~'lose that do occur can usually be attributed to the sulfon-
;i..mide component. Oral trimethoprim sulfonan1ide has
·ne advantage over other oral antimicrobials of causing
.ttlc disturbance among the normal intestinal anaerobic
;ilcroflura.
.,hibition of Protein Synthesis
:..xamples of drugs that inhibit protcin synthesis are tetra-
cyclines, aminoglycosides (am1kac1n, gentam1cin, kana-
mycin, neomycin, streptomycin, tobramycin, and others),
d11 1iuucyclituls (spectiuurnycin), cllloramphenicol, lln-
cosamicies (c:linciamyc.in, linromyrin), ;.ind macrolidcs
azithron1ycin, clarilhro1nycin, e1 yll110111yci u, tylu.siu, tia-
mul in, and othcrs). Because of the marked differences in
ribo5omol structurc, compo:;ltion, nnd function bctwccn
p rol<a ryotic and eukaryottc ce lis, many i mportant antibac-
terial drugs selectively inhibit bacteria! protein synthesis.
•.\11 LilJiutic.s affcctiI1g protein synthests can be dlvlded into
those affcc:ting tht> 10S rihoc;omi> (tPtr11ryclines, aminogly-
cosidcs, aminocyclitols) and those affecting the 505 ribo-
some (chloramphenicol, macrolides, lincosamides).
r:hapter 4 Antimicrohial Chemotherapy 33
Tetracydines. ·retracyclines interfere with protein synthesis
1Jy i11llilJitiug tl1t: lJir1ding of aminoacyl tRNA to the recog-
nition site. The various tetraryrlinPs havt> simi111r ;intimi-
crobial activity but differ in pl1armacologic characteristics.
Antinzicrobial Activity. Tetracyclines are broad-spectrum
drugs active against gram-positive and gram-ncgativc bac-
tena, 1ncluding ricketts1a aod chlamydia, sorne mycoplas-
mas, and protozoa such as Theilcria. Tetracyclines have
guod activity against rnany gram-positive bacteria, the
more fastidious nonentPrir h;irti>riil ~11rh ilS Actinohacil111s,
Dordetella, Drucella, Ilaen1ophilus, sorne Pasteurella, and
many anaerobic bacteria, but thcir activity against these
bacteria and against membcrs of thc family Entcrobactcri-
aceae are l1m1ted by acquircd resistance. I'seudomonas
aeruginosa is resistant, except in urinary tract infectiolls 1
whcrc, uecause of their t1igh concenttattons, tetracyclines
may he drugs of c:hoirt>.
Resistance. Widespread resistance to the tetracyclines l
has considerably reduccd their usefulness. Such resistancc
is high leve! and usually plasmid- and transposon-mcdi- · UJ
ated. Cross-resistance between tctracyclines is complete. :J:
Adverse Effects. Tetracyclines are generally safe antibi i-
u lit:)) witl1 a reasonably high therapeutlc index. The main W
adverse effects art> as~ori11ti>rl with thcir severely irritant CJ)
nature, with disturbances in gastrointestinal flora, wilh <(
their ability to bind calcium (cardiovascular e.ffects, teeth ._
or bone deposition), and with thc toxic cffccts of degrada- :J -1
tion products on liver and kidncy cells. Their use in horses
has largely been abandoned beca use of a tendency to pro-
duce broad-spectrun1 supprcsslon of the normal intestinal
flor;i ilnrl filtill superinfection with Salmonella or Clostri-
diu111 dif(lcile. -m
Chloramphenicol and Florfenicol. C hloramphenicol and flor-
fenicol are broad-spectrum, generally bacteriostatic drugs
that bind the SOS ribosome, distorting the region and in-
hlbltlng the peptidyl transfcrasc reactlon. They are stable,
1ipirl -~ol11hle, neutral compou nds.
A11ti111icrobial Activity. Chloran1phenicol and florfenicol
are active against gram-positive and gram-negative bacte-
ria, including chlamydia and rickcttsia, and sorne my-
coplasmas. Most gram-positive and many gram-negative
pathogenic aerobic and anaerobic bacteria are susceptible,
though restsrance ls increastng In memhers of the family
Enterobacteriaceae. Florfenicol is less active against mem-
bers of the family Ente1obacte1h1eceue bul has liigli aclivity
against Haernophilus, Mannheirnia haetnolytica, and Pasteu-
rclla. Thc drugs are gcne rally b acteriostatic.
J<esistance. Most resistance is the result of plasmid-
encoded acetylase enzymes.
Absorption, Distribution, and Excretion. In dogs, cats, and
preruminant s, chloramphcnicol is well absorbed from the
lutestine; in rumina11ts thc drug Is Inactivated after oral
acim in istriltion. Rer;.iuse of its low molecular weight, lípic.l
solubility, and modest plasn1a protcin bindi11g, lhe drug is
well distributed in rnost tissues and fluids, including the
cerebrospinal fluid and aqueous humor. The half-life of
chloramphenicol varies widely in an1mals from a low of 1
h our in horses to 5 or 6 hours in cats. In neonates the half-
life i)) cun:;idcra!Jly longer. The drug Is malnly ellminated
hy g lucuronicie c:onj11g11tion in the liver.
34 PART 1 Introduction
Advarse Effects. The fatal aplastic ane1nia seen in l in
25,000 to 40,000 t1urnans treatt:tl with chlura.iuvlrc11icul
clot>s not occur in animals, although prolonged high dos-
ing may cause rever:iiblc abnormnlitics in bonc mnrro~v
activity. ·rhc potential tor nondose-related fatal aplastic
anaemia in l1uma11s has led to its prohibitior1 for use in
food animals in most countries because of fear of the
presence of drug residues in meat products. Florfenicol
does not have th.is effect and so has selective use for foutl
animals.
Aminoglycosides. The aminoglycosides are bactericidal. The
mode of action of strcptomycio is best understood.
Streptomycin 11as a variety of complex cffccts 111 tbe bacte-
ria! cell: a) it binds to a specific receptor protein in the 30S
ribosomal subunit, distorting the codon-antlcodon inter-
actions at the recognition site and causiog misrcading of
the geuetk: cuue :>u Ll1aL faulLy prolclns are produced; b) it
hinct~ to "initiating" ribosomes to prcvcnt the formation
of 70S ribosomes; and c) it inhibits thc clongation reaction
of protein synthesis. ·rhe othcr aminoglycosides act SJ1n1-
larly to streptomycin in causiog mistranslatioo of the
gcnctic code and in irreversible inhibition of initiation, al-
though thc cxtent and type often differ. They have mul-
tiple blndlng sites on the rilJoso1nc, wl1erca:> :>Lreplo1nycin
has only one, ;-incl can also inhihit the translocation step in
protein synthesis. Spectinomycin is a bacteriostatic arni-
nocyclitol antibiotic that is believed to inhibit polypeptidc
chuin clongation at thc tra11slocation step.
·r11e aminoglycoside antib1otics are polar organic bases.
Their polarity largely accounts for the similar pharmacokl-
netlc properties that are shared by ali members of tl1e
group. Chemically, they con~i~t of a hPxosP nuclPus to
wl1ich an1ino sugars are attached by glycosidic linkages.
All are potentially ototoxic and nephrotoxic. The newer
uminoglycosidcs are more resistant lo plasmid mediated
enzymat1c degradation and are less toxic than the older
compouods. Amikacio > tobramycin > gentamicin >
neomycln = kanamyc.in > strcptomycln in potency, spec-
trum of activity, and stability to pl<ismi<i-mPrliat ed resist-
ancc. Tl1is acUvity 111irrors thc chronology of introduction
of the drugs, with streptomycin being thc oldest ot t he
ami noglycosides.
Antirn1crob1al Activity. Am1noglycos1c.les are particularly
active against gram-negative bacteria as well as against my-
cobacterla and sorne mycoplasma. AnaerotJic bacteria are
usually resistant. As a general rule, gr;im-positivt> hactt>ria
are 1esislanl lo older drugs (strcptomycin, neornycin) but
may be inhibited by newer drugs (gentamicin, ami.kaci n ). A
particularly useful property is the activity of newer arnino-
glycos1cles against P. aeruginosa. 'rhcir bacteridclal action on
aerobic gram-negative bacilli is markedly influenced by pH;
they are most active in an alkall11e environment. lncrea:seu
local acidity secondary to tissu(' damag<> may account for
ll1e failu1e of an a111i11oglycoside to kili usually susceptible
microorganisms at infection sites or in abscess cavities.
Combinations of aminoglycosidcs with penicillins are
oltcn synergistic; the concurrent admrn1stration of the
oewcr beta-lactam antibiotics with gentamicin or to-
bramycln has been usecl to trcat serlous gram-negativc iu-
fections, for example, those caused by P. ru n1ginnsa.
1
Resistance. Most clinically import;-int rt>sistanct> is
caused by a variety of R plasn1id-specified degradatíve cn-
zymes locatcd in the periplasmic space. Certain of these
cnzymc:; inactivatc only thc oldcr •uninoglycosides (strep
tomycin, or neomycin and kanan1ycin), but others are
broader spectrum. 'fh.e remarkablc property of arnikacin
is its resistance to many of thc c11zyn1es t hat inactivéltc
other aminoglycosides. Plasmid-mecliatt>d rPsistance to
:>lreµ1u111yci11 is v•>'idespread and con1monly linked to sul-
fonamides, tetracyclines, and ampicillin. Chromosomal
resistance to streptomycin, but not to the other aminogly
cosides, develops tairly readily during treatrnent.
Absorption, Distribution, and E:.:cretion. Aminoglycosides
are poorly absorbed from the gastrointestinal tral.t, 1.Ji11u Lu
a low extcnt to plasma proteins, and havt> limited capacity
to 1;;:11tcr cclls a11d penelrale ccll u lar barriers. ·r11ey do not
r0.adily attain therapeutic concentrations in transcellular
fluids, particularly cerebrospinal and ocular fluid. Poor dif
fusibility can be attributed to their low degree of lipid sol-
ubility. ·rheir apparent volumes of distribution are rela-
tively small, and their half-llves are short (2 l1uurs) i11
domestic animals. Even though these dn1gs have a small
volume uf uistril.Juliu11, seleclive binding to renal tissue
(kiclney cortPx) occurs. Elimination takes place entirely by
renal excretion (glomcrular filtration), and uncha.ngcd
drug is rapidly excreted in the urine. ln1pairecl renal func-
tion dccrcascs tl1cir rate of excretion and makes adjust-
rnent of the maintenance ctosage necessary to p revent ac-
cumulation with attendant toxicity.
Major changes are taklng place 1n recornrueu tlatiuus fur
in tra111uscular dosage with ami noglycosiciPs, wh ich is
r11uvi11g frorn ll1ree lin1es daily to a single daily dosagc.
This has the effect of increasing therapeutic efficacy, since
antibacterial activity dcpcnds oo both peak concentra
tions and total conccntration, and of reducing toxicity,
since the oephrotoxic effects depend oo a threshold effect,
concentrations above which have no further action. ·rhis
dran1atically changed understandíng of aminoglyco~icit>
uu:>agt! 111ay increase Lhe use of tl1e less toxic members.
Adverse Effects. All aminoglycosides can cause varying
dc.:grccs of otot oxicity ond ncphrotoxicity. "fhc tcndency to
produce vestibular or cochlear damage varies with the
drug: neo rnycin is the most likely to cause cochlear dam-
age ancl streptomycin to cause vestibular clarnélge. Ncpl 1ru-
toxicity (aCL1te tubular necrosi~) occ11r~ in association with
µrulu11ged Lherapy and excessive trough conccntrutions of
the aminoglycoside (particularly gentamicin) in plasma.
The arninoglycosidcs can produce neuromuscular block-
age ot the nondepo1anz1ng typc, wh1ch causes flacc10
paralysis and apnea. This is most likely to occur in associa-
tlon with anesthesia.
A1ninocyclitols. Spcctinon1ycin is an a111in ocyclitol antibi-
otic with a spectrum of activity and mechanism ot action
similar to that of kanamycin but without the toxic effects
of the aminoglycosides. lt is normally bacteriostatic and Is
not particularly active on a wcight basis. Its activity against
gram-oegative bacteria Is unpretlil.tabll.! 1.Jecau:>e uf natu-
rally resistant strail1s. Chromosomal resistance dcvelops
readily bul does oot cross-react ""ith arninoglycosidcs.
Plasmid resistance is uncommon but often extcncls to

r--:.-- ->myrin The clrt1g h¡¡s most of the ph;1rm;1coldnetic
'."'.X"-
~es of aminoglycosides but appears to penetrate
ü:!"E~.::rospinal flujd better. It has been used in agricultmaJ
~ - ce to trcnt snlmoncllosis nnd mycoplnsmn infections.
.;;,::::: des. Macrolide antibiotics are bacteriostatic with ac-
- partlcularly agatnst gram-posltlve bacteria and my-
..... -~ 'TTI" Thf'y bind to SOS ribosome in competition with
""'-""-ramphenicol and inhibit the translocation step of
;:ii:e·n synthesis. ·rhe precise mechanism of action is un-
- ·-n. Mncrolidc nntibiotics (azithromycin, clnrithromy-
- <'rythromycin, tylosin, tlamulin, and spiramycir1)
"'action and pharmacokinetic properties similar to the
....::... u~mldes. Llke the llncosamldes they are llpid soluble,
--·e rlr11gs th;:it ;:irP ronrf'ntr;:itr<I in ti s~11P compared to
:um and penetrate cells well.
.411ti1nicro/Jial Activity. Eryth ro mycin has an antibact er-
c. spectrum sín1ilar to penicillin G, but it includes activity
:;ainst penicillinase-producing coagulase-positíve staphy-
.._occi, Campyfobacter, Leptospira, Bordetella, rickettsia,
~u1yLlia, :;uu11; 111ycuplasrna, anti atypical mycobacteria.
; =iay he hact <'ricicla 1 ;:it h igh roncPntr;:ition<;_Tylosin ¡¡nd
~amycin are less active than crythromycin against bac-
~"ia but more active against a broad range of rnycoplasma.
-:u:iulin has better <tctivity than the other macrolidcs
__....::st anaerobes, 1nclucting Brnd1yspirn flyodysenterine,
- l i distinguished for its remarkable activi.ty against my-
~...-~rnas. Azithromycln and clarithromycin are particu-
-:\_ active against non-tuberculous mycobacteria. The ac-
. of Lht:::.e lwu Llrug::. agaiusl a variety uf ir1tracellular
~~::,-ia not only rl<'pf'nrl<; on the.ir intrin<>ic: ac:tivity hut
i:S:>on thc oftcn rcmarkablc conccntratioo of thc drugs in
•..o.. . including macrophages. Azithromycin may concen-
-., 200-500 fold inside macrophages compared to its
;:!ID concentrations.
ílesistance. Onc-step chromosomal resistance to eryth-
mvctn develops falrly readily, even during treatment ,
4< is generally unstable. Plasmid-mediated resistan ce is
-'11111011. C1 oss-resist a n ce bel ween eryl hro n1ycin and lin-
- ~mides and other macrolides is common. There is little
..n;"ormation about resistance of veterinary path ogen s to
-'!osin. Development of resistan ce to tiam u lin app ears to
oe relatively uncommon; organisms resistant to tian1ulin
-'-º"v une-way cross-reslstance wit h ot her macro liúes.
4hw¡rptinn, f)i~trihutinn, and F.xr.retinn. F.rythromycin
~ea rate and estolate are well absorbed after o ral adminis-
::::ation, but the base is not. Intramuscular injection of
c:ythromycin is very irritating. Thc nbsorption of tylosin
..:om the intest1ne var1es with the formulation. Tiamulin is
• '"ell absorbed. 1'hese drugs are weU distributed through
!Jutly tissues anti fluitl!>, except the cerebrospinal t1uld.
:-i'>~ue roncpntr;:ition<: oftPn PxC'PP<I SPr11m conc:entrations,
aotably for azithromycin and clarithron1ycin, '"'hich are
-herefore often dosed once a day or less frequently. In the
::ase of spiramycin, such tissuc conccntration is extreme
and is associated "v1th t1ssue b1nd1ng. A large proportion of
dlesc drugs is degraded in the body, but sorne is excreted
.i1ruugl1 tl1c kltlucy anti the liver.
Adverse F,ffert~. M;:irroli<IPs i'lrf> gPnPr;:illy safe drugs
though painful on injection . 'fheir potential for causing ir-
reversible diarrhea in adult horses mea ns th.at they should
Chapter 4 Antimicrobi;:il Chemotherapy 35
be <ivoided ln this species. ·ry1osin and tiamulin adminis-
tered intravenously to calves n1ay produce severe nervous
depression. The drugs should not be given orally to rumi-
nants bccausc of thcir potcntial for disturbing the n1men
flora .
Lincosamides. Llncomycin and clindamycin have antibac-
terial activity mainly against gram-positive aerobic bacte-
ria and against anae1obic bacle1ia. The d1 ugs bi11d Lltt: 505
ribosomal subunits at binding si tes that overlap with those
of chloramphcnicol and the macrolides. 'fhey inhibit the
peptidyl transterase reaction. ·rhe lincosamides, lincomy-
cin and clindamycin, are products of an actinomycete
with activlty and mechanism of action similar to t hat of
the macrolides. Lincomycin is most commonly used in
veterinai:y 111edicine, allhougl 1 i Lis lt:s:. active un a vveight
basis t han cl indamycin. Lincosamides are active against
gram-positivc acrob.ic and ali anaerobic bacteria, and
against mycoplasmas, but most gram-negative aerobes are
rcsistant. Clindamycin is more active t h an Jinco mycin
agalnst anaerobes and may be bactericida!. Chromosomal
stepwise resistance develops fairly readily, and plasmid-
n1ediated rcsistance is co111111011. Cros:.-n:::;i:;ta11ce between
lincosamides is complete and commonly occurs also '1-Vith
macrolidcs. Lincomycin is readily absorbed after oral or
intramuscular administration. Food delays and reduces ab-
sorption. The absorption of differcnt clindamydn com
pounds is variable. The lincosamides are >'lidely distrib-
uted in body tissues and fluids, including the prostate and
mil.k, but cerebrosplnal tluld concentrations are low. They
pPnPtr;:itp in tr;:icPll11 l;:irly hPr;;i11<;P nf thPir 1i poph i lir propPr-
ties. Most excretion is through the liver. The major adversc
ettect ot lincosamides is their ability to cause fatal diarrhea
in horses, rabbits, guinea pigs, and hamsters. In rabbits,
fatal diarrhea results from proliferation of Ctostridiunz
spiroforme or G'. difTicile. Oral lincosamides at low concen-
trations produce severe ruminal disturbances in adult
runlinants.
Antimicrobial Susceptibility and Drug Dosage
Prediction
The use of antimicrobial drugs in treating infections de-
pends on thc rclation of the quantitative susceptibility of
the mlcroorganlsm to tissue concentrations of drug. The
antimicrobial susccptibility of many veterinary pathogens
is predictable and clinical cxpcric11ce l1as eslablished effec-
tive dosages for infections caused by these organisms. In
many bacteria, howcvcr, thc prcscnce of various mecha-
nisms for acquiring resistance means that susceptibility to
a particular antibacterial drug may need to be tested.
Antimicrobial Susceptibility Testing
·rhere are two general methods for antimicrobial suscepti-
bility testing in vitro: the dilution method and the diffu-
sion method. The d!lution method gives quantitatlve in-
formation on drug susceptibility while t he diffusion
n1elhod ~ivc::::. 4ualilative (or at tJest serniquantltatlvc) ln-
formation. The tests m11<;t hf' pPrformed under standard-
36 PART I lntroduction
ized conditions. The description of a bacterium as suscep-
tible or resistant to a11 a11tiuli<.:ruulal urug depeuds ul Li-
m<'ltPly on clinical success or failure of treatment. Quanti-
tative informatio11 011 susccptibility is obtained in the
laboratory under artificial circumstances, wl1ich cannot
easily takc into account host defenses, the dynamics of
drug dísposition, or tl1e dynamics of interaction of a vary-
ing drug concentration with a bacterium in the host envi-
ronrne11t. Nevertl1ele5:., ioft<.:lio11s caused by bacteria clas-
sified as resistant in s11scPptihility tests rarely respond
successfully to trcatment, except under exceptional cir-
cumstances. Infections caused by bacteria classified as sus-
ceptible can be predicted to do so, dependíng on the dini
cal circumstances, nature of the lnfect1on, appropriate
dosage, and a variety of other factors-some of which are
discussed bclow.
L)i/ution A ntirnicrobial Tests. Antimicrohi;il clr11gs o f
k11uw11 (JUle11cy are prepared in doubling d ilutions of con-
centra tions similar to t hose ach icvable in the tissues of pa-
tients given usual drug dosagcs. Thc highcst dilution at
which there is no visible bacterial growth following inocu-
lation and incubation is thc mínimum inhibitory concen-
tratlon (Ml C), which is usually less than tl1e lllit1i111u111
bactericida! concentration (MBC) for cln1gs ('rahle 4 .1 ) .
1"he advantage of deterruiJú11g 4ua11tilative susceplibil-
ity of ;in nrganism is that this information can be related to
knowledge of drug conccntrations in particular tissucs in
the precliction of appropriate drug dosage. ln medicat
practice, Mi(-: rcsults are usually interpreted by the system
of categories suggested by the u.s. National Comrnittee for
c:tinical Laboratory Standards (NCC:LS) . 'fhese interpreta-
tive guldclines take into account tl1e irtl1ere11t :;u:;ct¡.>Liliil-
ity of the ore;inism to P.:!Ch cln1g, thP pharmacokinetiC and
pharmacodynamic properties of the particular drug,
dosage, site of infectíon, and drug toxicity. These cate-
gorics are 1) susceptible, meaning that the infecting organ
ísm 1s usually inhibited by concentrations of a particular
antibiotic attained in tissues by usual dosage; 2) intermedi-
ately susceptible, meaning that tht! ir1fet.ti11g orga1ús1u i:.
inl1ibited by bloo<l or tissuP concPntrations achieved with
n1¿1xin1un1 dosage; and 3) resistant, mcaning that it is re-
sistant to normally achíevable and tolerated concentra-
tions of antimicrobial drugs. /\ fourth category, flexible,
has recently been íntroduced by the NCCLS Subcommit-
tee on Veterinary Antimicrobial Susceptibility Testing
(1999). ·rhls indicares the availa!Jility i11 tl1e U1tlttu Stales
of the U.S. Food and Drug Aclmini<;tration flexible Iahel,
whicl1 allows veterinarians to adj u st the dose, within a
givcn range, based on the MIC of thc pathogen.
Diffusion Antimicrobial Tests. /\ standard concentration
of a pure culture of tl1e pathogen is placed on appropriate
agar and individual filter paper discs containing known
concentratlons of lndivi.dual antlfJiotics are ¡.>laced 0 11 Lhe
agar, which is l 11cubated for 18 ho11r~ at 1.')ºC. Th e zone of
i11hibilion arou11d each disc is n1easured and the measure-
ment is refer rcd to a chart that classifies tbe organism as
being susceptible, resistant, or intermediately susceptible
to the particular ant:tb1ot1c 1n eacn a1sc. 5tanaaras for per-
forming these tests are defined. Under standard condi-
tlons, t h ere is a li11ear iI1vtr:.1:: 1elalionship bclwcen the di-
ameter of the zone of growth inhihition and MTC. The
Ta b 1e 4. 1 . Mínimum Concentration of Tetracycline lnhibitory to
Sclected Vetcrinary Pathogcns
Minimum mhibitory
Concentrati\>11 (µg/ml}
MICso* MIC90•
Bordetella bronchiseptica 2 L
Bruce/la canis 0.25 0.25
Corynebacreríum pseudotuberculos/s 0.25 0.25
Escherichia co/i 4.0 64.0
Kfebsiella pneumoniae 2.0 64.0
Mycopfasma canis 4.0 8.0
Parteurella multodda 0.5 0.5
•Highest MICso of 50% of i:;olate< te<tcd, MI<:,¡,¡ of 90% of isolatos te<ted. The MIC of
~iflere11t u19•nC.111>.v•r.ie>wilh strain and spe<ies.
interpretation of zone diamcters as susceptible, resistant,
or intermediate relates to serum drug cu11<.:t11Lrations of
antibiotics in diffPTPnt :inim:il spPcips commonly achiev-
able u11der standard dosage rcgimens. From thcsc drug
con centrations, MIC breakpoints have been selected and
extrapolated to zone diameters in providing the intcrpre
tative standards. A specialized modilied diffusion system is
thc E test, wl1ich is a modified diffusion concentration gra-
dient strip system that gives quantltative results.
Design of Drug Dosage and Pharmacodynamic Properties
Ph a1111acokinelic descriptions of drug disposition in d.iffer-
ent animal species, wben combined with quantitative sus-
ceptibility (MIC) data and knowlcdgc of the pharmacody
namic properties of the antimicrob1al, allow predictíon of
reasonable drug dosage in animals.
Pharmacokinetic propertles lnclude the route uf au-
ministration , rate of absorption, rat(' of <1istrihution, vol-
un1e of dislribulio11, a11d route and rate of elimination.
Pharmacodynamic properties include concentrat ion ver-
sus time in thc tissuc and othcr body fluids, as well as at
the site ot i.ntection, toxicoJogic cffect, a11<.1 antimicrobJ.al
effect at the site of infection. The pharmacodynamic ef-
fects at the slte of Infectlon lncluue tl1c MIC, MBC, con-
centration-dependent killing pffect, post-antihiotic effect,
sub-MIC effect, post-antibiotic leukocyte enhancemcnt
(PALE) effect, as well as first exposure effect. for sorne
("conccntration-dependent") antimicrobials (aminogly-
cosides, tluoroquinolones), kllhng is a function of antimi-
crobial concentration relative to MlC, a function that may
perslst long after drug conct!11tratiu11:; are below MIC. For
these drugs the tota 1;imo11 n t of the drug above MIC ("area
uJ>.dcr ll1c curve") is also important. For otl1cr ("timc-
dependent") anti1nicrobials (bcta-Iactams, chlorampheni-
col, lincosamides, macrolides, tetracyclines, tri n1etho-
priro-sultonamides), bacteria! inhibition is a tunction of
the ti1ne that tissue concentrations exceed MIC; these
<Jrug:; tl1erefore llave Lu !Je uu:.eu Lo iudü1laü1 co.nccntra-
tions at the site of infection above MIC. For these drugs,
the total amount of drug abovc MIC is not irnportant since
no additional killing occurs with increased concentrations
 Chapter 4 Antimicrobial Chemotherapy 37

(an<.I in<.lee<.I for some drugs killing may actually decrease at in cxtravascuJar tissue fluids, principally the interstitial
lllgh co11cenLraLio11s). The 111axi111u111 i11terval uf drug dus- fluid. They penetra te capillary endothelium through pores
ing should preclude resumption of hacterial growth. that arlmit molecules with a molecular weight of less than
about 1000. Passage across biological 111e1111Jrancs suclJ as
Factors Affecting Tissue Drug Concentrations. Dosage. The dosage into tissue cells or across nonfenestrated capillary Pnrlo-
rcgimcn is made up of the siz.a of the dose, which is limitcd thclit1m <.lepends on drug ionization, lipid solubility, mo-
by drug toxicity, and the dosage interval, wh1ch 1s dcter- lecular weight, and the amount of free drug prescnt. Lipid-
mined by the half-life of the drug. 'l'he dosage interval re- soluble and nonionized drugs such as the macrolides and
qui1ed Lo n1aiuLai11 Llierapeulit: Lissuc cuuce11tratiuns by chloramphenicol distribute wcll and even concentrate 1n
intravenous dosing should not exceed twice the half-lifP ti<;<;11P, whereas ionized and weakly lipid-soluble drugs
for most antibiotics, but giving drugs by other routes such as penicillins a11d an1i11oglycu!>ille:. uistrilJute puorly.
lengthens the dosage interval. ·rhese physicochemical differences largely de.tC'rminP thP
Routcs of /\dministration. Antibactcrial drugs can be ad- pharmacoJ..;netic characteristics of the drugs; ll1us, anli110-
ministered by a wide variety of routes-for example, intra- glycosides and penicillins have small apparent volumes of
venoi1s, intramuscular, oral, subcutaneous, intramarn- distribution and short h;llf livc:: aftcr intravcnous injcc-
n1ary, inlraulerine, 01 respi1 alury: tlon and are eliminated tl1rougl1 the urinary tract, 'INhereas
m;icrolides and tetracyclines havc large apparent volumcs
1. Jntravenous injection of a drug gives immediate
of distributio11 a11d longe1 half-livcs aud are eliminated In
high serum drug concentrations, which rapidly de-
part through the liver. Penetration of srf'rial sitf>S in the
cline as thc d rug is disl1ibu Led. I11Lrave11uus <.lusing
body such as thc central nervous system, eye, and prostate
may he the only way to exceed the MTC. of somP
pathogens, but frequent dosing by this route is gen-
erally impractical in vcterinary medicine.
(which among other dilterences lack capillary pores) is
only by low molecular wcight, lipid-soluble, nonionized u
2. Intramuscular injection is commonly uscd in vctcri-
drugs.
Prntein Rinding ofDrug. Tn general, serum protein bind-
nary medicine because it gtves good serum concen-
ing of drugs up to 90 pe1ce11l i:, uf liltlc clir1icai importan ce.
trations within 1 to 2 hours of administration. The
Aminoglycosides and polymyxin hind extensivPly tn intr;i-
n1a jor advanlage is Lhal i11Lra111u.scular ir1jection
ccllular constitucnts a11d thus are inactivated by pus.
gives the highest serum concentration of ali routPs
Excretion A-1cchanisn1s. ·rhese determine thc concentra-
other than intravenous, a lthough subcutaneous in-
tion of drugs in the organs of excretion . Rc1narkably high
jection is a reasonable alternalive. Drug formula-
concentrattons of drugs may be achieved in urine or bile.
tion can be prepared to givc slow rclcasc of thc drug
J>hysiological Barriers. Anatomic-physiologic barriers in
after intramuscular 1nject1on and thus protong
lhe b1aiu, cere1Jru:.piI1dl flui<.l, eye, and mammary gland re-
dosage intervals to reduce handling of animals.
duce the entry of drugs from thP hloorl_ lnflammation re-
3. The oral administration of antlmicrobial drugs is
duces but does not abolish thcsc barriers.
limiten to n10J1ogastric and preruminant animals
Uuration of Treatrnent. Although it is axiomatic that a
and to young foals. Tl1e oral dose is genera11-y severa!
drug must be present for adcquatc time at thc sitc of infcc-
times greater tl1an the parenteral dose hecause the
tion, the variables affectinp; time of treatment havc not
drug is less well absorbed. Although the oral route is
been defined. The response of different types of infection
otten the easiest way to administer drugs, it is not al-
tu antibiotics varíes, and cllnlcal experience with differenL
ways the most reliablc. Sorne drugs (aminoglyco
tyr~s of infertion i-; impo rtan! in assessing response to
s!des, polymyxins) are not absorbed fron1 the intes-
treatment. ln general, if no response lo l1ealrneul i:. ulJ-
tine, others are destroyed by stomach acidity
servcd after two days, diagnosis and trcatment should he
(bcnzyl pe11icillin), ancl abso1plior1 1I1ay lJe iiupairc<.l
reassesscd. Trcatmcnt shoul<.I be continued for 48 hours
byfood (asoccurs with ampicillin, tetracyclines, lin-
aftcr symptoms have resolved, dcpending on the severity
comyci11). Admü1istration of antibiotics in water is
of in fection. For serious infections, treatment should last 7
ncvcrthelcss a particularly simple, convenient, and
ro 10 days. Sorne uncomplicatcd infcctions, such as cysat1s
inexpensive way to trcat livestock because it in-
in ff'males, have been successfully trcatcd with single doses
volves little if any handling oí animals and avo1ds
of antibiotics.
the expense of mixing antibiotics in feed .
4. lnfeclio11s of ll1e udde1, fe111ale ge11ilal trdLt, exter-
na! car canal, and skin are commonly treaterl hy
loc11l 11pplication of antibiotics. Tligh drug concen-
Use of Antibacterial Combinations
trations are obtained without systemic toxic effects.
Combinations of drugs sometimes have dramatic success
The concentration of free drug in the serum largcly
where individual drugs faiJ. An outstanding early example
determines the conccntration in tissue tluids, since
i:; ti 1e use uf penicillin-streptomycln combinations in ente-
pcnctration of drugs in to intcrstitial fluids in most
rococc.al enclor;irclitis in humans. However, early studics of
li:>$ues uf tl1e lJu<.ly is through pores iI1 capillary en-
thc outcoroe of con<bination lrcaln1e11L oí pHcu111u1..u1..1..<1l
dothelium.
meningitis in humans showed the serious clinical effects
Pl1ysicochernical Properties ofthe Drug. ·rhese charactcris- of mixing bacteriostatic an.d bactericida! drugs. Thc im-
tics largely delern1i11e Lhe e.>tleul uf tite distribution of a portancc of antagon1st1c interactions between drugs is
d rug in the body. Most antimicrohial rln1gs rlistribute well greatest in those infections or patients where immune de
38 PART I Tntroduction
fenscs are poor- meningitis, endocarditis, or chronic os-
teomyelltls- or where imrnunu<.leficie11cies are ¡Jrese11 L,
and whPrP a hact Pric:irlal action is needed. If a bacteriosta-
tic drug is ni.ixed witl1 a bactericida! drug, thcn thc formcr
may neutralize the latter, w!1ich may be crucial for clear-
ance of infection from certain sites or infections (menin-
gitis, endocarditis, chronic osteomyelitis). In other pa-
tients or diseases, because of the complexity of the
hosr-ba<.:tertal- a11tlllllcrulJlal l11Le1acliu11, il is l1a11.lt:1 Lu l.lt:·
tert PithPr synergistic: or antagonistic effects clinicaUy, and
it is likely that antagonistic effects of drugs are "laboratory
artifacts" with no clinical meaning.
A drug combination is additivc if the combined effect of
several drugs is the sum of their 1ndcpendent activities
measured separately, syncrgistic if the combined effect is
significantly greater than their independent effe<.:ts, anú
antagonisdc if it is significantly less than thf'ir inrlPpenrlPnt
effects. Syuergi:s111 a11d antagoois111 are not absolute char-
actPristic:s; such interactions are often <lifficult to predict,
vary with bacteria] species a11d strains, and may occur only
overa narrow range oí concentrations. No single in vitro
mcthod detects ali such interactions. l'he methods used
to determine in vitro intcractlons are generally time-
consuming and are not often available in the laboratory.
Antlmlcroblal combinatiuu:. art: írt:l!ueutly sy11ergistic
if they involvf' thP following mechanisms: 1) sequential
inhibition of successive steps in metabolism (c.g. 1
trimethoprim-sulfonan1idc combination), 2) sequential
inhibition of ccll wall synthesis (e.g., vancomycin-
pcnicillin, mecillinam-ampicillin), ;~) facilitation of drug
entry of one antibiotic by another (e.g., beta-lactan1-
amlnoglycoside1 polymyxln-sulfonaml<.lt:!), 4) il1liilJiLiu11
of inactivating enzymes (f' .g., ampici lli n-c:lavulanic acid),
and S) prevention of emergence of rcsistant populations
(e.g., erythromydn-rifampin combination against Rhodo-
coccus cqui) .
As suggested earller, to sorne extent antagonism be-
tween antibiotic combinations is a laboratory artifact that
depends on the method of measuremer1t a11<.l i:s tl1us, wiLh
son1e exceptions, unimportant clin ically. ·rhe antagonistic
effecls of son1e con1binations are, however, detected clini-
cally. Antagonism may occur if antimicrobial combina-
tions involvc thc following mechanisms: 1) inhibition of
bactericida! activity (e.g., bacteriostatic and bactericida!
drugs used to treat me11ingitis where, depending on the
time-dose relation, bacterici<.lal effl:!cts arl:! preve11Led),
2) competition far drug bin<fing sitf><; (P.g., macrolide-
1..hlo1an1pl1en.icol con1binations, "vhich are of uncicnr
clinical significance), 3) inhibition ot cell permeabi!ity
mcchanisms (e.g., chloramphcnicol or tetracycline-
aminOglycoside combinations, which are of unclear clini-
cal significance), and 4) derepression of resistance en-
zymes (e.g., new thlrd-generatlon cepl1alu:.¡.iuriu autibi-
otics with older beta-lactaJn <1r11gs).
Resistance to Antibacterial Drugs
The potential for mutation and for genetic exchange be-
tween aJI types of bacteria, comblne<.I \•vitl1 tl1e sl1orL lJacLe-
rial generation time, is of major importancp in límiting the
use of antimicrobial drugs in rontrollíng infPction in ani-
111a!s a11d l1un1ans. The use of antimicrobial d.r ugs <loes not
induce resistance in bacteria but rather eliminates the sus-
ceptible bacteria and !caves the resistant bacteria already
present in the population. The exposure of animals toan-
timicrobials is the basis of selection for the evolution ancl
spread of resistance genes anti resista11t lJacleria. The ge-
netic processes involved in thP rlPvf'lopment of resistance
i11 bacleria are prcciscly those involved .in thc cvolution of
bacteria generaUy. What has surprised people has been the
speed and scale with which resistance has emerged in
many bacteria! pathogens, a phenomenon that seems to
bave increased in recent years.
Resistance to antlmtcrobial úrug:. ca11 !Je classified as
constitutive or acguired .
Constitutive Resistance
Microorganisn1s may be resistant to certain a11tibiotics be-
ca use tbe cellular mechanisms required for antibiotic sus-
ceptibility are absent from the cell. Myc;uplusrna, for exan1-
ple, are resistant to benzyl penicillin G hecause they lack a
<.:t!ll wall.
Acquired Resistance
Acquired, genetically based resistance can arise because of
chromosomal mutation or, more importantly, througti
the acquisition of genetic material. Chromosomal mut;i.
tlons tend to produce changes iu lJaclerial cell sLructures,
whereas plasmid-mediate<I rf'sistanr.e tends to encode syn-
lhesis of enzyn1cs that n1odify antlbiotics. Chromosomal
resistance is often a gradual, stcpwise process, whercas
plasmid rcsistancc is oftcn high-level, all-or none, resist-
ancc. t::xamples of important mechanisms of resistance are
1) enzyn1atic inactivation of antibiotics, 2) failure of bacte-
ria! permeability, 3) alterat1on In target receptor:., 4) uevel-
opment of by-pass mcchanism<; in mPtaholic pathways,
5) developn1ent of enzyn1es with low drug affinity, and
6) removing antimicrobial drugs from the cell through et-
flux pumps.
(;11romosomal lvlutation to Resistance. Chron1oso1nal
mutation to resistance is generally a minor problem. Mu-
tatlons to anrlbiotlc resistan ce are :.µuu.La11eous evc11ts in-
volving changes in rhromosomal DNA sequences unin-
íluenced by the presence of antibiotics. Such mutations
may lead to other changes that !cave the cell ata disadvan-
tngc :;o that, in thc ab5cncc of antibiotic selection, these
mutants may gradually be lost. MutatJon to antibiotic rc-
sistance can be dramatic, as in the case of singlt>-step mu-
tatlon to streptomydn rt:sistara.:e vvhe1e MIC incre<1ses a
thousandfo.ld, o r gradual, as in the case of cbromosomal
rcsisla11ce to penicillin wh.cre a series of n1utational cvcnts
may gradually increase thc MIC: ot the organisms. ·1hese
diffcrcnccs occur bccausc when antibiotics affect one tar-
get site, chromosomal mutation is a single-step process,
whereas v..11en severa) targets are affected, mutatíon to rP-
slstance Is a multistep procc:s:..
ºfhe rate of m11tation rliffers for, and is characteristic of,
eacl1 antibiotic. Sometimcs antibiotics are uscd in combi
nation to overcome the possibility oí mutation to rcs1st-

~~---ne chance of mutation to resistance to two antibi-
the p1oducl o( lhc chances o( n1ulalion for eacl1 a11-
._...'"'., ... alone. Jn veterinary medicine, mutational resist-
-.r.t"""c: has Iimited the use of streptomycin, novobiocin,
~~-;nn, anct, to a lcsscr cxtcnt, crythromycin. It appears
~ increasingly limiling the use of fluoroquinolones.
~.. ~tingly, for fluoroqulnolones, there are dlfferences
:E:~~n <;pPrii>s in thP import;inrP of <;inglP-~ep muta-
- ín the dcvclop1nent of resistance. For Ca1npylobacter
-¡ and Pseudo1nonas aeruginosa, a single mutation ata
~cular si te in the gyrA DNA gyrase gene can Iead to clin-
:esistance whereas in E. coti a single mutation may lead
.cly a slight reduction in susceptibility.
., .. hrornosomal mutatlon resultlng in multiple antlbl-
-""'"" :esistance has hr@n <IPsrrihP<I for rlinirally rPlPvilnt
-:e:-ia. The region involved, thc Mar (multiple antibiotic
... stance) locus, controls efflux systems resulting in resist-
- ... e: to a varicty of drugs without modification of the
....-ugs.
Transferah/e Drug Resistance_ Gcnetic exchange as a
a ... se of aulil1iuli<.: rt::.i:;Lar1<.:t: i:; uf 111ajur irnport<:1nce in vet-
e.-L-iary medicine. (Jnlik<:> chrorri osomal resistanrP, whirh
ccurs in individual bacteria, transfer of genetic material
--oouces epideniic or in/ectious resistance. often to several
a=iñbiotics at one time and even, though rarely, in tl1e ab-
sc.-.1ce ot anCibiotic sclection. The extrachromosomal DNA
~ "'lecules rcspo11siblc for antibiotic resistance are usually
.i:.11útl:., wlú<.:h iI1 thi!) context are sometimes called R fac-
- "(or R pln~miri~) . ThP plasrnid DNA responsible for resist-
;?..'lce can reproduce itself withu1 a ccll a11d spread to oll1e1
.:ells by transduction, conjugation, transposition, or inte-
~:-ons:
l. Transduction. A process by which plas1nid DNA is in
corporated by a bacterlal virus and then transferred
to another bactcriu1n. An example is transfer of a
bt>t<:1-l<:1ct<:1rna!)e gene frorn pt>rlicillin resist<:1nt to sus-
<P[lti hlP <;tf'I [lhylororci. 'l"hP i m [lOrt;inrP of tr;insrl11C-
ti.on in sprcad of antlmicrobial resistance h as proba-
bly been underesti1riated but tl1e narrow specificity
of hactcriophagc~ may havc limitcd thcir role in re-
sistance transfer.
2. Conjugation. 'fhis refers to plasmid-mediated trans-
fer of reslstance. In th ts common process of gene
tr;in.<;fi>r, ;i <lonor h!!rtf'rium synthesizes a sex pilus,
which attaches to a recipient bacteriurn in a n1ating
process and transfcrs copies of plasrnid genes to the
recipients. Thc donor rctains copies of thc plasmid
genes but the rec1pient has now become a potential
donor. Conjugation can occur not only between
spc<.:ies of tll~ s<:1me gener<:1 but also across genera
an<I f;imiliP<;, <;o that <;imil;ir pl;ismids ca.n be found
in a wide range of unrelated bacteria. Plasrnids are
often partly constructed from transposons and intc-
grons (see below).
3. Transposition_ Short DNA sequences known as tra11s-
posons ("jumping genes") can transpose from plas-
rrútl to plasmld, pl<:1smid to chromosome, or chro-
mosomP to pl;isrnirl . A tr;in~poson copy usually
rc1nains at the original site. The freque11cy of Lrans-
position is characteristic of the particular transpo-
Chapter 4 Anti m irrohi;i 1 í.hi>mothPri!py 39
son and bactcrium. Thc importance of transposi-
lion as a key eh:11 u:11 l i 11 1c:.i:.La1 u.. c ll a11:.ft:1 i:. Lhal
transposition is independent of the recombination
proccss of thc bactcrial ccll- homology ~vith thc in-
teracting LJ_ A is not required_ l'lasmids trom di-
verse sources often possess identical antibiotic inac-
tlvatlng genes because of rransposons _ There are a
variety of diffcrent types of transposons that carry
resistance genes; sorne tra11sposons n1ay even cause
conjugation. Sorne transposons may contain inte-
grons (scc bclow). Thc simplcst form of a transpo-
son is a resistance gene flanl<ed on either side by an
insertion sequence, the smallest form of mobile ge
netlc element. Because of the widespread naturc of
in sertion sequences in bacteria, there is essentially
no gene in bactcrial genoines Lha Lca1uroLlJe 11101Ji-
lized and moved to othcr bacteria! geno1nes as a
transposon.
4. Integrons. lntegrons are increasingly recognized as
widcsprcad genetic elements associated with multi
ple ctrug rcslstance in gram-negative e11teric bacte-
ria An integran consists of an integrase gene anda
site-specific inlcgration sile inLo wl úclt Ll1t: inte-
grase can insert antimicrobial drug resistance cas-
settes. Ovcr ninc typcs of integrons and over 60
different gene cassettes llave been identified in
gram-negative enteric bacteria, with sorne inte
grons contatntng as n1any as seven dlfferent resist-
ance genes on their cassettes. In addition, sorne bac-
lt:ria 1uay <.:arry <.:a!)sctte!) in their genomes 'vhlch
have not incorporatP<I in to intPgron~ h11t m;iy bP !!S-
sumed to be capablc of so doing. lntegrons (with or
without resistance-gene cassettes) may be found in
the chromosome or in plasmids.
Clinical lmportance of Antimicrobial Drug Resistance
Arq11ired drug resistancc has become a majar p roblem in
pathogenic bacteria of veterinary in1por lance. lL is con1-
mon in many species, although sorne bacteria, particularly
gram-positive bacteria such as many streptococci and
coryncbacteria, have remained highly susceptible to com-
1nonly uscd drllgs. As a result, in many cases diseases pro-
d uced by these oacceria llave aeclínea cons1derably 1n im-
portance, to be replaced by diseases produced by bacteria
tl1at l1ave the abili ty lo evolve 111ort: rea<.lily. Acquiretl re-
sistance to pcnicillins is frequent in coagulase.-rositivP
staphylococci, and acquired multiple antibiotic rcsistance
to many common antibiotics seriously limits their use in
members of thc family Enterobacteriaceae such as Salmo-
nel/a an<l E. colí. Acquired resistance is increasingly ob-
served in nonenteric bacteria such as Pasteurella, Bordetella,
ru 1d H11er11upltilu:; <:111u h<:1s IJeen identified tn vlrtually every
pathogenic hacterial g<'n11<; a<; wf'll ;i<; in the normal flora.
'fhere is to sorne extcnt a causal relationship between
antimicrobial drug use and the dcvelopment of resistance
but its importance varies with diffcrcnt pathogcns and cs-
senrially rcflects their abil1ty to adapt to changing circu1n-
stanccs, i.c., to evolve. The dcvelopment of resistance has
lJet:11 p<:1rtl<.:ularly well c.Jocumenrcd among enteric bacteria
in VPtPrinary mP<lic·ine. Thc intestine is a major site of
40 PAl(T 1 Introduction
transfer of antibiotic resistance both because of the vast
numbcrs of bacteria present, the u~e of oral antimicrobial
dr11g~. ;:incl hPci'111sP nfthP npportunitiPs for sprE>ad of thesc
bacteria between intensively reared animals kept undcr
unhygienic conditions in closc associatlon with their ma-
nurc. Whilc tl1c sprcad of drug rcsistancc is not so well
documented in individual compan1on animals such as
horses and dogs, analogies to the situation 011 farms are
useful In understandiI1g llov• spread occurs.
Where multiple drug resistance is encoded on "pl;oismici
01 an inlegron, lhe use of any antirolcrobial to which re-
sistance is er1coded by onc of the genes present will help
maintain the entire collection of genes.
l11tesli11al Escherichia coli. Extensive stucly of antimicro-
bial resistance in intestinal E. coli in an imals has provided
information on the rnechanisms anc.1 ecotogy of anrtmi-
crobial resistance. These studics have shown the relation-
ship !Jetweeu tl1e exte11l uf re::.i::.La1 •Le a11d lhe degree of a11-
timi<'rohial use. For exa1nple, resistance iI1 E. coli from
adult ruminunt:s at pasture is slight, w!1crcas it is pro-
nounccd in intensively reared animals where antibiotic
use is common. These E. coli may be resistant to up to 10
clinically useful drugs as a result of plasmid-mediatec.1 re-
sistance. Among enterotoxigenic E. coli from farro ani-
mals, plasmid-111ediated resistance to tetracyclir1e:s, :sulfuu-
amidcs, and streptomy<'in is now prñ<'tirally universal, and
is incrcasingly coinmon to ampícillin a11d i1eo1nycin.
Ant ibiot ic-resistance encoding plasmids in enterotoxi-
gcnic E. coli in swine and calves may also include genes for
v irulcncc determinants such as toxin production or ad-
hesins. Although an unexplored area, antibiotic use may
thus potentially promote the transfcr of virulence genes
benveen bacteria.
\Vill1il1 ll1e intestine, R plasn1ids are found in E. coli and
in the more dominant anaerobic flora of the large bowel.
\\l'ithin a short time of trcating an animal "vitl1 an antibi-
Otic, the t . coli and much of the anaerobe populatlon bc-
come resistant to that ar1tibiotíc, principally beca use of se-
lectlon of resistant strains but also bccausc of enhancec.1
transfer of R plasmids. In the absence of antin1 icrobi;ils,
con.dlllo11s in lhe largc bowcl sccn1 to prevent the transfer
of 1{ factor:>. Short-term oral use of antimicrobials is fol-
lowcd by high lcvcls of E. coli rcsistancc, which fall once
the antimicrol)ials are removed beca use the ma¡ority of R
plasmic.1 bearing E. coli are not good intestinal colonizers.
However, the continuous presence of antimicrobials is as-
sociated with extensive resistancc, which persists long
aílt:r lhe anlinlicrobial is ren1ovcd sincc resistant E. coli
that are good ir1tcstinal colonizers have been selected.
Salrnonella Typhi1nuriutn. Multiplc nntimicrobial resist-
ancc is a major problem in Saln1011ella lyph1n1unum anc.1 is
often the result of chromosomally i ncorporated inte-
grons. Among s. Typhimuriun1 slralns, clones of certain
phagc types are characterizeci by thc presence of mn ltip lf'-
auliu1h.:1ul.Jial 1esislanl inlegrons. 'fhc cxte11t of resistance
is mosl rnarked among calf isolatcs bccause the extensive
use of antimicrobials in sorne typcs of calf rearing and the
na tu re of sal 1nonellos1s 111 calves apparently provide an op-
portunity for the development and spread of resistant
Salt11011ella. Recently, a multiply reslstant and highly viru-
1ent clone of S. 'fyphimurium DT 104 (a partiutlar phage
type) h<i:S di:sseu1ir1atetl witlcly t l 11uugh callle on a global
basis and has spread from this basic reservoir into othcr
anirnal species and to humans. 'fhis isolate has a florfcni-
col and tetracycline resistancc cncoding gene flankecl by
two integrons carrying a beta lactamase and streptomycin
resístance gene cassette. This cluster of genes form part of
a larger, distinctive, region called Salfnone//a genomic is-
lanc.11, which rnay have l.Jccu a pla:.111itl Lhal has inlegratcd
into thP chromosome; this island has also been identificd
in otl1er Sabnonella serovars, including Agona.
Hospital-Acquired Resistant lnfections
Acquired antimirrohial rf'sistance in resident hospital bac-
teria is a nlajor problem in hun1an ho:-;pitals and is incrcas-
ingly recognized in veterínary hospitals. ·r11ere is a causal
rclationship bctwcc11 antimicrobial use in hospitals and
the ctevelopment of rcsistance in bacteria. Colonization of
patients by rcsistant opportunist bacteria is hard to prc-
vent bccausc of sharec.1 air spal:cs a11u e11vi101u11enl, utcn-
sils, and veterinary staff. ln aciclition, patients with scrious
illnesses tnay be treated with broad-spcctrum antimicro-
bial drugs, thus removing the normal colonization resist-
ance of the body, including thc largc iI1tcstine, provided
by the normal microbiat ti ora, which are dcstroyec.1 by such
drugs. 'fhese paticnts 111ay also be immuoosuppressed by
their illnesses or by treatrr1ent t1y lmrnunosuppres:sive
drugs. Under sucl1 circLtmstances these patients can r1•ac1-
íly IJe colouized l.Jy l.Jat:Leria ll1al t11e eilher i11l1insically rc-
~ist;int to many antimirrohial drugs (e.g., Acinetobacter
spp., C'itrobacter spp., E11terococcus spp.) or that have ac-
quired resistance.
Public Health Aspects of Antimicrobial
Resistance in Animal Pathogens
'l'he use of ar1tirnicrouial age11t:. i11a1li111al::., µa1 licularly in
intf'nsivf'ly rearect livesto<'k, can rcsult in antimicrobial-
resistant bacteria rea ch i ng lhc hu man population through
a varicty of routes. The scale of the contribution via tl1ese
ro u tes has not been determincd and i ndeed, because of thc
comptexity of resistancc in bacteria! pathogens, would be
hard to determine preciscly.
Mo:st a11tiuliuouial rt:::.i:.lar 1Le in hu111a11 pathogens
<'omp_<; from antimicrohial use in human medicine. How-
ever, antimicrobial-resistant bacteria of animal origin,
such as Enterococcus spp. and E. coli, can colonize the intcs-
tines of people. Heavily cxposcd humans (e.g., farn1crs
who use feed containing antlmlcrobials, slaughterhouse
workcrs, cooks, and other food ha11dlers) oftPn hrivf' a
higl!cr lucitle11ce of re::.islanL /!,. coli in tl1eir feces than thc
gPnPral population. Contamination of mcat by intestinal
bacteria at slaugl1ter is extensivc and an importunt routc
by which resistant bacteria reach pcople. While many of
thcse bacteria are nonpathogcnic, 1nany pathogenic bac-
terlal species trom the lntestlne:s of ¡u1irr1al:; cau~e zuuuolic
infections in humans (e.g., Snll11011ella, Campyloharter je-
jur11) a11tl tl1e:sc infecliu11s 1nay be hardcr to treat beca use of

..-.-_~·=red resistance. Tb.e nonpathogenic bacteria of ani-
- acq uircd by human5 are a potential 5ource of re5ist-
-c sen es for human pathogerlic bacteria other than the
~e bacteria. ·rhese bacteria may reach humans not
_ Ihrough the food chain tJut also through vvater con-
~ :iatf'<i hy rf'sistant hactf'ria of animal origín.
:he topic of the contribution of antin1icrobial use in
=animals for growth promotion and disease prophylac-
_.,_ p':.l.Tposes to resistance in human pathogens has been
s-ubject of repeate<.1 review over many years, but the last
~.:: years has seen the issue subject to the most intense
~!tiny. Tl1e rnajor driving force for tl1e renewed deln1te un
-,,, !Jrudenc:e oft1sing antimicrohial drt1gs in food animals
~ growth pron1otíon and dísease prophylactic purposes
;._¿s been the alarruing increase in resistance in humar1
-tllogens, particularly those causing "community-
... ;~uirecl" (e.g., Streptococcus pneurnoniae) infections rather
c-a !l hospital-acquired in.fections. ·r11is 11as led to a total
e;:ppraisal of Llre use of ali a11ti111icrolJial urug:; i11 all cir-
.;:::..;n~a ncf's. Another factor driving the change is evidence
t-om Europe that vancomycin-resistant Enterococcus fi:te-
rzmz (VRE), a serious and essentially untreatable hospital-
,_,:quircd pathogcn of immunocompromiscd human pa-
-:ents, were being selected in intensively reared farm
~i mals by the use of avoparcin, a glycopeptide antímicro-
_:31 drug u:;eu et:> a gro1-vtl1 pruu1uter a11d di:;ea:;e propl1ylac-
::X. These VREs were alrnost universal in the intestines of
= eated animals and were found in a small proportion of
-.ealthy humans in Euro pe. By contrast, they werc not pres-
cnt in healthy humans in the United States, where the drug
~ not used. 'fhese findings \.Yere a demonstration of the
scale of the movement of resistant bacteria from animals to
2umans, which was clearly measurable. Another driving
factor was the entry of Sweden into the European TJnion in
1999. Since Sweden had banned Lile use of all anlin1icrobial
C:ugs as growth promoters and disease prophylactics for
""Iany years, in order to bring its practices into line with
:t°!.ose of otl1er European Union (EU) countries it either had
:o changc its rcgulations orto changc thosc of thc EU. lt
.l.Sed the VRE evidence to persuade the EU to change poli-
d es on the use of antiinicrobial gro°l'. th prornoters, most of
1
·\-h1Ch (avoparcln, bacltracin, splramycln, tylosln) were
banned in F.urope in late 1999. This han has withstood
court challcnge and seems certain. to remain. A similar ban
h as occurred in Australia, and seems likely in Canada and
Japan.
In the United States, there has also been extensive reex-
amination of the use of antimicrobials in animals. One no-
talJlc deci:;iu11 wa:; to rever:;e tl1c approval uf fluoro-
q uinolones for treatment of F.. cnli sept ic:em ia in c:hic:kens
because of the rapid develop1nent of fluoroquinolone-
resistance in C. jejuni. Tt was estimated that as many as
14,000 people annually treated for campylobacteriosis l1ad
their treatment affecte<.1 because they were infectecl with
resistant bacteria acquired from fluoroquinolone-treated
c ltil:ken:;. Tri addltiou, tl1e Center for Veterir1ary Medicine
of the TJ.S. f.ood and Drng Arlm in istration has recently de-
veloped a guidance document for the evaluation of lhe in1-
pact of resistancc as part of the regulatory approval of new
antimicrobial drugs. 'l'hc documcnt takcs into account the
Chapter 4 Antimicrobial Chen1otherapy 41
relat ive importance o f different antimicrobial drug classes
in human medicine and analyzes the r.i:>l<s of production
of resistance and its acquisition by humans, depending on
the proposed usage of the drug. ·rhe Center for Veterinary
Medicine ir1tends to reassess the safety of ali antimicrobial
dr11gs 11sf'<i in fooct anim::ils in a prioriti7ed man.nc~r de-
pending on the type of risk analysis propo:;ed for new drug
approvals.
It seems likely that antimicrobial drugs important in
human medicine will be removed from use as growth pro-
n1oters or long-term. disease p rophylacti.cs throughout the
world. 'I'his is in keeping \.Yith the important prudent- use
principlf' that antimicrobial drugs should only b e used
when the benefits are clear and subst<1ntial.
Control of Antimicrobial Resistance
Avoiding the use of a drug is the best '"'ªY to control an-
tirnicrolJial resistance. Most ma¡or national veterinary
medica! associations have published p rudent-use guide-
liI1es Ior ll1e oplünal use of auLi1uicrolJial age11ts tl1at are
rcadily available on their web sites. Prudent use is definf'ct
as optimizing the efficacy of antimicrobial drug use
while minimizing the developme.nt and spreacl of resist-
ance . .A.lthough t hese guidelines are general in scope, they
are ir1creasingly supplemented by specles or veterinary
prac:til.f>- type g11i<1f'lini>s that are sometimes case-specific.
Further refinement of such guidelines will occur in.to the
fu tu re.
Antifungal Chemotherapy
'l'he susceptibility of ft1ngi to dífferent di·ugs is often, but
nol always, prediclable. Fungal drug suscepLibiliLy Lesling
is technically complex and simple methods parallelingthe
disk diffusion antibacterial susceptibility test are not gen-
erally available.
Antifungal Agents for Topical Use
Many chemlcals have antlfungal properties and are used
for topical treatment of fungal infections of the skin ancl
sometimes of the rnucosal surfaces. 'l'hese include pheno-
lic antiseptics such as hexachlorophene; iodides; quater-
nary a.m moniurn antiscptics; 8-hydroxyquinolinc; salicy-
lamide; propion ic, salicylic, and undecar1oic acids; and
chlorphenesin . .A.mong the more effective topical broad-
spectrum antifungal drugs are natamyclo (a polyene an-
ti microhia l), clotrimazole (an im_idazole compound), r1ys-
tat in (a polyene antin1icrobial), <u1d ketocon azole and
miconazole (see below).
Antifungal Agents for Systemic Use
The recent development of the imidazoles (ketoconazole,
itraconazole, a.n d fluconazole) has been a major advance
in sysLe111ic fu11gal tl1crapy because of thei.r oral adminis-
tration. relative lack of toxicity, anct effectiveness. The ear-
lier 1najor antifungal drug for systen1ic use, an1ph0Lericif1
42 PART 1 lntroduclion
B, h;id thf' dis;idvantagcs of toxicity and requiring intra-
venous administration, but it did have the advantage of
fungicida! activity.
Griseofulvin. Griseofulvin is a fungistatic antimicrobial
that inhibits mitosis and is active only agalnst dermato-
phytes (ringworm fungi) . Resistance in sorne dermato-
phytt:::. Ita::. ue1::11 11::po1 led lo develop during treatn1ent.
Griseofulvin i~ f'fff'ctivP against rin&'-vorm fungí only if ad-
n1i1listered orally. The drug is incorporated into keratin in
thc basal cells of the epidermis and rcaches the superficial
dcud und pnrnsitizcd kcrntlnized epithelium through pro·
gressive maturation of thc basal cells.
Arnphotericin. An1photeri<.:in B is a polye11e a11tiinicro-
bial, lil<e nystatln, whlcll blnds ergosterol, tl1e prlr1clpal
sterol of the fungal 1ne1nbrane, causing lf'a kage. of the cell
cu11l1::11L::.. IL is a b.road-spcctrun1, generally fungicida! an-
ti rn icrobial. lt is active against Blasto1nyces derrnatitidis,
l Tistoplas111a caps11/at11111, Cryptococcus ncoforrnans, Candi da
spp., Sporot/1rix schenckii, and Coccidioides immitis. Strains
of fi lamentous fungi, though commonly susceptible, vary
from extrcrnc susceptlbllity tu rc~1::.ra11ce. A111phuL1::1iLiu J3
must be ad1ninisterPd intravpnously. Renal toxicity is an
inevilablc sidc cffc<.:t of such treatment and must be mon-
itorf'<1; th<> <'ffpct is reversible if the drug is stopped.
Amphotericin is the most important drug availablc for
treating systemic mycoses caused by dimorpllic tungi and
by ycnsts. lts prime place is increasingly being challenged
by thc 1midazoles, \Vhich are less roxic and easier to ad-
mü1ister. The drug is given by slow intravenous injection,
usually cvery other day over 6 to 10 week::.. Lipid fo11nula-
tlons cont<1ining amphotPricin B, though expensive, show
grcat promise clinically becau;5e of lowcr kidncy toxicity.
Flucytosi11e. 5-ilucytosine is deaminated in the fungal
cell tos fluorouracil, which is incorporated into messen-
ger KNI\ to produce garble<l codons and faulty proteins. lt
has a narrow spe<.:trum of activity, which includcs most
Cryptococcus neo(c1nnc111~ aud 11lé111y Curuliclu., !Jul wosl fila-
1nentous fungi are resistant. Resistance dcvelops readily
during treatment. ·rherefore, flucy tosinc is oftcn uscd only
in con1bination witll other ctrugs, usually an1photericin.
lmidazolcs: Kctoconazole, Miconazole, ltraconazole, Flu-
conazo/e. 1n1idazolcs in terfere wtt h the b iosynthesis of er-
gosterol and bind fungal cell rnembrane phospholipi.ds to
cause leakage of cell contents. Ketucouazule, 111ico11azole,
itracona7ol<', ;1 nd f111con;i70Jp ;i rf' fu ngistatic against a
widc rangc of yeasts, dimorphic fungí, and dermatophy-
tes; thcy also have sorne antibacterial and antiprotozoal
activity. Kctoconazolc, itraconazole, and flu<.:onazole ap
pear to be more active than m1conazole and are the fa-
vored drugs for systcmic ad1ninistration because they can
be given orally rather than intriiver1ou::.ly. Kctucu11azole,
itraconazole, an<1 fl11con<17.0IP ilppPar to produce few sig-
nifican! advcrsc cffects in humans and animals, but liver
damage has been reported in people given ketoconazole.
'l"hey appcar to be an cffcctivc trcntment for many sys
temic tungal infections in <logs and cats, but there !las
bccn little experience with their use in other animal
species. ·rhey havc thc dlsadvantage of fungistatic actiu11;
prolonged trcatment may hf' nf'rPss;iry in serious infec-
lion.s Lo preven 1 l he rclapsc3 that have occurrcd, and this
is expensive.
Antiviral Chemotherapy
Antiviral Drugs
Thc dcvclopmcnt of nontoxlc chemicals for therapeutic
use 111 v1ra1 wseases is far more ólfflcult than tne deve1op-
ment of antibacterial drugs, but the Iong-term prospects
for antivlral che1nothcraµy iJ1 a11i111als are encouraging.
Human immunodeficiencyvirus (HTV) inff'rtion in people
has led Lo lhe dcvclopment and introduction of antiviral
drugs effective against retroviruses. Viral replication de-
pends largcly on thc active pnrticipntion of the metabolic
pathways ot the host cell, and the balance between pre-
vcnti ng viral replicatlon and wrecking cellular rnetabolism
is delicate. Only a relatlvely small number of uscful urug::.
have been described, and thcir spectn1m of an ti viral activ-
ily b ofl1::11 11arrow. Work has concentrated on selective
drugs that use virally encoded enzymes e.i ther as specitic
targets for inhibition orto nctivntc drugs v.rithin virally in-
fected cells. Recen tly these have included many drugs pref-
ercntially phosphorylated by virus-specific thymidinP ki-
nase and further phosphorylatcd by cellular enzyn1es; the
resulting trlpl1osphatf'S of thPse second generation antivi-
ral urug::. iulliuil viral DNA polyn1erase, actas bogus sub-
stratf'S for this rn1.yme, or both.
Basic anti viral targets include the following:
l. Inhlbltlon uf viral allacl1111enl to cclls (im-
m11nogloh11lins, rpce.ptor homologues)
2. Inhibition of uncoating of vlruses (amantadinc, ri-
mantadine)
3. lnhibition of DNA or RNA synthesis
4. Nuctcos1de anatogues t1óoxur1órne, Victarabine, rri-
fluridine; acyclovir, ganciclovir, ribavirin; foscarnet
5. Inhlbltion of rt!vcr~c tra11::.cripla::.e (zidovudine)
6. Inhibition of mRNA translation (;intisf'nsf' oligonu-
cleolidcs, Lnlcrfero11s)
7. Inhibitors of posttranslational modification (pro-
tcnsc inhibitors, glycosylation inl1ibitors)
Antiviral drugs are generally only effective prophylucti-
cally or in thc carly stages of disease vvhen viral replication
is occurri ng. Rapid diagnosis is thcrcfore required. Al
tho ugh no antiviral drugs 11avc been approved for vctcri-
nary use, ·rabie 4.2 summarizes the potential of currently
availal)le medica! anllvlral compounds for tupical ª'id sys-
temic use in animals. Thc desir<ihlf' ch;iracteristics of vet-
e1 inary an UviJal co111pounds are broad-spectrum efficacy,
Jow cost, ease of actministration, and lack of drug residues.
Few of thc antivirul drugs nvailablc possess these charac-
teristics, although sorne of the 1mmunomodulators may
have them.
 r :haptP.r 4 Antimicrobial Chemotherapy 43

Table 4 . 2 . PotP.nti;il Topkril anci Sygpmic Applic;ition of Antiviral nrugs in Veterinary Medicine

Drug Topkal Systemic Possible Veterinary Use

Acyclovir + + Herpes lnfectlons

Amantadine + EquinP infl11P07il rrophyl;ixi(
2-deoxy·D-glucose + Herpes infectioos
Foscarnet + + Herpes infections
Ganciclovir + Cytomcgalovirus infcctions
tdoxundme + Herpes infections
lnterferon + Feline herpes, feline leukemia, feline infectious peritonitis
Methisazone + Bovine vaccinia or pseudocowpox
Rih;wirin .. + Influenza, para influenza, bovine herpes, canine distemper,
feline infe<.tious peritonitis, feline calicivirus
Vidarabine + + Canine herpes
Zidovudine + Feline leukemia virus, equine infectious anemia, feline
immunodeficiency virus
 Antilllicrobial Drugs: A
Strategy for Rational U se
and the Ralllifications of
Misuse
D WJGHT c. HlRSH
Antiffilcrob1al drugs are ttsed to treat (therapeutic) or pre-
vent (prophylactic) disease produced by infectious bacteria!
agents. Most of the discussion that follows will deal witl1
therapeutic use of these drugs, though commPnt will hP
1uau1: rcgi:11Lli11g p1opJ1ylaclic use wl1en appropriate. fur-
thcr, the discussion will deal only with bacteria! agents.
Strategy for Rational Use
1'111:: <.lc<.:biu11 tu u~e a11li11Jic1obial d1 ugs therapeutically in-
volves the determination of ivhether there is a11 infectious
agent present. Some considcration (though this rarcly oc-
curs) should be given to whcther the intectious process ac-
tually poses a threat of sufficient seriousness to outweigh
the rlsks of use of these drugs (dlscussect below), and
whcthcr the infectious process will resolve ivithout thcir
u:.c. Tlic :>i:1111e con1111ent should be n1ade about prophylac-
tic: use: tonsideration should be given as to wl1ether th ere
is an unacceptably high prevalencc of in fcctious com plica-
tions following a certain procecture to justify use of antimi-
crobial drugs.
Central to the decision to use antilnicrobial drugs is the
demonstration that an infectious agent is part of thP clis-
t:'a~-=: µroct:::.:. u11der conside1aLion. ·r11e gold standard for
this determination, of course, is the result of microbiolog-
ical culture. Strict application of this standard is unrcalis-
tic, however, because the dccisions to use antimicrobial
drugs are made severa] days before culture data are avail-
able. Therefore, as an aid in determining that a particular
proccss has an infect ious component, certain cl11Ps ;:i rP
u:;cu. 1'111:: I11feclion Con trol Con1n1lttee at t h e Veterinary
Mt>dical Teaching l-Iospital, University of California, has
drawn up guidelines to be used as aids in t hc dccision-
making process (Table 5.1).
The following scl1eme is use<l to justify the therapeutic
use of an antimicrobial drug the day tf1e patient is first seen.
·rhe whole purpose of the exercise ls to answer thP follow-
ir1g 4uc:.tiu11:.: 1:. there an iniectious agcnt present? What is
thf' hest antimicrobial drug to use?
44
Is There an lnfectious Agent Present?
Apart from having the results ot bacteriotogic culture in
hand, this question can be answcrcd by experience (e.g.,
ali similar cases have hact an infect1ous component) or mí-
crobiologically. It is the microbiological aspect of the deci-
sion process that will be the focus of the discussion here.
Unless noted otherwise, all subsequent remarks w ill hf' fo-
cuseu Ofl in feltiOUS (JfOCt::>:>t:S i llVolving 110Il1lally Steri le
(or nP;:irly '\tf'rilf') sitf's.
One of the most rewarding methods that can be uscd to
determine the presence or absence of an infectious agent is
thc dircct smear (see Chapter 3). Examination of a direct
smear answers two very important question.s: Is there an
infectious agent present? And, if so, what is the agent
llkely to be? Ans~vers to lJutlt uf thc:.t: yut:slions help justify
the use of ;:in <intimicrohi;:il ;:igcnt, and, what is equally im-
portant, help determine which one is likely to be effectivc.
After it has been dcmonstrated that an infectious agcnt
is prcscnt, Le., m icroorg¡1nisms are seen in t he d irect
smear, tlle next step in the process is to make an educatcd
guess as to their identity. 'fhis is the most importa11t step in
thl:! proce:;:; ir1 thc rG1tio11G1l u:.t: uf anU1nicrobial drugs-to
h;:ivp in mind what it is you are going to be treating. Expe-
rience and rctrospective data are keys to this dctcrmina-
tion, and allow the clinician to tormulate a "microbiolog1-
cal differential list" with the proper hierarchy. By noting
the shape of the microorganisms seen in the dlrect smear,
certain members of thc differential list can be "r11lf'd in" or
"rulcu out." Ge11erG11ly, bacteria come in. two shapes; rods
;:ind tocci. Filamentous forms (Actinom.vces, Nocardia) are
trcated as a separat e category. Of coursc, if 110 infectiO\.lS
agcnts are seen but other clues point to infectious process,
a mlcrobiological differential list is still constructed, but it
w11l be more difficult to "rule In" or "rule out" certain rni-
croorganisms or groups of microorganisms bt>ca11sf' of thc
la<.:k uf vi:.ual clue::..
More sophisticated mcthods involve tl1e detection ot
prokaryotic (bacteria!) DNA in a normally sterile site. A
polyn1erase chain reaction (l'Cll) that uses universal
 Chapter S Antimicrohial l)r11gs: A Srrategy for Rational TJse and the Ramifications of Misuse
-.:ole 5 .1 . Guidelines for Rational Use of Antimicrohi~I AgPnts
• Demonrtration of an infectious agent
-·
• Clinical data (at lean two of the following)
a. Fever
b. leukocytosis
c. Localized inflammation
d. Components ot the sample
e. Radiographic evidence
f. Elevated serum fibrlnogen
- -im Pr~ for prokaryotic L)NA allows for this detennina-
tion. "fhe disadvantage to such a detern1i11ation is the lack
: an "isolate" to identify and to test for susceptibility to
.L"ltimicrobia 1 agcnts.
Observation of bacteria in a direct smear may seem to
-esult in the formulation of a n1icrobiological d ifferential
~-:.t uf entlless posslbllitles. Experience and retrospective
±!ta allow for thP p;:iring clown nf the list, keeping only the
most con1n1on in p1ope1 ltie1a1cl1ical urtlt:r. Fur exarnple,
-"""'. samples from dogs, Bordetella is almost never founcl ex-
("{>pt from thc rc:;pirotory troct, ond hcrc it is not com-
monly found tn samplcs obtained from the lower tract ot
:>atients with clinical signs of pneumonia. Likewise, in
.Lmples from dogs, Pseudo111onas Is hardly ever found in lo-
-;¡tions othcr than thc externa! ear or the lower urinary
=act. Bordetellu 01 Pseutlorr1011us would be very u11lik<;:ly
.:andidates for rod-shaped bacteria in a sample of exudate
f!om the peritoneal cavity of a dog. Aside from "common
-:icrobiological sense," there are other clues that are help-
-U:. For example, misshapen or long, slender rods are al-
n!OSt always members of the anaerobe group (especially
~ seen in malodorous fluid obtained from a norrnallv
, ster-
..:e si te).
4/im1•ntary Canal-A Specinl Case. Seeing a n1icroorga11-
~1 in a norn1ally sterile site does not pose tnuch of a prob-
..., in dctermining whether there is an infectious process
:::.cnt. Infcctious discascs of norrnally contaminatcd sitcs
-~th, vagina, gastrointestinal canal), however, pose spe-
.:,, problen1s. '!'he alimentary canal is the only contami-
- -:ed slte where the dlrect smear can help determine
"'i:-ther thcre is an infcctious bacteria! d isease. ·rhere are
_:--ce conditions in which direct sincar ca11 aid il1 Ll1e diag-
s~s and treatment: I) diarrhea associated wjth Campylo-
xu:r, 2) Hclicobactcr-associatcd conditions of stomach and
--:estinal canal, and 3) JJracflyspira (Serpulina)-associated
w...seases. In the first condition, the observation in stained
=.g., Wrlght·Gtcmsa) smears of curved rods, in the second,
.-,, observation of hclical-shaped rods, and in the third,
"!e observation of spirochctal shapes suggesl lhal an infec-
~us agent may he associated with the ahnormal signs oh-
crved. In ali three instances, the presence of a microorgan-
:n \vith a uniquc characteristic gives the visual clue
e<:essary to makc an cducated guess as to the etiology of
:..'1e abnotmal s\gns. More sophisticated techniques (PCR)
iave heen shnwn to be useful in determining the presence
o_f other pathogcnic 111ic1001ganisn1s, e.g., Clu:;lridiurn di((i-
:ile or thc genes cncoding its toxins.
45
What Is the Best Antimicrobial Agent to Use?
If an infectious agent is observed in the direcl so1ear, Ll1e
answer to the first qucstion posed ahove has hf'en an-
swcrcd. If nonc are seen, yet the other clucs are present
pointing to an in!cctious process (see Table 5.1), the first
question has also bccn answered in the affirmativc. Thc
next step in the declslo11-making process is "'1-vhat antimi-
crohial drug <:ho11l<I hP 11.;;pci?" To ans"'1er this question, it is
important to have in mind the "n1icrobiological ta1geL"
Depending upon thc site from which the sample was ob-
tained and what was sccn in thc dircct smcar, an educated
guess can be made as to the identity ot the agent(s) in-
volved. Retrospective data are uscd to suggest what antimi-
cru\Jial ag<;:JJ t~ wuultl lJe t:>fft:>ctive for the 1nicroorganisms
on the microhiologic;:i l <lifferential list. Then, depending
upon distribution, toxicity, and expense, a final cl1oice is
made.
The ncxl day, the microbiology laboratory should b e
able to furnish infor1nation that is useful in determining
the appropríatencss of first-day choice of antimicrobial
age11t(~)- TI 1c lal>uratury can, for instance, tell you whether
;:i roci-~h;:ipPd bacteria seen in direct smear is a 111ember of
the family Entcrobacteriaceae (e11le1ic g1ou1i) wltlcl1 i:; a11
cxtremely important piece of information since memhers
of this group are unpredictable with respcct to susccptibil-
ity pattcrns (due to the propens1ty of this group of mi-
croorganisms to possess resistance-encoding genes, see
~haplcr 4 a11tl l>l!luw) and, a!> a const>quence, more expen-
s1ve and morr toxic antimicrohial drugs are used if their
prescncc is likcly.
Oblígate anacrobic bacteria pose a special challenge. It
is important to suspcct thcir prcscncc carly bccausc thc
niethods used to conf1rm them are lengthy; and, just as
importantly, most laboratories do not perform suscepti-
billty tests on thcm. Even if they did, the results would
not be available for at least a week after sample submis-
sion. Thcrc are, howeve1, son1e clues lhal help Lielerr 11i11e
whether membcrs o( this group are present. A major clue
is odor. Tf an cxudatc or fluid smells fetid, there is a good
chance that obligate anaerobes are present. Without
smell, an educated guess can also be madc as to their pres
ence dependtng upon the si re fron1 >vhich. the sample was
obtained and the shape of the rnicroorganism (mis-
shapen, slende1 1rids).
Ramifications of Misuse of Antimicrobial Agents
Tn aclclition to ohviou~ hi>nefit~ ciPTived frorn antimicrobial
agents, thcrc are risks involved with their use. Tl1ese risks
involve the patient as well as others that share the same
environmcnt, includlng oursclvcs. Thc risks concern re-
sistance to antimicrobial agents.
Resistance
ln general, therc are t\vo mechanisms whereby bacteria be-
come rcsistant: mutation and acquisition of genes encocl
ing the reslstant phenotype. Mutations occur randomly
(i.e., thcy are unrelated to the prescnce of t he drug). Tf ~
46 PA RT 1 Tntro<l11rtion
mutational cvent ipvolves DNA encoding a target for a par-
ticular antimicroblal drug, then n:sistancl:! to that <.lrug
may rPs11lt. Otht>r mutational cvcnts may have more
"globul" effects, however. for ín:stuncc, thc chromosomol
gene encoding the transcriptional activator residing Ln the
Mar (111ultiple antibiotic rcsistance) locus results in 1nulti-
ple drug resistance. TI1c 111ccl1anism for this pheno1ne11on
is the deregulation of control of chromosomal ge11es gov-
crnlng the flux of drugs across rhe bacteria! cell •vall. 'fl1u:.,
the "mttltidrug resistance" ph<>notypt> i~ <lut> to changcs in
t l 1t: L1ansporl systems of thc ccll wall, leading to resistance
to just about any clinically useful antibiotic (e.g., fluoro-
quinolones, beta-lactams, tctracyclincs, and chloram
phenicol). Orugs are pumped out of the bacteria! cell be-
forc they reach therapcutically useful concentrations. 1'his
phenotype has becn described for J!,scflerichia coli, Salrno-
nella, Pseudornonas aeruginosa, Prote11s v11lgaris, K/P.hsiP.lla,
and (~arnpylubcu.:ler.
ThP majorway in which bacteria bccon1e resistant, l1ow-
ever, is through the acquisition of DNA that encodcs rcsist-
ance to antimicrobial drugs. 'J'hcsc genes may conta1n the
information for enzymes that inactivate certain antimicro-
bials by acetylat1on or phosphorylation (aminoglycoslde
and chloramphenicol modifying cnzymes), for enzy1nes
rt1at JnaL-rlvalt: l.Jy l.J1eaki11g uv11u::. (l¡ela-la<:ldillases and
ct>pha lospori nases that inactiva te pen icillin/ampicillin
and ccphalo::;porins, respcctivcly), or for protcins that are
involvcd with transport of an antin1icrobial (tetracyclincs),
or thcy may cncodc a targct protein different fro1n the na-
tivc (susceptible) one (a diffcrcnt tetrahydrofolate reduc-
tasc is responsible for triincthoprim resistance).
DNA encoding these varlous enzyn1es can be ¡¡c4uiretl
by bacteria il1 several ways: 1) thPy c;in t;ikt> up ONA from
thci1 cnvironn1ent (called tra11s(on11atio11); 2) thcy may be-
come "infected" by a bacteria! virus tl1at contains the re-
sistancc genes the virus had acquired from a previously re
sistant bacteria! host (called transduction); or 3) they can
"receive" it from other bacteria by a sexual process (called
conjuga1ion) . Although thcrc ar<:! examples uf each uf tl1e:.c
occurring in nature, the n1ost common mt>thorl of f)NA
acquisllion is Lhrough conjugation, at least for tbe acqui:;i-
tion of resistance genes by bacteria in an environment
containing antimicrobial drugs.
Acquired UNA may exist within the bacteria! cell sepa-
rate from the chromosomc (extrachromosomally). This
exrrachromosomal DNA ls calletl a plasmid. lf the µla:.u1itl
contains genes encoding resist;i nct> to ;intin1icrohial drugs,
t l 1t:11 Lhese plasnlids are callcd R plas111ids. R plasmids may
encode information for conjugatio11, and if so, such plas-
mids are transmissible and thcrcfore have the capacity to
move trom one bacteriun1 to another, either witl1in a fa m-
il y (e.g., Ji. coli to E. coli, or F. coli to Salrnonella) or outside
of a family (e.g., E. coli to Pasteurellc1). 'l'hi:s tra11:s11li:s::.ilJiliLy
has been noted for gram-positivP ;;ind gr;im-nt>gative hacte-
ria, [~11 vbligale aerobcs, and for facultativc and obligate
anaerobes. lt appears to be most clinically relevant among
members of thc fan1ily Entcrobactcriaccac (e.g., E. coli,
Sa/111011ella, Klebsiella).
A particular bacterium may contain a numbcr of differ-
cnL plasmids, severa! of whlch may be H. plaslllitl:.. Tite
number of resistance genes enr«1<1f'<I on ;i plasmid is vari-
ablc, from two (which seems to be ;iho11t tht> mínimum) to
1uu1~ ll1a11 seve11. ·r11e genes cncoding resistance lo almost
ali of the co1nmonly used antimicrobial drugs are found
on p la::;mid Dl'V\. 'fhc cxccptions are resistance to thc fluo
roquínolones, the polymyxlns, and 1netronidazol e.
Consequently, after conjugation, a susceptible str;iin of
bacteria may have acqulred r<:!slstancl:! to a 11u1111.Jer o( a11-
timicrobial agents and h;1vf' thi> pntt>ntial to pass these re-
:>blai1ce ge11es 011 Lo yet another, whilc keeping a copy for
themselves.
ln additio11 to bcing mobilc by means of conjugation,
resistance genes are mobile 1n their own rigl1t. Mobile
genes, called transposable genetic elernents or transposons,
move from one piece of DNA to anotl11:!r. Mo::.L ofle11, Lhe
information encoding resistan et> to ;i p;irticular antimicro-
bial age11L is on a lransposon. The importance of this is
1·hat, in the hospital environment where bacteria are cx-
posed to a nurnbcr of diffcrcnt nntü11icrobial agents (and
by detinition theywill have acqu1rcd the gene neccssary to
cope with each of these antimicrobials), the genctic pool of
resistance genes is very la rge. Antl since tl1t: gt:11es lhe1n-
selves are mobile, they v.•ould have tht> tt>nrlency to insert
themsl:!lvl:!s 011 a11y 11u1nber of plasn'lids, formi.ng plasmids
with many and varied resistance genes. Bacteria (both
gram-positive and grum-ncgotivc) containing such R plas
mids would be extremely difficult to kíll or suppress wirh
antimicrobial drugs if they were to becon'le Lnvolved with
a disease process.
Another mechanism whereby bacteria may becomc re-
sistant is through "trapplng" of seg1uc111::. vf resislance-
encoding DNA (called cassettt>S) hy ~t>gments of DNA (inte-
g1011s) 1esiding il1 lhe chron1oson1e, on a plasmid, or
witl1in a transposon. Integrons contain the DNA needed
for the insertion of thc cassette (an integrase), a recogni
tion unit that is "recogni.z ed" by the cassette, and a pro-
moter (the cassettes do not usually have one). 'fhcrc are
many rypes of cassettes, but the most cornrnunly vccur-
ring are those that contain rf'~i-;t;inrt>-t>ncoding genes.
Seve1al casselles co11taining severa! resistancc cncocling
genes can associate with a particular integron, of which a
bacterittm may bavc 1nany. Intcgrons are very common in
gram-negative t)acteria, and 11ave been reported iI1 so1ne
gra111-positive species.
Integrons contalnlng anthnicru!Jial re:.i:. La11ce-e11cod-
ing genes, and R pl;ismiris St>t>tn to he quite common
a111011g 111cn1bers of the family linterobacteriaceae. lt is cx-
tremely difficult; therefore, to prcdict which resistance
genes will be prcsent, espccially if the R plasmid l1as been
"constructed" from the res1stance gene pool in a hospital
environ1nent. For this reason, a major effort shoulci be
made i.n "ruling out" or "ruliug i11" 111e111bers of Lhis group
on the microbiologir::il <liffer<' ntial list for a particular site
or co11dltlon.
Within 24 to 48 hours aftcr the initiation ot antimicro-
bial thcrapy, dramatic changes oc<..-ur in onc of the 1najor
host dctense systems of the patient, the normal flora.
'l'hese changes are reflccted in the replacement of the nor-
mal flora with bacteria resi:.ta11L Lo Lhe a11linúcrobial drug
being used. To undt>rst;ind why this is a risk, it is important
LO u11dcrstand the role of the normal flora as a host dcfcnsc
barrier.
 Chapter s Antimicrobial Drugs: A Strategy for Rational Use and the Ramifitations oí Misuse
--~ Risk
- 'onization resistance (see Chapter 2) is a term that de
!IU:Ut!:. Lite lur1C1te iu11nuntty C1ffortle<.l tt1e host by lts nor-
-~1 flora. Tt is thP normal flora ;inc1 thP mechanisms
::creby thc norrnal flora is maintained that prot:ct thc
5: from colonization (infection) by extraneous m1croo r-
':r'-isms. This is an importa11t conccpt sincc p rcvcntion of
~:on i7.ation by agents with pathogenic potcntial (e.g.,
~'1nont•lla) or prevention of colonization by resistant
...:3_j'ls of 111icroorga11is111s is key to 1ni1J.i11J.i;dng the risk we
--=-our patients take in living in an environmPnt ront;im-
- -""d by mícroorganis1ns.
....olonization resistance can be decreased by a num ber
..2\ltside influences, principally stress, and by antimicro-
,,· agents. Jf t he cotonization res1stance is decreased suf-
ently, then replaccment will occur (a surface <loes not
"' - unoccupied!) .
..\nti m irrohi;i l ;igi>nts decrease colonization resistan ce
~spí talized dogs were 38 times n1ore likely lo have ac-
- ..rl:ed resistant Safmonella if they were first given an an-
L:ñotic). Within 21to18 hours after administration of an
-:.rtmicrobial, the normal flora is depressed to the degree
~ -..a.t resistant strains start to recolonize since those mi-
oorganis111s Llial rt::plact:: tl1e 11orrnal flora will be reslst-
-¡ to the antin1icrohial drug arlministPrPrl. 1'hpsp ri>si~t­
~t strains may be a part of the normal flora (e.g., norn1al,
.~:nedicated dogs pcriodically shed large num bers of R
- asmid containing E. coli i.n their fcces-thc rcason for
-;:;sis unknown) or from the animal's environment. ln ei-
~-:cr case, the replacement strain wíll be resistant to the an-
-=:!!icrot>lal belng used. Having rcslstant bacteria occupy-
~g ;i ~11rface is not harmful in itself, unless the resistant
.:::ain has the ability to il1vadc Lhc hosl cell Lo which il has
':'.'ai-'1ed access. But as mentioned above, if a normally ster-
~ sitc contiguous to the recolonized surface becomes
mpromised, bacteria (now resistant bacteria) ~.ll con-
i2minate, and the resulting disease will be more d1ff1cult to
:.:eat. This recolonization effect is the reason beh1nd the
-~ommendation that prophylactic use of antimicrobial
__.:ul::> t::xL1;:11tl 110 lu11ger tl1C1u 24 to 48 liour~.
Colonization resistance is rcduced hy thP. strPss of ill-
'.€5-S and the stress of nevv social/envi.ronmcntal experi-
e:ces. ·rhese changes occur secondary to changes in the
- ormal flora. What appears to transpire are changes in thc
=.en1ents responsible for the maintenance of t l1e stable,
::wrmal flora. Although these changes have been defined
~osl p11::cisely i11 Liie orc1l cavity, tl1ere are uata that suggest
~at they al so occur in the inte.<;tínal r.ana 1. l n thP oral r;iv.
;::--. thc cpithelial cells are coated with fibroncctin, a glyc~­
~rotein . i:ibronectin contains receptors for streptococc1,
=i..:e most ab undant microorganisn1 found on tl1c bucea!,
•:igual, and gingival surfaces. It is the strcptococei that ex-
~'.lde olher, potentially more dangerous microbes from as-
~ia liug will 1 llie oral cavity (u1ost woultl agree t~at these
- ·ould be n1icroorganisms h elonging to thP PntPnc grollp
such as E. coli or Klebsiclla). The amount of fíbronectín
.:oating these cells decreases in the stressed animal, leaving
.r·ailablc underlying attachrnent sites (for gram-ncgativc
::!icroorganisms) either on the cells themselves or on gly-
47
coproteins that co;it thP rPll~ ;ifti>r fihronectin is gane. It
should be notcd, too, that if the animal is treatcd wilh an-
timicrobials as well. thc strcptococci would also be re-
moved since the oral streptococci are very susceptible to
most antimicroblal agents. There is recent evidencc that
shows that sorne gram-negative enteric bacteria possess
acthesins fo1 rect::plors 011 fiuronectin, underscorlng the
importance of keeping the normal floríl int;ict . Th<> reslllt-
íng changc from a flora composed of relatívely ínnocuous
bacteria (nonhemolytic streptococci) to one with patho-
genic potential (members of the enteri~ group) becomcs an
important issue lf compromise occurs 1n a normally ster1le
contiguous site (e.g., the lung).
Rísk to Others
Antimicrobial agents select bacteria that contain genes en-
coding resistancc to t hat particular d rug. ilecause the
genes encod1ng res1stance are almost always tou11d on R
plasmid DNA, antimicrobial use selects for resistance
01;:u1;:::. Lo other ali Ul>iotlcs a::. well. In the hospi~al cnvuo~­
ment where antim icrohials ;i rp 11sP<1, thi> env1ronment 1s
contaminated ~vith m icroorganísn1s containin g very n10-
bile genetlc material encoding resista11ce to a variety of an-
timicrobial agents. We are a part of this cnvironmcnt, and
therefore also partake in this gene pool. Even with an in-
tact colonization resistance, we are transiently colonized
wil1 1 uacleria tlt:riveu fro111 anirnals placed ln our care.
-rransient coloni1.ation is c>no11gh for con¡ngation and sub-
sequent passage of resistance genes to our resident norn1al
nora. If by chance the host human is also being medicated
with antimicrobials, th e chance of colonization with rc-
sistant animal strains increases, as docs the possibility of
passage of an R plasmid.
Summary
ln outlining thc rational use of antimicrobíal agents, this
chapter emphasizes the importance o1 two questions: Is
there an ínfectious component to the disease process
under conslderatlon? And, tf so, what anrirnicrobial drug
would be cffcctíve? The direct smear is a tool that is useful
in asccrlai11iJ1g whelhe1 a11 i11fi;:cliou::. co111µo nent exists.
Once it has becn reasonably established that there is an in-
fcctíous component , a "microbiological differential líst"
(in hierarchícal order) is created anct further shapcd de-
pending upon visual clues obtained from the dircct smear.
Experience and retrospectivc susceptib1hty data are used. to
determine which an timicrobial drugs would be effect1ve
for Lhe n1osLlikel y uf Llu:: co11stituents of t he mlcrob lologi-
cal differential Jíst. A final rhoicP is m;iclP after considering
distribution, toxicity, and cost.
Antimicrobial drugs should be treated as an environ-
mental pollutant. They exert very powcrful sclcctivc pres-
sures o n thc gene pool in which we all partake. ln atlditíon
to being potent selective agents, antimicrobíals are a risk to
Lhuse palii;:nt::. receiving them by dlmlnlshing host defense
barriers.
 Vaccines
N. JAMES MACLACHLAN
lntroduction
Vaccines are substances that are used to elicit immune re-
sponses to prevent or minin11ze dist!ast:: pruLlult:Ll l>y infec·
tious agcnts. Vaccines can be cnn1po'\t>d of the infectious
ageut il~t:lf (t:ilher livc or killed), a portion of the agcnt
that induces a protcctive immune response (subunit vac·
cine), or a product of thc agent. Products containing a
killcd bacteria! agcnt are more propcrly called baccerins.
Products that have toxic activities are called toxins, and
toxrns that have bccn inactivated are calleo tuxuids.
To be effective, vaccines must eliclt <tn imm11nt> rc~­
sponse that ir1tcrfcre~ vvill1 Lhe "lifc style" ofthe infectious
<18Pflt.
Humoral Immunity
Antibodies function im1nunologically by binding to epi·
topes on the surface of the infectious agent and/or one of
its products. By binding to the surface of an infectlous
agcnt, antibodies interfere with attachn1ent to host tareí•t
cells by stearic iutl'.rfcre1 Jlt: a11d/01 by cha!1gü1g the charge
or hydrophohiciLy of the surface of the agent, and trigger
the complement cascade generating products that are op·
sonic and products that are damaging to agents that have
surface membranes. Antibodies that bind to products of
infectious agents can block tl1e attachment of the product
to receptors on cell u lar targets and/or change the co11fie11-
ratlon of the protlu1.;l rt:::.u!Liug in a change in binding
;.i ffi ni ty.
Cell·Mediated lmmunity
Cell-mediated immunity is an immune response that re·
sults in the gcnt~r•1tion of "activated" macrophages a11d/or
specific cytotoxic 1· lyinphocytes. Th!s aspect of the im·
mune response concerns agents that live insic1t> of cPlls,
vvhich are tl1us prolt:1.:Lt:d fro111 interaction with the ele·
mt>nts of the humoral components of the system.
Activatcd macrophages are mononuclear phagocytic
cells that havc come in contact with lnterleukin 1 (IL· I)
un<l interfcron. y (l1'lF gamma). Such cells have incteased
phagocytic and enzymatic actlvity, contaiu iI1lrt:ast:d
amo unts of n itrie oxide, a11d h avt> incrPased production of
Lytukiues, such as lurnor necrosis factor (TNf) and IL-1,
and incrt><ist>d expression of MHC class 1(MHC· ll). ·rhis in·
crease in activity is thought to be responsiblc for thc de
struction of infectious agents that nonactivated mononu·
48
DWIGHT C. HIRSH
clear cells cannot destroy following uptake. Sorne terrn tl1i~
im1nune state (i.e., activation of macrophages) ce/111lnr hy·
persensitivity.
Cytotoxic 'í lymphocytes rPcogn ize affected l1ost ce lis
(c.g., 1.:ells iofeclcd with virus or bacteria) . In so doing,
these lymphocytcs secrete substances that result in the
<leath of thc affcctcd host cell. If the affectcd bost cell con-
tained an inlcctious agent, that agent would now be llbcr-
ated and in contact with other host immune participants
(e.g., antibody, complement, activated ruauovhages).
Generation of the lmmune Response
Antigens that are processed by antlgen·processil1g 1.:~11::. via
the exogenous pathway elicit antibodit>s. ·rhus, extracellu·
lar bacteria (live or killed), inactivated viral particlcs, por·
tions (s11h11nit'\) of virus, and products are processed by the
exogenous pathway. Epi topes are presented to thc immune
system in context of MHC-11 by an ailtigen·presenting ccll
that secretes IL· 1 and little, if any, !L 12. T helper cells (fHz
subset of C:U4 lymphocytes) respond to this stimulus by sc-
creting cytokincs that trigger an antibody response (U.-4,
1L·5, IL· 13).
Sorne infpctious agt>nts rcplicate within cells. Tf the
agent multiplies within a roononuclear phagocytc, thcn
antigens are processed by"vay of the exogenous and/or en·
dogcnous pathways (see below). /\.s outlined above with
extracellular antigens, antigens of intracellular agcnts
are presented in context of Ml-TC-11, but tl1e <tntigf'n.
prcsentlng c.:ell Selretes 1L·1 a11Ll IL·12. 1L·l2 stin1ulates ·r
helpPr ct>lls (I'H 1 suhset) while turning off cells of thc TH 2
subset. ·rH 1 cells secrete INf·gamma, rcsulting in the acti
vation of mononuclear phagocytic cells. Sorne of these
"intracellular" agents (some viruses, bacteria, fungi) repli·
cate in the cytoplasm of mononuclear phagocytic.: cell:>.
A11tige11s from these agents are also processed hy thC' en·
dogenous pathway, as art: auligens liberated within non·
phagocytic c.c>lls, so that epitopcs are presented to the im·
n1une system in context of MHC·l. .Epitopes presented in
this fashion are recognized by CU8 cytotoxic Iympho·
cytes. These lymphocytes function by lysing infected tar.
gets, i.e., cells expressing epitope-MliC·I complexe:>.
DNA Vaccines
DNA vaccines are those in which the gene encoding the
antigen in qucstion is inserted lnto a pla::.111i<l vector 1hat

'has a strong pro1noter (e.g.. cytomegalovirus immediate/
early promoter; SV 40 early promoter) that will result in ex-
tnession of the target gene, a termination seque11ce (poly.A
tail), and a number of cytidine-phosphate-guanosine
CpG) motifs. The function of tl1e CpG 111otlf (a II1otif
"nmmon in bactPrial gPnomPs) is to dirPc.t the antigen
p rocessing cell to secrete lymphokines that favor T Hl lym-
phocytes. ·rhe construct is administered in any number of
..\-ays (intramuscular, intradermally, upon a mucosa! sur
face), but intramuscularly is the most con1mon. Myocytes
:hat become transfected serve as antigen-presenting cells
&id express a11tige11 in co11text uf MHC-T (lur11 011 CI)8 T
'ymphoc.ytPs). Tt is unc.lear ho1,v antigen is expressed in
context of MIIC-II (for CD4 T lymphocytes). Possibilities
include MHC-JT antigen-presenting cells (macrophages/
dendritic cells/B lymphocytes) becoming transfected, or
rransfected myocytes transferrtng the plasmid consn·uct to
~fH C-II antigen-presenting cells.
DNA vacci11es have been successful in eliciling proLec-
tive immune responses (both humoral and cellular) to a
<ariety of bacteria!, viral, and protozoal microorganisms;
h-01-vever. commercial DNA vaccines are not yet available in
•·eteri11ary medicine.
Adjuvants
Adjuvants are used to influence the nature of t he immune
response elicited by an antigen. The response is influenced
at various stngcs, dcpc11ding upon the adjuvant. Sorne ad-
juvants function as depots, so that antigen is slowly re-
leased over an extended period of time to maximize the
immune response. Examplcs lnclude water/oil emulsions,
.minerals/salts (bentonite, aluminu1n), and inert p;irtic:les
1n icrospheres). OLher adjuvants direct activity to t he
processing step in the initiation of t11e im1nune response.
Examples in.elude "imrnune stimulating complexes"
<TSCOMs) composed of cholesterol-phospholipid struc-
tures that contain the immunogen, and liposomes (lipid
vesicles) . "Targeting" various cornponents by using
various cytokines as adjuvants can influence imrnune
responses. For example, TL-1. activates T lyrr1phocytes, IL-
1/. and INF-gamm:i infh1i>ncP thP hPlpPr ·r lymphoc.ytP
subset selcction, an,d gra.nulocyte macrophage colony-
stimulating factors activate macrophages and increase effi-
ciency of antigen processing.
Viral Vaccines
Immunization of animals with viral vaccines is critica! to
t he prevention of many viral diseases. 'fhe basis of an effec-
tive vaccine is its ability to induce an imrnune response or
responses capable of eliciting protection to subsequent
field exposure to patl1ogenic viruses. A multitude of vac-
ciu1;: preparatio11s l 1ave 1Jee11 tlevelupetl a11tl us1.:d ovt::r tlie
years. with variable success. The success of a potential vac-
cine hinges primarily on safety and efficacy; h<n.Yever, eco-
nomics also continue to dictate vaccine design, develop-
n1ent, and ultimate production on a comn1ercial basis.
Various approaches to vac(.ination have been employed
Chapter 6 vaccines 49
over the years. ·rhese inelude 1) adrninistering .1 ive virulent
virus in an anatomical site so that the target tissue or tis-
sues are not infected, 2) administering Jive virulent virus
to animals ata time of relati ve resistan ce to disease expres-
:;iu11, 3) cu11curreul ad111i11islraliu11 uf live virule11L virus
and immune serum, which no longer is acceptahle for oh-
vi.o us reasons, 4) use of live avirulent viral strains (e.g., at-
tenuated or "modified live" viruses). and 5) use of inacti-
vated virus. ln recent years, additional approaches to
vacctne development have becoine avaU.able. These in -
clude subunit, synthetic peptide, and recombinant prod-
ucLs. Regardless of vacci11e Lype, Ll1e desi red resulL is Lo
induce immune responses specific for viral antigens ex-
pressed on the virion surface or on the surface of infected
cells, so that the immunized host is immune when ex-
posed to the virulent virus. 'fhe rational development of
an efficacious viral vaccine requires an u11derstanding of
viral pathogenesis, o.f p.rotective ilnmune responses in-
duced followi11g i11fecl.ion, a11tl of Ll.ieir pruleil1 :;p1;:ciJici-
ties. The latter point is of obvious importance for develop-
ing reco1nbinant and synthetic peptide vaccines.
Concerning pathogenesis, the followtng three general
types of viral infections occur:
l. Infections that are confined to the mucosa! surfaces
of the respiratory or gastrointestinal tracts. In such
instances, local in1munity in the form of secretory
antibody (e.g., IgA) is important. The role of cell-
mcdiated immu11ity (CMI) is less i.vell characterized
in :;ucl1 iufectior1s.
2. Tnft>c.tions that he.gin at m ucosa] s11rfac.es l1ut then
cause a systernic i1lfection with viremia, and subse-
quent infection of distant target tissues. In these in-
fcctions, botb immunity at the mucosa! surface as
well as system1c 11nmunity are important.
3. Infections that gain direct entry into the host's cir-
culation via lnsect bite (arthropod-born.e viruses),
inadvertent inoculation, ora traumatic break in an
epllhelial surfacc. Jn sucll. infections, systemic im-
munity is the primary line of defense.
'fhese mechanisrns of viral il1fection and subsequent
llisse111i11ation 111ust IJe consideretl iu vacciue tlevelop-
ment. Modified live (attenuated) and inactivated (killed)
virus vaccines dominate thc vcterinary vaccinc markct
fiable 6.1).
Live Attenuated Viral Vaccines
Tlie <tLLeuuaLed viral vacci11e~ iuclucle <trlificially aLLeuu-
ated (1nodified-live) strains of virus or naturally occurring
viruses vvith reduced virulence for the host. The origin of
t hese naturally attenuated isolates may be the natural
host, or a closely related virus isolated from a different
host; for example, the cowpox virus was initíally u sed to
vaccinate humans against smallpox. The major require-
1ue11ts uf sucl1 an i1pproacl1 are that it induce adequate im-
n1unity and that the attenuation (lac.k of v in 11PncP) of thP
vacci ne straLn be stable. The majority of vaccines currently
u sed today in veterinary medicine are attenuated virnses.
The most common approach to viral attcnuation is thc. dc-
velopment of host-range mutants. Other approaches in-
50 PART I Introduction
Table 6.1. Types ot Viral Vaccines
l. Uve Viral Vaccines
A Attenuation for low virulence of viruses that produce natural
diseases.
B. Host rangc mutants- use of different viral strains infecting
d1fferent host spec1es that are relate<! antigenically to the virus
strain that produres a natur;il rli<eaSP in the original ho~.
C. Relu111bi11dnl hele1oloyuu) vi1dl valor Vdtti11e>--to11struction of an
infectious viral recombinant that expresses protective antigen(s) of
another virus that produces a natural disease. Construction of a
recombinant virus with insertion of genes with known antiviral
activitics or with known immunorcgulotory functioos.
D. Recombinant homologous viral strains attenuated by targeted
mutations on deletions of genes coding for specific virulence
factors tllat produce a natural dlsease.
E. Nonreplicatíng recombinant viral vector vaccines capable of
replicating to high titer in vitro but unable to grow effidently in
vivo.
11. lnactivated Viral Vaccines
A. lnactivated viral vaccines by chemical methods.
B. lnactivated viral vaccines by physical methods.
C. Purlfied vira! antlgens ustng monoclonal antibody immunoaffinity
chromatography.
D. Cloned viral protein )Ubunit Vdttines µ1udutal in eukd1yotíc or
prokaryotic cells by recombinant DNA technology.
C. Synthetic viral polypeptide vaccines representing immunologically
urpident domains of viral surface antigens.
F. Direct injection of plasmid ONJ\ encoding viral protective antigens
into tissues in vivo.
G. Use of anti·idiotypic antibodies as antigens to induce an antiviral
antibody response.
elude development of temperature-sensitive and cold-
adapted 1nutants (missense mutations), deletion mutan ts,
and recombinant viruses.
Host-range mutants are developed by serial passage in a
host system different from the natural host to be vacci-
r1ate<..1, usually lal.Juratury a11i11ictl:., e1ul.nyv11aled chicke11
eggs, or, increasingly, cell cultures. Upon serial passage in
these system s, vin.1ses often lose their virulence for thc
natural host dueto accumulation oí mutations in the viral
ge11on1e that result in changes in virus specified proteins.
The basis of attenuation of many modified live virus vac-
cines, however, is poorly characterized and the possibility
of reversiun tu virul~IH.:c aftcr gru~vt11 iu l11e 11alural hosl is
a lways a concern.
Conditional lethal mutants have been generated \-Vith
the intent that such viruses wou!d exhibit limited replica-
tion in the host and so serve as vaccines. Temperature-
sensitive mutants are typically created by mutagenesis
and phenotypically selected on the basis of temperan1re.
Cold-adapted mutant!> are ge11erateu l.Jy J!IUJ!agaLion al
surrPssivPly lowpr tE>mpPratures, thc cnd product being in-
capable of rcplication at normal temperatures. The cold-
adapted mutants typically acquire rnu!tip!e mutations in
genes encoding virulence and are relatively more stable
than temnerature-sensitive mutants.
A unique approach to expression of cloned viral genes
is the use of hetcrologous viral expression vectors. Vac-
clnla virus has wldely been used as an infectiuus va<.:<.:i11e
expression vector hpc·a11'\P it is a virus that has widely heen
used as a human vaccine and the fact that at least 22 kilo-
bases of the vaccinia genome can be deleted without loss
of infectivity. ºfhe latter attributc givcs rcscarchcrs amplc
space in which to rnsert tore1gn c1onea genes and nas the
potential to permit inscrtion of multiple foreign viral
genes for the purposc of designing rnu1tivale11t a11u iuulli-
viral vaccines. A major potentia! advantage of surh infPc-
tiuu:. va<.:1.:i11e veclv1:. i:. lhe pole11lial for lnduction of cellu-
lar immunity by inserting exprcssed viral proteins into the
host cell membrane in contcxt with histocompatibility
anti gens.
Co n struction of delction mutants is another potential
mechantsn1 of virus attenuation; thus, tl1e selelllve dele-
tion of genes that expn~ss f;:ictors for vi rulence, persistence,
or iIIuuuuu:>u¡.¡¡.¡re:.:.ivn ca11 oflen be accon1plished with-
out compromising viral replication. This approacl1 re-
quircs a thorough understandil1g of thc virus and the
pathogcnesis ot the dísease it causes; a deletant vaccine
has bccn dcveloped to prevent pscuc.lorabies in swi11e. The
thymidinc kinase gene is deleted ln t.he pseu<..loral.Jies vac-
cine strain, which is able to induce an immune response
wlthout producl ng dl:>eilse. Furtl1cru1ur1::, Ll1e genes encod-
ing virus glycoprntPins gpl, gplll, or gpx are deleted in the
vaccine strain. The presence of antibody to these specific
virus antigcns can be used to differentiate field-strain in-
fected animals from vaccinatcd animals, an important ad
vanee iJ1 detining the epidemiology and control of this
disease.
Nonrepllcatlng recombinant viral vectors that are not
capable of replication in vivo but that can express forPign
p10Lei11s du1 i11g abo1 live i11feclio11s can induce humoral
and cell-mediated immunity in immunized hosts. Experi-
mental studics show that dogs or cats are rcsistant to wild-
type rabies viral challenge when inocuiated vvith avian
pox-rabies glycoprotcin recombinant viruses. 'l'his type of
vtral vaccinc has the distinct advantage of belng safe in the
immunosuppressed host.
l'here are adva11tages a11d disadvantages to attenuated
viral vacclncs. Table 6.2 lists the general characteristics of
livc attcnuatcd as comparcd to killcd viral vaccines. A
major advantage of líve viral vaccines is their ability to
replicate within the host and thereby elicit both humoral
and cellular tmmune responses. In the case of viral ir.Lft::<.:-
tions attaching primarily to the m11cosal surfaces of the
respiratory and gastrointestinal tracts, administering at-
tenuated viruses by the nasal or oral routes stimulates local
immunity. Economic considerations also favor attenuated
vaccines due to the lower cost of production and the typi-
cal absence of a requiremcnt for adjuvants, immunopoten-
tiatlng agents, and the need for ruultiple lI11111uJiizaLions.
While attenuated vira I vaccinPs continue to provide the
1nainslay of veterinary vaccines, there are a variety of po-
tentially serious disadvantages to their use. Significantly,
only a fine linc scporatcs n1odification and !oss of im-
munogenicity. ·l hus, modified Uve vaccine strains of virus
require a compromisc between loss of virulence of the
virus and loss of lts lmmunogeni<.:ity l.Jel:au::;e u1a11y viruses
 Chapter 6 Vaccines 51

_ Relative Advantages and Disadvantages of Live vs. lnactivated Virus Vaccines

-act1vated Virus Vaccines
Many inactivated vaccines have been developed for use in
vetertnary n1edicine. Virus inactivation has most com-
Criteria Live lnactivated
monly employed formalin, beta-propiolactone, acetyleth-
fmmunity Long Short ylenei1nine, 0 1 bina1 yeth yle11c::i 111i111::. Atltliliu11al u11::tl1utls
Adjuvant No Yes include ultraviolet light, gamma irradiation, psoralcn
satety Variable Usually very safe compounds, and ozonc gas. Thc primary advantage of
Complications (potential} Reversion to virulence, Sensitization kHled virus vacdnes is safety-many potential disadvan-
spread to susceptible tages of live-virus vaccines are climinated since no virus
anima Is rcplication uc<..urs. Virus for lnact!vat!on has becn pro-
Potcntial contamination Possiblc Mínima! duced in Jahoratory animal~. !'mhryonatP<1 rhirken eggs,
lnterference Poss1ble Mínima! and, most commonly today, in ccll cultures. From an eco-
Cort Mini mal Significant nomicviewpoint, viruses that grow to high titer in cell cul-
lmmunomodulation Not requlred Required tures and exhibit first order inactivation kinetics providc
Vaccine marker Possibly genetic marker Serologic marker the best candidates for vaccinc prcparation. Adjuvants are
Stability Poor Good typically required to induce good immunity 'l-vitl1 killed
CMI induction Yes No viral products, a11d n1ultiplc doses a!'e usuall y re~ui ri;:tl.
local secretory immuníty Yes No With thc continued develop1nent of better adjuvants and
Reassortment/recombination l'ossible No immunopotcntiating complcxcs (ISCOMs), inactivated
Persirtent Yes No vaccines will prove more effcctivc. Inactivated vaccines are
also relatively stable under advcrse conditions, and their
pulc11tial fur strain interference In mult!valent prepara-
tions is reduced compared to att<'n11atf><1 varrinPs.
Thcrc are, ho\•vever, certain disadvantages associated
exhibit reduced immunogenicity as thcy become attenu with the use ot inactivated virus vaccines. Most inactivat-
ated. furthermore, accurate assessment of viral attenua- ing agents are toxic, an<l sorne are carcinogenic. Unlike live
'ion can often be difficult since consistent experimental vlrus, tnactlvated vaccine virus IS not quantitatively am-
repioduclion of clinical disease i:-. difficulL wiLIJ c1::rtaiu plified, so it reguires adjuvant <ind multiple inoculations.
Yiruses. In such instances, a vaccine virus considered to hP. J:lurthern1ore, such preparations do nol ü1duce strong cel-
attenuated may produce clinical diseasc under special cir- lular immunity, since inducing such responses requircs
cumstanccs involving stress, physíologic imbalancc, or that thc antigcn be prcscntcd in association with histo-
concurrcnt infections with other organisms. Viruses that compat1b1hty ant1gens on ccll surfaccs (processed via the
exh1b1t a w1de host range are also problematic. Vuus atten- endogenous pathways, see above). Nor are such vaccines
uated for one animal species may reta in virulence for more a~~uciatetl ~.Yitl1 c.levelopruent uf local secretory immunity,
\Usccptlble species, and since attcnuated vlruses cause an du<' to the usual parentPral routf> of varcination.
::1ctivc infection, transmission to other species may occur. Thc succcss of viral inactivation depends on tl1e inacti-
A n1ajo1 concer11 h1 ll1e use of attenuated viruses is 1e- vant and viral characteristics. While most viruscs can be
·:ersion to virulcnce. This phenomenon has plagued vac- successfully inactivated, the retention of critica! antigenic
cinc development and licensing ovcr thc years. Reversion intcgrity is variable. Antigens responsible for inducing
to virulcnce is a more serious possibility with those viruscs protective imn1unity must be preserved. A possible com-
that enjoy a wide host range or that are biologically trans- plica Lion of inactiva ted vacciue use i~ tl 1e JJOLe11Lial fur <u 1-
mlttect by arthropod vectors. Whlle the virus may appear i rnal sensitization such that an exacerbated clinical disease
stablc in the host far '"'hich the v<iccine was intended, re- is cxpcricnccd upon cxposurc to virulcnt field virus. 'fhis
vcrsion to viJule11ce n1ay occu1 in Lhe vectoi 01 in othe1 sensitization is not well understood, but it is apparently
specics. Vaccination of pregnant animals must also be of immunologically precipitated by an unbalanced immune
conccrn, sincc attcouated viruscs may be pathogenic for response such as humoral vs. cellular immunity, immune
the developing tetus. Vaccinating animals with rcduced rP<;pon<;P to nonnentr;¡Ji7ing epi topes, or preferential stim-
immunologic responsiveness can result in expression of ulation of lgE.
cllnlcal disease. Development of subunit vaccincs is currently an area of
AdditionaJ negative features of attenuated virus vac- extensive research¡ it includcs purification of viral sub-
cincs includc 1) tl1e polenlial Coi 1easso1 L1nenL (viru~i;:~ units, rccombinant technotogy, and peptide synthcs1s.
\vith scg1ncnted genomes) or recombination betvveen vac- Thc basis of a subunit vaccine is an in11nunogenic protein
cinc strains or with wild-type virus to create new viruses, (01 pt::ptitle .s1::que11ce.s) capalJle uf eliciting protective tm-
L) tack ot a vaccine marker tor scrologic differentiation of munity. Such proteh1s would typic.ally he founcl on thP
vaccine and wild-type virus expnsure, 3) development of virion surface and contain epitopes capable of inducing
persistcnt infections, 4) poor stablllty of vaccine virus, cs- neutralizing antibody. Vaccincs can be prepared by dis-
peria lly in hot tropical areas, and S) replication interfer- ruption of tl1e virus followed by protein purification. 'fhe
ence betwccn viruscs in n1ullivalcnt vaccines. Viru:;i;:~ tl1at potentlal cost of such preparat1ons has precluded their
exhibir continucd antigenic drift present another dilem- commercial development, but recent biotechnological ad-
ma sincc ncw isolates must be continually attenuated and vances have offered alter11atives via recon1bi11a11L DNAa11u
tested tor satcty and etticacy. peptide synthesis technologics.
52 PART 1 lntroduction
·rhe approach to dt>vf'loping rec.omhinant vaccines in-
volves inscrtion of DNA that contains lhe desired viral ge-
nomic coding seque11ces into an appropriate expression
vector. Thc viral or complementary DN/\ (cDNA) is in-
sertetl into a plasm1d or bacteriophage followed by infec-
tion of susceptible prokaryotic cells such as Jischericliia coli;
yeast cells, or n1ammalian cells. Multlple strategie:. 11dve
been used to construct vaccinc PxprPs~ion vectors, and
IIHJ:>t i111.:lu<.le a st1ong pron1oter (constitutivc or in -
duc.ihlc). Following infection of the cell with the plasrnid,
the cloned cDNA can be cxprcsscd and the desired gene
product purified tor vaccine use. Recent attent1on has also
becn focused on the inoculation of plasmld DNA encoding
viral antigens direetly into animals. Such DNA vaccine:.
offer tl1e hypothetical advantage of having viral glycopro-
tei11s cxpressed on thc :.urfa1.:e uf L1ansfected cells and in-
ducing imm11nity without interferencc fro m passiveJy ac-
y_uired viral antibodies.
Synthctic peptide vaccines have also been developcd.
As with cloncd viral vaccines, the successful development
ot synthetic peptidc vaccines requírcs extensive knowl-
edge of the viral proteins involved in inducing protective
immunity. Two basic approache:. are dvailable for dctcr-
mining critica! peptide sequencPs: 1) indirectly, fro1n nu-
cleuti<.le ::.ey_uences derived fron1 cloned viral genes, and
2) directly; by sequcnci n g purified peptides. Immunologi-
cally bascd pcptidc and epitope mapping to deter1nine
the regions involvcd 1n protective immunity fadlitate thc
latter approach. An additional approach to determlning
critical peptide scqucnces is based u pon the projected tcr-
tiary structure of the viral protein, wlth the areas that
demor1stratc lty<.lrovliilic cl1araclcristics serving as candi-
rl;ite sec¡uences. One major drawback of synthesizing pep-
t idc vuccincs i:> thc pote n ti al for criticol cpitopcs to be
lormed by the tertiary structure (an epitope formed by
juxtaposition of two separated peptide sequences). Such
epitopes confound attempts to deduce the pepti<.le :.c-
quence and realize such complex configurations in thC'
syntht>tic µru<.lu1.:l.
'l"h<> use of anti-idiotypic antibodies as immunogcns to
stimulate the production of viral ncutraJizing antibodies
has also been explored. ·rhe advantage of tl1is type of
immunoger1 may overcome viral variability problems by
inducing broaclly neutraliZing anllbodles. However, tl1b
type of vaccine induces only antibody r<><:ponses, not c.cll-
mediatec.l resµuu:.c::..
Toxoids, Bacterins, and Bacteria! Vaccines
As with viral vaccines, the basis of an cffectlvt> toxoid, lJac-
terin, or bacteria! vaccine is the ;1bility to induc.e an im-
mune respo11:.e ur re:.punses capable of eliciting protection
from fiPld exposurc to the pathogcnic microorganism.
Most of the principies outlined abovc with rcspect to viral
vaccines apply to products designcd to induce protective
i mmunity to b;1cterial age11ts. The development of an effi-
cacious product depcnds on understandlng the patl1oger1-
esis of the bacteria! disease that is to be prPvPntP<L
In general terru:., <.li:,ea::.es produced by bacteria can be
group<>d into threC' categories: 1) those that result from as-
sociation with a bacteria! toxin, 2) those that result from
t hc scqucluc of extraccllular multip lication of tl1e bacteria!
agent, and 3) those that result from tl1e sequelae of lntra-
cellular multiplication.
Toxoids
Bacteria! toxins are of two kinds: exotoxins and en<.1otox-
ins. E11dotoxins are strictly defined as the lipopolysaccha-
ride portion of t h e gram-negative cell wall (it is the llpld A
portion that is specifically responsible for t he "toxic" man-
ifestations). Muran1yl dipeptide, wlii1.:h is presenl in gram-
positive cell walls ancl to a lcsser extent in gram-negative,
al::.o I1as "loxic" properties. Wc have used quotation marks
around "toxic" because both endotoxin and muramyl
dipeptide elici l thcir ''toxic" activities by inducing the pro-
duction ot a variety of cytokines t)y 11osr cell5. It is tJ1c de-
gree of vigor of the host response that defi nes the toxicity.
Exotoxins are proteins that interact will 1 hosl cells (usually
after binding to a specific rPct>ptor) resuJting in deregula-
tiu11ufl1u::.l cell fui1ction without undue harm to the ccll,
intt>rference of thc normal physiology of the host cell(s),
o r death of the host ccll.
Antibodies elicited to various cp1topes on toxins that
rcsult in neutralization are sometimes called antitoxins. As
mentioned previously, an antibody may block intcn:11.:Liuu
betwee11 a toxin and its c<•ll11 lilr r~ceptor or chan.gc the
co1lfigurdl iur 1uf Lhe loxi11 so that it no longer has an cffcct
on the host cPll. Antihodies to exotoxins have been shown
lo be efficacious in preventing disease. Antibodics to cndo-
toxins havc had n1ixed results as far as preventing disease.
·1oxoids are toxins without toxic activity tl1at can elicit
an immune rt!Sponse, i.e., a11t1bodies (see above for expla-
nation). Toxoids can be produced by cl1e.m ir;il in::irt iv;.1.
tlon of the native tuxiti or IJy n1anipulations of the gene
enrocling the toxin so that the toxin is inactivated. For ex-
ainple, iJ1 lhe case of A-B toxins (sce Chapter 8), whcrc thc
A subunit is rcsponsible for the toxic activity ot thc toxin
and the 13 subunits are rcsponsible for binding of the toxin
to the l1ost, lhe gene encoding the A subunit can be elimi-
nated and a toxoid produced that is con1posec! of 13 SL1b-
u11its. Antibody to llpopolysaccharide (e11dolox in) i~
elicited by immuni7.ation with 111utants (called "rough"
1uula11ls) that produce vcry little of thc 0 -rcpcat unit of
the lipopolysaccharide (see Chapter 8).
·rhc main advantage of toxoids is that they are safe.
Toxoids adm inistered parenterally el icit antibody (IgM
and IgG) that interfercs with toxin-host cell intcractions
tllat are not ata mucosa! surfa<.:c. 0 11 lhe otl1er hand, the
administration of toxoids on a mucosa! surface elicits an ti-
body (slgM and slgA) t hat interferes with toxin-host cell
interaction at the mucosa! surlace. ·1 he main disadvantage
of toxoids used for immunization by way of a mucosa! sur-
tace is their extremely short half-llfe.
Bacterins
Baclerins are killed pathogenic bacteria. Thcy are usually
produced by chemlcal killing ol the infectious agent, with
the aim to preserve bacteria! structures expressing epi topes
important in eliciting a protective lmmunc response. ·r11c

-:unune response is almost always antibody (see above for
'planation).
Thc advanlage of bacle1i11s is Lhal lhcy are :;aft!. If au-
;:¡1niste.reci parenti>r;:illy, thi> ;:intih()rly elicited (IgM and
;G) will be effective if the bacterin is 111ade fron1 a
""'Jthogen that has an extraceUular life style. If the bacterin
_ administcred byway of a mucosa! surface, the antibody
-.llclted (sTgM and sTgA) "l>Vill intcrfere with interactions of
;i.athogcn with host cells. The disadvantage is that the
:nain immur1e respo11se is a11Libody so tltat u11ly ¡u1tiuuuy-
mediated protection \Vill oc:r11r. Th11s, hPtrtPrins adminis-
tered parenterally are not as cffective against intracellular
oathogens. Bacterins placed on a mucosa! surface have ex-
·remely short half-lives, a scrious disadvantage. J\nothcr
úlsadvantage is that the pathogen is usually grown in
•itro, and cpitopes expressed in vivo may not be expressed,
-,·h ich ca11 resull in a pruuuct tliat rnay elicit antibodles
~vith inappropriate specificities.
Bacteria! Vaccines
Bacteria! vaccines are composed of attenuated versions ot
·he pathogen; i.e., they are live but reduced in virulence.
Chaprer 6 vaccines 53
Attenuation may be accomplished in a number of ways: se-
lection of a naturally occurring attenuated strain, repeated
passage on artificial media, or elimination óf a virulence
trait by mutation of the gene encoding the trait.
Tl1c 111ajur a<.lvautcigt::; uf IJacterlal vacclnes are directly
related to th.eir being alive. Live vaccines not on ly h::ivi>
longcr half-livcs than their dead counterpa:rts (regardless
of Jocation), they will cxpress epitopes that may only be
expressed in vivo, thus eliciting antibody to epitopes that
the pathogen wlll also express follo\ving infeetion. An-
other advantage is that Uve vaccines v.>ill elicit antibody
and cellular hyp1;;r:;1;;u:;itivity (:;t!e above for explanation). A
major disadvantage is th;:it livP v;:ircini><; m;iy procl11rP cli<:-
casc, for cxamplc, throug.h reversion to the virulent phe-
notype. Also, if the vacci.nated host has reduced resistance,
then the vaccine is more apt to produce discasc.
 PART 11

Bacteria and Fu11gi

 Fainily Enterobacteriaceae
DWIGHT C.
,--!>ers of the family Enterobacteriaceae ("enterics")
-=disease in both food animals (e.g., neonatal diarrhea
- sal11101it::llusis) a11d <..:olllµa1Iiu11 a1Ii1ua.ls (e.g., urinary
=::r L"Úections, abscesses). Approximately 35 genera com -
-:=:s._ me family, but only .a few are consistently involved
-- disease ot animals crable 7.1).
: : 5{ri ptive Features
:'"j;iology and Staining
~bers of the family are similar in morphology and stain-
" ...11a ra<..:teristi<..:s, being pleomorphic, gram-negat!ve,
---spore-forming rods that measure 2 to 3 µm by 0 .4 to
- t1111 (for an exarnple of gra1n-negaUve rod:., see Fig
-~-\l. It is difficult to tell memhers of one genus fro1n
se o f another by visual observatiot1.
::e jlar Anatomv and Composition
.::-:= cell wall is typically gram -negative and consists of
--er and outcr mcn1branes separatcd by pcptidoglycan.
.::...;.1ous proteins are found in each membrane, son1e tra-
~-sing both. C apsules, flagella, and various adl1csins are
-uetimes present.
-he capsule (K-antigens) is the outern1ost structural
_ :::!ponent of the bacteria! cell. Capsules of enteric organ.-
-.; ;ire composed of c;:irbohydr;itf'S. Thf' v;irions types of
,-.. ~bohydrates, together with the types of linkages be-
--een the sugars, form the antigenic determinants that
::efine capsular antigcns. Encapsulated entcric bacteria are
'.'Elatively hydroph.ilic, a characteristic imparted by the
.::apsule.
The so1natic antigens (O-antigens) are composed of
a.1tigPnic cletPrmin;ints formed by the different configura-
-;ions of sugar types, and the linkages bet\-veen sugars
"ound in the 0 -repeat portion of the lipopolysaccharide
Figs 7.1 and 7.2).
Flagella, which are cellular organelles used for locomo-
~on, are composed o f protein subunits (flagellin).
<:nding upon the type of flagellln, dlfferent antigenic
- ·nants arf'. formf'<1. Thesf' ;intigenic determinants
...,prise the 11-antigens. In cells of n1ost Salnionella and
~e other species, one or tbe otl1er of two sets ("phases")
_:°antigenic determinants are possible. In culture, sponta-
~rous pl1ase var1at1on occurs, that is, a shift from phase .l
2 o r vice versa. The antigens of both phases, if present,
-=-P d efine the serotypes.
HIRSH
Fimbriae or pili are protein adhesins that are co1nposed
of subunits- pilin- and assembled in various configura
tions using dtfferent pilln molecules, which results in the
ge.nf'.r(!tion of clifferPnt types defined by their affinity for
various carbohydrates. The ni.ost con1111only found fin1-
briae have affinity for mannose-containing compounds.
These fimbrine are called typc 1 or comrnon fi1nbriae (also
termed F 1) . Type 1 fimbriae have not been concJusive1y
shown to be virulence determinants. On the other hand, a
variety of other vlrulence-associated fimbriae have been
de.sc.rihe.cl th;it (!ggl11tinate erythrocytes in the presence of
mannose. txan1ples of such n1annose resisla11l (MR)
hemagglutin ins are f 4 (K88) and FS (K99), tvvo virulence-
associatcd adhcsins tl1at are important in the pathogene-
sis of enteric disease produced by certain strains of
Escherichia coli.
Most members of the family possess mucopeptide anti-
gens in commo11, the so-called enterobacterial common
a11lige11.
Cellular Products of Medica! lnterest
Cellular products of medica} interest con1mon to ali or
most of the mcmbcrs of thc family are cndotoxins and var-
ious siderophores.
Endotoxin. Endotoxin is the term given the lipopolysac
charide (LPS) that is part, and extrudes from, the outer
membrane of t l1e gram-negat ive cell wall (see Fig 7.2). 'l'he
lipid portian of this substance is en1beddcd il1 tl1c ouler
membrane and has the toxic properties associated \"1'ith
cndotoxin. Thc most important constitucnt oí LPS as far as
the toxic n1anifestations of the molecule are concerned is
the lipid portion, called lipid A. LPS binds to lipopolysac
charide-binding protein (a serum protein), which in turn
transfers it to the blood phase of CDJ4. The CD14-LPS
con1plex bi11ds Lo ·roll-like l'eceplor prolein;s (:;ec Cl1apter
2) 011 the surface of macrophage cells triggering the release.
of proinfla1nmatory cytokines. It is these cytokines that
are responsible, in part, for the ahnonnal signs associated
with endotoxin.
Síderophores. Siderophores (Greek for "iron bear1ng") are
iron-carrying molecules (catechols or hydroxan1ates) of
bacterial origin. They fun.ction in Lhe solubilizalio11 a11d
transport of ferric ions. Tl1ere is very little free iron in
hosts; nearly ali is associatcd with the iron-binding pro-
tein.s (ferr1tin, transferrin, and lactoferrin). Since iro11 is an
absolute requirement fo( almost all bacteria, ·parasitic
strains, especially invasive ones, must compete for iron.
57
58 PART 11 Bacteria and Fungi

Ta b 1e 7. 1 . Members of the Fam1ly Enterobacteriaceae

lmrort;int in VP.tP.rio;iry MP.dicinP. F 1G U RE 7 . 2 . Mo/pr11/~rorgani7ation of the outer membrane
Citrobacter Providencia of gram-negative bacteria. The chains of rectunglcs rcprcscnt thc
0-repeat units determining 0 -antigen specíficity. They are attached
Enterobacter Salmonel/a
to the po/ysaccharide core (irregular shapes), which is linked to
Escherichia Serratia
lipid /\ (fringed ob/ongs). These three components constit ute the
Klebsiella Shigella lipopolysaccharide (LPS) of the yrarr1-negdlive t ell wall. With t/Je
Morga ne/la Yersinla underlying zone of phospho/ipid (frinqed circles), LPS makes up the
Proteus outer membrane, an asymmetric bilayer that a/so contain5 protPint:
A-outer n1embrane protein A; PP-pore protein; DP-nutrient-
binding protein. Interior to the outer membrane lies the perip/asmlc
t(larP (PPS); the reptidoglycan /ayer (PG); and the cytoplasmic
membrane (CM) with carrier protcin (CP). (Rcproduced by permission
of Lugentberg B, Van Alphen L. Molecular architecture and function-
ing of the outer membrane of Escherichia coli and other gram-
F 1G U R E 7 . 1 . Anatomy of an enterobacterial ce// showing negative bacteria. Biochim Biophys Acta 1983;737:94.)
lv~d/i¿d(ion of ce// svrface antigens of Escherichia coli. Only one of
many peritrichous tlagella is shown. (Reproduced by perm1ssion of
B<irntim DA. et al íolihacíllo~i~. (IRA VPtPrinafY Monograph Series
19G7;2:8.)

Cytoplasmic
mi:tmhr;ine - - -----.,.-

Cell wall - - - - - --

- - - - K-Capsular

Fimbriae --<< \ ';-/ PPS

• - - - - -- - - H- Flagella

Flagellum -----------=::::::===~

Most utilize siderophores that remove iron from the iron- is thc abscncc of cytochrome c, rnakiI1g tl1c111 uxiLla~c­
bindi ng protei ns of the host. ne¿yitive.

Growth Characteristics Resistance

Members ot this group ot microorgarusms are facultatlvc
anaerobcs. 'fhey utilize a variety of simple substratcs for
growth. Under anaeroblc con<Jltluns, they are depeulleut
upon thc presence of fermPntahiP carhohydrate_ (Jndcr
aerobic conditions, the range of suitable substratcs in-
cludes organic acids, amino acids, and carbohydrates.
'rhe end products of sugar fcrmentation are useful it1
making a11 identjtjcation. Al111ost au mcn1bcrs of tl11s
group fermcnt glucose to pyruvic acid via thc En1bden-
Meyerhof pathway. Sorne, such as B. coli and Salmonclla,
produce succinic acid, acetic acid, formic acid, and ctha-
nul l!y way uf tlle r11ixed acid fer1ne11latio11 pathway.
OthC'rs, 'iuch ª"
Klcbsiel/a and E11terobacter, produce buta-
nediol from pyruvic acid, thcrcby rc<lucing thc rclativc
amounts 01 ac1d1c by-proaucts.
An important diagnostically useful biochemical charac-
terlstic of all the members of ttte fanilly EriLerubac.leriuc.eue
Sunlight, drying, pasteurizarlon, and the common dlsln-
fectants kill members of thc fan1ily Enterobacterif1Cef1e. In
u rubl, :-.l raLleLl e1 l vilu1u11eul:>, :>uLI 1 d:> J!<lslures, n1anurc, Ji t-
ter, and bedding, they can survive for many months.
Though many are susceptible to broad-spcctrum antimi
crobial agents, their susceptibility is not accurate!y pre-
dictable and can change rapidiy through acquisition of R
plasmids, or resistance-encodlng DNA cassettes (which
may ir1sert into numerous integrons J.ocaterl in thP
genou1e <:u1t1 iI1 µlas111 iu!)) (see Cha plcrs 4 and 5).
Variability
Variability of one isolate of enteric as compared to another
wlthin the samc spectes or geuu:. llcpc11Ll:; upuu lite ~e­
netic basis for the trait unrlPr considcration. Differences in
lhe capsular, son1atic, or flagellar antigens account for

_~zbility among isolates of the same genus and species.
'"' variation among members of tl1e same ge11us and
1:!2.t::> in tlJt: (¡uuily, as wt:ll <ts atuong u1t:utl.Jt:rs of <.liffer-
-...=:genera and species, is accounted for hy the presencc~ of
~-=:es residing on. plasmids encoding certain phenotypic
~:s. Such traits as resistance to antimicrobial agents, pro-
_......~on of toxin, or secretion of hen1olysin may be plas-
- ;:: encoded and will vary depend.ing on the presence or
~ce of a particular plasmid.
7ransiUou fn)111 lhe s111ooth to Ll1e rougli ¡>ltt:uotyi.11:: oc-
-·_s ~\'ith all members of the family. Likewise, change in
-- - O-a11tigens 11as been sho,.Yn to occur following ly.sog-
~- by certain bacteriophages (lysogenic conversion).
Susceptibility to various bacteriophages (phage typing)
:.ametimes useful in demonstrating differences in iso-
:res (strains) of the saine genus and species. Phage typing
~ useful epidenliological Lool.
-=..:>oratory Diagnosis
__-:.-!'.' farnily
is composed of a large number of related, facul -
=:_-iyely a11aerobic, oxidase-negative, nitrate reducing
=-am-negative rods. No clear divisions exist between the
=-~ogn ized genera. Differentiation within tl1e family is ac-
;nplished by a combination of cultural, biochemical,
~ serologic LesLs. A nurnber of r11a11uals <.leal exclusively
-.-b this family, and because of the extreme clinic.al i m -
-{l:tance and prevalen ce of these organisms, an increasing
r: .::".ll) er of programmed a11d/or co1nputerized identifica-
--:1 schemcs are commcrcially availnblc.
~·phology and Staining
-- are gram-ncgative rods. Ali look süniJar in the grarn-
it.:ained smear.
:~ tura! Characteristics
_!ethods used to isoJate enteric pathogens vary depending
-:>On ,.v hether the source of the sample is intestinal or ex-
-:alntestinal. When the source is extraintestinal, isolating
"ly of the family from normally sterile sites is significant.
_..,_ culLurt: u1t:<.liuu1 with wide aµµt:al is use<.l. The 1ne<.liurn
:or this purpose is an agar rnediurn c.ontaining red hloo<l
.:ells (usually sheep or cow). Incubation is at 35- 37ºC.
When the source is intestinal, two consistently patho-
;enic genera may occur in fecal samples: Saln1onella and
'-nigella. 1'11ough patllogenic strains of E. coli might be pres-
"'lt, there i.s no easy way to determine a pathogenic strain
:..on1 norn1ally occurring, 11011palhoge11ic slrains of E. coli.
.!\ll enteric media are devised to favor the identificatior1
of Salmonella, Shigclla, or both. Thc bases of the inedia are
3.S follovvs :
l. An inhibitory substance, usually a bile salt ora dye.
Thcsc substanccs inhibit gram-positi vc organisms
from grovving.
? . A substrate utilized or not by Salmonella, Shigella,
and by few others.
3. A pH indicator to tell if the substrate has been
cha11ged.
Chapter 7 Family Enterobacteríaceae 59
·rhe following are useful selective media for isolating
enteric pathogens.
MacConkey Agar.
lnhibitor: Bile salts and crystal violet (inl1ibil gra111-
positive microorganisms).
Substratc: Lnctosc- Salmonc/la, Shigella, and l)roteus do
not ferment lactose.
Neutral red: If lactose is fermented (acid), colonies will be
µink; if lactose is not fermented (peptides digested-
hasic.), c.olonif's will be colorless.
Usefulness: Very pern1issive 111ediun1. Salrnonella a11d
Shigella readily grow on this medium (as do most
othcr cntcrics and Pscudo1nonas).
Xylose Lysine Deoxycholate (XLD) Agar.
lnhibilor: Bile salts.
Substrate: 1) Xylosf'- not fermented by Shigella (Salmo-
nella ferments xylose). 2) Lysine-isolales ll1al íer-
ment xylose, but not Iactose and sucrose, and are
lysine decarboxylnsc-ncgativc (Proteus rnirabilis)
produce colonies that will be amber-orange. Salrno-
n.ella decarboxylates lysine. "J'he ratio of xylose to
lysine is such. that an alkaline pH predominates
(more <lecarhoxylation) . Shigella does not decar-
boxylate lysine. 3) Lactose and sucrose-Salr11011ellu
and Shigella do not ferment these sugars rapidly.
4) Ferric salt-colonics of organisms producing H 2S
(Salmonella; Proteus) Will have black centers (iron
sulfi.de).
Phenol red: Acid colonies (non -Salm.onella or non-
Shige.Tla) will be yellow. Alkaline colonies (possible
Salnzonella or Shigella) will be red.
Usefi¡/n.ess: An excellent all-purpose medium for both
Sahnoncl/a and Shigella.
Hektoen Enteric (HE) Agar.
ln.hibítor: Bile salts.
Suhstrate: Lactose and salicin- Salmonella, Shigella, and
son1e species of Pruleus a11<.l Pruvicli::.ru..:iu are lacto:;e-
negative and salicin-negative.
Ferric salt: Organisms producing H 2S will forro black-
ce11tered colonies.
Bromthyrnol bluc: Fcrmcntors of salicin and/or lactose
will form yellow to orange colonies; isolates not ter-
menting these sugars 1.¡ill forrn green or. blue-g reen
colo 11 i.t::;.
Usefi¡/ness: Excellent for Salmnnella. ancl Shigella .
Brilliant Green Agar.
Jnhibitor: Brilliant green dye (supprcsscs thc growth of
most members of the family except Salmonella).
Substrc1te: Lactose and sucrose- Salrnonella (and so1ne
:;traiu:; of Proteus) does not ferme11t these sugars.
Neither does Shigella, hut Shig1!lla grows poody (if at
ali) on t his medium.
J>fleriol red: If sugars are not fermented (alkaline),
colonies \·Vill be red; if sugars are fcrmcntcd (acid),
colonies •ViJI be yellow-green (dueto the color ot the
background dye).
Usefulness: Excelle11t for isolating Salmonella.
60 PART II Bacteria and Pungí
Enrichment Media. At times, the numbers of Salrnonella or
Shigella in fecal samples may be too low ( <104 /gm) to be <ie-
tecled 011 Ll1e prin1ary plating media discussed above.
'fhcrcforc, in addition to being plated directly to a selective
mediurn, thc fecal samplc is also placed in an cnrichmcnt
mcdiu1n. l'o detect Saln1onel/a by utilizing enrichment
methods, at least 100 sahnonellae/gm are needed.
l;or Saln1onella, enrichn1ent may be achieved by incu-
bating Ccccs for 12 to 18 hours in selenite F broth. During
Llli~
llu11;:, l lrt:: g1owlh of 01 ganisn1s oLher Ll1a11 Sal111011ella is
suppressed, whereas growth of Saln1011ella is not. After the
12 to 18 hours have elapsed, an aliquot of thc broth is
streaked onto a plate of selective medium (e.g., brilliant
green agar).
E111 icl1111eul fo1 Shigella is nol easy becausc it is rather
sensitive to commonly used inhibitory substances found
in selenite f and tetrathionatc brotl1s. An cnrichmcnt
broth called gram-negative (or <.JN broth) is used in the same
manner as selenite is used for Sa lmonel/a.
Men1bers ofthe genus Pseudomonas (especially P. acrug-
inosa) inay b e found in feces, but tl1is is probably an in-
~ig1Jiíicanl fi11di11g. Pseuúorr1or1us (11ol a .u1en1ber of the
fam ily Enterobacteriaceae) will grow on enteric m edia. This
microorganism does very littlc to thc substratcs othcr than
the peptides and peptones and thus mimics Saln1onella
and Shigella on selectivc media. Pseudomonas is oxidasc-
posítive, a useful distinction.
 Enterobacteriaceae:
Escherichia
DWIGHT C. HIRSH
The genus Escherichia, a i11en1ber o! tl1c fan1ily Enlerubutle-
riaceae is composed of severa! species, but only E. coli is an
important pathogen of anirnals. 'fhis species, the major fac-
ultative gram -negative spccies comprising the normal flora
of the gastrointestinal tract, is thc cause of septicemic dis
ca~t: in foals, calves, piglets, pupplcs, and lambs; of diarrhca
in nc>Wh()rn f::irm ::inim::ll<;; ::lnrl nf PrlPm::i rli<;P::l<;P in pigs. It
rnay also be opportunistic in almost ali animal species (e.g.,
in urinary tract disease. abscesses, and pneumonia).
Descriptive Features
Cellular Anatomy and Composition
The anatomy of the n1emJ)ers of the genus Escherichia is
typical for the family J:..nterobacterit1ceae. They may possess
capsules (K antigens), 11agella (H a 11lige11s), or auhe:siu:s
fimbria or pili), and ali possess a typical gram-negative
cell wall composed of lipopolysaccharides (0-antigen) and
proteins.
Cellular Products of Medica! lnterest
Adhesins. Adhesins (also knovvn as fin1bria or pili) are pro-
tcins that mediate adherence to target cells in the gastroin-
testinal tract and to cells comprising the niche for the
strain. Bccause of their relative hyd rophobicity, adhesins
111ay abo ¡.iro1note i:l$~U<.:iatiou wlth the memhrane of
phagocytic cells. Adhesins are imp()rt::int vi r11lPnce f::ictors
\\'hen the microbe is <>n mucosal surfaces. Escherichia cofi
produces many different types of adhcsin, most being
!inkcd with strains associated with a spccifíc discasc.
..\lmost ali of thc adhesins promote adherence to glycopro-
teins on thc surface of epithelial cells of the intestinal
úaL l:
1. F4, FS, F6, F41. 'l'he fimbria! adhP.sins F4 (f()rmPrl y
known as K88), FS (K99), f.6 (987P), F17, and F41 are
uscd by cnterotoxigenic E. coli to adhcre to target
celIs in the sn1all intestine. F1, FS, and F6 are usunlly
plasrnicl-encoded, while f4 1 is chromosomal.
2. F17. '!'he protein Fl7 (llke CS31A) is a plasmid-
cncoded adhesi11 re:.µ011:.iulc for atlherence o.f sep-
ticemic ("invasive") strains of /~. cnli to their small
intestine target cells.
3 . CS31.I\. The protcl11 CS31A (like Fl 7) is a plasrnid- r
cncoded adhesin respon.~ihlP. f()r ::icihf'rence of scp· r
ticcmic ("inv asive") strains of E. coli to the sn1all in -
testi ne target ce lis.
1. AAF. The adhcsin AAF (aggrcgative adherence fim -
hriae) is responsible for the adhercnce of enteroag-
gregative E. coli to their small in testinal epithelial
cell Largets.
S. Bfp. '!'he Bfp adhesin (for h11ncilP forn1ingpilt1s, dt1e
to its propensity to tangle togcther and form "bun-
dles" when viewed under thc clectron microscope)
is responsible for the adherence of cntcropatho-
genic E. coli to their small intestinal epithelial cell
targets.
6. Curli. Curli are auhesins that promote adherence to
extrace.Jlular matrix pr()tf>in~
7. On1pA. Omp...\ (o utermembrane protein A) is a pula·
tive adhesin used by enterohemorragic E.coli in ad-
herence to large intestinal cpithclial ccll targcts. )
Capsule. Capsular polysacchar1dcs (K-antigens) are im-
portant for those microorganisms (such as invasive strains
of E. cofi) that come in contact l"llth elements of the in na te
imm11nf> ~y~tf'm of the host. Capsula r substances protect
thc oute r membrane fron1 the n1c111brane allack con1ple.x.
o! thc complement cascade, and inhibit tl1e microbe from
attach mcnt to, and ingcstion by, phagocytic host cells.
The capsule is thought to endow a degrec ot hydrophilic-
ity relative to the membrane of phagocytic cells. Most cap
sules are ncgatlvely charged, as are thc membranes of
ph::lgocytic cells.
Ceff Waff. Th.e cell wall of lh~ 111e1111Jer:s uf tlli:s gl:!nu:. i:.
one typical of gram negatives. The lipopolysaccharicic>
(LPS) in the outer membrane is an important viculenc.e de-
terminan t. Not only is thc lipid A co1nponent toxic (endo-
toxin), but the lengt h of thc ~idc c hoin in thc 0-rcpcot
unlt hlnders the attachment of the membrane attack co1n-
plcx of the complement systcm to the o uter membrane.
LPS binds Lo lipopoly$a<.:<.:l1ariue-1Jlntling protein (a seru1n
protci n), which in turn transfrrs it t() thf> hlood phasc of
C:Ol4. Thc CD14-LPS complex binds to 'loll-like receptor
proteins (see Chapter 2) on the surfacc of macrophage cells
triggering the relea se of proinflam matory cytokincs
Enterotoxins. Enterotoxins are usually plasmid-encoded
proteins. Escherichia co/i produces at least three: labilc
Loxi11 (LT), :staule toxin (ST), anti Enteroaggregative E. cofi
61
62 PART 11 BacLeria a11d Fu11gi
heat-stable enterotoxin l (EAST l ). ·rhese protein exotoxins
affect the control of cyclic nucleotide activity within ~he
"targ~" <.:ell, vvllicl1 results i11 <.leregulatiu11 uf \vater a11d
e\Prtrnlytf' sPrrPt1nn hy thf' afff'rtf'<i hnst re\\:
l. Labile toxi11 (L'f). L1' affccts the ade11ylyl cyclase sys-
tcm. LT is composcd of two subun its, A and B. Th e B
subunit is a 1nultiincr that b1nds to gangliosides on
the surface of the intestinal host cell, followed by
translocatlon of the A subunlt across the host cell
membrane. Thc A subunit, ;1fter artivation, rlt><ivt>s
nicoli11an1ide from nicotinamide adenine dinu-
cleotide (NAD) and then couples the remaining ri-
bosyl adeninc diphosphatc onto thc e rcgulatory
protein ol the adenylyl cyclase enzyme system. The
result is deregulation of adenylyl cyclase, causing
overproductio11 of cyclic AMP (cAMP) followed by
opcni11g of chloride channels in crypt cells (sn -
<.:alleu 1.:yslic fibtosis lransn1cn1bra11e con ductance
regulator chloride channels) and the blockage of
NaCI absorption in apical tip cclls. As a consc-
quence, water and electrolytcs (chloride, sodium,
and bicarbonate ions) are Iost into the intestinal
lumen. These events lead to díarrhea, hypovolemia,
metabolic acidosis, and, if the acidosis is severe, hy-
perkalemia. Tt1ere are twu ::.ervlugi<.:ally ui::.Li11cL 5ub-
classes of LT. LTI is plasmid-encoded and neutral-
ized by anticholcra toxin antibodics; LTII is ncithcr.
Lfl has been isolated lrotn t:. coli affecting h umans
(L'l'h-1) a11d swine (LTp-1). LTIJ has been isolated
fro1n cattle, water buffalo, hurnans, and food.
2. Stable toxin (ST). Tl1ere are n~·o kinds of ST: STa and
STu. The genes encoding STa are lucated un a tra11::.-
pos;ihlt> t>lt>mi>nt. Tht> gPnf's for s·rh are not. STa
causes fluid accumulation in the intestincs of suck-
ling mice and piglets; STh causes fluid accumulation
onJy in piglets and weaned pigs. The toxins are not
rclated antigenically. STa affects the guanylyl cyclase
system by deregulating cyclic GMP (cGMP) synthe-
sls, resulting in flui d and electrolyte accurnulation iu
thf' hnwt>l l11mi>n snhSP<]11rnt 1·0 hlockage of sndium
and chlorid e ion (and thus water) absorption (tip
cel!s) a11d Joss of chloride ions (crypt cells). 1'he re-
ceptor for STa is a 1nembrane bound guanylyl cy-
clase. This receptor, when bound, results in the syn-
thesis of cGMP. Increase in intracellular cGMP leads
to the opening of chloride chaiu1els witll tl.te r~ull­
a nt flow of chlori<lf' ancl watt>r into the intestinal
lumen. The STa receptor is normally the target for
guanylin (a 15 amino acid paracrine regulator),
which is produced by goblet cells. Guanylin appcars
responsible for hydration of mucus that is also pro-
duced by goblet cells. STa and guanylin have com-
mon C-terrnini. S'f l> l>iu<.ls lo a sulfal.ide cecepLor
followt>d hy syntht>sis of rt\MP activating a G regula-
tory protein. This leads toan increase in intracellular
calcium concentration, which in turn activates pro-
tein kinase C. Protein kinase C phosphorylates pro-
teins composing the chloride channels resulting in
loss of chloride and water into the intestinal lumen,
as well as the pt10::.pt1orylatiu11 uf tite 111e111b1ane-
associated ion transport proteins resulting in block-
age of absorption of NaCl. In wild strains of entero-
tuxige11i<.: E. c:uli, STa i:> 111tn e co1nn1ot1ly found.
~- F.nteroaggregative E. coli heat-stable enterotoxin 1
(EASTl) is an en t erotoxin similar in actio n to STa by
being functionally similar to guan ylin.
Other F:nteríc Tox íns. Escherichia coli produces oth er pro-
teins that affect t he cells of the intestinal tract . Though
these products have "enterotoxic" activity, the term "en-
terotoxin" is reserved for LT, ST, and EASTl as discussed
above. ·rhe other enteric toxlns lnclude shiga and sh1ga-
like toxins, cytotoxic necrotizing factor, a plasmid en-
coued tuxi11, ar1<.l a cyLoleLhal-disLending toxin:
l. St:r (for Shiga and shlga-/ike coxin). SLTs (also
known as Vero tissue culture toxins because of their
characteri:;tic cffe<.: L::. 011 V1::10 cells) are p.rotein tox-
ins similar to shiga toxin prod uced by Shigella (see
Chapter 11). These toxins are composcd of an A sub-
u n it and a B subunit. ·rhe ~ subunit is responsiblc
for binding of the toxin to endothelial cclls. The A
subunit inhibits protein synthesis of the target cell
(an endothelial cell) following interaction with the
60S ribosomal subunit. Tl1erc are lwo lypes of shlga-
like to.xins, ST:f-Tancl s1 ;r.11. Sl.T-T is neutralized by
antibody specific for the shjga toxin produced by
Shigella spp.; SL'f-11 is not. SI.;f-1 is p robably ictentical
to sh iga toxin, whereas SLT-IJ is a variant. A family
of b acterioph ages has been shown to encode the
sl1iga an d shiga-like toxins. A varian t of SLT-TI,
called SLT-Ile, is respons!ble for the vascular da mage
characteristic of edema disease of swine. The genes
e11<.:ulli11g SLT-Ile do 11ol appea1 lo be bacteriophage-
assodated.
2. CNF (for cytotoxic necrotizing factor). CNFs are pro-
teins that interact with epithelial cell small G'l l'-
binding protein Rho, resulting in membrane "ruf-
fles." There are rwo types of CNF, CNFl and CNF2,
which are im munologically related and simila r in
si;(.e. The gt:11e e11coding CNfl is located on the
chromosome in a Pathogcnicity Isl.and (a clu ster of
genes encoding v írulcncc dctcrminantfsl, an inte-
grase protein , a specitic insertion site, and mobil-
ity). The Pathogenicity lsland that contains the
gene encoding CNl;l al so contain:i tl1e genes c11<.:u<.l-
ing a nun1ber of othcr rhrnmosomally encodcd vir-
ulence lrails, e.g., hcmolysin, serum resistance, and
the adherence protcin Pap, needed by sorne strains
of E. coli to adhcrc to urinary troct epithelium an
tecedent to urinary tract drsease. The gene encoding
C:NF2 is plasmid-based.
3. Pet (for pJasmtd-encodeu tu.x.h1). Pct i:s <t :seri11e .fJlO-
tease enteri.c-arting toxin that affects the cellular
cytoskeleton of affected intestinal epithelial cells,
resulting in d am age to the cell a nd stimulatin g an
inflammatory response. Diarrhea is thou ght to re
sult trom prostaglandin synthesis by the recruited
PMNs and affected epithelial ceUs, as well as activ;:i-
tiu11 uf variou::. i11osilol-signaling pathways within
affected host cells. ThP nPt rcsult is the secretion of
ch.loridc ions and water.

-l. Cdt (for cytolethal áistending toxin). (~dts are a
tam-
ily of related toxins that affect the mammalian cell
cycle. A role in pathogenlclty has not been pro-
vidcd.
Hernolysins. Escherichia coli prodl.lCe at least threc he
--:olyslns: alpha hemolysin, enterotiemolysin (Ehx for en-
,.rol1emorragic E. coli to.xin), and cytolysin A (Cly):
l.Alpha hemolysin. Alpha hemolysin (Hly) as well as
enterohemolysin (scc bclow) belongs to the R·rx
(rPpPats in toxin, so called because of the common
fearure of repeats in glycine-rich seque11ces willlli1
the protein) family of toxins (see also Pasteurella!
Mannhcimia lcukotoxin in Chaptcr 12, Actinobacil-
lus haemolysin in Chapter 13, adenylyl cyclase
tox.in of Bordetella in Chaptcr 15, lvforaxel/a cyto-
tuxin in Chapter 19). r-ny ts secretcd by many viru-
lent stra ins of Ji. r.nli. l .oss or g;.iin of hly by genetic
manipulatio11 produces corresponding chan.ges in
virulence of E. coli strains. Hly damages cell mem-
branes, and is detcctcd by growth in vitro on a
med1um conta1n1ng red blood cells.
2. Enterohemolysin. Enterohcmolysin (Ehx, for en-
tt:roheruorragic E. culi toxln) as well as Hly (see
ahove) helongs to thP n'f'X (rPpP;.its in toxin, so
callcd bccause of the common fearure of repeals in
glycinc-rich seque11ces within the protein) fam ily of
toxins (sce nlso Pastcurclla/Mannhcimia lcukotoxin
in Chapter 12, Actinobacillus haemolysin in Chaptcr
13, adenylate cyclase toxin of Bordctella in Chapter
15, Moraxella cytotoxin In Chapter 19). Evidence
suggests that Ehx may be the result of a defect in the
secretion system for Hly. Ehx is secreted by many
virulent strains of E. coli (especially those that pro-
duce SLT, see above) and is detected on media that
contains red blood cells and added calcium.
3. Cytolysin A. The gene cncoding cytolysin A com-
monly occurs among strains of E. coli. Expression of
cytolysin .A. (Cly) occurs following infection of the
t.>acterial cells with the temperate bacteriophages
F.hyl ancl 2 (inc.orrPrtly tPrmrcl "f.ntProht>mol)ISins
1 and 2").
lrun llcquisiliun. lron is an alJsolute growth requirement
r most, if not ali, living things. Sic1Prophores (e.e., aer-
bacti n) that remove iron from host iron-binding proteins
.-re necessary if a microbc is to have invasive capabilities.
',.scellaneous Products. M1scellaneous ccllular products in-
._;udc the following:
l. Acid tolerance. RNA polymcrasc containing RpoS
(the sigma factor associatcd with stationary phase)
preferentially transcribes genes responsible for acid
tolerance (survival at pfi, 5), allowing safe transit
through the stomarh .
2. lntimin. lntimin is a protein encoded by eae (for E.
coli attaching lffacing) locatcd within a Pathogeni-
city lsland (a cluster of genes cncoding virl.llcncc dc-
terminant(s), an integrase protcin, a specific inser-
tion site, and mobility) callcd LEE {locus effacing E.
culi) . Intimin anaches to another secreted bacteria!
Clzapter 8 Enterobacteriaceae: Escllerichía 63
protein, 1'ir (translocated intimin receptor) inserted
into the host cell membrane.
3 . Type III secretion system. ·rhe genes encod1ng a
Type ITI secretion system (an assemblage of pro-
Lei11~-1uore th<1u 20-thdt form a hollow tube-Ul<e
struc.turP. th ro11gh whirh pffrrtor proteins are "Jn-
jected" into host "target" cells) also located within
LEE (see above).
4. Esp. The genes encoding Esp (for EPEC signnling
protein, also located \\'ithin LEE, see above) act1vatcs
a tyrosine phosphokinase in the affected cell result-
i11g i11 <.:ytoskclet<1l rearrangements leading to "col-
Japse" of thC' mirrovilli (PffarPmPnt). Diarrhea oc-
curs secondary to increases in intracellular calciun1
ions and activation of protein kinase C. Protein ki-
nase C is responsible for phosphorylation of pro-
teins composing the chior1de channels resulting in
loss of chloride and water into the intestinal lu1nen,
as well as ll 1e ¡.¡l 1os¡.¡horyl<1tlon of the membrane as-
sodated ion transport protPins resultine i11 block-
age of absorption of NaCI.
Variability
The variability of E. coli resides in the antigcnic makcup of
the 0-repeat units (type of sugar subun1ts, how the sub-
units are hooked togcthcr, and the length of the chain),
Lhe con1posi Lio11 uf Ll1c fl<1gt:ll<1r protein (flagellin), and the
composition of the capsule~. ·rhrr<' arP at lPast 80 distinct K-
antigens, approximatcly 165 serologically distinct 0-
groups, and at least 50 scrologically different tlagellar (H)
antigcns. 'fhc 0 -, H-, and K-antigcns are used in serotyping
a particular isolate. ror example, 0141:K85:H3 describes
an isolate with antigens of the 141 scrogroup, capsular
<1utigeu numt>er 85, and flagellar antlgen number 3.
Ecology
Reservoir and Transmission
Strains of E. coli capablc of producing clisease reside in the
Jower gastrointestinal tract and are abundant in environ-
ments inhabited by aillinals. ·rransmission is throl.lgh the
fecal-oral route.
Pathogenesis
Mecl1c.1r1isrr1s unú Diseasc: Pullc:rns. It is very diffic.ult for most
strains of E coli to produce <liseasP hrrausP thPy clo n()t
havc the ne.ce.ssary genes encoding thc proteins needed to
do so. lt the genes are acquired (by transduction, conjuga-
tio11, or transformation), the nonpathogcnic strain rnay be
changed to one with pathogen1c potential. -i he type of dis-
ease produced would depend upon the genes acquired.
Er1tc10Loxi¿;enic: Diurrheu. ·rhis disease occurs In neonatal
pigs, calves, and lamhs, ancl in wPanling pigs. Jt has been
reported in dogs and horscs.
Enterotoxigenic diarrhea is caused by strains of E. coli
that produce adhesins that promotc attachmcnt to glyco-
proteins on the surface of cpithelial cells of the jejunum
64 PART ll Bacteria and Fungi
and llcum, anct an cnterotoxin(s) that affects the epirhelial
cell (to which the enterotoxigenic strain of E. coli is ad-
!icrctl), rc=>ul Li11g i11 Jlwtl :-.eLrcliu11 a11d dia11hea. Bolh
traits are necessary for disease to result, since unless the in-
gested strain adheres to these cells, peristalsis will move it
into the large boweL 1'he cells ot thc jcjunurn anct t11c
ileum are susceptible to the action of enterotoxin; tl1e cells
of the large bowel are not.
At least four adhesins may be found on enterotoxigenic
E. coli, F4, F5, f6, arH1 f41. 'fhey µu:.:.e:.:. ~uurc lru~l ~µct.:ic~
spt>cificity: F4 anct F6 are almost always associated •vith iso-
lates frorn swine; fS \Vith isolates from cattle, sheep, and
swine; and f41 with thosc from cattlc. The epithelial cell
rcccptors for thcsc adhcsins rcgulatc the age incidence of
this disease as well. ln calves anct lambs, the receptors
appear transiently during thc first wcek or so of life.
Analogous receptors are presenr In plgs throughout the
first six wf>Pks of life. There are rnany uncharacterized ad-
hesins that probably play a role.
Aside from the adhesins outlined above, some entero-
toxigenic strains of E. coli cxprcss curli. So, in addition to
adhercncc to g!ycoproteins on the surface of ep1thelial
celis, somc strains adhere to extracellular matrix protcins.
-r11c prcscnce of curli may explain the mercase ln the win-
dow of agc susccptibility to enterotoxigenic disease in ani-
1nal:. cuut.:urreully i11feclcd wilh iotavirus or crypto-
sporictia, two agents that may cause enough tissue darnage
to lcad to exposure of extracellular matrix protcins.
Tn addition to ad11erence to the target tissues ot the
small intestine, enterotoxigenic strains inust have the ge-
net1c capability of synthesizing enterotoxin. Strains pro-
ducing only s·r are the most common, followed by those
secretlng uotll S'l' arH.l L1~ autl tllc11 uy lllU=>C :,ccrclil1g LT
only.
Sorne of the adhesins and enterotoxins are encoded on
plasmid DNA. As a consequence, it is ditticult to predict
which strain of E. coli posscsscs thc gcnetic information
necessary to produce disease. Sorne adhcsins prefcr to be
associatcd with ccrtau1 serotypes. In particular, the genes
encotling the µruteiI1 fur the F4 l al.llic:.i 11 are al1110::,t ah-vay~
fo11nct vvithin strains of F.. coli of the 09 and the ()101
scrogroup:;. A:; m ight be cxpcctcd, thc genes cncoding thc
proteins tor f41 adhesi11 are locatcd on chron1oson1al
DNA.
Followlng lngestlon by the host, cnterotoxigenic strains
of F.. coli adhere to target cells, 1nulli ply, and secrete entero-
toxin. Fluid anti elettrulyte~ at.:t.:u111uldlc i11 lhe luu1en of
thP inti><;tinP, rpsulting in ctiarrhea, dehydration, and elec-
trolyte imbalances. In time, thc infecting strain is moved
distally away from the target cell and the disease process
stops, due probably in part to thc ccssation of expression
of the adhesin along with a decrease In available substrate
following the aln1ost explosivc multiplication of the strain
in the sn1all iutesti111::. Uule:-:, sttµ:. ate laken Lo correcl the
fl11id ;:ind PIPrtrolyte imhalanccs, the disease has high
rnortality.
"I"he diarrhea produced is watery and nonbloody. There
are mini mal, if any inflammatory changes in thc small in-
tcstine. Bacteria will be observed histologically coating the
villi of thc rnid to distal portions of the small intestine.
Entcruaggregalive Diarrheul Di:>eu:>c. Et 1lc1oa~g1egalive
stralns of E. coli (EAggEC) assoclated wlth this ctisease are
isolated from weaned pigs and calves with di;irrhi>a .
.EAgg.EC adhere lo cells li11ü1g lhc s1nall intestine by way of
thc AAr adhesin. l~ollowing adhesion, EAggEC secrete a
prot-cin (cncodt~d by a gene tcrmcd agg for aggrcgation)
that promotes the fom1ation of a sheet of microorganisms
strongly adherent one to another (so1nc have referred to
Thls as a "biofilm"), thus the descrlprlon, "enteroaggregél-
tive." EAggEC produce EAS'fl and Pet, both pott>ntial
Lauses of diarrhea.
'fhe diarrhea is usually watery (though blood and
leukocytes may be obscrvcd in sorne cases). Histologically,
sheets ot bacteria (entrapped in mucus) will be seen cover-
ing the small intestinal epithelia.
lnvasíve Dísease. Associatlon of susceptible animals
(usually a neonate that has received inadequatf' i1rno11nts
uf t.:ulu~lrur11 vr coloslrun1 of inadcquate quality) with in-
vasive strains of E. coli may occur by way of the conjuncti-
vuc, inadcquatcly treatcd un1bilicus, or ingestion. If the in-
vasive strain associatcs via ingestion, it first adheres to
target cells in the distal small bowel. Adherence is probably
related to expression of any number of acthesins, L>ut
c:S31A is one that is comrnonly associated with inv;isivP F..
coli. Likewi~e, tlre adhc=>i11 Fl 7 01iginally described on a
pl:i<;micl tPrrnPct Vir (so-called hecause of its association
with virulent or invasivc E. coli) is prevalent on invasivc E.
coli. following adherence. invasive strains "induce" their
own uptakc by cxprcssion of either CNfl or CNF2, result-
ing in the forn1ation of "ruffies" that entrap adhering bac-
teria and "pull" them into the intestinal epithelial ccll.
Entry into the lymphatics and subscquently the L>lootl-
stream follovvs. Extensive multiplication within thP intPs-
Linal epill1elial cell probably docs not occur. The mecha-
nism by which the invasive strain gains access to
lyrnphatics aftcr uptakc by thc cpithclial ccll is unknown.
l.ikewise, the mechanism of entry into lymphatics after as-
sociation with conjunctivae or thc un1bilicus is unknown.
Once the epithelial surface is traversect, expression of ad-
hesins is repressed (otl1erwisc adhesi n-expressing hartPria
could adhe1e Lo l1osl pl1agocylic cclls with disastrous con-
sequences for the bacteriu1n).
Thc infccting strain 1nulti plic~ in thc ly1nphatics and
blooclstream ano endotoxcmla develops (Fig 8.1). Death of
the host occurs if antibacterial therapy, the iinmune sys-
tc111, ur !Jull1 Ju uuLrcJ1tOVt: Lhe n1icroorga11ism.
lnvasive sttains h;ivP <;pPci:il qualities, e.g., they n1ust
escape pl1agocytosis, cornplernent-mcdiated lysis, and
havc a mcchanism to acquire iron. Capsule and various
outcr mcrnbranc protcins confer rcsistance to comple
ment-mediated lysis (serum res1stance). How capsules pro-
tect tl1e outcr mcmbrane frorn insertion of the membrane
attack complex is not k.r1U\'Vll. Ccrtai11 t.:apsules (such as
Kl) ilrP rhPm ir;:illy simih1r to the surface of host cells in
that t h cy are composed mainly of sialic acid. Con1plemcnt
components associating with surfaces composed ot sialic
acid are shunted to degradative pathways rather than am-
plif1cation and formation of membrane attack complexcs.
Escape from phagocytosis is also related to c;ipsult> anct
certaln outer membrane prutci11::.. Huw ouler n1embrane
proteins function as antiphagoc:ytic factors is not known.
Thc genes encoding the adhesin (e.g., CS31A, F17) and

F1G U RE 8 . 1 . Cascade of biologically active m ediat ors
following interaction o f lipopolysaccharide (LPS) with thc bo dy.
W/ie11 gt d111-negative 1nicroorganisms grow In the body, LPS is
released (no t on purpose, but when a bacteria/ ce// makes LPS, sorne
of ít escapes). Jf LPS is in the b/o odstream (endotoxemia), then a
generalized cytokine "storm" p lus activation of sanie o ( (/11:: e11¿y111e
cascades (comp lem ent is one) results in intravascular clotting, an d
increased vascular permeahility, whirh /p;uf( tn rlecreased organ
pcrfusion and mu/tiple organ failure, and very serious pH problems.
Thís Nstate* is cal/ed *septic shock. N /he same occurs 1n a limited
area if LPS is liberated "locally" (as might occur when a gram-
negative bacteria grows in tissuc). (•JL 8 vttracts PMNs, and MCAF
{111dl1uµlidy1:: lf11::111utdllil fdttor] atuacrs macrophages.)
lncrPa~P va~nrlar
1 SomnogeniL ¡. -_,,.
Br....,
ai,....
n- @U- -.,,Co-m- p.,..le-m-en"""t- . permeability {attract
inflammatory cells)
~ 1LP5 bindíng protein
1Macrophage 1
*
f TNF ll-1 ll-6 1
'. In crease vascular permeability- arachidonic acid pathway (TNF on endothelium)
l lncrease intravascular clotting- ara(hidnnir arid rathway {TNF on endothelium)
1 Attract inflammatory cells- (TNF/ll-1 induce endothelium to produce IL-8 and MCAF)*
4. Release of acure phase proteins (liver) (IL-6)
5. Endogenous pyrogen (TNí, IL-1, IL-6) (brain)
6. Somnogenic (TNF, IL-1 )(brain)
th ose responsible for siderophore production reside on
plasmids. As mentioned above, the genes encoding fl7
h ave been associated with the plasmid Vir, as has t he gene
encodlng CNF2; rhose responsiblc for siderophore produc-
•ion havP hPPn as~ociated with thP. plasmid pCoJV. In the
,attcr instance, t h e siderophore genes aJe linked closely
-,,ith t he gen es for the pro duction of colicine V. ·rhe
siderophore, aerobact in, has a high affinity for iron.
Many of the strains with invasive capabi lity, except
~hose from foals, p roduce a hcmolysin (Hly) an d are he
.11ulytic un bloou agar.
Histopathologically thf'rf> are inflammatory changes in
;J\·er, spleen, joints, and meninges. There n1ay be he111ur-
:hages on pericardium, peritoneal surfac.es, an<l a<lrf'nal
corticcs.
Nonenterotoxi~enic JJiarrheas. Enteropathogenic strains
o f R. colí (EPEC) produce diarrhca in ali animal species, in
cluuiug l1un1an !Jeings. EPEC do not produce ST, LT, or any
other d ia rrhea assoc.iatf'd toxin . Thcy do, h owever, p ro-
duce a characteristíc lesion in the in teslinal lfacl ll 1al i:> d e-
scribed asan attaching and effacing lesion. The characteris-
tic lesion occurs because of thc "collapsc" of the microvilli
of the affccted cell giving the hlstopathologic appearance
of "effacement" (Fig 8.2). The location in the tract is the
distal :>111all i11testine, and upper large.
EPEC: have a nnmhi>r of attributes that are involved in
pathogenesis. 'fhe first is the produclion of Lhe adhesin
Chapter 8 Enterobacteriaceae: t,scherichia 65
F 1G U RE 8. l . EfPrtron photomicrograph of an enteropatho-
genic strain of Escherichia coli (CPCC) showing (a) b undle f orming
plll, (b) the arraching and effacing les1on produced by this strain,
and (e) a d iagramma tic depiction of the genpr;itinn nf thi~ IP~ion
See t ext for expl<>notion of Tir Jnd intimin. WASf': Wiskett-Aldrich
syndrome protein; Arp213: nuc/eat/on s/te protein. (Reproduced
with permission of Donnenberg MS, Whittam TS. Pathogenesis and
evolution of virulence in enteropathogenic and enterohemorr<>gic
Escherichia coli. Journal of Cli11ildl /11ve)liyation 2001;107:539.)
a b
· nr
~
-~40
1 3 4
Bfp. Bfp is responsible for "targeting" the particulaI ü1les-
tinal epithelial cell that will become involved in the
proccss. After association with an intestinal epithelial cell,
a more intimate attachment occurs hy way of intinlin,
which binds to the protein Tir that har. heen insertcd into
LI 1c "t<trgete<.l" cell. Esp protel os are produ ced and are "in-
jected" in to thf' targf't cell by way of t he 1'ype IIT secretio11
system . Th e Esp proteins produce tl1e effacernen L 1 1::~iu11,
and diarrhea. Many EPEC also produce enterohemolysin
(Ehx). It is unclcar what role Ehx plays in enteropatho-
genic disease.
Sorne attaching and effacing strains of E. coli are lysoge-
ni:t.ed with the bacterlophage(s) that cncode the shiga-ltke
toxins SLT-I and/ or SLT-11. These strains are termed entero-
ller11oriagic E. e,uli (EHEC) IJe<.:<tuse, In addition to producing
attaching and effacing lesion .~, thf'y alc;o p rnrl11rP hPmnr-
rhagic dia rrhca. Howcvcr, the target cells are those of the
large intestine. 'J'hu s, the Btp adhesin is not involved, and
evidence strongly suggests that it is Ompi\. 1'h c prototypc
EHEC ts a strain of E. coli of the serotype o l57:H'/ that pro-
ci11rf's disease in human beings, and calves given the strain
experimentally. Followiu~ altacl 11nent (the large intestlne
via OmpA), an attaching and effac.ing lf'sion is produced
(thc intirnin produced by EIIEC strains is slightly dilfeienl
from that produced by EPEC, reflecting the different target
cell), and SLT is produced. The SLTs affcct cndothelial cells,
leadlng to rheir injury and Ioss of integrity. ·rhe ettects of
66 PART 11 Bacteria and Fungí
.SL'l's are local, i.e., the endothelial cell under the cell to
which EHEC is attacl1ed, and systemic, i.e., the endothe-
lial cells elsewhere in the body but mainly in the kidney
and brain. 'fhe local effect is hemorrhage. ·rhe systemic ef-
fecls or SLT, al lea:.t i11 l 1ur n<111:s, rt::sult iu a :sy11<.lrurne calle<.l
the hern0/)1tic uremic syndron1e (HlJS), characterized hy 1ni-
croangiopathic hemolytíc anemia, glomerulonephritis,
and thrornbocytopenia. How SLT is absorbed locally or
systeru.ically is not understood. HUS does not appear to be
a significant sequela of EHEC-based disease in nonhuman
animals. Approximately 5% to 10% of human patients af-
fected with EHEC (almost all are 0157:1-I?) will develop
rllJS. Virtually all strains of 0157:H7 procluce F.hx.
All affected anin1als acquire EPEC:/El-IEC by way of the
oral route. Tt is not clear whether EPEC have zoonotic po-
tcntial, but anin1als (including humans) probably acquirc
the infecting strain by the fecal-oral route. Strain 01.)/:H/
is a part of the normal flora of nonhuman animals, espe-
cially bovines. Huma11 bei11gs become lnfected folloWing
ingestion of contan1inated food, mainly beef. At slaugh.ter,
the surface of Ll1e carcass becon1es co11tan1inated with
fecal microorganisms. 'fhe surfaces of cuts of meat derived
fro1n an infected carcass are readily sterilized by cooking.
When the meat is ground, the n1icroorganis1ns on the sur-
face become mixed throughout. Though improper cook-
ing will readily kill surface microorganisms, including
0157:H7, thosc insldc may not be killed.
Dia.rrhea a:ssucialed will1 EPEC will be walery, usually
without blood and inflarnmatory cells. 111e characteristic
histopat.bologic les ion .i s an "attaching and effacing" one af-
fecting cells of the small intestine. Attachment is localized.
Diarrhea associated with EHEC will be hemorrhagic. The
characteristic histopathologic Jesion will also be an "attach-
ing and effacu1g" one, but affecting the large intestine.
Edernu Di::;eu::;e. E<.leu1a di:sea:se is ar1 acute, often fatal
"enterotoxemia" of weaned pigs. The disease is character-
ized by subcutaneous and subserosal edema, caused by ab-
sorption of SI.:f-IIe produced by certain serotypes of E. coli
(e.g., 0111 :K85, 0138:K81, and 0 139:K82). The toxin at-
taches to and affects enclothelial cells throughout the pig,
resulting in extensive edema. 'l'he toxigenic strains inhabit
tlI<:: large l!ow<::l uf r1uru1al pigs, anu tl1e:se strains are
thought to increase in numhers during nutritional, social,
or physical stress.
·r11e typical lesion is generalized edema of various or-
gans and tissues (e.g., head, neck, colon, stomach, intes
tine, brain).
Colibaci/losis of Fo1vl. Colibacillosis of fowl is an eco-
nornically irnportant disease caused !Jy invasive strains of
F:. coli. 1..he disease takes many fo rms in fowl, depending
upon the age of the host and mode of infection.
'fhe egg surface can be contaminatect vvith potentially
pathogenic strains at the time oflaying. 'fhe bacteria pen
etrate the st1ell and infect the yolk sac. lf the l)acterta grow,
the embryo dies, usually late in incubation. Embryos that
survive rnay die shortly after, with losses occurring as late
as:~ wf'f'ks aftf'r hatching.
fowl may also be infected by the respiratory tract and
develop respiratory or septicemic disease. The course may
be rapidly fatal or chronic, inanifested by debilitation, di
arrhea, and respiratory distress.
Other clinical syndromes seemingly caused by E. coli in-
clude cellulitis, synovitis, pericarditis, salpü1gitis, and
panophthalmitis.
The E. coli responsible for this disease have been shown
to pos:se:s:s sorne of tl1e sarne virulence <.leter111i11a11ts as
those isolatecl from mamn1als, most notahly adhesins,
production of aerobactin and associated iron-regulated
outer 1ne1nbrane proteil1s.
lmmunologic Aspects
Immunologic defense against diseases produced by patho
genic E. coli occurs at two levels: at the site of attachment
to the target cell and through destruction of the bacteria or
neutralization of its protluct:s.
P.nterntoxigenic /)iarrhea. Specific anti-adhesin antibody
(slgA and slgm) found in colostrum and milk, prevent at-
tacbment to "target cells." Likewise, specific anti-Ll' neu-
traUzes LT enterotoxin.
Enteroaggregative Diarrhea. Specific anti-AAF (adhesln)
antibody (sigA and slgM) found in colostrum and lnilk
prevents attachment to "target cells."
Jnvasive I>isease. The neon.ate acquires im1n11ni1y from
tl1e dan1 a11d, depc11dil1g upon the isotype of the im-
munoglobulin (IgA, IgG, or IgM) the type of protection
diffcrs. for tl1c first 36 hours or so of lifc, ingcstcd lgG and
lgM attach to receptors on the surface of epithelial cells of
the small intestine. Transfer across the cell into the sys-
temic circulation follows attachment. If the antibodies are
specific for a virulence determinant, then disease may not
re:sult if tl1e neo11ate e11cou11ter:s a patl1ugeuic st.rai11 ex-
pressing that virulence determinant. For example, anti-
capsular antibodies acquired from the dam will protect the
newborn from fatal invasive disease by strains of E. coli
possessing that particular capsule.
Nonenteroroxigenic Diarrhea. Specific anti-Bfp antibody
(slgA and slgM) found in colostrum and milk preve11ts at-
taclu11e11t tu "target cells." A11til!ody :specific for SLT will
neutralize this toxin, preventing its activity on endothelial
ce lis.
Edema Disease. Antibody specific for sr;r lle will prcve11t
the edema associated with this condition.
It is imperative, tberefore, that the dam be exposed ei-
ther naturally or artificially to the microorganism and its
vi ruler ice delerr 11ina11 Ls befo re parluri Lio11. Sucl1 exposure
allows for antibodies to be made for secretion into
colostrum and milk.
Laboratory Diagnosis
Demonstration of Enterotoxigenic Strains of Escherichia coli
Enterotoxigenic strains multiply to numbers approaching
1os tu 109/uil uf luuli11al conle11Ls. If the anin1al survives
the fluid and electrolyte imbalances, large numbers are
shed into the environment. Diagnosis is based on tbc sus-
picion that the disease is dueto enterotoxigenic J:::. coli. ·rhe
least troublesome and least invasive procedure (and also
the least reliable) for verification of this suspicion is to

.:a::::;.~:lStrate large numbers of specific adhesin-expressing
in the teces. Den1onstration e11tails plating a portian
_ :'ecal sao1plc onto a selective n1ediun1 (MacConkey
__: •or example). As adhesins are expressed poorly on se-
• c10: media, a number of colonics are subculturcd onto
--aa tJ1at will promote the expression of the various ad-
"'""!.S: for F4, E medium; for FS and F6, Minca medium;
.....__ :or F41, E or Minca n1edium. Slide agglutination tests
~un on each colony using antiserurn specific far the
=ous adhc.sins. An enzyn1e-linked in1111unosorbent
..__ __ has been developed to measure directly the presence
~ and F5 adhcsin-cxprcssing bacteria in feccs. Such a
-~t'!od eliminares many of the problems inh.erent in the
;;:::a.;-sis of feces for fimbriated bacteria. Gene-specific
_ ..::1ers have been developed so that demonstration of ap-
--i:iriate genes in isolated colonies can be made by use of
-e polymerase chain reactio11.
~more reliable n1ethod to verify the clinical diagnosis
.onterotoxigcnic E. coli-induced diarrhea is to quantitate
:.::: number of Ji. coli in the small intestine. Nonnally,
...::.c:-e should be very few E. coli in such sites, especially in
.....;r: 1ejunum, and the presence of large numbers of bacte-
- 'n these locations is highly suggestive of enterotoxi-
-:r.pjc E. coli disease. San1ples are plated 011lo differenl
-e<lia chosen to promote the expression of the various ad-
-esins, and colonies are p icked and testcd '~'ith thc mono-
::>eei.fic anti-acthesin sera. Examination of stained smears
: ::.be contents of the small intestine is another methocl
b3.5ed upon the increased numbers of enterotoxigenic E.
_ -1 in this location; find.i.I1g > 100 per oil immersion field
iplies > 106 /u1l oí co11te11ts. Altl1ougl1 this 1netl1od lacks
pecificity, it strengthens the diagnosis.
n uorcsccnt-labeled a11tibody techniques provide the
=::siest 1nethod and are probably the most reliable except
- ~ demonstration of the toxin. S1nears of scrapings taken
-om the small intestine are flooded with antisera that
.Le specific for the various adhesins. After treatment "''ith
~;.iorescent-labeJe(1 secondary antiserum, preparations are
-=xam ined for labeled bacteria adhering to the epithelial
cells.
Enterotoxin production by isolated strains of E. coli is
:-cst dctcc'tcd by utilizing an ELISA test spccific for ST and
LT. 1'his test is repu ted to detect 140pg/ml of Sr (> 100
:imes more sensitive than the suckling mouse assay) and
.,90 pg/ml of i.:r.
E. coli containing tl1e genes encoding the various ad-
::esins, as "''el! as the entc.rotoxi ns can be detected by using
9~A probes or polymerase chain reactio11 (PCR) primees
spccific for thc corresponding base sequcnccs cncodiJ1g a
specific trait (e.g., an adhesin or an enterotoxin) . .Such
f>TObes or primers have been used to deteet the genes (in
bacteria)·in feces as well as in culture.
)emonstration of Strains Producing Enteroaggregative Disease
"'.solates suspcctcd as being capable of p roducing entc.roag-
~.regative diarrheal disease, can be tested for their ability to
associate wi.th. HEp-2 tissue culture cells in aggregative pat-
tern (the "gold sta11dard"). However, demonstration of the
:'.1rese·n ce of [ )NA associated with the genes encoding
EASTl and/or AAF is cerlainly easier and n1ore cost effec-
Cltupli:r 8 Ertli:tubut.lt:r iut.i:ui:: E.~t.ht:r it.hiu 67
tive. Histopathologically, a diagnosis of enteroaggregative
clisease is su pported by the presence of sheets of bacteria
associated with the intestinal epithel.iun1.
Demonstration of Strains Producing lnvasive Disease
The 1nicrobiological diagnosis of invas.ive disease is based
upon the demonstration of E. coli in nor1n.ally sterile sites
or locations (joint, bone marrow, spleen, or blood). In fo1Nl,
the sa111e siles are cullured, plus lhose grossly affected
(lung, air sac). Dead in-shell embryos are cultured. Culture
of th.e livc.r is to be avoided even though the Kupffer cells re-
move bacteria trom tl1e blood , because retrograde move-
ment of enter.ic bacteria during the. agonal stages of the dis
ease complicates tl1e n1icrobiologic find.ings.
Demonstration of EPEC/EHEC Strains
Thc. presence of genes encoding shtga-like toxli1s can be
determined by specific DNA probes or by PCR. More cum-
bersome is the demonstration of cytotoxin activity for tis-
sue culture cells (vero cells).
The demonstration of attaching and effacing strains as
Lhe cause o[ Lli:;ed:;e il1 the live anirr1al is more clifficult.
Aside from biopsy of intestinal mucosa an<l thf> ñnding of
attaching and effacing lesions, detection of genes associ-
ated with EPEC/EHEC:, eaeA, bfp. or slt have been used
(specific DNA probes or polymerase chain reactio11 with
scquence-specific primers), or function assays testing for
SLT activity for tissue culture cells. Fecal isolates obtained
frorn a selective n1edium (e.g., MacConkey agar) can be
testeo for thf> genP.s or proci11cti nn of <;higa-likP toxin in
culture supernatants tha t are tested for cytotoxicity for tis-
sue culture cells. Most of these isolates have been shovvn to
produce urcasc, an uncommon trnit for E. coli. Eschcrichia
coli 0157:H 7 does not ferment sorbitol. t-.1acConkey agar
containing this sugar instead of lactose is used to examine
teces for the presence of sorbitol negative isolates, vvhich
are then tested for antiserum specific for ()157 and/or H7.
Since Ehx produclio11 is found will1 co11siderable nun1ber
of SLT-producing strains of E. coli, den1onstration of these
:;train:; can be n1adc on blood agur plat cs supplemcntcd
\.Vith calciurn.
Demonstration of Strains Producing Edema Disease
'l'he n1icrobiological cliag11osis of eLle r11 a Llisease ueµe11us
upon the isolation and demonstration of certain serotypes
that have been shown to play a role in this diseasc. Tl1e
characteristic gross and m icroscopic tissue changes inake
this disease relatively easier to diagnose pathologically
than microbiologically.
Treatment, Control, and Prevention
·rreatment of an animal that has diarrhea dueto an infec-
tious cause centers on correcting fluid and clcctrolyte im-
balances. If t he an1m.al is in shock due to cardiovascular
collapse, then the fluid and electrolytes (sodium bicarbon-
ate, I<CI) are given TV; if not, oral electrolyte solutions are
68 PA1tT Il Bacteria and fungl
given. Since the animals are acidotic, soctium bicarbonate
is included. Adding glucose to oral electrolytes will en-
hance the absorptioo of the sodium ions lJeix1g ex<.:rt:tt:u.
The use of a11tilnicrobials is controversia!. Beca use the cnn-
<.:entratio11 uf a11li111iL1obic acllievablc (and available) in
thP lumen of the howel is not known. the results of in vitro
susceptibility tests to guidc thcrnpy nrc of doubtful reliabil
ity. Administration of nonabsorbable antimicrobics (such
as r1eomycin) will sufficiently reduce the numbers of E. coN
in the upper small bowel to allow c.:orrectiox1 uf fluiu auu
electrolyte imbalances. Sucl1 rec111ction occurs even
though in vitro tests sl1uw tl1al sLrains of E. coli con1n1o nly
test "resistant" to n<>omyci n. The fact that in vitro tests
1ueasure susceptibility to microgram amounts \-vhcrcns
milligram amounts may be available locally accounts for
the discrcpancy.
J\ntimicrobial agents, fluid, and electrolyte augmenta-
tion are necessary to successfully treat septicemic diseasc
produceel by invaslvc strains uf/:!,. luli. Iuvasi ve disease re-
sults in an endoto:xemi;:i progressing to a Iactic acidosis be-
<.:ause of decreased organ perfusion secondary to hypotcn-
sion and disscminated intravascular coagulation. ·rhis
should be taken into account whcn the electrolyte replacc-
ment is chosen. Antim icrobial agents shoutct be chosen ac-
cordi ng to susceptibility trenels ln the practice area.
Usually E. co/i isolatcd f:rom farm ::inimals are susceptible to
ge11la1nicin or an1ikacin, trimethoprim-sulfonamides, and
ceftiofur. They are usually resistant to tetracyclines. strep-
to mycin, sulfonamides, ampicillin, nnd kanamycin. Th.:
severity of the sütns ot endotoxemia has been rectuced e:-..-
perimentally by administering antibodies to the lipid r..
portion of thc LPS.
Prevention a nd c'on tro l of thP e ntcric diseases produced
by pall1ogenic strains of E. coli are one and thc samc. ThL
key is sound husbandry practices. It is important tl1at thc
dam be exposed to the antigcnic dctcrminants of the vari
ous virulence tactors cxpressed on or by the infectin~
strains. Exposure can be provided naturally by placing the
dam into the environment in which parturitiur1 will Lak1.:
place or artificially by vaccinating the dam wíth prepara-
tion s cor1taiui11g tlie a11Lige11ic d etenninants pcrccived te.
bf' ;:i threat to the newborn. Commercially produ ced pre-
parations containing monoclonal antibodies to the ad
hesins (for ETEC) can be given orally to the neonatal an.-
mnl. Although this practice will not significantly reduet.-
the incidence of diarrhea, it will reduce the severity au-.
mortality.
 Enterobacteriaceae:
Salmonella
DWIGHT C. l lIRSH
The genus Sal1nonella is a member of the family Hntero/Jacte-
riaceue, and is composed of two spccies, S. bongori and S. en-
.r:ritu. ·rt1ere are six subspecies w!thln S. enterica, enrertca
somet imes clesignatecl as s1 1hsperiP~ T), salarnae (subspecies
~I), arizonae (supspecies lila), diarizo11ac (subspecies Illb),
1011tenac (subspccics IV aI1d VII), and indica (subspecies Vl)
those bclongi ng to subspccics V wcrc placed in to S. bon-
~on) . There are numerous serotypes (serovars), more than
1000, within S. bongori, and thc subspecies of S. enterica.
Tlic 111ajority of these serovars have been glven names that
::irc capitalized and depicted in roman print. Others are
-:ncrely denoled by anlige11ic fv111 1ulat:. Thuse 1.Jelu11giI1g tu
S. bongori and S. enterica subspecies JI through VII are
mainly associatcd with cold-blooded vertebrates, while
those belonging to ~. enterica subspccies I are more com-
monly found in mamn1als and birds. However, each serovar
is capa ble of produciI1g discasc rcgardless of the host.
In this chapter, the subspecies designation will not be
u:.t::ú u11l1:::.s i1upurta11l lu tl1c Lliscussiu11. fur exa1uple,
Sal1nnne/la enterica suhspecir.c; e11terira serotype 1·yphim11-
ri um will first be denoted as Sall11011ella enterica serotype
Typhimurium, and then, simply S. Typhimurium .
Descriptive Features
Cellular Anatomy and Composition
Thcrc is onc capsular typc, Vi (for vir ulence), Lh(Jugh n1o:;L
members of thc gcnus do not produce one. ·rhe cell wall is
typical of gram-negatlve bacteria, composcd of lipopoly-
saccharide (LPS), and prote1n. Thc antigcnic composition
of the polysaccharide portion of the LPS in part deter-
111 i 11cs tlle serotype. The kind an<.l number of sugars to-
gether with the linkage between them determine the anti-
genic dctcrn1 i naJ1ts con1prising Lhe 0-au Lige::11s uf the
particular isolate. The 0-antigens, together with the
antigenic determinants on thc surfacc o f thc flagclla
(H-antigens), which are posscsscd by most salmonellae,
help to define an isolate serologically (Table 9 .1). 'I'his clas-
:.ificaliu11 :;cl1eu1e is call~d the Kauffman-White schema.
Cellular Products of Medica! lnterest
.1dh esi11s. Adhesins (also known as (in1bria or pi/1) mediate
adherence to target cells in the gastrointestinal tract and to
cells comprising the niche for the strain. Because of thcir
rclativc hydrophobicity, adhesins mny nlso promotc asso-
c1ar1on with the membrane of phagocytic cells. Ad!1esins
are important virulence factors only ~vhen the microbe is
on 111ucosal surfaces. 1'here are at least thrcc different ad-
hesins implicatecl in the intPr~rtion between salmonellae
and target cells (M cells, intestinal epithelial cells) of lhe
host-Pef, Agf, and Lpf:
1. Pef (plasmid encoded fimbriae) are responsible fo1
attachment to small intestinal epithelial cells.
2. Agf (thin aggrcgativc fimbriae, or curli) are responsi-
ble for attachment to small intestinal epithelial
cells.
3. Lpf (long polar {imbrlae) are responsible for attach-
ment to M cells.
Capsule. The role ofthe capsule (Vi) is unclear. Since sal-
monellae are primarily intracellular parasites, possession
uf a capsule uoes not seem to be a srrategy that Is co11sls-
tPnt with the role thic; c;tr11ch.1rP has in other microorgan-
isms (i.e., antiphagocytic). However, Vi prolecls lhe oule1
membrane from effective interactions with membrane at-
tack complcxcs gcncratcd by thc complernent system. ·rhis
is useful in protecting salmonellae when extracelluiar.
Cell Wall. ·rhe lipopolysaccharide (LPS) in thc outcr
me.m branc is an important virulence determ1nant. Nol
only is the lipid A component toxic (endotoxin), but thc
lt:11~ll 1 uf 1l1e :.iut:: Llirtiu iu LIJt:: ()-1t:!pt::al u11lL l1lI11Je::rs ll1e
attachmcnt of the membrane attack complex of thP. com-
plcmcnt system to the outer membrane. LPS binds to lipo-
polysaccharide-binding protein (a serum protein), which
in turn transfers it to the blood phase of CD14. Thc CD14-
LPS complex binds to ·roll-like receptor proteins (sce
Chapter ?) on the surface of macrophage cells triggering
tlie ndease of proinflammatory cytokines.
J!,ffector (Tnxin) Prnteins. SP.vPral of thP gPnes responsibic
for Sahnnnella virulencc are located in clusters, called
J>athogenicity Islands, a cluster of genes encoding viru-
lcncc deter1ninant(s), an integrase protcin, a spccific inscr-
tlon sitc, and mobility. There are at least five ~almonella
Pathogenicity lslands (SPis). While SPI-1 is found in both
spt:!cit:s uf Sulmunella, SPI-2 (an<.l presumably SPI-3 thiough
S) is found only in S. P.n/Prirn Ali five SPis contain genes
encoding the proteins necessary for the ·rype 111 sec1e::Liu1J
system (though the genes and thcir products are differcnt
69
70 PA!{f II Bacteria and Fungí
Table 9.1. Representative Antigenic Formu las tor Salmonellae
0..g)'OOP. Sl!rovari~ty At\tig~nic wrmulaª
B 5.)/mo11el/¿¡ Typhimuriuni 1,4,S,12:i:1,2
B Salmone//a Agona 4, 12:f,g,s;-
D Salmonel/a Dublin 1.,9,12:9,p:-
E Salmoriella Anatum 3, 10:e,h:1,6
G Salmonel/a Worthington 1,13,23:z:l,w
a 0-antigens: boldt.ace ournerals. Phase 1 1-!-antigen: lowerr>1e letter. Pha~"' 7 ti-anti·
gen: Aumcral (or lowcrcosc letter).
for each island), a11d tnay or n1ay not contain thc cffcctor
proteins (tl1ose proteins that interact v\Tith the 11ost "tar-
get" cell). The Type lII secretion syste1n consists of an as-
semblage of proteins (more than 20) that form a hollow
tube-like structcire through wl1icl1 effector proteins are
"i11jecled" ü1Lo 11osL "Larget" cells.
The effector proteins associated with SPI-1 include Ssps
(Sal111011el/a secreted proteins1 encoded by a numbcr of ssp
genes located within SPI-1), and Sops (Salmonella outer
protein, encoded by a number of sop genes, located outside
of 5PI- l) . Ssps and Sops are invoJved \Vith uptake of salmo-
nellae by the target cell(s) by inducing membrane "ruffles"
that fulluw tl1e rearrauge111e11L or lhe aclin cyLoskeleto11
following the activation of the small GTP-binding pro-
teins, CDC42 ar1d Rae. In addition, Ssps also interacts with
caspase-1 causing the death of activated macrophages.
The effector proteins associated with SPl 2 include Sses
(secret\on system effectors, encoGeG ny a number of sse
·genes located within SPI-2), Ssas (secretion system appara-
Lus, cncoded by a 11un1ber of ssa genes, located wi lhin SPI-
2). Both Sses and Ssas are induced hy low pH (stomach,
phagosome), and interfere with inacrophage functlo11.
The effector proteins associ.ated '"'itl1 SPI-3 iI1clude Mgts
(ma.i,>nesium lransport syste1n1 e11codedby a number of mgt
genes located within SPI-3). These genes are induced by
low concentration of magnesium ions (as occurs \·vithin
rnaLTuphages), aud the encotled proteins ctppear to be im-
portant for survival inside of macrophages.
The effector proteins associated with SPl-4 and SPl-5 are
associated with intracellular survival, and as yet are not
clearly defined.
Enterotoxin. Members of the génus Salmonella secretean
enterotoxin, Stn (Salmonella enterotoxin) associated with
vvater and elec:trolyte secietion by t1ost target cells. Stn dif-
fers fro·m cholera toxi n and 1:r of l!sr.herichia r.nli (see
Chaptcr 8) by being a peptide rather than being composed
of subunits. 'I'he role (if any) of Stn in the production of di-
arrhea is unclear.
Jron Acquisition. Salmonellae produce siderophores (e11-
terobactin) when growing in iron-lin1iting conditions.
Stress Proteins. Stress proteins are definell as proteins
macle when the mirroorganism is placed 11nder conditions
of stress (e.g., 11eat1 cold, low pIJ, high pJI). RNA poly-
n1erase containing RpoS preferentially transcribes genes
responsible for acid tolexance (survival at pH 5) and regu
lates genes found on Spv plasmids (see belo\-v). RNA poly-
merase containing Rpo.E is involved with survival \.Vithin
phagocytic cells.
Virulence Plasmids. Salmonellae possess plasmids ofvar-
ious sizes, son1e of which have been associated with vi.ru-
lence. 'l'l1e n1osl 11olable is a fanuly of large (approximatelr
SO to 100 kilobases [kb]) plasmids, termed "Salrnonella vir-
ulence plasmids" (Spv plasn1ids) that are found within
those species of salmonellae with potential to produce dis-
se.nlinated disease. Sorne of the genes (spv genes) carried by
tl1ese plasmids are necessary for intracellular growth and
are regulated in part by RNA polymerase cont<'lining thf'
::;t(;ltiur 1ary vitase sigrna factor, RpoS (sec "Stress Proteins,"
ahove). Other genes on these plasmlds are responsible for
serun1 resistance and may be involvcd with adhcrcncc and
invasion of the cellular target.
Misce/laneous Products. The transcriptional regulato;,
SlyA (for salmolysin), is in part responsible for survival of
salmo11ellae within 111acrophages, perhaps afforrling prn-
tectiou fru1u ll1e loxic products ge11erated by oxygen-
dependent pathways. 1'he products of the phofY/phoQ ope-
ron are responsible for resistancc of salmonellae to de-
fc11sins found in the Iysoson1al granules of phagocytic cells
(by dirccting the remodeling of the salmonella outer mem-
brane). Tl1e product of the shdA (for sheddi11g) gene governs
fecal shedding of saln1onellae by an infected host. This gene
is restricted to serotypes of S. enlericu ::;uu:sped.e::; en.ler icu. A1 L
(for lll'robic reg11 hition r:ontrol), is a h\To-con1ponent globa:
regulator systen1 involved witl1 intraccllular survivaL
Ecology
Reservo\r
'fhe reservoir for members of the genus Salmonella is the
gastrointestinal tract of lvarm- and cold-blooded ani1nals.
Sources of infection include contaminated soil, vegeta-
tion, water, and compor1ents of animal feeds (such as
bone, meat, and fish meal), particularly those containing
n1ilk- 1 n1eat-1 01 egg-derived co11stituei1ts, and the feces oi
infected individuals. Lizards and snakes (usually asympto-
matic) are comn1only infcctcd, somctimcs with scvcr:~
serotypes. Sallnonella enterica subspecies enterica are aln1ost
exclusively found in 'INar1n-blooded mammals and birds
(evi<.lence suggests that the possession of the :;ful.A. ge11e
prod11ct is rPsponsihle).
Transmission
lnfection occurs following the ingestion of salmonellae.
The outcon1e of the interaction between host and
Salmonella depends upon the state ot the colonization re-
sistance of the !1ost (a measure of the "bar.ri.er" produced
by the norn1al flora), the infectious dose, and the particu-
lar species of Salmonella. Disease mayor may not occur fol-
lowing ingestiou. lf it occur::;, it ruay du su i1u1uedialely or 1
at sorne later date. In the later instance, the initial interac-
tion may rcsult in the colonization (without disease) of
the host, but with a change iI1 the intestinal enviionmenl,
brought on, for example, by stress or antibiotics (activities
that affect the normal flora), and disease may follow.

-:-=:mogenesis
:ZChanisrns. The most common clinical man ifestation of
2lalonellosis is diarrhea. In certaJn 1nsrances (clef!ned by
.'.:DSt factors, the strain of Salmonella, and dose) septicemia
xcw·s. Ho:>l fac lors include age, ü11111u11e sta tus, co11cur-
::2!1t disease, and "health" of the norn1al flora (coloniza-
~o:i resistance).
Stationary phase salmonellae appear best suited to ini-
=te disease, because under these conditions, R.NA. poly-
-:ierase containing the alternative sigma factor, RpoS, ini-
:iates transcription of genes responsible for acid tolerance
~t.l subsequent survival t11rough the stou1acli. Alsu, RNA
""Olympr;isp cont:iining RpoS is ;i positivP. rP.gnlato r for t hP
;enes found on the Spv plasmids.
The target cells are the M cells atop the lymphoid nod-
~es and thc epithelial cells of the distal small intestine
...:!d the upper large bowel. If the target cell is "vacant" rel-
::.::ve to the numbers of salmonellae (a reflection of the col-
ontzation resistance), disease may result. Vacancy of the
-;>Jget cell depends u pon the stah1s of the normal flora. If
::":e flora ls disrupted (stress, antibiolics), tl1e11 ll1e in fec-
tious dose does not have to be as high for salmonellae to
5'tin access to t he target cell. It appears that tl1e M cell is
:'.le preferred target, and it is t his cell tl1at is affected first.
.-.dl1esion is the first step in the disease process, mediated
':>y one or more of the adhesins Agf, Pef, Lpf, o r by others
:et to be determined. Follo\.ving adhesion, salmonellae are
:nrernallzed following the induction of membrane ruffles
!U the target cells t riggered by Ssps and Sops subsequent to
~l)eir "iujecliou" by L11e 'I'ype III secretion systern . 1'he lar-
5t:t cell is irreversibly damaged by t his interaction, under-
5oing apoptosis. Sahn.o nellae are now found "vithin the
:arget cells, the lymph nodule, and submucosal tissue. An
...c"lflammatory response is initlated by release of various
c!:temokines from affected host cells, as well as release of
?roinflarnmatory cytokines following host int eraction
'\'ith cell >vall LPS- activities that result in an i11flux of
?Qlymorphonuclear neutrophil leukocytes (P1vfNs) and
:nacrophages. Tl1e influx uf PMN~ i11ay !Je reflected i11 a
::;ansient peripheral neutropenia. PMNs are highly effi.
dent iI1 phagocytosing and destroying salmonellae, the
macrophage less so. If the immune status of t he host and
the charactcristics of the salmonellae are such, the infec-
tious process is arrested at t his stage. Diarrhea is thought to
:-esult from prostaglandin synthesis by the recruited PMNs
and perhaps by the affected host cells), as well as activa.
don ofvarious inositol-signalingpathways within affected
hosl cells. The nel resull is the secretion of cl1loride ions
and water. ·rhe role of Stn in the production of diarrhea is
w1clear.
lt the infecting strain ot Salrnonella 11as properties that
allow disscmination (posscssion of SPI-2, 3, 4, and 5-
associ.ated gene products that allow growtl1 within
:nacrophages; Spv plasmid encoding ability to gro~" intra.
cellularly and serun1 resislance; PhoQ/PhoP syste1n alluw-
ing resistance to defensins; SlyA allowing resistance to
oA-ygen-dependent by-products; arcA), septicemia may
result. 'fhe likelihood ot th.is occurring is increased if im-
m une status of the host is diminished. Salmonellae dis-
seminate and multiply within phagocytic cells (macro-
Chapter 9 tnterobacteriaceae: Salmonella 71
pbages mainly) witl1in. phagoso1nes. Not only are the inva-
sivc strains bcttcr ablc to withstand thc action of lysoso-
m al contents, sorne "sort" to phagosomes that do not fuse
with lysosomes. The presenting signs are usually, but not
always, septicemia and shock (see Fig 8.1). Strains produc-
ing thi<: form of clisP:ISP Psc:ipP rlPstr11ction by thf> host :incl
n1ultip ly within macrophages of the liver and spleen, as
well as intravascularly. During the dissemination process,
salmonellae are occasionally outside of the intracellular
environment and therefore at risk from the formation of
complement membrane attack complexes on their sur-
faces. 'fhis occu rrence is cliscouraged by at least two n1ech-
i"lnisms: a proc'h 1ct of thP Spv p l:isrnicl ;inci t he IPngth of the
0 -repeat unit of the LPS (there is a direct correlat ion be-
tween 0 -repeat length and viru lence).
Invasive salrnonellae are capablc oí sccrcting a sidcro-
phore, enterobactin that ren1oves iron from the iron-bind-
ing proteins of the host, although it is doubtful whether
tllis i~ 11eeded within the cells of the host.
Multiplic.;ition of thP organism res11lts in endotoxemia
(see Fig 8.1), which accou nts for most signs and t he course
of illness.
Pathology. If the infcctious proccss is limitcd to thc in-
testinal tract, the Jesions vvill consist of a hemorrhagic in-
flammation of the distal small intestine and large bowel.
·r11ere 1nay l.Je superficial necrosis. In the septicemic form
of the diseasP., thP.tP. ilrf> inflamm:ito ry c.h:ingt>s in livPr,
spleen, and intestinal tissue. 'fhere may be hemorrhages
011 pericardium, peritoneal surfaces, and adrenal cortices.
Disease Patterns
Ruminants. Salmonellosis 1s a slg111flcant dlsease of rumi-
nan.ts, mainly cattle. Th.e d isease affects young (usualJy 4
to 6 weeks of age) as well as adult anin1als. Anin1als in feed-
lots are commo11ly affected. The disease may be a septice-
mia orbe lirn ited to the enteric tract. Pneurnonia, he1nato-
ge11ously acquired, is a comrnon presenting sign in calves
with septicemia due to S. enterica serotype Dublin (S.
Dublin). Abortion may follow septicemia. S. enterica sero-
type Typhimurium, S. Dublin, and S. enterica serotype
Newporl are Lhe sero Lypes con11nonly isola led íron1 callle,
S. Typhimurium the serotype from sheep.
Swine. Salmonellosis in swine can p resentas an acute, ful-
minat ing septicemia oras a chronic debilitat ing intestinal
disease. The form depends upon the strain of Salmonella,
the dose, and the colonization resistance of the infected
a rLiu 1aJ. The <liseast'. is ~eeu 1nu~t ufter1 in pigs t11at l1ave
heen stressed. Such conditions occ.ur oftP.n in fP.edP.r pigs,
an age group in which salm.011ellosls co1nmol1ly occurs. S.
Typhimurium and S. enterica serotype Cholerae-suis are
thc prcdominant scrotypcs.
Horses. Adult horses are comrnonly affected with
Sulrnunellu. '!ºhe pattern is úiarrhea, though septicemia i.s
seen oc.c.asionally. r:olic, gastrointPstin;il sui:gery, and an-
tlmicrobial agents predispose the horse to the develop-
ment of clinical signs. 'fhe agent is either carried normally
(as in approximatcly 3% of clinicully normal horscs) or ac-
quired from other sources (e.g., a veterinary hospital). ::i.
72 P 1\ltT 11 Bacreria and Fungi
Typhimurium and S. enterica serotype Anatum are most
commonly isolated.
Dogs and Cats. Salmoneliosis is uncommon in dogs and
cats, although carriage is rcµurtculy lLigh i11 cli11ically 11or-
m;il po11n<1 <logs (11pwar<1s of 3.)%). When outhreaks occur
they are usually associatcd 'vith a common :source, such as
contaminated dog food or "treats" (e.g.• dried pig's ears).
Saln1onclla should be high on the microbiological clifferen
tial list for cats w1th s1gns of septicemia.
Poultry. Scc "Salmonel losis of Poultry."
Epidemiology
Sabnonella spp. a re ubiquitous gcographically and zoolog-
ically. Sorne scrotypes are relatively host-specific (S.
Dublli1-cattle; S. cnl'erica serotype Typhisuis- swine; S.
enterica serotype Pullorum-fowl), whlle others, notalJly
S. Typhimurittm, S. Anatt1m, and S. Nf>wport, afff'c.t a wide
!Ju~Lra11ge a1nong whicl1 fcral birds and rodents play im-
portant roles in interspecific dissemi.J.1ation of infection.
Long periods of asymptomatic and convalcsccnt shcdd-
ing ensure widespread, unchccked distribution of the
organisms.
Clinical outbreaks are correlated with depressed im-
mune states, as in newborn animals (calves, foals) and
:.l1e:.:>eu duulls, f.ld1 lu1i1:11l 1..vvv:., eY.uiue swgical palie11Ls,
and swinc with systcmic viral diseases. Ali animals are at
increased risk of developing discasc if thcir normal flora is
disrupted (stress, antibiotics). These circumstances render
anima Is susceptible to exogenous exposure or activation of
silent infecttons.
Humans appear to be susceptible to all Salmonella
:-.e1ulypes, lhe n1osl in1porla11l sourcc for wl1icl1 are ani-
m;ils an<l thPir hy- products. Poultry and poultry products
(cggs) are a major source of Saln1011el/a in humans. Sa.lmo-
nella enterica scrotypc Entcriditis (e.g., phage type 4) is es-
pecially adaptcd for egg transmission. Whether a person
dcvclops discase following ingestion of salmonellae from
tl1c environrnentdepends upon the dose of organisms, tl1e
serotype of Salmonella, anti t11e 1.:uluniz<1tio11 re:sista11<.:e uf
thf' inff'Ctf'<i i n<l ivi<111al. Salrnonella Typhjmttrium is most
common, usually producing gastroent eritis. Sorne
serotypcs havc greater invasion potential, for example, S.
Cholerae-suis (from swine), anc.l S. Dublin (ir1fected milk).
·rhou~h S. Typh1murium DT104 (the Definitive Type
designation specifies a particular phage type), which is ac-
qulrcd fro1n cattle, appear!) more pru11e to sy!)ten1ic <.lis-
ease, tho11gh thi~ h;is hPPn <liffic.ult to prove experimen-
tally. Asymptomatic reptiles have become an important
sourcc of Saln1oncl/a in humans.
lmmunologic Aspects
Proter.tion <lf'pf>n<l<; 11pon spf'cific ancl nonspccific im-
n1unological factors and microbiological defense mecha-
nisms. Discasc may be prevented by an intact colonization
rcsistance at Lhe leve! of the target cell (see Chapter 2).
Whcthcr 1nfecnon can be prevented is not kno\-vn.
Antibodies specilic tor surtace structures of Safmone/1
possibly adhesins, prevent adherence to target cells. Tui::
newborn is protected passively by ingesting speclflc sl;.-
or IgG 1 (bovine). The immunologically mature animal_
vruLecLed by ex.udaUon of specific io1n1unoglobulins (Ig'
and IgG) at the si te of invasion or by the production of Sc-
cretory immunoglobulins.
Another. more novel approach has been to feed animL
microorganisms that out-compete salmonellae for nich_
along the gastrointestinal tract. Competitive exclusion. :...
this phenomenon is called, may be quite usPf11l hf'c;iu-
Llit:urt:Lically, saln1011ellae of a11y serotype would be e.;
cluded as long as they shared the same niche as the co:=-
peting strain.
Antibodies in the circulation act as opsonins and pr --
m ote the phagocytosis of the organism . Destruction of th.,
salmonellac that have been phagocytosed follows the i .....
munological activation of the macrop.hagPs by spf'rifira
sti1uul<1lt:d 1y 111 J?llocyles cr cells). though activa te_
macrophages are damaged or killed by Ssps. NK cells l;:
Sal111onella-infected cells.
Acquired immunity revolves around activation
macrophages, which takes place as follows . After initiaI ::"'-
teraction bctween salmonella and macrophage, IL-12 iS :=-
leased by the affected macrophage. IL-12 activates the ;
subseL ofT helper cell!) (see Cliaptt:r 2). TJ 1is subsel sec1e_::-
amone othc>r cytokinf'S, intprferon gamma, which ac-
v11tc.:s n111crophages. Activated macrophngcs are cffici~­
killers of intracellular salmonellae.
1\rtificial immunization against salmonellae is diffict.-
Ilacterins have had limited success. Apparently, they ~
not stimulate strong cellular immunity, even thoi.:_
abundant antlbody is produced. Antibodies that are p: -
duc:cd loc:ally or p;i.<;sf'<i in colostr11m or milk interfere \\-;-
adsorption to the target cell and protcct against diseasc
this location. Macrophages can be activated and antibP-;:
production stimulatcd in response to modificd livc ·~
cines. lt given o rally, these vacci nes stimulate local secr ~
tory immunity and ccll-mediated activation of phago0-
cells. Aromatic-dcpendent mutants of Sa.lmonella sh_
promjse as effective modified live vaccines, especially _
calvt'.:>. uruA u1ula11Ls of Salrnonella cannot n1ultiply v.itt:
the host since vertebrate tissue does not contain :.
11ccdcd prccursors for aromatic acid synth.e sis.
Laboratory Diagnosis
111 cases of intestinal üúcction, fecal samples aie coliect.::_
in systemic disease, a blood sample is collected for sta-
dard blood culture. Splccn and bone marrow are cultiLI:"_
tor the salmoneuae when postmortem diagnosis of s:-
temic salmoneliosis is required.
Fresh fecal samples are placed onto une or mure ~elt:"...-
tive media, including MacConkey agar, XT.f) ;igar, Hf'ktnt>-
t:lllt:ric 111ediun1, and brillia11t green agar. For enrichmen:
Selenite F. tetrathionate, or gram-negative broth (GN) is
recommended.
Salmonellae appear as lactose-nonfermenting colon1es
on lactose contai ni ng media (lactose-fe.rmenting strains oi
Salrrzonella have been reported, but these are rarely en-

:::ountered). Since most serotypes of salmonellae produce
~1$, colonies on iron-containing media (e.g., XLD agar),
-!ley will llave a b lack ce11ter. Suspiclous colonies can be
::ested directly with polyvale11t an.ti-Salmonella antiserum
J r inoculatetl into differeutial iue<.lia a11Ll LI 1e11 Lesled wil11
antisPra _
·ro cultivate salmoncllac from tissuc, blood agar can be
~ed.
De.finitive identificatior1 involves determination of so-
;;iatic and flagellar antlgens and possit)ly bacteriophage
::ype.
Va1ious Sa/rnonella-specific DNA probes and primers for
:he polymerase chain reaction have been developed for
_dc1,tification as \-VCll as dctcction in samplcs (food, fcccs,
ater) containing other microorganisms.
A multiplex polymerase ch.ain reaction (PCR) assay
".lSing pr.i1n ers designed to detect the common diarrhea-
as~oci atPd 111icroorganisms of swine (Rrachyspira hyodysen-
:eriae, La:t-tlsonia intracellularis, and Saflnone/la) 11as been
d.escribed .
Treatment, Control, and Prevention
'\ursing carP is thf> princ.ipal treatment for the enteríc. form
ofsalmonellosis. ·rhe use of antirnicrobial agents is contro-
>ersial. Sorne studies sho\.v that antibiotics do not alter the
course of the d isease. In addition, thcre is cvidcncc that
a11tibiotics promote the carrier state and select tor resistant
~n:ains . Proponent f- of antibiotic usage .recommend a
:nen1ber of the fluoroquinolone class of drug (e.g., en-
:ofl oxacin or ciprofloxacin).
TJeaL1ue11 L of Lhe sysLen1ic forn1 of saln1onellosis in-
d udes nursing care and appropriate antimicrobial therapy
'.lS determined by retrospectively acquired susccptibility
<lat a. Since saln1onellae survive in the phagocytic ceJl, t he
:i.ntirnicrobial drug should be one that penetrates the ccll.
Exan1ples of those that ciistribute in this manner include
:?lllpicillin, e11rofloxacin, trimethoprim-sulfonarnides,
~nd chloramp11en1col/florfen1col. 'freatrr1eut uptlu11s 111ay
"'<' compron1isPcl cl11P to ac.quisition of R plasmids o r inte-
~ons encoding rcsistance to n1ultiple antibiotics. A serious
;iobal epidemic of S. TY11him urium D'f104 (the Definitive
7°ype dcsignation specifies a particular phage type), a type
:' salmonella that affects h umans and other animals
~ ·orld>vide, contains a "cluster" of antibiotic resistance-
encocling genes withi11 its chru111osuu1e. Tbis ''<.;luster,"
nilli>cl "SalmonPll::i genomic ísland 1" (S<~TI), contains the
::i'enes for resistance to arnpicillin, chloramphenicol/
'Jorfenicol, strepto1nycin/spectinomycin, sulfonamides, and
=etracyclines bounded by n-vo integrons. SGil has moved
mto S. enterica serotype Altiany (fish in Southeast Asia) and
5. enterica serotype Paratyphi B (tropical fisl1 in Singapore).
Salmonellosis is controlled through strict atteutiun to
protoc.ols clesigne.d to curt::i il the spread to susc.eptíhle an í-
:;nals of any contagious agent found in feces. Artificial im-
;nunization with modified live products has shO\'Vn prom-
~e (e.g., aroA mutants, see above). Atten1pts have been
:nade to treat and prevent the endotoxemia produced t)y
<he syste1nic form of the disease by adn1inistering serum
...-untaining antHlodies to the core LPS. Like\<Vise, the ad-
Chaprer 9 Enrerobacrertaceae: Salrnonefla 73
ministration of JS, a rough variant of E. coli, h as been
shown to stimulate the p roduction of antibody to the core
LPS. Both rnetl1utls appear to µrevcn t a1H.l <.:011t rul tl 1e sig1 is
of disease produced by systemic salmonellosis.
Salmonellosis of Poultry
Paratyphoid
"Paratyphoid" is salmonellosis produced by any of the
motile strains of Salrnone/la (all but S. enterica serotype
Pullorum and S. enterica serotype Gallinarum are motile).
The disease produces its h ighest losses in the first?, v1eeks
o[ life as <t sepli<.:e11Ilc Llisease. Survivur:. uecu111e <t::.y111plu-
matic excretors.
Infection is through ingestion. 'l'he source is usually
teces or tecally conta1ninated materials (litter, Uutt, water).
Diagnosis is made by culturing the organism from af-
fectecl tissue (spleen, joints) fron1 b irds that had bccn
showing clinical signs of disease. Tt is more difficult to de-
LecL a11 a~y111plo111alic carrier !Jecau:se :sucli carrier:s u11ly pe-
riodically shed the organism in feces. So1ne have suggested
thnt culture of fluff nnd littcr could be usc.d to dctcct cnr-
rier flocks.
Treat ment <loes not elim inate carriers, alth.ough it does
control mortality. T'reatment regimens have inclucletl
avoparcin, lincom ycin, furazolidone, streptomycin, and
ge11la11 Iicin. Exclusion of sal1nonellae by feeliing "cock-
tails" o f normal flora has heen used •vith sorne success to
reduce thc nu1nbcr of salmoncllac shcd b y carricr birds
(competiti ve exclusion).
Pullorum Disease
Pullorum disease, causeli by 5. Pullorum, ls rare in N orth
America but not in the rest of the -vvorld. The disease has al-
most been elin1inatet1 in the Unitetl States tlue tu a l>reetl-
in.g flock testing progr::im.
Sal111011ella I'ullorun-i infects the ovil of turkeys and
chickens (see fig 74.7}. 'fhus, the e1nbryo is already in-
fected when thc egg is hatched. The hatchery environ-
ment is contan1inated follovving hatching of an infected
egg, leading to infection of other chicks and pottlts.
Morlality is tlue Lo sepli<.:e111ia a11d is grealesL i11 lhe seconcl
to third week of life. Surviving hirds carry the hacterium
aJ1.d may pass it to their offspring. Tt is difficult to detect in-
fected breeding hens by bacteriologic n1eans. Agglutina-
tion titers, produced 3 to 10 days after infection, are used
to detect carrier birds.
Eliminating infected breeding birds detected serologi-
cally controls this disease. 'l'reattnent with a11tirnicrobial
agents (ma inly sulfonamiclP<;) rP-rl11c.P.s mortality in in -
fected flocks .
Fowl Typhoid
Fowl typhoid, caused by S. Gallinarurn, is an acutc scp-
ticemic or chronic disease of domesticated adult birds,
mainly chickens. Fowl typhoid is rare now in t he United
States dueto control programs.
74 PART 11 Bacleria aud Fuugi
The clisease is cliagnosed by culturing the organism
fron1 liver or spleen. It is treated witl1 antimicrobial agents,
IIldinly :;ulfo11a1Hilie:; (:;ulfd<{UÍ.UOXdUilC) auu !IÍ trofura11s.
Fowl typhoid is c.ontrolled hy management and eliminat-
i11g infected birds. A bacterin made fron1 a rough variant of
S. Gallinarum. 9R, has been shov"n to decrease mortality.
Avian Arizonosis
SwI.~.ar.ena enl:erir.o. ~nbspecies arizonae and S. entericct suf)
species diarizoniae (Arizona htnsftLtwii) o.re most often iso-
lated fro1n reptiles and fowl, although these species can be
isolated from any animal. 'lürl<eys are most commonly af-
fected. There are 55 serologic types affecting fowl, with
type 7: 1, 7,8 most comrnonly isolated in the United States.
Salmone/la enterica subspecies arizonae and S. enterica
sub:>.¡Jecie:; diurizuniut: dre 111ai11t ained in turl<ey flocks via
hatching eggs, which become infected follow ing ingestion
by the 11en. Feces also spread it.
Diag11osis is 111ade by culluring sali11011ellae fron1 tl1e
liver, spleen, blood, lungs, or kidneys of affected birds, or
from dead poults and hatch dcbris.
Most serotypes of Salmonella entcrica subspecies a.n -
zonae and S. enterica subspecies diarizoniae possess R plas-
n1ids, which sometilnes makes ít difficult to prevent and
t·reat tllis disease. Various antimicrobial agents such as fu-
razolídonc and sulfa1nerazine added lo feed llave !iÍIO\'\".C
some success in lowering morta\ity. ln)e.c.tion of c'lay-old
poults with gentamicin or spectinomycin decreases n1or-
tality, but survivors still harbor (and shed) the organism.
Control measures should be aimed at prevention rathc:-
than treatrnent. .Because of tl1e multiplicity of serotypes,
no effective vaccine is available.
•
 Enterobacteriaceae: Yersinia
DWl<..TH'J' c. Hl!ZSH
;;enus Yersinia is included in the family Enterohacteri-
.._ There are 11 species, one of '"'hich, Y. ruc:kcri, affec."ts
rtsn. Yersinia enterocolitica and Y. pseudotuberculosis are
ciated with mesenteric ly1nphadenitis, terminal ileiti.s,
z=::,........·~e gastroenteritis, ancl septicemia. While Y. enterocolit-
"fects mainly don1estic animals and primates, Y.
:-intu berculosis affecls largely birds and rodenls, and
occasionally domestic animals and prin1ates. Plague,
-5C'd by Y. pestis, is a rodent-based zoonosis. T'he zoono-
;ospects of Y. enterocolitica, Y. interrnedia, Y. frederiksenií,
- ·: k ristensenii are uncertain. Yersinia aldovae, Y. rohdei,
::JllareNi, and Y. bercovieri are without k11own patho-
-~.. ~ potential.
: ~cr i ptive Fea tu res
:-:r.ology and Staining
=bers of lhe ge,nus Yersinia are gran1-negalive coc-
.:_cilli. Bipolarity is cornmon in tissue smears. Most
des are flagellatcd at ambient temperatures.
'::.. •th Characteristics
:r:s_~Jae grow on ordinary laboratory media, including
;:;Conkey agar, although they grow slower than rnost
- ~ - mf'nlhf'rs of thf' f;imi ly l~nterohar.teria1:eae. (~olony <li-
~~-:ers rangc from under l 1nm (Y. pestis) up to 1.5 mm for
~ other yersiniae. They are not hemolytic when grown
;:;:ood agar.
~ b iochemical activities and resistance, yersiniae re-
.e::: ..,!e other members of the family Enterobacteriaceae.
=::>IN/A PESTIS (PLAGUE BACILLUS)
~- .;:ia pcstis is
thc cause of thc rodcnt-bascd zoonotic dis-
;;c=-:: caUed plaxue. In human patients, and susceptible do-
..="2c animal specics (mainly cats), plague is manifest as a
CiL ~ymphadenitls (bubonlc plague), pneun1onia (pneu-
- --e p!;igue), or septicemia (septicemic plague). Analysis
:J'\A sequences of the genes encoding the 16S riboso-
- R."lA indicates that Y. pestis is a subspecies of Yersinia
-.Jot11bcrculosis.
Descriptive Features
Cellular Composition and Products of Medica! lmportance
Yersinia pestis contains the plasmids pYV (encoding the
·1ype 111 secretion system, Yops, and LcrV), pMTl (encod-
ing the capsule, and Ymt), and pPCPl (encoding pcsticin,
coagulase, and plasminogen activator). A chromosomal
locus called the High Pathogenicit)' Island contains the
ge11e:; eucoding iron uptake, and the H1ns phenotype. Also
located on the ch rornosomf' ilrf' thf' gt>nf's f'nroding the r
protein Gsr.
Capsule. The capsule plays many roles, the most impor-
tant of which are interference with phagocytosis (an-
tiphagocytic), and the protection of the outer membrane
from the deposition of membrane attack con1plexes ge11er-
••
ated by activation of the complement system. The capsule
of Y. pestis is <illlf'rl (~;ifl (for r.apsular antigen fraction 1),
and is encoded by genes that reside on pl<15n1id pMT1
(which also carries the genes encoding the toxin Ymt, see =
bclow).
Cell 1t\Tall. The cell wall of Y. pestis does not have 0-
antigen (rough phenotype). T'he lipopolysaccharide (LPS)
in the outer membrane is an important Virulence determi-
nant. LPS binds to lipopolysaccharide-binding protein (a
serun1 proleil1), wl1icl1 in lurn Lransfers il lo lhe blood
phase of CD14. 'I'he CD14-LPS complex binds to loll-like
receptor proteins (see Chapter 2) on the surface of macro-
phage ceus triggering the release ot prointla1nmatory
cytokines
High Pathogenicity lsland. High Pathogenicity Island
(HPI), so-called because its presence is related to lncreased
virulence as com.pared to strains that do not contain it, ls a
cluster of chromoson1al genes involved >vith iron acquisi-
tion (see below, "!ron Acquisition ").
Htns Phenotype. The Hms (for hcmin storage) phenotype
is associated with iron acquisition and colonization of
fleas (sce below, "!ron Acquisition").
[ron A cq11isition. Iron is an absolute growth require-
u1e1tl aud 111u:;t lJe rernoved frorn iron-binding proteins of
the host. Thf' genf's f'nro<ling proch1rts involved with i ro11
acquisition reside on a chromosomal _p athogenicity is-
land I-IPI (a cluster of genes encoding virulence determin-
ant(s), an integrase protein, a spccific inscrtion sitc, and
mobility.) 1'he rIP1 of Y. pestis encodes the genes for the
iron-acquiring sideroph.ore yersiniabactin, and the Hms
(for hernin storage) phenotype. Colonles growing on the
s11rfac.f' of hloorl ;igar plates that displ<iy the Hms pheno-
75
76 l'ARr II Bacteria and .Fungí
type appear pigmented (they are not) dueto the binding
of hcmoglobin (or Congo Ilcd if prcscnt). It is probable
that bound hemoglobin is used as a source ot iron. '!.'he
genes responsible are encoded by the pgm (for pig1nent)
locus. in addition to playing a role in iron acquisition, the
Hms phenotype is someho\'\• involved ~vi t h colonization
and blockage of Lhe provenlriculus of fleas.
1ype 111 Secretion Systern. ·rhe 1'ype III secretion system
consists of an assemblage of p roteins (more than 20) t hat
torm a hollow tube-like struch1re through which etfector
proteins (Yops, LcrV) are "injected" into host "target" cells.
1'he genes encoding tl1e proteins needed by the Type III se-
cretío11 syste1n reside on plasmid pYV (along with the
gerH:'.::i er1couiI1g Yop:; a11u LcrV, :;ee IJeluw) .
Tnxin~. Yersinia fJP.stis pro<iuces a numher of toxins that
are secreted by way of a Type III sccrction systcm (see
above):
J. Yops. Following thei r "injection" int o macro-
phages, Yops (for Yersinia outer protein) interferc
with the actin cytoskeleton, therel.Jy l.JlockiI1g
phagocytosis, and down ri>g11lating thi> inflam -
n1atory response::; by the inhibition of N.F-KB.
"lnjection " into neutrophils, results in a decrease
in thc cxprcssion of cndothclial ccll-adhcsion
proteins, theret)y reducing ettective inflamn1atory
responses. The genes encoding Yops reside or1 the
plas1nid pYV (also known as pCDl) . 'fhe genes
residing 011 this plasmid encade Yops, LcrV, and
Lhe ·rype lll secretion syslen1. 'l'hey are down regu-
lated at 26ºC, and up regulated at 37ºC and low
calcium.
2. LcrV. LcrV (íor lovv calciun1 response virulence, also
known as Factor V) is a protein located on t he sur-
face of Y. pestis. LcrV has severa! roles: it aids in the
"injection" of effector proteir1s (e.g., Yops) into tar-
get cells; after "injeclion" i11to pl1agocylic cells, il
reduces the excret ion of proinflamn1atory cytokines
and inl1ibits ncutrophil chcmotaxís. 'fhe genes en-
codi ng LcrV reside 011 tl1e plas1nid PYV (also kI1own
as pCDl) . The genes resid.i ng on this plasmid en-
cocle Yops, LcrV, and the Type III secretion systen1 .
·rhey are down regulated at 26ºC:, and up regulated
al 37ºC and Io'-v calciu111.
3. Ymt. The genes encoding Y1nt (for Yersinia rnouse
toxin) reside on the plasmid pMT (vvhich also co11-
tains the genes tor tl1e capsule, see above). Y1n.t is a
phospholipase D that is expressed at 2SºC (and
poorly at 37°(:), and p lays a role in protecting Y.
pestís from t he digestive enzymes within the midgut
of fleas.
4 . Pestlcin. Pesticin is a bacteriocin produced by Y
pcstis v.•itl1 an uncertain role in the production of
disease. Ge11es on plasmid pPCPl en.c ode JJesticin
(along with Plasminogcn Activator).
5. Plasminogen Activator. Plasminogen Activator is re-
spo11sible for tl1e coagulase, fibrinolytic, ;ind C3
degradative activity of }~ pestis at 37ºC. Gen.es 011
plasmid pPCPl encode Plasminoge11 Activator
(along with Pcsticin) .
6. Coagulase. See "Plasminoge11 Activator," above.
7. Gsr. Gsr (for global stress requirement) is expressed
at 37ºC while Y. pestis is within the macropl1age
pl1agolysosome. 'fhe Gsr protein is responsible tor
thc survival of Y. pestis within this environ_men t .
Variability
Yersínia pestis is serologically uniform. 'rhere are three bio-
Lypes (1Jased on ca1bohydrale fern1enla lio11 a11d abílily Lo
reduce r1itrate): Antiqua, Medievalis. and Orientalis.
Ecology
Reservoir
1'o lerant rodent:; in enuernic areas (see IJelvvv) conslilule
thP. plague rP.servoir. 'rhey rarely develop fatal d isease and
are called 1nai11te11a11cc or enzootic hosts. In North A.lncrica
a long the coastal regio ns of California, the meado'"' mouse
b1icrotus californícus is such a host.
Transmission
1'ransmission is by fleas . They may carry Y. pestis to more
susceptible, eplzootlc, or an1p lification hosts, :;uch as
grou11d squirrels or ra ts. Wh1>n thPSP dii>, still nthi>r hosts,
sucl1 as 11u111ans, are attacked. Tnfected mammals 1nay
spread plague by the airborne route. Oral acquisition is by
predation, cannibalism, and scavcnging.
Pathogenesis
Fleas feed on an infected host . Yersinia pestis inside fleas
proliferate until they b lock (obstruct) the flea's proven-
triculus (function of Ymt and Hms). "Blocked" fleas in-
fes l a new hosl and conlan1inale lhe feeding site with
Y. pestis ~vhen attempting to feed . At flea ten1perature,
the 1'ype lll secretion syste1n, Yops, LcrV, Cafl, and Gsr
are not produced. ·rhe bacteria, thus introduced into a
vertebrate host, lack defcnse against the host 's innate im-
n1 u ne sy5tem, ancl are killed when ingested by neu-
trophils (an inflam1natory response is generated due to
products of tl1e flea bite along w ith t he gram-negative
cell '"'al i components of Y. pestis) . In mononu clear
phagocytcs, ut iuam1nalian tcmpcraturcs, nnd low-
calcium ion concentrat.ions, and while protected by Gsr,
Y. pestis activates the Type 111 secretion system; produces
Yops, Lcrv, and Cafl; and is released from t he phagocytic
cell following initiation of apoptosis. Yersiniai> acquiri>
res istan.::c to furthc-r phagocyt osis and int racellular
killingby neutrophil and mononuclear pl1agocytes (LcrV
reduces the excretion of proinflammatory cyt<)kines, and
inhibits neutrophil chemotaxis; "injecte(1" Yops prevent
phagocytosis; Cafl prevents phagocytosis and promotes
serum resistance). ·rhus, early in the d isease process, Y.
pP.stis is an intracellular parasite, and later, an extracellu-
lar one.
Extracellular mult iplication made possible by iron
acquisition systems and capsule production elicits a hem-
orrhagic inflan1matory lesion, followed by local lymph

"?IX.: involvement (bubo). This form is called bubonic
-e intection commonly becomes septicemic and, if
...::::cc.:
-uted, terminates fatally (an endotoxemia aided by
-= -unction of Plasminogen Activator, which accelerates
..:;i:ition of disseminated intravascular coagulation, see
_ .í ) . Su1nc i11<.livi<.lual:. <.lcvcluµ plague µ11euu1orlia an<.l
:::e.:. r. pestis in sputum and droplet nuclei. Others con-
~"L primary pneumonic plague from this source and
. ....:lsmit it by the samc routc. Under epidemic conditions
~ :orm is nearly always fatal.
•.-...'llong do1nestic animals, cats acquire natural clinical
~on, often by ingestion of infected prey. Signs in-
_\!e 1egioual (p<trlil.'ularly 111a11<.lil.Jular) lyruµha<.lenitis,
- .:r. depression, anorexia, sneezing, coughing, and occa-
nally central ncrvous systcm disturbances. Most cases
==.e fatally. Lesions, main ly in the respiratory and alimen-
--- tracts, include lymphadenitis, tonsilli.tis, cranial and
"ical e<.lema, and pneumonia.
Human plague has been traced to feline infections.
oecled i1101.:ulaliuu ruute:; are via cuts, bites, scratches,
=:.!. airborne and flea-borne pathways, although the latter
~"llikcly sincc thc cat flca (Ctenocephalides felis) does not
">::-....;me blocked.
=~ ::miology
""'"-zue is concenlrated in ce1 lain ende1nic areas iu :;uutl1-
-
_- and southeastern Asia, southern anct \-vest c:entral
- -'ca, western North America, and north central South
-..:::e:ica. Endemicity largely parallels presence of enzootic
-¿ epizootic rodent hosts. Human plague epidcmics havc
- -orically been prec1pitated by ímportation of infected
-i.. -s on board ships coming from endemi.c regions. Today,
h t human cases result from infectton following contact
-;., n1r;i l wild anima Is (sylvatíc plague).
Plague is a discase of roden ts. The orga11isn1 is n1ain-
~"led in enrlem ic hosts (c:erta in spec:ies of Mir:rntus ;incl
~nzyscus, i.e., voles and mcadow mice). Endemic hosts
...:e infected following the bite of infected fleas. Though
.:-demic hosts are fair¡y resistant to development o f
::c:-ious dlscase, when thetr populattons rapictly increase
~<l <;preaci of Y. pestis is rapid, die off of endemic hosts oc-
--..:s. Infected fleas, having a shorlage of preferred hosls,
~ on epidemic, highly susceptible species such as
- ;;;iiric dogs, rats, micc, und ground squirrels. What con-
tutes an endemic host and an epidemic host is some-
::at clouded since there is considerable overlap between
~-e n·vo.
:n f'nciemic areas, infcctions are clustered during the
-:!.." ID months. "Off-season" plague affecls n1osUy persous
-..3..1.dling infected rabbíts, bobcats, and occasionally house
- ts. Carnivorcs such as canids (wild and domestic), bears,
::.ccoons, and skunks, as well as raptors, seroconvert fol-
""·;ng infection (ingestion, flea bite), but rarely develop
't4....'1.ical disease. Cat fleas and dog íleas, Ctenocephalides felis
&:ld C. canis, respectively, do not transmit Y. pestis effi-
~ enUy becau~t: 11t:illier ílca 1.Jecurnes "blocked." Thus, ln-
·::ce-J.on of humans from an ínfec:ted rat (rilrf'ly clog) occ:urs
tton1 infcction3 of scrutchcs or bites with infected saliv¡i or
- a m inhalation of in tected droplets.
Chapter 10 Enterobacteriaceae: Yersinia 77
lmmunologic Aspects
Specific resistance to plague probably requires antibody
and cell-medtated responses. Capsular antigens (Fl anti-
gen) evoke opsonin formatíon. Antibody to the LcrV anti-
gen is p1vl1:cliv1:. Di:.pu:.al of intracellular organisms de-
pends on activated macrophages. lmmunity follo\-ving
rccovery is good, but temporary. Dctcction of antibodies
to Y. pestis in rcsistant species (e.g., canids) is a way of de-
termining the presence of the organism in a particular
environment.
Laboratory Diagnosis
lJiagnostic attempts should be supervised by qualified
public health personnel (see below). Sa1nplcs from affcctcd
sites (i.e., edematous tissues, lymph nodes, and nasophar-
ynx), transtracheal aspirates, cerebrospinal fluid, and
1.Jluud (fur culture and scrology) are collected.
Di Tf'Ct smf'ilr~ ilTP px;im i nPci fol lowing immunofluores.
cent, Wayson's staining, or Gra1n staining. Culture is do11e
on blood or intusion agar. Tdentífication is confirrned by
immunofluorcsccnce or bacteriqphage susccptibility.
Mlce or guinea plgs ínjected subcutaneously With Y. pestis
die within 3 to 8 days. DNA techniques utilizing molecular
probes 01 Lhe an1.11lificaliu11 uf sµecific DNA sequences by
the polymerase chain reaction are avail;ihlf'.
Serologic tests (hemagglutination, hemagglutination-
inhibition, enzyme-Jinked immunosorbent assay [ELTSAJ)
are useful for retrospective studícs.
Treatment and Control
If plague is suspected in domestic cats, tl1e follov,rü1g rec-
ommendations from the Centers for Disease Control
apply:
l. Arra11g1:: i111r11c<.liatcly with local and state public
health officials for lahoratory cliagnostic assistancf'
and stcps to prcvcnt sprcad and contarnination.
2. Place all suspect cats in strict isoJ.ation.
3. When handling such cats, wear gown, mask, and
gloves.
4 . 'freat every suspect for íleas (SO/o carbaryl dust for
residual effecl).
flea elimlnat1on should precede rodent control.
Aminoglycosides, ch loramphenicol, fluoroquinolones,
and telracycli11e a1e efft:t.:live a11lí11licrobic.s.
No vaccines for animals are availahle. Prntf'ction of hu-
mans by bacterins is transient.
YERSINIA PSEUDOTUBERCULOS/S
Yersínia pseudoruberculosis is associated with mesenteric
lymphadenitis, terminal ileitis, acute gastroenteritis, and
septh::cn1i<.l, affecling 111<ti11ly l.Ji1u:. a11u 1ulle11L:., auLI uuly
occasionally domestic animals and primates. Yersinia pseu-
78 PART 11 Bacteria and fungi
dnt11herculnsis is closcly rclatcd to Y. pestis, and many con-
sidcr }~ pestis to be a subspecie:;.
Descriptive Features
Cellular Composition and Products of Medica! hnportance
Yersinia pseudoh1berculosis contains the ptasmid pYV (en-
codes the 'l'ypc 111 sccretion system, Yops, and LcrV). A
chromosomal locus called the High Pathogenidty Jsland
contai11s thc genes for iron uptake. Also located 011 thc
chruu1u::.u111e a re ll 11:: g1::11t:::. 1::11coding Lhe proleins Ail, I11v,
YarlA, ancl Gsr.
Adl1esins. l'ersi11ia pseudotuberculosis produces t hree ad-
hesins that are rcsponsible for adherence to~ integrins 011
thc luminal surfacc of M cells and the basolateral surface
of ileal epllhclial cells -Ail, Inv, and Yad:
1. Ai l (for attachment invasion /ocus) adheres to re-
ceptors on thc surface of M cells. Ail al:su ¡.¡rute<.:Ls
the outer mcrnbrane from insf'rtion of the mem-
l;1a111:: allack co1nplcxcs gcnerated by activation of
the complement systcm.
2. lnv (for i11vasin) adhercs to rcccptors on thc surfacc
of M cells and thc basolateral surtace of ileal epithe-
lial cells.
3. Yad (for yerslnla adhesin) adheres to receptors on
thc surfacc of M cells and the basolateral surface of
ílea! e¡.¡it l 1elial Lt:ll:.. Yad aJso pro lec ls ll1e o u ter
m<'mhranc from insertion of the membrane attack
complcxcs gcncrated by activation of the comple-
rnent system .
Ccll Wall. 'l'hc ccll wall of Y. pseudotubercu/osis has O
antigens (smooth phenotype). The cell wall of the mem-
bers of this genus is one typical of gram r1egatives. Thc
lipopolysaccl 1aritle (LPS) í11 tht:: uuter 111e1111Jra11e i:s au iu1-
pnrt;:int vir11 lf'n<'f' c1PtPrminant. Not only is the lipid A
con1ponent toxic (endotoxin), but the length of the side
chain in the 0-repeat unit hindcrs tl1e attachment of the
mcmbranc attack complcx of thc co1nplem ent system to
thc outcr mc111brane. LPS bi n<ls to lipopolysaccharide-
binding prote in (a ~erum protein), which in turn tr<insfers
i L lo lht::: blood phasc of CD14. Tl1e CD14-LPS con1plex
hinds to 'lbll-like receptor proteins (see Chapter 2) on the
surface of macrophage cells triggcring the release of proin-
flammatory cytokines
High Pat'1ngcnicity Jsland. See above, "Yersinia pestis,
<.~ellular Composition and Products of Medica! lmpor-
ta11ce.".
Tro11 Acquisition. !ron l:> an alJ:>ulut1;: gruwth rc4uirt:::111c11L,
;:inc1 m11~t hf' rPmovPrl from iron-hinding proteins of the
host. The genes cncoding product:> involved with iron ac-
quisition reside on a chromosomal pathogenicity island.
Thc HPI of Y. pseudotuberculosis encodes the genes for the
iron-acquiring s1derophorc yersiniabactin.
T)1pe 111 Secretion Syste111. See above, "Yersinia pestis, Cell-
ular Composltlon ancl Proclu<.'t:> of Me<.li<.:al Jru¡.¡urta11<.:e."
<Jsr (Glo/Jnl Stre~~ Rr>r¡11irP.rnmt). SPP a hove, "Yersinia pestis,
Ccllular c:omposition and Products of Medica! Im-
portance."
Toxins. Yersinia pseudotuberculosis produces toxins thac
are involved with pathogenicity:
1. Yops-See above, "Yersinia pestis, Cellular Con1pos~­
tion and Products of Medica! Irnportance."
2. LcrV-See above, "Yersinia pestis, Ccllular Composi-
tion and Products ot Mec1ica11mportance."
Variabi 1ity
Tlrt:re are av¡.¡rvxiu1al1::ly 21 serolypes based 011 variabilit:-
of the somatic (O) antigens.
Ecology
Reservoir
Ycrsinia pscudotubcrculosis is a parasite of wild rodents,
tagomorphs, and birds, but it infects other mamrnals and
reptiles and persists in the environment. The cat is the
most commonly lnfected dome~tic u1au1u1al. M inor eµ i-
demics occur among sheep, pigs, nonhuman primates,
íuwl, a11u JJt:::l bi1ds.
Transmission
Exposure is primarily through ingestion.
Pathogenesis
Yersinia pseudotuberculosis attaches to M cells of lymphoic
nodules of the distal small intestine following cxprcssion
of the cell surface proteins lnv, Ail, and YadA. Attachment
triggers actin cytoskeletal changes resulting in a "zipper-
ing" pl1enomenon that Ieads to the enclosure of the cen
membranc around the attached yersiniae, resulting in
llreir iI1Ler11alizatio1 1. Iule1 nalized n1icroorga11isn1s pass
through to the lymphoi<.I nodule where they are pl1agocy-
tosed by macrophages within the nodulc. Exprcssion of
Gsr permits intracellular survival. Within rnacrophages
(37ºC, low calcium), the 'fypc 111 secretion apparatus is ac-
tivatcd, Yops and LcrV produced. Ingested yersinae are re-
l e:i1:P.ll :iftPr initi;itinn nf ;ipnptn~i~ nf thf' n1;:icroph;:igp
Now extracellular, ycrsiniac lnterfere with furtl1er phago-
cytosis. Invasion of thc basolateral surface oí ileal epithe-
lial cells occurs following attachmcnt of YadA and I11v, and
internalization tollows. J ntlammation induced by extra-
cellular yersiniae (lps), together with interferon secretion
by natural klller cells and gamma delta T cell-recognitior.
of infected epitheliaJ cells (as well as lipopolysac.chariclf'),
1esulls in a11 inll ux of polymorphonuclear neutrophil
leukocytes (PMNs). Extracellular yersiniae avoi<.l phagocy-
tosis (Yops), and dcstruction by complement mediated
mcchanisms by expression of Atl and YadA, both ofwhich
impart complement resistance. Yersinia pseudotuberculosis
acqui res lron from lron-blndlng proteins of t!re hu:st ful-
lowing secretion of yersiniabactin (f'ncoc1f'rl within the
I-IPI). Dia11hea i.s lhoughL to result from prostaglandin
synthesis by the recruited PMNs (and perhaps by the af-
fected host cclls), as wcll as activation of various inositol
signaling pathways within affected 11ost cells. The net re-

s the secretion ot chloride ions and water. Septicemia
-IS fro1n the host's inability to "clear" yersiniae from
~ed sites (scrum reslsta.nce, antlphagocytlc tralts, and
"-"lvenginrr). The rt•sult is enteritis and septicemia.
~:-sfnia pscudotu/Jcrculosis causes il1testi11al infectio11s
- :orn1ation of necrotic foci in the intestinal wall, ab-
.cr=. nal lymph no<lcs, and víscera, particularly livcr and
--,,~. There may be vomiting, ctiarrhea, or constipation,
·eight loss, pale to subicteric mucous Inembranes,
~ ..!.epression. Fever is inconsistent. Few cases are diag-
-d clin ic<illy <intemortem . Mastitis is seen in cattle a11d
\io n i11 ruminants ar1d n1.or1keys. h• in1111unocon"1pe-
-- humans, the disease is generally an enteritis and ab-
-~nal lymphadcnitis that is sclf-limiting or rcsponsivc
~-=-atment.
~ =~-niology
~otuberculosis occurs worldwide. Cases tend to clus-
"' tbe cold months. In cats, prevalence is biased toward
__ :. rural, outdoor cats.
- :-iunologic Aspects
"""-al infection lea.ves surviving individuals in1111une.
-.Lent live vaccin.e protects against homologous chal-
=-~- It is not available co1nmcrcially.
= "'Oratory Diagnosis
--":-:tosjs involves isolation of the agent antemortem
reces or lymph nocte aspirates. lsolation, particularly
_,,_ m ixed sources, is enhanced by cold enrichment,
-- ~, incttbation of a 10% mixture of inoculum in a
- -·~al medium. for severa! weeks at 4 ºC. DNA tech-
....es u tilizing molecular probes or the a1nplification of
'"':ir DNA sequen ces by the polymerasP chain reacti.on
: .!"'ailable.
-·=-atment and Control
'"""otut>erculosis responds to the same antimJcrobics as
_.__e.
: : s'NIA ENTEROCOL/TICA
_ -;:a enterocolitica is associated with rnesenteric lym-
~Lnitis,terminal ileitis, acute gastroenteritis, a11d .sep-
-~ia in domestic animals and primates.
:;;criptive Features
==. _ ar Composition and Products of Mediral lmriort;incP
:i•n,z enterocolitíca contains the plasmid pYV (encodes
:-· pe III secretion system, Yops, and LcrV). A chromo-
Cllapter 10 Enterobacteriaceae: Yersinia 79
soma! locus called the High Pathogenicity Jslanct contains
the genes for iron uptake. Aiso located on tl1e chromo-
some are the genes encoding the proteins .1-\il, Inv, YadA,
Gsr, and Yst.
Adhesins. See above, "'r'e1siniu pseucluluberc,ulu::;í::;, Cell-
ular Composition and Products of Medica! fmportance."
Cell V\fal/. See above, "Yersiuia pscudotuberculosis, Cell-
ular c:omposition and Products of Medical Importance."
Gsr (Global Stress Requiren-1ent). See above, "Yersinia
pestis, Cellular Composition and Products of Medlcal Im-
portance ."
High Pathogenicity Island. See above, "Ycrsíníc.t ]J<:.Slb, Ce\l-
ular Composition and Products of Medica! lmportanc~. "
/ron Acquisition. See above, "Yersinia pseudotu!Jerculosis,
<..:ell.ular Co1nposition and Products of Medica! In1por-
tance."
T'ype 111 Secretion Systern. See above, "Yersinia pestis, Cell-
ular Composition and Products ofMedical !Jnportancc."
Toxins. Yersinia erzterocoliticu produces a 11 ur11 l.Jer uf tux-
ins that are involved with pathogenicity:
l. Yops- See above, "Yersinia pestis, Cellular Cornposi-
lio11 and P1oducls of Medical Irnporta11ce."
2. LcrV-See above, "Yersinia pestis, Cellular C~ornpnsi ­
tiqn and Products of :tvledical Importance."
3. Yst (for Yersinia stable toxin)- Yst is a chromoso-
mally encoded enterotoxin unique to Y. enterocolit
ica. Yst affects the guanylyl cyclase systen1 by dereg-
ulating cGMP synthesis (il1creases in intracellular
cGMP leacls Lo tite oper1i ng of chluride cl 1<:111nel:;
with the resultant flow of ch Inri de. a nd watf'r i ntn
the intestinal Jumen), resulting in fluid and elec-
trolyte accumulation in the bO\-vel lu1nen subse-
quent to blockage of sodium and chloride ion (and
thus w ater) absorption (ti p cells) and loss of chlo-
ride ions (crypt cells) (see also, ST enterotoxin of
Escherichia culi, Chapter 8).
Variability
"fhcrc are approximatcly 34 0 -antigcn une! 20 I-1-untigcn
serogroups. O groups 3, ~. 21, 8, and 9 are associatect with
classical disease of the gastrointestinal tract. ·rhere .are five
biotypes.
Ecology
Reservoir and Transmission
Water. food, soil, fruits, vegetables, and asympton1atic in-
dividuals from 11umans to mollusks have been proposed as
reservoirs for Y. enterocolitica. Expression of certain viru-
lence determinants at 22ºC to 2SºC suggests that mam-
mals acqulre Y. enterocolitica from a "cold" source (water
and food, for example) rather than a warm -b looded ani-
rnal. Infection follows lngestlon of organisms expressing
thP acl hPsin .
Transmission
Exposure is primarily through ingestion.
80 PAl~T Il Bacteria and fungí
Pathogenesis
The pathogcnesis of disease produced by Y. enterocolitica is
thc samc as Y. pseudotuberculosis (scc abovc). In addition to
<11arrhea assoc1ate<1 w1th 1nvas1on ot ep1the11a1 cells an <11n-
flammation (as with Y. pseudotuberculosis) , Y. enterocolitica
produces Yst.
Epidemiology
Certain serotypes are geographically restricted. 0:8 is in-
digenous to the United States but not the rest ot the world.
():3 until recently 'V. as rarely isolated in the United States
1
but is common in the rest of the world, and is becoming
more so in the United States. 0:9 has not been reported
outstde uf Europe.
Tht> rh iPf intPrPst in ;in imal yPrsin iosis derives from its
possible epidemiologic relation to hun1an yersinial infec-
tions. Outbreaks lin ked to anin1als and animal p roducts
havc bccn rare. Serotyplng and biotyping su ggest that
there is littlc relationship between animal strains and
human disease isolates.
lmmunologic Aspects
Ycrsinia cntcrocolitica is an extracellular microorganism.
l'hagocytic cells readily destroy it, even t hough the mi-
croorganism excretes proteins (Yops) that in terfere with
this p rocess. Most o ften, Y. entcrocollttca disease is self-
limiting dueto the innat e immune respo n se: phagocyto-
:.i:., ly:>i:> uf i11fecled epilheli<ll cell:>, i1 011 sequesl1alion, a11d
complement proteins.
A serologic relationshlp between 0:9 serotype, com-
mon in swi11e, and Bruce/la spp. has complicated swine
brucellosis eradication programs.
Laboratory Diagnosis
Samples of feces, lymph node biopsy, and biopsy from af-
tected t issucs are cxamincd microbiologically. Selective
media containing bile salts are somewhat inhibitory to i:
enterocolitica; especially at 37°C. MacConkey agar is least
inhibilory. ·r11ere are special inedia designed for tl1c isola-
tion of Y. enterocolitica (c.g., CIN mcdium). Cold enrich -
mcnt of t hc samplc at 4ºC a ids in attem pts to isolate smal
numbers of Y. enterocolitica from a contaminated environ-
mcnt. Isolatio11 from t issue necessitates t h e use of blo0<..
agar plates in.cub ated at 37ºC. DNA techniques utlJizln ~
molecular probes or t he amplification of specific DNA se-
quences by Lhe polyn1erase chaiJ1 rcactio11 are available.
Treatment and Control
Antimicrobial agents useful for treating disease product;...
by Y. enterocolitica a re the fluoroquinolones, tetracyclinc:
triluet h u pri111-:>ul f u11'11u id es, a nd c11!01 an1pl1e11icol. -
plasmids are comn1on in Y. enterocolitica, and genes ene.oc·
ing resistance to tetracyclinc and strcptomycin are mO'
commonly tound.
Y ERSINIA RUCKE RI
"F.ntPrir rPrlmouth" is a hemorrhagic inflammation of tÍ'"~
perioral subcutis of frcshwater fish, particularly rainlx..
trout. Infection is systcmic and causes significant morta..
ity in 11atcheries of North Amcrica, Australia, and Euror-~
'l'he agent is disseminated by asym ptomatic carrier fi<.
and possibly rip arian ma1nmals (muskra ts). Outbreaks a~
pear to be related to massive exposure.
·rhe patl1ogenesis of this diseasc 11as not been o
scrilJeu. A prutease, Yrp (Ycr:.il1ia ruckeri prule<l:>e) <1¡.>pc.._
to play an important role since inactivation of the encr
ing gene significantly reduces virulence.
Outbreaks are brought under control with antimicr
bies (e.g., sulfonamides, tetracycline, tri1nethopri-
sulfonamide), and bacterins ha ve been successful in 10'' ""-
ing mo1ta\ity .
 Enterobacteriaceae: Shigella
DWTGJ-TT C.
~"'"lwr~ of thf.> genu$ Sfiigella bPlong t o t hf.> f<imily
~cteriaceae, and cause bacillary clysentery in pri-
......::5,. The discussion that follows is limlted to the disease
-,,.,n.,'1uman primates. The disease occurs aln1ost exclu-
!n captive primates and appears to be relatect to
~":l.I situations (c.g., transportation, crowding) or im-
~~u~ogical tly~functiuns (e.g., silnian acquiretl irnmun-
n::·cency syndromr.). Hurnans may he affec.ted hy a li
- ~cies of shigellae, S. dysenteriae, S. flexneri, S. boydii,
_ ~ sonnei, whereas nonhuman prin1ates are affected by
zrr.eri, S. boydii, and S. sonneí.
: a ::riptive Features
-= ~· Anatomy and Composition
..!!atuu1y uf tl1e u1cu1l.Jers uf tl1e ge11us Shi:5t:llu is typi-
- the family 'RntP.rohar.teriar.P.ae. However, they rossess
~' capsules (I< antjgens) nor flagella (IT anti.gens). All
=-~=ss adhesins (IpaD) and a typical gram-negative cell
.:omposcd of lipopolysaccl1arides (O antigen) and
-ens.
~-;.ere are four serotypes, identification of which is de-
ie=.~nt upon the con1position of the 0 -repeat units of
-popolysaccharide (LPS). Each of the four serotypes
~n given species desig11alion. Will1iI1 each species
-o: are a number of serotypes (Table 11.1). Conversio.n of
.>crotype to anothcr is undcr thc rcgulution of thc
= ...:es::::i con tained within a l'athogenicity Jsland (Shi-0 for
~a lsland) a cluster of genes encodingvirule11ce deter-
--r2,..l\S), an integrase protein, a specific insertion site,
::rrobility. ln t he case of Shi-0, the genes are contained
.na defeclive (in1n1obile) lysogenic bacleriophage, so
- crological traits are stable.
•,._ ar Products of Medical lnterest
".!SinS. A su rface protein, IpaD (for invasion protein anti-
·s responsible for adherence of shigellae to g integrins
~e surface of M ceUs, and lhe basolaLeral surface of
~ -e ~ ntestinal epitl1elial cells.
::dJ Wall. The cell wall of the 1nembers of this genus is
~-ypical ot gram negatives. ·rhe lipopolysaccharide
fS in the outer membrane is an important virulence de
""'"..:;,,nant. Not only is the lipid A com.ponent toxic (endo-
•: x1a,, b ut the length of the side chain i:n the 0 -repeat
~ - hin de1s Lhe aLLacl1111.eul of Llte 111e1111Jraut'. attilck cuu1-
- _ of the complement systen1 to the outer memhrane.
HTRSH
LPS binds to lipopolysar.r.haride-binding protein (a serurn
protein), which in turn trans(e.r:s il lo tl1e blood phase of
CD14. "l'he CD14-LPS complex binds to Toll-like (eceptor
protcins (scc Chaptcr 2) on thc surfacc of n1acrophagc cclls
triggering tl1e release of proinflammatory cytokines
"lnvasion" Proteins. Virulence factors (invasion pro-
teins) produced by members of this genus are mainly en-
cocti:>ct on l;:irge p lasmi.ds (terrn.ed invasion plasmids). 'fhese
genes, for the n1ost parl, are regulaled by al leasl si.Ji. chro-
mosomal genes. The plasmid gene products cncode pro-
teins that are rcsponsiblc for a 'fypc III cxcrction apparatus
whereby son1e i11vasion proteins (lpa for invasion plasmict
antigen) are "in jected" in to host cells, namely lpaB,C. 'fhe
·rype III secretion system con sists of an assemblage of pro-
tf'ins (morf> than ?.O) that form a hollow tube-likc structure
through vvhich effector protein.s are "il1jected" into hosl
"target" cells:
l . lpal3. "Injection" of lpal3 (like IpaC) into the host
target cell leacts to activation of small CiTP-binding
proteins of tl1e Rl10 family resulting in cytoskeletal
changes anct the for1nation of "ruffles." IpaB also is
responsible for lysis of the membrane of t he pl1ago-
so1ne contalnlng entrapped shigellae. Wlthln
macrophages, IpaB activates caspase-1 resulting in
a popto:;is.
2. lpaC. "lnjection" of lpaC (like lpaB) leads to activa-
tion of small GTP-binding protcins of thc Rho fam-
ily resultíng in cytoskeletal changes and the forma-
tion of "ruffles."
3. IcsA. The surface prot ein IcsA (for intracel.Jular
spre;id) is required for intracellular sp.read . lcsAis re-
quired and is sufficien.t for actin deposition and
motility \\7hen shigellae are within host epithelial
cclls.
4. lcsB. 'l he surface protein lcsH (tor intercellular
spread) is for intercellular spread.
Enterotoxins. Two e11terotoxins have been described,
ShETl (Shigella entcrotoxin, encoded within the chromo-
sor ual Pa Lhugeuicily Isla11<.l, Shi-1- a cluster of genes en-
coding virulence determinant(s), an integrase proti=>in, a
specific insertion si.te, an.d mobility) and a 63kL)a product
of the sen gene (Shigella enterotoxin, encoded on the inva-
sion plasmid). How these proteins elicit fluid sccrction is
not known.
Exotoxins. Shigella clysenteriae is the only member of the
gruup that has t he genes necessary for production of shiga
toxin . Shiga toxin is chron1oso1nally encoded . 1'he toxin is
81
82 PART 11 Bacteria and fungi
Table 11.1. Species of Shigella
Species Group {Types)
S. dysenteri.ie A (1-10)
S. ffexneri 8 (1-6)
S. boydii e c1-1 s¡
S. sonnei D (1)
a prolein of 70,000MW composed of an A subunit
(32,000MW) and íivc ident ical B subunits (each about
7700MW). The targct cclls fo r thc toxin are thc cndothclial
cells that linc blood vcsscls. lleceptors on these cells are
recognizcd by thc B suhunit. The toxin, by vvay of the A
subunlt, inhlblts peptlde ct1a1n elongation at the level of
the rihosome by affecting elongation factor 1-clf'pf'ndf>nt
processes. 'l'his actlon rcsults in the death of the cell. ·rhe
production of toxln is iron-regulated (by way of Fur. see
bclow), more bcing produced in conditions of low iron
concentration. 1he v1rulcncc of a particular strain or iso-
late is directly related to the amo unt of toxin produced.
Other Exotoxins. Shigcllac produce at Ieast two other ex-
otoxins that, at least theoretically, play a role in pathogen-
esis. 'l'!Je~c twu ¡.¡rulciu:> i:1fl.'. SigA a11Ll PiL:
1. SigA (for ShigeUa lgA protease). SigA is postulated ro
play a role by inactivating Shigella-specific lgA in the
intestinal tract, therelJy i:1lluwiug tl1e Llisease ¡.¡ro<.:<:::>:>
to progress. SigA is encoded withln the chromoso-
mal Pathogenicity Island, Shi-1.
2. Pie (lor protcin involved in intestinal colorúzation).
Pie is a scri ne pro tease, v•lhich is thought to digest
intestinal mucus that overlays the intestinal epithe-
lial cells so that shigellae have access to host "tar-
get" ccll~. 'J'l1c gcuc:1 c11<.:uui11g Pi<.: ar<:: willli11 ll1e
chromosomal Pathogpnicity lsland, Shi-1.
Regulatory Gen es. Shigellae possess a number of genes
whose actlvatlon results frorn certain envirunmental cues.
Thf>SP inchu1P i:nr ano íln RNA polymPrasp containing
RpoS:
l. Fur ((erric utilit:c1tiuu rcspu11:1c-iru11 ltvels). Tl1e Fur
systPm "sPnsps" avai lahlP iron conccntration, and
when low (as would be inside the host, since most
iron is bound to host iron-binding proteins), acti-
vatcs thc synthcsis and sccrction of the sidero
phorc, acrobact1n, and sh1ga toxin (see above).
Genes \AJithin the Pathogenicity Island Shi-2 encode
Fur.
?.. RNA polymPra<;P. R A polymerase containing RpoS
prefercntially transcribe:> gene:> re:>ponsible for acid
tolerance (surviva1 at pH 5), pern1itting safe transit
t hrough thc stomach.
Variability
Slúgellae are "typP<I" h;ispc1 11pon thP makeup of the 0-
antigcn (spccies designation). Within each specie:>, there
are a number of serotypes (see ·rablc 11.1).
Ecology
Reservo ir
The reservoir for members of the genus Shigella is thc largc
bowel of clinically ill, recovered, or asympton1atic pri-
mates
Transmission
'fhc discasc is transmitted by the fecal-oral route, but the
iufcctivc du:>c i:> :>V :>111all lllal fon1iles n1ay also play a role.
Memhers of the genus Shigella are grcatly jnfluenced by
thc colonization resistance (a n1easurcmcnt of thc "bar-
rier" effect the normal flora has in cxcluding ''foreign" mi-
croorganisms from the tract) in the large bowel. Antimi-
crohial clrugs, stress, or dictary changes pron1ote rlsk by
reducing colonization resistance (by decreasin8 the n1.11n-
l.Jers uf liH.: J1ori11al l1v1a resulling i11 a reduction of the
"harrier"), which may lead to diseasc in asymptomatic car-
ricr animals o r by lowcring thc orul do:;c nccdcd for infcc
tion and subsequent disease.
Thc 1node of transm ission of S. fle,Yneri 4 associated with
periodontal dísease of nonhumar1 primates is unknown,
but it is assumed to be feces.
Pathogenesis
Envíronmental cues triggt>r thP Pxprf>ssion and excrction
of virulence protcins. Ingested shigellae safely traverse the
stomach (RpoS dircctcd acid tolerance response). 'fhe tar-
gct cclls are thc apical surface of M cells in the large íntes
ti11c to wruch sh1gellae attach by way of IpaD after diges-
tion of thc ovcrlying mucus !ayer (Pie) . Cell-associated
bacteria are rrappe<l ln the "ruffles" formed follow111g the
"injection" of IpaB,C by way of the Typc IIT secretion sys-
le1n. Son1e of lhe shigellae witl1i11 the vacuole are trans-
ported into the nodule, where the uptakc is again trig-
gered, but by macrophages within thc nodulc. Ingcstcd
shigellae may initiatc apoptosis o~ the macrophage (lpaH)
and/or other abnormalities reflected in a drop in concen-
tration of lntracellular adenosine t ripl1ospl1ate (ATP).
Either effect results in the dcatl1 of the m~crophagP. Thr
libcratcd shigcllac adhere to the basolateral surfacc of thc
colonic cpithclial cel l (TpaD) and induce their own uptake
as bcforc. Shigcllac escape.: thc.: vacuolc by sccrctio11 of IpaB
resulting in thc deposition of the microorganism into the
cytoplasm of the epithelial cell. Expression of les and for-
matlon of actln at one pole of the bacterlum results in in-
tracellular movernent. Movement appears to hf> more con-
centraled along actin "stress fibers" and shigellac with
their actin "tails" move toward cadhcrin proteins marking
intcrccllular bridgcs. Thus, shigellae n1ove laterally. IcsB
lyses the aouble membrane resultmg úom movement be-
tween cells. Affected host cells together with dead and
dying macrophages secrete varlous chemokines (i11<.:lud-
ing TL-1 and IL-18), which rcsults in an intPnSP inflamma-
lo1y 1esponse. Likcwisc, cxtracellular shigellae also initiate
inflammation (by virtue of their LPS). Transepithelial mi-
gration of polymorphonuclcar ncutrophil leukocytes
(PMNs) is stimulated l)y Ll'S. Natural Killer cells llave been

-plicated in lysis of infected epithelial cells, a11other
-:iamn1atio11-inducing process. l'issue destruction with
-.;...11erous PMNs is the hallmark of dysentery caused by
¡¿jgellae. 1'he origin of hemorrhage p roduced by non-
~ga toXin-producing shigellae is not known, but the tis-
-t destruction, ~vhich includes endothelial cell damage,
;¡robably responsible. PMNs phagocytose and d.estroy
~ gf'llae, lim.itiog the process to the intestinal tract.
!:>iarrhea is brought about by the activation of phos-
-::-;olipase e (perhaps dueto "ruffle" formation) leading to
-creases in intracellular calcium io11s, activation of pro-
~::::: : kinase e and subsequent pnosphorylation ot proteins
-= rhe chloride ion cha11nels a11d those of tl1e membrane-
~d ated ion transport proteins involved In NaCl absorp-
ti. Thf'Sf' activitif's Jp;id to cii;irrhf'a .
The role of enterotoxin in this disease is unclear. The di-
::.dlea, sometimes watery, may be caused by interactions
"'e11terotoxin \vith small intestin.al epithelial cells and in
- -'-r b e due to changes in t he colonic epithelium brought
mout by tl1e inv asion/ir1flammatory process.
Stti¿;c:llu dy~c:nleriue pro<.luces shiga toxin. This toxin
:a;nages suhmLJc.osal P.nclot ht>lia l ct>lls (ht>morrhage) and
,....ay cause t h e development of hen1olytic u ren1ic syn-
..::om e (HUS) (see Chapter 8).
Shigella flexneri 1 has been en.c ountered i n periodontal
sease of monl<eys. A causal role is suspected but unde-
:::::ed.
;:: ;:;:miofogy
--¿ disease is seen almost exclusively in captive primates.
- ""IU nologic Aspects
- ~ion from bacillary dysentery is by specific secretory
:mmoglobulin fOUild Oil tl1e lu111iual Side Of lhe inles-
- - G1na l. ·rhPsP. antihorliP.s prevent adherence and suhse-
n-t u ptake. Shigellae are serum-sensitive (i.e., they are
~:.Jy destroyed by complement-generated membrane
~ck complexes), and PMNs deal with them effectively.
,;. :-esult, extracellu lar shigellae (those outside of the M
ma crophage, and colonic epithellal cell) are dealt
effectively !Jy coruµleu1e1rt µrotei11s iu Ussue OuiJ:;,
......:. the c.onstit11P.nts of t he inflammatory exudate (com-
=ent proteins and PMNs).
- n ether n onbuma11 primates after infection and dis-
......_-.¿ are resistant to reinfection ( exacerbat ion) is not
--11. Reinfection or exacerbation occurs in human be-
;s in st ressful situations such as in prisoner-of-"1-,rar
"'"""p5. Bacterins gi ver1 o rally or pareuterally llave IJeen in-
--::rtve. Somf' protP.ction has hP.en demonstrated follo,.,-
-:: '"accination with avirulent, live oral vaccines. These
-= =ot univer.sally available.
-=.:ioratory Diagnosis
-=-r~l swahs or samplP.s of ff'C.f'S ohtainf'cl from thf' rf'ct1 1m
=oo!lected for laboratory diagnosis. Direct exan1ination
;.:2ined smears of fecal material V'.1ill reveal the presence
Chapter 11 Enterobacteriaceae: Shigella 83
of inflammatory cells, cellular debris, and red blood cells
(RBCs). Such a fi11dil1g is 11ol diagnoslic, however, since en-
teritis produced by Campylobacter results in the same signs.
Thc prescncc o,f curvcd rods in t he direct smear suggests
that Campylobacter is the cause ot the disease.
The sa1nple is plated onto a selective medium that is less
inhibitory than media for isolat.lon of salmonellae.
Suitable media include MacConkey agar, XLD agar, and
Hektoen. E11teric n1ediun1. SS agar is often Loo i11hihiLory
for sorne strains of shigellae, and hrilliant grf'f'n clot>s not
work at ali. Por enrichmen t, GN bro th is preferred. Selenite
or tetrathionate broths do not enrich for shigellae.
Shigellae appear as lactose-nonfermenting colonies on
lactose-containing media. Although sorne species (S. son-
nei and S. boydii 9) ferment this sugar, n ot enough is fer-
n1e11ted will1in lhe 24- to 48-h o u r incu1Jalio11 µeriotl to
affect the selection of appropriate colonies for further test-
ing. Suspicious colonics are tested directly with shigellae-
specific antisera or inoculated into ditferential media and
then tested with antisera.
Primers have been desigoed to amplify (by the poly-
merase chain reaction) D N A specific for shigellae. This
assay 11as bee11 used lo detecL aud i<le11 ti (y rnernlJers uf this
genus.
Treatment, Control, and Prevention
Treat1nent of siligellosí.s involves nursing an d supporting
care. A11lin1icrobics are iI1dicated in serious cases buL uul
routinely since their use in an anin1al facility selects fo rre-
sistant strains. 1rimethoprim-sulfonamide, or a fluoro-
quinolone, is effective against most strains and these drugs
do not disrupt the normal flora (colonization resist ance)
as much as other antimlcroblals. The yeast Saccharomyces
boulardii has been shovvn to reduce susceptibility of exp er-
i111enlal anin1als to challenge by Shigella flexneri, suppos-
edly hy interfering with attachment of shigellae to host
target cells.
 Pasteurellaceae: Pasteurella,
Mannheimia
DWIGHT C. HlRSH
'fhe family l'asteurellaceae contains the genera Actinobadl-
lus, Gallibacteriu1111 Hae11·1ophilus, Histoplúlus, Lonepinella,
Mannf1eirnia, Pasteurella, anct /lhocoenobacter. AH but Lone-
pinella (L. koalaru1n-a tannin degrading bacteriurn iso-
lated from normal Koala feces) anct (iallibacteriurn (G.
anatis-isolated from the intestine of n orm al ducks) con-
Lai 11 :)¡Jt::L'it:::) l l 1<:1 l <:11 e VÍ 11 11;:úi\.:al illl¡JOita11ce.
All the memhers of the family Pasteurellaceae are gram-
negativc coccobacilli. Thcy are facultative anaerobes, and
typically oxidase-positivc (which sets them apart trom
me1nbers of the family Enterobacteriaceae). Most are com-
mensal parasites of anlmals.
·rhe genus J>asteurella, particularly P. haemolytica, has
u11dergone extensive taxonomic changes over the past sev-
<'ral yPars. l'n~tl'11rPlla hnPmnlytirn ¡¡tone time contained 17
capsular types, and 2 biotypes (A for arabinose ferme11la-
tion, and ·r for trehalose fermentation). Thus, each strain
of P. haen1olytica was idcntificd by a lcttcr (cithcr an A or T)
aesignating tne b1orype, a11d a number (l- 1/J aes1gnat1ng
the capsular type. For exa1nple, P. haernolytica of the A bio-
typc posscssi11g a lype 1 capsule was úe:)ig11<:1ted as P. hue-
rnnlytica /\ 1. C.i>nPtic :in:ilysis <;howed th¡¡t all of the A bio-
typcs con1priscd a new and separate genus, dcsig11ated as
Man11heirnia. 1'hus, al! but one of the P haernolytica A bio-
types beca me Mannhcirnia hacrnolytica. Thc onc cxccption,
P. haemolytica A11, beca me a diffcrent species, M . Xlucosida.
All o f the ·r biotypes wcre p laced i11to a new species, P. tre-
hulusi. C0Hse4uently, "Pasteurclla haenzolytica" is obsolete.
Discusse<i in this chaptPr arf' thf' g<'nf'ril PasfP.11rella and
Ma1111hei111ia, whose men1bers p lay an in1portant role in
discascs of severa! animal species (Table 12.1). Photobacte-
riu1n da111scla (prcviously Paslcurc//a piscicida) and Rieme-
rella anatipestifer (previously l'asteurella anatipesti(er) wi 11
be discusscd briefly, as will Eugonic fermentor 4 (EF-4) a
bacterium that resembles Pasteurella.
Descriptive Features
Morphology and Staining
Members of the genera Pasteurella and i\4.annheimia are
gram-negative coccobacllll measuring 0.2 µm by up to 2.0
pnL Bipolarity, that i~. thf' <;tainíng of only the tips of cells,
is demonstrable with polychron1e stains (e.g., Wright's
stain).
84
ERNST L. BIBERSTETN
Structure and Composition
Capsules contair1 acidic polysaccharidcs. ·r11e capsule of ]).
multocida typc A is made of hyaluronic acid, the capsule of
type D is made of hl:!parin, and the capsule of type F is
1nade of cho11droilin (see below, "Variabilily," for discus-
sion of P. multocida capsule nomenclature). Sorne P. multo-
cida and M. hae1110/ytica strains express adhesins.
Cell walls are typical of gram-negative microorganisms
consisting mainly oí lipopolysaccharides and proteins.
Sorne of the latter are iron-regulated (Le., they are ex-
pressed undcr iron-poor conditions).
Cellular Products of Medica! lnterest
Adhesins. Sorne, and probably all members of the famil y
Pasteurellaceae p1oduce adl1esins (aud possibly 111ure tha11
one kind). A type 4 fimbria (adhesin) has been descrihed
for avían :;train:; of /'. 1nultocida, nnd nn adhcsin (tcrmcd
Adhl) 1s expressed by M. haernolyttca. As with other mi-
croorganisms, the expression of adhesiJlS probably de-
pends upon envlronmental cues. That is, adhesins are ex-
pressed while the microorganism inhabits an epithelial
surface, bul repressed when lhe 1nicroorga11isu1 is iuside
the host where adherence to a phagocytic cell could he dis-
astrous. M annheirnia haernolytica and I~ trehalosi produce
fibrinogen-binding p roteins. 'lhe role of t hcse proteins is
unclear at present, but in the streptococci (see Chapter 28)
flbrlnogcn-blnctlng protelns lrnpart a11 antiphagocytic
propPrty to thc~ strcptococcal particle by "coating" the bac-
teria! cell, thercby "coverillg" sites for con1plen1e11t activa-
tion (and thus dccrease opsonization, and the gcncration
of functional mcmbranc attack complC){cs) as wcll as those
recognized by serum proteins (collectins/ticollins) thatop-
sonize foreign particles. 1·he hyaluronic add capsule of
type A strains P. mu/cocida serves as an adhesin probably
simil:ir to hyaluronic ¡¡cid encapsulated Streptococn1s pyo-
gc11cs (Chapter 28), which binds to human epithelial cells
via CD44, a hyaluronic acid-binding glycoprotein.
Capsule. Thc capsule plays many roles, thc most impor-
tant of which are interference \vith phagocytosis (an-
tiphagocytic) and the protection of the outer membrane
from thc deposltion of membrane attack complexes gencr-
ated by activation of the complement system. The amount
of capsule produccd is .in.versely proportional to the
amount of available iron . Tn vivo, where the amount of

Ta b 1e 1 2 . 1 . Memher~ of the Genera Pasteurella. Mannheimia, Gallibaeterium, and
Phocoenobacter and Their Usual Source or Associated Condition
Species
Pasteurel/a aeroqenes
P. avium
P. bettyae (CDC Group Hli·!>)
P. caballi
P. canis
P. dagmatis
P. gal/inarum
P. langaa
P. Jymphangitis
P. mairi
.f . 111ullvtitld
P. pneumotropica
P. stomafis
P. ~kyenu~
P. 1es1uúi11i>
P. trehalosi (P. haemo/ytica T-strainsl
P. volantium
Mannheimia haemofytica (Pasteurella
hacmolytico)
M. granulomatis
M. glucosida
M. ruminalis
M. varigena
Gal/ibactcrium JnJtis (PJstcurc//¡¡ anatis)
Phocoenobacter uteri
.._ 11hle iron is low, the amount of capsule forn1ed is lcss
.ililcient to protect the microorganism from phago-
- -.,., and comple1nent-medialcd lysis). Pastcurclla multo-
t -pe A capsules are made of hyalurontc acid, type V
~sw.~s are made of heparin, and type F capsules are inade
nd rol t ln (see below, "Variabillty," for discussion of P.
~-·""11 capsule nomenclature) . These substanccs are
~~¡ if not ide11tical) lo hosl Lissue cu111pur1er 1l:;, au<J arl:'
¡oorly antigenic, as wcll as binding complement
'1Cnts poorly (and are therefore, antiphagocytic).
·-a1uron1c ac1a capsule also serves as an adhesin tor
-_rory tract epithelial cells as in thc case of capsule
---..... :.Lrai11:; of !>. multocida (see above).
' \'n i/. Lipopolysac.chariclP (1.PS) i>li rifs <1n i11flamma-
-.. sponse following binding to lipopolysaccharide-
n~ ; protein (a serum protein), which in tur n transfers
e blood phasc of CD14. 'J'he CD14-LPS complex
~:.:;. · Toll-like receptor proteins (sce C hapter 2) on lhe
i macrophage ce lis triggering the release of proin-
:..:::=~ . ~u ry c.ytokines. In addition, LPS is not only direct:ly
-t>c;piratory tract epithelial cells, but also acts syn-
_ .;.....;;a ·~ \vith thc M . liur:rnofylit.u h:ukvtvJliu t!JaL aífe<.:L:>
- - - macrophages (see belO\>V).
CfzaptPr 17. Pnsteurellaceae: Pasteurella, Mnnnheimia 85
Usual Soun;e or As50ciated Condition
Associated with gastroenteritis in swine; abortion in swine; fowl
Rcspiratory tract of fowl
Human Bartholln gland abscess; septicemia in immunocompro-
mised patients; periparturient septicemia in human infants
Pneumonia and assoclated conditions in horses
PnP.umnni;i in n09s; nther •mouth" related conditions in dogs
Respirato1y lldtl ol doy~; 1~pi1dt01y lract of fowl
Respiratory tract of fowl
Respiratory tract of fowl
Bovine lymphangitis
Abortion in swine; septicemia in piglets
Pneumonla and resp1ratory condltlons in ruminants, swine, dogs,
and cats: other "mouth" related conditions of dogs ;md rats;
septicemia in ruminants; septicemia in fowl; absces~s and
pneumonia in rabbits
Pneumonia in rodcnts
Pneumoma m dogs; other "mouth" related conditions in doc¡s
Septicemia in farmed Atlantic salmon
Respiratory traas of desert tortoise and box turtles
Pneumonia and septicemia in niminants
Wattles of fowl
Pneumonia and respiratory conditions in ruminants
Panniculitis in ruminants; respiratory conditions in ruminants, deer,
~nd h~rQs
Resplratory condltions of ruminants
Norm;il flor;i nf r11mPn
Respirato1y tract condition~ ul 1u111i11dr1l> anu swin~; ~~pticemia in
ruminants and swine; mastitis in cattle
Intestinal contents of normal duck5
Uterus of harbar porpoise
Toxi11s. Jvfannheirniu aud Pu:,leurellu µrotlu<.:e a number
of protcins witl1 toxic activity. At least two of th ese are im-
portant in thc pathogenesis of disease (i.e., reduction of
virulence results trom inability to produce these toxins):
an R'l'X, and a Rho activating toxin:
l . RTX toxin. The R'l"X (repeats in toxin, so called be
cause of the common feature of repeats in glycine-
rich sequences within the protein) type of toxi11 is a
leukotoxin (Lkt) produced by M. /Juernulylica, P. Lre-
/1a/osi, and P. g/ucosida (a comparable toxin is pro-
duced by P. aerogcnes, P. 1nairi, and M. varigcna).
Though similar 111 amino acid sequence to otl1er
mcmbers of the RTX family (sce also l!.scherichia he-
cuulysins ir1 Ctiapter 8, Actinobac//lus haemolysin in
C:haptP.r 1'.~, a<IPnylyl cycl<isc toxin of Bordetella in
Chapter 15, Moruxe//a cylulox.i11 iu Chapter 19), Lkt
specifically affects bovine leukoc.ytes, an<I Prythro-
cytcs. Thc cffect on erythrocytes probably has little
importance in vivo, but is responsible for the he-
molysis observed when M. hacrnolytica, P. trchalosi,
an<l P . glucostaa are grO\>VD on blood agar ptates. LKt
hinrl.; to CD18 (a í?.2 integrin), which is expressed on
86 PART 11 Bacteria and Fungi
thi> s11rfr1ci> of hovinc leukocytes (neutrophils, ma-
crophages, platelets, and lymphocytes). At Jow con-
centrations, Lkt causes target cell activation, at
highcr conccntrations, apoptosis is initiated, and at
very h1gh concentrations, necrosis is produced (due
to production of transmembrane pores). Upon con-
tact wlth Lkt, neutrophils degrauulate releasi11g vo-
tent enzymE'~ th;it proc111ce ;is well as aggravate in-
fian1n1ation, macrophages rclease proinflammatory
cytokines (magnified in the presence of LPS), Jym-
phocytcs undergo apoptosis and necrosis, and
platetets respond by increased adhesion. In addi-
tion, tissue mast cclls degranulate releasing vasoac-
tive amines. In summary, LKT drivt:s CJ ti:>:>ue-
dcstroying inflammatory responsf'.
2. l\hu CJcli valing Loxh1. 'f!1e Rho activating toxin is
procluced by l~ rnultocida capsule type D, (see below,
"Variability," for discussion oí P. multocida capsule
1101nenclature) associated with Atropl1ic Rhil1itis of
swinc. 'fhe toxin, Pmt (P. rnultocida toxin), stimu-
lates two dlfferent signallng proteins: the :>111011
GTPase Rho, and heterodimeric G proteins. l JnlikP
similar tuxio:. (CNF p1uduced by Escherichia coli, see
Ch;iptPr 8, ;incl dermonecrotic toxin produced by
Bordctclla bronchiseptica, see Chapter 15), Pmt docs
not enzymatically affect either ot these regulatory
protcins. However, the interaction of Pmt with ei-
ther results in toxicity mediatcd by increases in ln-
tracellular calcium. ln addition, Pmt binds to vi-
men tl n, an intract!llular iuler111ediale filan1ent
respon.,iblP for provirling mechanical strength to
the cell.
3. Miscellaneous toxins. Sorne strains of P. multodda
produce hyaluronidase and neuraminidase. The
role played by thcsc cnzymes in the pathogenesis of
disease is unclear. It is tempting to speculate that
hyaluronldase Is actlVt! ÍIJ vivo auu 111ay be 1espon-
sible for the m icroorg;inism 's ahi 1ity to "spread"
Lhrougl1 tissue. Neuraminidase has been postulated
to play a role in the colonization of epithelial
surfaccs by rcmoving tcr1ninal sialic acid residues
trom rnucin, thereby n1odlfying normal host innate
ilnmunity.
lron Acquisition. Rpc;iusP iron is an ahsolute growth re-
quire111enl, n1icroorganisms 1nust acquire this substancc
if they are to cxist within the host. Sorne avian strains ot
P. 111ultocida produce a sidcrophore, multicidin, which is
neither a pnenotate nor hydroxynate type siderophore.
Siderophores have not bcen demonstrated in Pasteurella
or 1\lfannheirnia fru111 ul11t:r sources or species. However,
Pasteurel/a and MnnnhPimia hind transferrin-iron com-
vlexes by virLue of iron-regulatcd outer membrane pro-
teins expressed under iron-poor conditions (so-called
transferrin-binding proteins, or Tbps). Iron is acquircd
from the transterrin-iron complexes that bind to the sur-
face of the microorganism. Pasteure/la multocida also
binds heme and hemoglobln, which are utlier pu~~ible
sources of iron.
Mi:.1..elluneous Products. Sorne avían strai ns oí P. 1nulto-
cida express an outcr-membrane protein that is toxic to
phagocytic cells. lt is unclear what role this protein might
play in thc pathogcncsis of disease. If capsule íormation is
down regulated in vivo dueto lo"v available iron concen-
trations, thcn the toxic outer membrane protein might
serve to protect the m icruurgo1J.i:>1u fru111 vhagocylosis.
Growth Characteristics
Pasteurell11 and Ma1111llei111ia grow best in the prcscncc of
scrum or blood . After overnight incubation (35- 3/"C),
colonics are about 11nm in diameter, clear, and smooth or
mucoid. Mannl1eirnia flae"1olytica, P. trehalosi, and P. glu-
cosida produce hemolysis on ruminant blood agar. Ali are
gram-negatJvc, nonmotíle <.:u<.:culJat:illi. Tl1ey are faculta-
tive anaerobes, typic;i lly oxirlasP.-positive, reduce r1itrates,
and allack carbohydrates fermcntatively.
Resistance
Cultures die within 1 or 2 weeks. Disinfectants, heat (.SOºr.
for 30 minutes), anti ultrCJviuh.:t ligill are p10111ptly lethal.
Pasteurella n111/tocida survives for months in bird carcasses.
Pu:,leurellu and Mannhei111ia, especially from food ani-
mals, have become increasingly resistant to penicillins,
tctracyclines, and sulfonan1idcs, to which they were origi~
nally susceptible. The gene encoding tetracycline resist-
ance is unique to J>asteurella and J...fannheitnia. Resistance
encoding genes are frequently assoclated with R pla:.uliu:..
Variability
Pasteurella multulida cuu:>i:.t:. uf five capsular (A, B, D, E, F)
and 11 ~om;itic (1-11) serotypes occurring in 20 different
con1binations. Serotypes are often related to host spcci-
ficity and pathogcnicity. The serotype is designated with a
lcttcr dcsignnting the capsule type anda number designat-
ing the soma tic type, e.g., A: l.
Mannheirnia haetnolytica consists of 12 capsular types (1,
2, 5-9, 12-14, 16, 17), P. lrehulu:;i, fuur. (3, 4, 10, 15).
Ecology
Reservoir
Pasteurclla and Mannheimia are carried on mucous mf'm-
branes (most commonly in the uruvl1a1yngeal region) of
susceptible host species (;iciherence to these surfaces is due
lo adheslns). Carri11ge m11y be widcsprcad, a:; with P. multo
cida in carnivores, or exceptional, as \-Vith aVJan cholera-
producing strains in birds or hemorrhagic septicemia-
producing stra1ns 1n ruminants. In avian cholera, one ho:>t
species may serve as reservoi r for another.
Transmission
Infection is by inh;il;ition, ingestion, or bites and scratch
wounds. Many infections are probably cndogcnous. In
bovine hemorrhagic septicemia and avian cholera, envi-
ronmcntal contamination contributes to indirect trans-
mission.

;;thogenesis
cc1zanisn1s.In general, there are thrcc manifcstations of
- ~teurella/ Mannheimia-inctuced disease: respiratory tract
-;;oJvement, septicemia, and trauma-associatcd condi
- ::>ns:
l. Respiratory tract involven1ent is e ither pneumonia
or uppcr tract disease (Atrophic Rltinitis in swinc).
P11~u111u11ia is seen wu:st frequently in ruminants
and is usually associated wi th M. liaP111nlytir.a, P. rnul-
tocida, or I'. trehalosi. Environmcntal stress (e.g.,
shipping, weaning), virus infection, or other bacter-
ia! infections (e.g., Mycoplas111a) usually precede
pncumonia, and are thought to decrease host de-
fenses of the tract allowing commensal bacteria liv-
ing in Lile up¡;er Lra<.:l (u1usl <.:uuuuunly M. haemoly-
tica, P. multocida, or P. trehalosi) to infec.t tht> lt1ng
(1nblc 12.2). Mannhei1nia liaen10/ytica/P. trehalosi
trom the upper respiratory tract is deposited in the
Iung, and secrete Lkt and, along with LPS from thclr
ccu walls, initiate a11 intense inflammatory re-
sponse with fihrin deposition. Though P. 111ultocida
has nol bee11 showu Lu ¡;ruLlu<.:c Lkt, it:s deposition
in thc lung initiates an inflammatory rP_<;ponsP hy
virtuc of havlng LPS. Capsule, iron-scavenglng abil-
ities, and pcrhaps binding oí fibrinogen enhance
survival of the bacteria within the Iesion .
Upper tract disease is lirnited to Atroph1c Rl1initis
of swinc. The serious (or progressive) form of this
ubcasc is une in which Bordetel/a bronchiseptica (see
C:hfl ptt>r 1.'i) fírst ;iclhPrf'<; tothf' n;;¡~;il mucosa, and se-
cretes a toxin, called dermonecrotic toxin, wl1ich
mildly damages the epithelium. Capsule type D
strains of P. multocida adhcre to this mildly damagcd
epithelium and secrete l'mt (thesc strains do not
readily adhere to a normal epithelium). It is Pmt that
is responsible for the destruction of the nasal turbi-
nates. The action of B. bronchiseptica toxin alone,
wilhoul Lhe parUci¡;aliou of P. r11ulluLidu, results ir1 CI
1nild, nonprogressive turbinate hyp()plasia.
2. Scpticcmic disease produced by members of the
family l"asteurellaceae is almost cxclusively caused
by Pasteurella in ruminan ts (hcmorrhagic septice
mla In cattle; septicemia in shccp) or in avían spe-
cies (avian cholera). Why these strains produce
septlcernia is not understood. HO\\l'ever, capsule
-i:ile 12.2 . Etiologic ond Possiblc Contributory Factors in Bovine Shipping rever (after C.A. Hjerpe)<
Bacteriaª
•.fannheimia haemo/ytica Mycop/asma bovis
"'osteurella multocída Mycoplasma dispar
- IS!ophllus somni Ureaplasma
O-Jier bacteria uncomrnonly associated: Salmonell~. Streptococcus. Staphyloroccm aur1>1K, F<fhl>rirhi;, rnli. Arrñnobactetium. pyogenE<, obfrgate anaero~, Ch~mydfa.
"'he< viruws uncommonly associatod: bolñne virus diarmea virus, >donoviru<, rhinoviM, reovirus, hcr¡>C$VÍ<U$, cntcrovirus, ca!i<lvirus, and coronavinn.
"crpc, CA. Bovinc respiratory disease complex. Curr Vet Ther 198ú;2:G70.
r.ltapter 12 Pa~tr.11re1laceae: Paste11rella, Ma11nheirnia 87
and iron scavenging are crucial for survival of the
organisms within the host. Outer membrane pro-
tPins on ;ivi;in <;tr;ii ns of P 1nultocida appear to play
sorne role by decreasing phagocylosls. Signs aud
outco1ne of this disease are attributable to endo-
toxcmi.:i (scc Fig 8.1) .
3. Trauma-related conditions are those in which
"mouth" m icroorganisms (Pasteure/la is the most
con1111on) are iuo<.:ulatcLI i11tu tite site of infection.
Examples include bite wounds, licking compro-
miscd sites (e.g., a surgical wound).
Pathology.Lesions vary wilh sile of infecLiu11, virulc11<.:e
of strains, and host resistance. In septicemias, vascul;ir
damagc rcsults in hemorrhagc and fluid loss but little ccl-
lular 1n flammatory response. Focal necrosis in parenchy-
matous organs or ulcerations of mucous meinbranes may
u<.:<.:ur. Mammals develop generallzed hemorrhagic lym-
phadcnopathy.
Tn most pneun1onias, inflan1n1ato1y cells are pruu1i-
nent, with erythrocytes, neutrophils, and mononuclear
cells appcaring and predominating successiveJy. The tissue
appearance changes accordingly trom reddish-black to
red, pink, and gray. Necrosis, abscess forrnation, and fibrin
uc¡.>u!>ltion vary in severlty. In chronic fo\•\Tl cholera,
cascop11r11lfnt inflammation occurs in joints, middle ear,
ovarics, or wattles.
Atrophic rhinitis of pigs (see also under Bordetella bron-
chiscptica, Chaptcr 15) is a chronic rhinitis accompanying
dlsturbcd osteogenesis adjacent to the inflamed areas.
Tne rea sed osteoclastic and dimin ishcd osteoblastic activity
destroys thc turbinates and bones of thc snout, resulting in
<iistortions of facial stn1ctures (see f.ig 73.1 ). Histologically,
fibrous tissue replaces osseous lissue. Bu11e atrupl1y is Cl<.:-
companicd by inflammation of varying acutencss.
The pathology of lesions that are "mouth" related is un-
remarkablc with neutrophils predominating.
Disease Patterns
Cattle. The most common form of hovine
Pnt'11mnnia .
"pasteurcllosis," involving M. haernolytica, P. rnultocícJ.u, or
P. trehalosi, is shipping fever, a fibrinous pleuropneumonia
or bronchopncumonia sccn whcrcvcr cattle, especially
calves, are transported, assem blcd, and handled under
stressful conditions (see Figs 73.6 and 73.7) . Whilc thc ul-
timatc cause is uncertain (see Table 12.2), the agents pro-
Envlronmental Stress
lnfectious b<ivine rhihotracheitis virus) Exhaustion. starvation, dehydration. weaning. ration <h~ngf!s,
PJrJinflucnz¡¡ 3 overcrowding, chilting, overheating, excess or irregular
Bovine respiratory syncytial virus high·energy feed, poor ventilation, excess humidity, non-
respiratory disease, castration, dchorning, sociul maladjustment
88 PART II Bacteria and Fu ngi
ducing severe illness and death are bacteria, most com-
n1011ly and aculely M. haernolytica capsule type l .
'fl1e onsct, 1 to 2 weeks after stress, is marked by fever,
inappetence, a11d listlessness. Respiratory signs (nasal dis-
chargc, cough) are tew and variable. At more advanceá
stages, the fever may drop but respiratory distress will be
obvious. Abnormal lung sounds can be detected, espe-
cially over the apical Jobes, which are first and most se-
verely (:lffe<..:l eu.
Hemorrhagíc Septicetnia. Hemorrhagic septicemia is an
acute systemic infection with J>. 1nultocida, serotype B:2
(Southern and Southeast Asia) or E.:2 (Atrica), occurring in
tropical areas as seasonaJ epidemics Y\1ith high morbidity
and mortality. Signs include high fever, deprcssion,
subcuta11eous edema, hypersalivation and diarrhea, or
sudtle11 tle(:ltll. Ali ex<..:relions and secrelions are l1igbly
inf~~ctious.
Mastitis. Mann'1ei1nia haernolytica may cause mastitis
with much tissue destruction, J1emorrhage, and systemic
toxic co1nplications, which are often fatal. A somewl1at
less severe form is caused by P. multocida.
Sheep and Goats. Septicernia. Septicemic pasteurellosis, usu-
ally dueto P trehalosi in feeder lan1bs and M. haemolytica in
11ur~i 11g lar11bs, 1esen1bles bovh1e l1en1orrhagic septicemia,
although intestinal involvement is o ften absent and the
morbidity rate is much lower.
1!.nzootic JJneurnonia . .Enzootic pneumonia of sheep 1s
the avine equivalent ofbovine shipping fever. Mannheirnia
haernolytica is most often involved.
Mastitis. Gangrenous mastitis due to 1'vf. haemolytica
(usual) or P. lrehulusi uccurs Jale in laclalion, 1"7l1e11 large
lamhs h ruise the udder and provide the inoculun1 from
t heir oropharyngeal flora . Acute systen1ic reactions ac-
compa11y disease oí the udder, parts ot which unáergo
necrosis ("blue bag") and n1ay slough.
Swine. Atrophic Rhinitis. Atrophic rhinitis of young pigs (3
weeks lo 7 n1on lhs) leading Lo Lurbh1ale destructio11 a11d
secondary co1nplications results from synergistic nasal in-
fection by J>. 1nultocida (usually capsule type D) and
Bordetella bronch.iseptica. ln addition, ammonia (in concen-
tratio11s so1n etimes found in swine-rearing houses) acts
synergistically with Pmt.
Signs include sneezing, epistaxis, and stainins of the
face due 1.0 lear-ducl obsLruclion. Skeletal ab11orn1aliti.e s
rroduce lateral deviation of the snout or wril1kling due to
rostrocaudal compression. Secondary pneumonia is duc in
part to the elimination of the turbinates as ctetenses of t he
respiratory tract (see Fig 73 .l).
Pneurnonia. A fib rinous pneumonia, associated with f'.
multocida, often follows viral infections.
Rabbits. Respiratory Tract Conditions. "Snuffles," a muco-
purule11t rhinosinusitis of rabbits due to J>. 1nultocida,
develops under stress of pregnancy, lactation , or n1isn1an-
agement. Complicat ions include bronchopneumonia,
m iddle and iru1er car infection, conjunctivitis, and sep-
ticemia.
Genilul Truc;L Diseuse. Ill Lhe genital lracl, J>. rrzultocida
may cause orchitis, halanoposthitis, and pyometra.
Avian Species. Avían Cho/era. Avian cholera, a systemic in-
fe<..: tiu11 uue tu P. rnullotiúu (111osl corurnonly capsule lype
A), is acquired by ingestion or inhalation and affects
mainly turkeys, waterfowl, and cl1ickens. Tbc pcracutc
form kills about 60°/o ot intected birds without preceding
signs of illness. The acute type, marked by listlessness,
anorexia, diarrhea, and nasal and ocular discharges, may
last several days and be about 30o/o lethaL The subacute
fur1u is u1uslly re:>J!iralury and is n1a11ifeslcd by rales and
mucopurulent nasal discl1arges. In chronic fowl cholera,
there is localization of caseous lesions. Pastcurclla galli-
narum. is sometimes isolated trom chronic cases.
Dogs and Cats. Mouth Related Conditions. Pusleurellu rnullo-
cida (cats) and f> canis (dogs) are fo11ncl alongside predon1-
iI1a11lly anaerobic flora in wound infections, serositides
(e.g ., feline pyothorax), and foreign body lesions.
Equine. Respiratory Tract Conditions. J.Jasteurella caballi oc-
curs in equine respiratory disease, usually in association
with Streptococcus equi subspecies zooepídemicus.
Laboratory Rodents. l'asleurellu pneurnulropica, a co1nn1011
commensal, may contrih11te to opportunistic infectio11s
such as p11eun1onia. Phenotypically simila r organis1ns in
other hosts p robably helong to different species (e.g., P.
dagmatis).
Human Beings. Pasteurella rnultocída (a11d rarely other pas-
teurellae) causes wound infections in hunians resulting
fron1 a11ilnal bites or scratches. A second form, especially
Íll tl1c resviralury lracl, is usually 110l directly traceable to
animal sources.
Epidemiology
·rabie 12.3 shows the relative roles of exogenous reservoir,
dissemination, a11d stress in various conditions associated
with Pasteurella and Mannheimia. In avian cbolera, feral
sources are sometimes implicated.
lmmunologic Aspects
Basis of lln1nunity
(;irculating antibody is significant in protection against
hemorrhagic septicemia and fowl cholera. The type-
specific capsular antigens are esse11ti(:ll i111111u11ogens in he-
n1orrhagic septicemia . With other forms of disease associ-
ated with Pasteurella and Mannhcinúa, the picture is lcss
clear. Both antitoxíc and antibacterial antibodies are im-
portant in protection .
Artificial lmmunization
Pastcurella adjuvant bacterins are effective in preventing
bovlne hemorrhagic septice111ia, coufe1ring p rolection for
up to 2 yi>ars. Antiserum is useful for short-term protection.
The essential attributes of an effective avian cholcra
vaccine are not k.nown. Field perfor1nance of l)acterü1s has

Ta b 1e 1 2 . 3 . The Relative Roles of Stress, Agent lntroduction, and Agent Disscminiltion in
Various Types of Pasteurel/a lnfections
l11l1U\lui.llu11 ur
Agent into
Population
Avian cholera
BovinP hPmorrhagic septicemia
Bovine )hiµµi11y fever, uvine enzootic
pneumonia. avine septicemic
pasteurellosis, rabbit pasteurellosis
Human pasteurellosis
Sporadic pneumonia in ruminants,
Pasreurella infection in dogs and cats
l"!Jeml. • =irrelevant; + =contributory; ++ = Slgnifleant; +++ =criúcal; ++++ =paramount
- inconsistent, even with autogenous preparations.
~ pro1nising havt: l.Jt:t:u live vaccines contalning atten-
-...o..~=" orga nisms (e.g., CU or M-9). Attf'n11;ition appears in-
e;- rclatcd to immunogcnicity.
•ost shipping fever vaccincs are bacterins of mixtures
. haen10/ytica, P. rnultocida, and P. trehalosi combined
s>..ISpect viruses and other bovine bacteria! pathogens.
o::::J:ts are inconsistent. Vaccination of calves with bac-
, prepared from M . haernolytica reduces the degree of
:-ization of the upper respiratory tract by this organ-
::he effectiveness of n1odified live vac1.:i 11 1::~ await.s fiel ti
~ation. Recombinant Lkt alone does not protf'rt
e from 1\lf. haemolytica-induced pneumonia. ·rhe gene
:c.., g Lkt has bee11 cloned into thc genome of- and
;:-s¿quently cxprcsscd by-white clover, the strategy
- : m 1mmun1ze by teed1ng. No data are available on the
---..r-1mess ofthis method.
=er1rellu rr1ull1u..iúu ;111u B. brunchiseptica bacterins with
t:X::::'._C. are val uable in the control of atrophir rhinitis.
_::~ratory Diagnosis
;e- :.xamination
:..:._.:...-..=.
~te,
tissuc imprcssions, scdiments of transtracheal
-..-~es. and, 1n birds, bloocl smears can be stained with
-:-chrome stain (e.g., Giemsa, W right 's, Wayson's)
cé - ""111li11eu for bipolar stalning microorganisms. The1r
":'."3é'!1Ce is s11ggf'sti ve but not unique to Pasteurella/
1eirnia . On Gran1 staln, Pasteu.rellu/lvfunnheimia clo
o;:i~ disti nctive.
,......,,..-~= - and ldent ification
- - ~lla/ Munnhein1ia grow overnight (35-:37ºC) on
-:? o r shc'<"P hloocl agar and are identified using diffcr-
=~ :ests . Capsule a11d son1aLic Lypi11g are <.lone in a rcf-
=:""X'..-.: '..aboratory. DNA probes and primf'r<; rlf'.;igned to
Chapter 12 Pasteurellaceae: Pasteurella, Mannhcimia 89
lmportance of:
Obsemlnatton of fnVlronmental or
Agent Wrthin Individual Stress
Population or lnjury
+++ ++++ ?
+++ ++++
+ ++ +++
+? ++++
+++·+
ampli fy spccific rcgions of the bactcrial chromoso1nc by
the polymcrase chain reaction are available for demonstra-
tion and identification of Pasteurella/ Mannheimia.
Treatment and Control
Disea.;p<; rroclncf'cl by Pnsteurella/Mannheimia respond to
timely an timicrobial therapy. St1ai115 fror11 1.:anlivores and
humans are generally susceptible to almost all antimirro-
bials. Most Pastcurc/la/Mannhein1ia show moderate rcsist-
ance to a 1ninoglycosides, w t1ich is probably not significant
clinically. Those from food- producing animals vary. In
these species, cost and withdrawal requirements before
slaughter are additional considerations. Consequently,
sulfonan1ides, pe1licilli11 G, ceftiofur, tllmicosin, florfeni-
col, and tetracyclines are preferre.rl. ·rh Pir appropri<1tcness
should be confirmed by susceptibilíty tests. Sulfonan1ides,
pe11icillin li, fluoroquinolones. and tetracycline are used
to trcat avian cholera.
Sullonamides, penicillin G, and tctracyclines in swine
and poultry, are suitable for mass medication via feed or
water, the1apeuli1.:ally or p rophylactlcally.
Managementpractic:P.<; rlirrcted at rcd ucingstress are im-
portant in preventing Pastcurclla/Mat1nheitniu-assu1.: iateu
disease in Uvestock.
Immunization has been dcpcndablc o nly in bovine he-
morrllag1c septicemia and atrophic rhinitis control.
RIEME RELLA ANATIP ESTIFER
Rie1nere/la anatipestifer, the age11l of "new uu<.:k disease,"
produces asevere polyserositic disease especially of rl 11rk-
lings (duck septicemia, infcctious serositis of ducks). At
various times in the past, thjs microorganism has been
called Pasteurclla anatipestifer or Moraxella anatipcstifcr.
90 PART 11 Bacteria and Fungi
Descriptive Features
Rietnerellu uuutipestifer is a gram-negative, norunotilc, cn-
capsulated, occasionally bipolar coccobacillus. It grows
best under increased carbon dioxidc tcnsion on blood or
chocolate agar (see Chapter 14 for a descr1pt1on of tl1is
agar). Colonies are nonhcmolytic, transparent, and may
exceed 1 mm in diametcr after 24 hours of lncubation. The
organism is oxidase and catalase-positive, proteolytic, fPr-
1nents no carlJuhyllrale:::., <111d survivcs for \'Veeks in litter.
ThPrf> ;irf> 21 s0rotypes (1-21) occurring in severa] combi-
nation.s.
Ecology
Reservo ir
'l'he agent occurs in ducks and otl1cr fowl. Carrier blrds
constitute the rcservoir.
Transmission
lnfection occ11rs via respiratory, percutaneous, and possi-
bly other routes.
Pathogenesis
Rie1r1erella anatipesti(er septicc1nia causes sudden mortal ity,
or, less acutcly, respiratory sigu:. wiLll s11eezing, coughing,
and discharges fron1 no<;P anct eyes. ~entra! nervous in-
volven1enL produces head tremors, torticollis, and ataxia.
Diarrhea is also seen.
Fibrinous polyscrositis is observed in the acute disease.
·rhe exuda te Is mostly m ononuclear. ln proLracted cases, fi-
broblasts and giant cells are sccn.
Mortality rates vary fru1u 5º16 Lo 75%; duraUon is fron1
one day to i;Pvf>ril 1 Wf>Pks.
Epiderniology
()nly ducks are consistently a t risk. Stress factors play a
significant predisposing role. Young birds are most sus-
ceptible.
lmmunologic Aspects
.Kccoverea 01rds are res1stant fouowing rc-expostue ro tlH.:
bacterium. lmn1unity is serotype-specific anrt may he in-
duced lJy 1Ja1.:leri11:..
Laboratory Diagnosis
lsolation and identificat1on ofthe agent are mandatory for
diagnosi~. ()rga.r1is1ns 1nay be demonstrated in organs and
fluids by Gram stain or lmmunotluore:-.ce111..t:.
Culture plates are incubated under i11creased c;1rho~
dloxlde tension.
Treatment and Control
Sulfonam1dcs (sulfamethazi11c, sulfa<..limethoxlnc, sul-
faquinoxaline) may be given prophylactically in fppc1 or
water. Penlcillin G, tt'tracycli11e, e1ylhron1ycin, and novo-
biocin arP 11st:>c1 therapcutically. Corticosteroids adminis-
tered along with antibiotics (penicillin C) do not iníluence
resolution of lesions.
Vaccination with killed or live avirulent bacteria of ap-
propriate serotypes is beneficia!.
PHOTOBACTERIUM DAMSELA
Photobacteriurn damsela contains two subspecies, pisricida
and da111sclac. Pholobactcriu1n dan1sela ssp. piscicida (pre,-¡_
ously classilicd as l'asteurella piscicida) is the agent of fish
pasteurellosis. Photobacteriu1r1 damsela ssp. dan1selae was
previously classified as V/brío darnsela. Botl1 :.ubspecies
produce septicemic disease in farmer1 m;i ri nP fish of a vari-
ety of spe<.:ies. Plrulubc1Lle1 iurn d.a111scla ssp. da1nselac is capa-
hlf> of rroctucing septicemia in n1ammals (dolphins and
h1.1mans).
Pholobacteriu111 da1nsela is a gram-negativc, oxidase-
positivc, halophilic rod. Differe11tiation of the subspecies
is done using trad1tional biochemical tests, or by dctcrmi-
nation of DNA sequences of the gene encoding lhc 16S
rRNA (subspccies-speclftc priI111::r:. are available and the
test is done by using thP polymPrase chain reaction).
Both subspecies are susceptible to a variety of anti-
microbials, including tctracyclines and beta lactams. h
plasmid-mcdiated resistance has rnadc treatment difficult
in sorne outbreaks.
Group EF-4 (Eugonic Fermentar)
A gram-negative i1onmotile bactcriu1n, superficially rc-
scmbling Pastcure/la spp., occurs in the mouth of carni-
vores and sporadically causes apparcntJy endoge11um
fatal, a cu te focal, pyogranulon1atous pncu mnn i;:is. 1n hu-
ma ns lt Is 1n1plicatec.J iu aui111al hi Le wou11d infections.
lt is oi<idasP, c;italase, and nitrate-positive; it fer1ncnts
glucosc slovvly and h11:s no othcr dingno~tically dcp('ndable
characteristics.
lsolates frorn humans stemming from animal bites test
susceptible to the macrolldes (erytl1ro111yciu, cla1ilhron1y-
cin, azithromycin), ccphalosporins, ;1mpicillin, tetracr-
cline, aminoglyco~ic.lt::., a 11d fluoroquinolones.
 Pasteurellaceae:
Actinobacillus
Dw1GHT C.
~e family l'asteurellaceae contains thc genera Actinobacíl-
Gallihacteríurn, H(./ernophílus, Histophilus, Lonepinella,
.;.:s,
'a11nheimia, Pasteurel/a, and J'hocoenobacrer. All but Lone-
_-:nella (L. k()afarum-a tai1nln degrading bacterium iso-
_re<l fron1 norn1al Koala fcccs) and Gallibacleriurn (G.
~:..itis-isolated from the intestine of normal ducks) con-
:=!~!1 spccics that are of medical importance.
.-\lthough thcy are genetically disti11ct, tl1e genera
ctinobacillus and Pasteurella overlap phenotypically. As
rhe case wlth the gcnus Pasteurel/a, the genus Actinoba-
- '}IL<; i~ 11nrlPrgoing a considerable amount of taxonomic
~~ange. As of this writing, tl1e genus Actinobucillus L"OIJ-
~"1S 20 species, many of which are associated with dis-
~ in severa! animal species (Table lJ.1). Discussed in
'C.:5 chaptcr are A. equuli subspecies equuli (previously A.
~uli, the cause of neonatal septicemia of foals), A . cqu-
~ subspecies haetnolylica (previously A. suis-llke, and oc-
=---s mainly in respiratory tract disease of horses), A. lig-
,tt:.')ii (the age11t uf pyogranulomatous processes,
-:imarily of n1min;.ints), A. s11i~ (;.issoci;ited wlth respi-
-:;:ory, septicemic, and locali7.ed infectio11s of swine),
...::d A. plcuropncumnniae (the agent of porc:inf' plt>11rop-
::-umonia).
·\11 the members of the family Pasteurellaceae are gram-
-:::-gative coccobacilli . ·rhey are facultative anaerobes, and
-pically oxldasc-postttvc (setting them apart fro m mem-
- :<; of thf' f;.imily P.ntf1rnhacteriaceae) . Most are commensal
~arasites of animals.
)~scripti ve Features
• rph ology and Staining
.e_rnbers ot the genus Actinobacillus are gram-negative
--.ccobacilli, approximately 0.5 µm wide and variable in
~gth.
:··Jcture and Composition
_..apsules are polysaccharide. The cell wall is typir;.il of
::-::lm negativc microorganisms consisting mainly of
,opolysacchar1dcs and proteins. ~on1e ot the Jatter are
~n-regulatcd (i.c., they are expressed under iron poor
:iditions).
HIRSH ERNST L. BTBERSTEIN
Cell Products of Medica! lnterest
Adhesins. The role of adhcsins, as in other microorganis1ns,
i ~ to alJuw the bacterlum cxpressing them to ad herc to
cells liníng ;i p;irtic11J;ir niche, as well as to t he surfacc of
so-callcd "target" cells prior to ll1e i11itialion of d i$t::a!>e (in
sorne cases, niche and target cclls may be the same). Th<>
expression of ndhcsins depcnds upon various environ-
mental cues. Sorne, and probably ali, members ofthe fam-
iJy Paste11rellaccac exprcss adhesins (a11d possibly more
tliau 011e kind).
Exccpt for A. plr>11rnp11r1J1noniae, virtually nothing is
known about the adhesins of thc otl1er acli11obacilli assu-
ciated ~vith diseases of animals of vcterinary irnportance.
/\ctinobadllus plcuropncu111oniae adheres to two different
ccll types, those lining the nasal cavity and tonsilar crypts
(as is the case with carrier animals), and the cells of the ter
minal bronchloll and alveoli (antecedent to disease).
Artin()harilf11s ple11ropne11moniae produces two structures
that function as adhesins. '!'he fi1sL is a11 adl1e:;iI1 i11 tl1e
classic sense, i.c., a structure composed of protci n suh11 nits
''\'hose main function is to bind to tl1e surface of hosl ceUs.
'rhese adhesins are type 4 fimbria, and it is unknown to
which ccll typc thcy adhcrc. Thc othcr, ccll wall lipopoly-
sacchandc, 1s not usually cons1aerect an actt1esin . Never-
theless, thc lipopolysaccharide (in particula r tl1c core
po1 lio11) lJf A. µleu1uµneu1T1uniue i:s re:;pon:;ible for tbe ad-
herence of this microorgan is1n to c:Plls of thf' lo11vPr respira-
tory tract of swine.
c.:apsules. 'fhe capsule plays many roles, t he most impor-
tant of ~vhich are intcrfcrcncc with phagocytosis (an-
tiphagocytic) and protect1on of the outer mernbrane ITom
the deposition of membrane attack complexes generated
!Jy aL"tivation of the complement system. The amount of
capsule procli1rf'<l i~ invPr~Ply proportional to the amount
of availablc iron. ln vivo, where the an1ou11l of availablc:
iron is low, the amount of capsule formed is less (but suffi-
cicnt to protcct thc microorganism from phagocytosis and
complemcnt-mediated lysis). It is difficult to reconcile
capsule expression and lipopolysaccharide as an adhcsin
in adherence in A. pleuropneumoniae.
r.r>ll Wnll 1 ipr.polysaccharide elicits an inflammatory
response following associalion wilh lipopolysacL"l1aride-
binding protein (a serum protein), which in turn transft>rs
it to thc blood phnse of CD14. 'fhe CD14-LPS complex
b1nds to ·1011-like receptor proteins (see Chapter 2) on the
91
92 PART II Bacteria and Fungi
Table 13.1. Members of the Genus Actinobacillus and Their Usual Source or Assoc1ated
Condition
---Species
A. actinomyceremcom/tans
A. arthritidis
A.capsulatu;
A de/phinicola
A equuli ssp. equuli
(A equu/1)
A. cquuli ssp. haemolytica
{A suis·lll<e)
A hominis
•A. incJolilu; •
A. lignieresii
"A. minor•
A muris
A. plcuropncumonicJc
•A. porcmus·
A. rossii
·A. salpingítldts•
(will hP<omp Gallibaáerium sp)
A )l.u(iae
A seminis
A succinogenes
A. suis
A. ureae
surtace ot macrophage cells trigger1ng the release of proin·
flammatory cytokines
Toxins. Members of the genus Actinobacillus produce at
least two products th;it h;:ivp toxic activity. The most i m-
porlant is an RTX type of toxin, and thc other is the en-
zyme urease:
l. RTX type toxin. The RTX (repeats in toxin, so called
bccause of the common fcaturc of repeats in
glycine-rich sequcnces wlthln the protein, see al.so
Escherichia coli hcmolyi;in in \.hartPr 8, Pasteurella/
Mannheirnia leukotoxin in Chaptcr 12, adenylyl cy-
clase toxin of Bordetella in Chapter 15, Moraxella cy-
totoxin in Chaptcr 19) typc of toxin is produced by
A. pteuropneun1oniae, A. suis, and A. rossii. The toxin
is termed Apx (for A. pleuropneumoniae toxin). Ac·
tinobacillus equuli ssp. hae111olytica produces a very
similar toxin, Aqx (for A . Pq1111li toxin). Even though
A. lignieresii contains the genes cncoding Apx, it
does not express them (lacks a functioning pro-
motcr) . As with othcr RTX type toxins, there is a
dose effect. ;\t Jow concentrations these toxins in-
tcrfere with macrophagc and neutrophil fur1ction
by trtggertng degra11ulallu11, a11ú al lligl!e1 LUuL1::11-
t rations tht>y arf' rytolytic for macrophagcs, neu-
trophils, and alveolar epithelial cells. In addition,
the RTX toxins lyse erythrocytes (explaining the !1e-
molytic phcnotypc of actinobacilli expressing them
whcn grown on blood agar), and are responsible for
Usual Sourcc or Associated Condition
oral cavity, perlodontal dlsease in primates; endocardi1ís in primates
SeptirPmia of foal1 and horses; normal flora of oral cavity of horses
Althritis of rabbits
Septicemia in sea mammals
Septicemia in neon~tlll foJls
Respiratory tract conditions in horses
Respiratory tract conditions in humans
Pleuropneumonia end pneumonia in swíne
Chronic granulomatosis in ruminants ("Wooden Tonque")
PleuropneumoniJ Jnd pncumonia in swine
Respiratory tract conditions of rodents
Pleuropneumonia in swine
Pleuropneumonia and pneumonla In swine
ReproductivP trart of 1ow1
Condition; uf lht! 1espiratory and reproductive tracts of chickens
Septicemia in harbor porpoises
Reproductive conditions of sheep (epididymítis in lambs); normal flora
of gcnitJI tract of sheep
Bov1ne rumen
Respiratory tract conditions, and septicemia in swinP
Resplratory tract condltlons in humans
a CAMP reaction when cross-streaked wit!1 a !Jeta
toxin- producing strain of Staphylor.nr.cu.~ (Sf'f' l.hap-
ler 28). The effecl on crythrocytes probably plays
little if any role in disease production. There are four
types of Apx toxin (Apxl, ApxII, ApxIII, ApxI\.
each with a ditterent degree of cytotoxicity (ApxI 1.
thc most potent of the four, followed by ApxIII, anc!
then ApxII- the poterH.: y uf ApxIV i~ unknown
Actinobacilli may conta in the genes encoding an~
co1nbir1a lio11 oí thc four toxins. In thc case of A
pleuropneun·1oniae, serotypes (see "Variability," be·
Jow) l, 5, 9, 10, and 11 produce Apxl, al! thc sero
types (except serotype 10) produce ApxII, serotype.
2, 3, 4, 6, and 8 produce Apxlll, and ali of th ..
serotypes produce ApxIV. Ac:linubcllillus rossii pro-
duces Apxll and Apx!TT, wherea<; A. suis produce
Apxl a11d ApxJI.
2. Urcase. Urease produced by A. pleuropneul1'1onia.
has bccn shown to be important in disease pru-
CluceCl by this m1croorganism (it is unkno\':
whether this cnzyme p lays a role in the diseases pre
d uced by the other Ufl:!a~1;:-¡.¡u:.i Li ve species of act.·
nobacilli). TJre;isP is rPsponsihle for the liberation
ai11111onia from urea (thc nssocintion con:;tunt of
pleuropneumoniae ureasc is extremely high allO'\o\1r _
effective association with the very low concentr:.
tions of urea found in blood and tissue fluids). In a
dition to attracting and activating neutrophils a!"
macrophages, ammonia il1hi!JiL:; phagolysosor-

fusion and increases the pH within phagolyso-
somcs, thcrcby rcducing tl1e effectiveness of various
acid hydrolases. ·rhese ettects result not only in a de-
crease in the host's ability to clcar the microorgan-
i:.111 from tl1e lung, but also to assurc effeetive colo-
nization of thP 11ppPr rP<;piratory tract, thereby
promoting the carrier state (mutants unable lo p10-
duce urease are clearcd rapidly from the respiratory
rract).
-tcquisition. Bccause iron is an absolute growth re-
-ent, microorganisms must acquire this substance
dre t o exist wlthln the host. Actlnobacilli bind
--:::=::_.tin -iron romplPxes by virtue of iron-regulated
lr:~ ~en1brane proteins expressed ui1dc1 i1on-JJOor 1.:ou-
so-called transferrin-binding protcins, or Tbps).
.icquired from the transfcrrin-iron complcxes that
rhe surface of the microor.ganism. ln adclition to
_, ng iron by way of 'fbps, Actinohacillus pleuropneurno-
- •o ulili:t.e::. ::.iuerophores (both hydroxamates and
:m~ '.s) produced hy othPr spPries o f bacteria even
_ - •t does not produce them itself.
. ~laneous Products. Other products produccd by ac-
.::.:::- ~!Ji (spccifically A . plcuropncu1noniac) that 1nay play
~ disease production include Ig/\ and Jg0 proteascs
""'~ adherencc, and opsonization, rcspcctively), and
_s111ic SUJJt:roxitle tlis1nutase (suggcsted as a strategy
-~"'~t digestion within thP phagolysosome by inacti-
....__ uperoxide molecules).
:il:::C- Characteristics
~acilli grow on b lood and scrum-containing media
ro 42ºC. Colony sizes at L.4 hours reach 1- 2 mm.
...-rtnobacilli (A. indolicus, A. rninor, A. pleuropneurno
rype 1, and A. porcinus) require nicotinamide ade-
!!udeotide (NAO) for growth, while A . pleuropneu-
biotype 2 uucs not. Hemolysis depends upon
o n of Apxl, ApxTT, or Aqx (toxin~ with hernolytic
- 'Jhydrates are fermented without gas production.
o rthon l troph cnyl beta d galactopyranosidasc
--:ase, "beta-gaiacrosidase"), a11d nitratase are typí-
' r-sent. No indole is produced, and most strains grow
""'-=-.Conkey aga1 (l¡ut µoorly). Cultures die Within a
~,. -riey are facultative anaerohPs, an<l typirally oxidase-
e:::::--.re
.xillus pleuropneu1noniae contains R plasmids en-
- ·csistance to sulfona1nides, tetracycline, and peni-
.. :r
i..i llus /ignicrcsii and A. equuli are antigPnirally di-
.,e six soma tic types of A . lignicresii have son1e rela-
.:wgraphic an<I host species predilection. ·rhere are
-'-'-.;..-tic serotypes of A. pleuropneutnoniac (1- 15), and
-~ ~ (biotype 1 rcquires NAO for growth, biotype
Chapter 13 Pasteurellaceae: A r:tinnhar ilfus 93
?. rloes not). Artinobacill11s suis has at least two somatic
serotypes (Ol and 02).
Ecology
Reservoir
Ac:tinoharilli (Pxcept possibly A. caps11lnt11s) are commen-
sal parasites on n1ucous n1en1branes. Ac.linubucillus pleurup-
ncumoniae are rnore closely associated with the respiratory
tracts of sick or recovcrcd animals.
Transmission
Except in neonatcs, most uctinobacilloscs are probably cn-
dogenous infections. Neonatal toats acqutre A. equuli ssp.
eq1111li before, at, or shortly after birth from their dams,
co111u1u11!y vi<1 the umbilicus.
Pathogenesis
,Vfechanis1ns. Most disease produced by 111e1111Jer::. uf the
genus Actinobacillus result from "contamination" (infcc-
ti<>n) of a normally steríle, compromiscd sitc by microor-
ganisms living on a contiguous site (niche). Common
compromises are viral infections, trauma, or stress. Sorne
Lin1es il b uiffi1.:ult to determine the nature of the ante-
cedent event (P<;pPri ~ Il y true for n eonates with septice-
mia). Deposition o f actinobacilli into a nonually :.tcrile
site results in initiation of an inflammatory response due
to the lipopolysacchnridc of thc ccll wall, and urease if the
infecting stra1n produces this enzyme. ·rhc capsule inter-
feres with ph.agocytosis (antiphagocytic) and protects the
outer membranc from the deposition of me1nbrane attack
romplexes generated by activation o f the complement sys-
ten1. Transferrin-binding pr0Lei11:. µarticiµate in iron ac-
quisition. The R'fX toxins (J\px, Aqx) int<'nsify the inflam-
mntory response by activating and damaging neutrophils
and macrophages. ll the infectious process involves the
Jungs, alveolar epithelial cells are also damaged. Urcasc-
produc111g actinobactlli that are phagocytosed res1st dc-
~trHc:tion by generating an1monla, and perhaps superoxidc
disn1utase. Opsonit.a Lio11 i:. reuuceu IJy tlle productlon of
lgG protease.
In the case of neonatal septicemias (A. cquuli ssp. equuli,
in particular), actinobacilli gain access to the systemic c:ir-
culation. Capsule and 'fbps permit mtlitiplication within
the bloodstr.eam rcsulting in an endotoxemia (see Fig 8.1).
J>ntholog)'· Lung lesions (usually produced by ,•\. equuli
ssp. haernolylic.u, A. pleuropneumoniae, and A. suis) are sup-
purative. lnflammatory rPlls are promincnt, with erythro-
cytcs, neutrophils, and mononuclear cells appeadng auu
prcdominating successively. 'fhe tissuc appearance changes
accordingly from reddish-black to red, pink, and gray.
A soft tissue process, mainly produced by A. lignieresii, is
a chronic granuloma in the ruminant tonguc. At its center
is a colo11y uf A. li8nieresii, rlnged by eos!nophilic, club-lii<e
processes forming a "ro~ette ." Tl1e complex is surrounded
by neutrophils and gra11ulation li:.:.ue 1.:ontaining
macrophages, plasma cells, lymphocytes, giant rPlls, and
94 PART JI Bacteria and Fungi
fibrohl;ist<;. Plant fihPrs ilrP oftP.n prcscnt. Through coales-
cen ce, larger granulomas (1 cm or more in diameter) may
be formed. lnfectioo sprcads to lymph nodes, producing
granulomas along thc way. The proliferative tissue reac
tion causes thc tongue to protru<.le from the mouth. 1Jrher
tissues in the vicinity, and occasionally along the gastroin-
testinal tract, may be lnvolve<.I, along with i:1.Llji:1.ce11t lyu1µ1J
nodes. Superficial lesions oftf'n 11lcPriltP. In shePp, suppu-
1aliv1: i..nfeclio11s occur around the head and ncck and in
the skin and mammary gland. 'fongue involvement is not
typical.
Disease Patterns
Ruminants. "Actinobacillosis" or "Wooden Tong11P" involv-
i11g A. Ii;sriiere~ii uccu1s in run1inanls a11d, rarely, dogs a11d
horses. ln ruminants, the agcnt (normal inhabitant of the
nasopharynx) is p robably inoculatcd by trauma (plant
fibers). initiating the process described. Thc course is pro-
tracted and 11ealing slow. Interfercnce with feed intake
causes weight loss and dehydratlon.
Swine. Pneumuniu. Ac;linuúuc.illus pleurop11eur11oniae (sero-
typP~ 1 ;inrl .'i ;:irp more common in North America; sero-
typc 2 i3 more common in Europc) cousc:; n primary pncu
monia ot swine, particularly young p1gs 2 to 6 months old.
Sprcad is favored by crowding and poor vcntilation. Early
signs incl ude fever and reduced appccitc, followeLl witl!i11 a
day by acute respiratory distress. Anilnals m.ily rliP within
24 liuur:;. Morbid ily 1nay reacl140%, witli n1ortality up to
24ºAl. Survivors show intermittent cough and u nsatisfac-
tory v.•eight gains. Chronic infections, oftcn v\'ithout pre-
ceding acute episodes, are the source of persistent herd
problcms. Lesions consist of fibrinous pneumonia and
pleur1t1s. Arthr1t1.s, men1ng1ns, and abortion occur as co1n-
plications. Pneumonia in older aniroals may be caused by
A. suis.
Septir<~mia.
Si>pticPmi;i of young pigs and arthritis, en-
docarditis is sometimes associatcd with A. suis.
Horses. Pncumonia. Bacteria! pneumonia in horses (of any
age) is usually associ.ated witll a mixture of beta l1en1olytic
strcptococcus (usually S. equi subspecics zooepidemic11s)
anda gram-negi:ltive rnicrourga11i:.u1 (cv111111u11ly A . equuli
ssp. ha<'mnlytir.a)_
Septicernia. Foal septicemia due to A. e.quuli ssp. equuli
("sleepy foal disease") occurs within a few days ot birth
and is markcd by fever, inappetence, prostration, and diar-
rhea (see fig /4.1). Anima Is surviving the first day typically
develop lamcness due to (poly)arthritis. Sorne foals de-
vclop um bl 1ical infection~ wlth tlt i:. 111icroorganisn1
("n;ivpl il l").
Epiderniology
Actinobacilli are opportunistic pathogens produci.ng dis-
ease when their host's integrity is compromised, as by
trauma, lmmaturity, or otht:!r :.trc:.:.. Tri:lwua tu wucous
mcmbr¡¡nes of rnmin;ints hy rough feed may cause 11erd
outbrcaks suggestive of transmissiblc diseases.
In the case of Porcine Pleuropneumonia, c h;
asymptomatic carricrs are thc apparcnt reservoirs. D!s
occurs when nonimmune an1mals are exposect to sutx..
ically infected individuals. That prevalence is híghes
the colder m o nths Is due probably rnure tu i11i:1.11i:1.ge::-••
(i.e., the mixingof individll<llS with rliffpre nt exposure
luri1::.) Lha11 clin1alic factors.
lmmunologic Aspects
The pathology of "Wooden Tongue" suggc:.l:. d
mediated hyperscnsitivity. AntihorliP.s of no knO\.\'Tl -
Leclive fu11clio11 appcar during infection. The benef
hacterins have not bccn established. Antibody to A. ,
ropnc:u1noniae is opsonizing and colostrum protects p ie
Antibody to KrX-type toxin is protcctlve.
Laboratory Diagnosis
Actinohacilli can oftcn be demonstrated in gram-sta
exudates. They grow best on blood agar under incre_
amounts of carbon dioxide (35-37"C). Colonics are e _
:;ticky. Spcciation is accomplished by cultural and
chcmical tests. DNA prlmers have been <.levelupctl tu a__
identification by means of thc polymi>r;ise. chain rea~
Treatment and Control
Porcine pleuropncumonia is controlled an d treated ~
combination of management pract1ces, immunopror
laxis, and ai1timicrobial therapy. Management pracn
lnclucte minimiZing contacr of piglets with mi:lture .._
rier) animals (e.g ., "ali in, ;ill 011t11 ri>aring practices; ide-
ficalio11 a11d elin1ination of carrier sows by serological ~ ­
ing, culture, and/or by polymerase c11ain reaction u
primers dcsigncd to dctcct thc genes encoding one of
apx gcnes- apxlV appears to be the most useful since
unique an d in all serotypes). Vacclnation strategies incl ...
bacterins (these are serotype-specific ar1d d o 11ot µre'
carrier states) and mociifiPrl livP v;:ic.c.in es (A. pleuropnl!!r
11iae -v.rith Apx and ureasc dclction mutants). Potent:_
useful antimicrobial agents include penicillin G, tetr~
clinc, gcntamicin, kanamycin, cephalosporins, tilmico
tiamulin, florphen1col, ceftiofur, enrofloxacin, "
trimethoprim-sulfas.
In "Wooden Tongue" iodides givc11 vrally or inlr_
nously promptly ri>rl11cP the inflammatory swelling, \.\"!"!
is the n1ain clínica! problcm. Avoidance of harsh, dry
reduces the likelihood of developn1cnt ot t h is conditic. -
Cood foaling hygiene, including nave! disinfection
duces the likelíhood of foal septicen1ia. Potential effec-
antimicrobials for treatment of septice1nia produced ¡,,
equuli ssp. equuli in elude µer1icilli11 G, cefliofur, and ~~
tamicin.
Actinobacillus equuli ssp. '1ae111olytica responds to !!'
antimicrobics.
 Pasteurellaceae:
Haemophilus and
Histophilus
DWIGHT C. HIRSH
Jmily Pasteurellaceae contains thc genera Actinobacil-
-:Ullibacteriu111, Haen,1ophilus, Histophilus, Lonepinella,
.eimia, Pasteurella, and Phocoenobacrer. Ali but Lone-
and í.allihacteri11m contain species that are of med-
"""'!PO rtan ce.
e::1bers of the genus Haemophilus, beyond sharing thf'
uaits of thc farnily J>asteurellaceae, require for prop-
n one or both of two 6>TOwth tactors: porphyrins
:==- or nicotinamide adenine dinucleotide (Nl\D,
- • originally called X (heat-stable), and V (heat-
'actor, respectively. Sorne members of the family
_ _...~_llaceae showi11g Lht::>t: 11eed:; are genetically unre-
-v t11e type species T-1. influmzaP. (a~~ociated with
:!ryngeal, middle car, and respiratory tract co11di-
~ hurnan patients) .
.cussed in this ch•tpter are Haemophilus paragallina-
· 'ie cause of infectious coryza in chickens), H. para-
·he cause of a septicemic disease called Glasser's dis
'polyserositls, and secondary respiratory disease of
;ind Histophilus so1nni (the cause of septicemic, res-
.=:.~~. and genilal Lracl disease iu calUt'. a11d :;l1e::ep).
.:!us somni is the name now given to those n1icro-
- sms prcviously dcnotcd as "Hae1nophilus sornnus,"
-npllilus agni," "Histophilus ovis." Hrietly discussed is
_.._. b.icteriu111 rhinotracheale, a bacterium that is not a
::r:=: ~: of the famlly Pasreurellaceae, but phcnotypically
'inically) resembles some strains of H. paragalli-
-'1e members of the family PasteurP.llar.eae ;irp gr;im-
~ coccobacilli. Th cy are facultative anaerobes, and
-~ ox1dase-positive (setting them apart from mem-
:he family Enterobacteriaceae). Most are commensal
---- o uf IDimal:s.
.. -~= ..·ptive Features
.._.,...._- : ogy and Staining
e-:. uf tl1e genera Haetnophílus and Histophilus are
·~- :cegative roct.~. IPSS than a micrometer wide and 1 to 3
-::; . but sometimes forn1 longe1 fila.rut:.ut:>. Sorne !>-pecles
- :.1/linarum and H. infiuenzae) are encars11l;itPrl.
EKNs·r L. B1BERSTETN
Cellular Constituents and Products
Capsules are polysaccharide. The ccll wall is typical of
gram-negative microorgan1sms consisting mainly of
lipopolysaccharides and proteins. Sorne of the latter are
iron-regulalcd (i.e., Llit:y are expressed under iron-poor
conditions).
Cellular Products of Medica! lnterest
Adhesins. The role of adhesins, as in othcr microorganisms,
Is to allow the bactenum expressing them to adhere to cells
lining a particular niche, as well as to the surface of so called
"Largel" <.:t:ll:s prior tu the initiation of dlsease (in sorne cases,
niche and target c~lls m;:iy hf' the samc.>). 'fhe expression of
adhesins dept:nds u pon various environ111enla1 cues. So111e,
and probably all members of thc family Pasteurellaceae
express adhesins (and possibly more than one kind).
Histophilus somni also produces a particular surface pro-
tein, appearing as ñbrils when viewed with an electron mi-
croscope. Thesc structures are responsible for the b1nding
of thf' mirroorea nism to endothelial cells (in which apop-
tosis is triggered "vilh subsequeul fil;rin de::pu:>itiun, see
below, "Mechanisms"). The protein comprising this fihril -
lar network is onc of two immunoglobulin-binding pro-
te1ns (lgBPs) produced by Histophilus sornni- in particular,
the so-called high molecular wcight IgBP (scc below).
Little else is known about the adhesins ot the haemo-
phili and H istophil11s, except for the human pathogen H.
í11fluenzae, wl1ich product::s :se::vcral (also called fimbriae or
pili) that are responsihle for adhPri>ncP to human upper
rcspiratory t ract epitheliurn.
Capsules. ·rhe capsule plays many roles, the most impor-
tant of which are interference with phagocytosis (an-
tlphagocytic) and the protection of the o uter membrane
from the deposition of membrane attack complexes gener
atcd by aclivalil11 1uf the complemcnt system. Haemophilus
influenzae and H. paragallinarum produce capsules.
Ce// Wall. Lipopolysaccharide (LPS) clicils an infla1111ua-
tory response following bínding to Jipopolysaccharidr-
binding protein (a serum protcín), which in turn transfcrs
lt to the blood phase of CVI4. ·rhe CD14-LPS complex
binds to Toll-like receptor proteins (see Chapter 2) on thc
95
96 PART 11 Barteria and Fungi

s urface of macrophage cells triggering the release of proin-

flammatory cytoklncs F1G U RE 14.1 . Satellitic growth of Hocmophilus on a feeder
The cell wall lipopol y~arrharic!C' of Histophilus somni is streak of Staphylococcus. Haemophilus was lnoculated eveniy over
Le1111ed lipooligosaccharide (LOS) . LOS, under the direc- the entire surface. The staphylococcus was then inoculated in a
tion of the gene lob (for LOS biosynthesis), undergoes single streak.
phase variation rcsulting in diffcrcnt cpitope expression
subsequent to changes in the carbohydrate portions of the
LOS. Histophilus son1ni that phase vary are more virulent.
Explanations for thts lnclude lmmune evasion, aud phasi.:
varying isolates are more serum r<>sist;int (i!n in vitro rncas-
un;: uf n~sistance to con1plen1ent n1ediated lysis) than
thosr that do not phasc vary. Clinical isolatcs (from cen-
tral nervous system, lungs, joints, and aborted fetuses)
phasc vary, most isolates trom carrier sites (prepuce, va-
gina), do not (or cannot).
·1 herc are nvo different il11munoglobulln-blnding pro-
teins (IgBPs) expressed o o the st1rface of Histophilu.~ .~nrnni,
a 41 l<Da p roteiu, <111u a liigh n1olecular weight protein
(100 - .1:10 kDa). lloth fgBPs bind imrnunoglobulin, but thc
high inolecular wcight p rotein prcfcrcntially binds IgG¿.
Immunoglobulin molecules bind to lg!H's by '"'ªY of the Fe
portion rcndcring thc antibody ineffectual in opsonization
or triggering comp!cment activation. In addition, the hlgh
molecular vveight lgBP serves asan adhesin (see above).
fron Acquisltíon. BC'cause iron is au a!J~ulu le g1owll1 rc-
quiren1ent, microor1y111isms rn11st acqu ire this substancc if
L11ey are Lo cxist within the host. Ifacmophili and Histo-
pl1ilus bind transferrin-iron complexes by virtue oi iron- Resistance
regulated outcr mcn1branc proteins expressed undcr iron-
Haen1ophilus and Histophilus are readily killed by heat an ...
poor conditions (so-called transferrin binding proteins, or
die rapidly in culture and storage unless freeze dried -
·rbps). l ron is acquired from the transferrin -iron com-
stored at minus 70ºC. t\t cool temperatures, H. paragaf1 -
plcxcs that bind to the surface of the rnlcroorganisru.
narutn survives in exudates for severa! days.

Growth Characteristics
Mcmbcrs of thc genera Haernophilus an<l Histophilus are
tacultative anaerobes, typ1cally oxidase-positive, and ar-
tack carbohydrates fermentatively. Carbon dioxide en-
hances growth of sorne stra.i.ns. On ade4uate rueuia i11c111-
bcrs of this genus produce within ?.4 tn 4R hours turhidity
i11 bruLh ai.1d colo11i.cs 1 1nn1 in diameter on agar (35-37º<~) .
c;rovvth factors may be supplied as hcmin (X factor) and
NAD (V factor) . A mcdium naturally containing them is
chocolate agar, a blood agar prepared by addition of blood
when the n1elted agar is at 75- 80°C (rather than SOºC
when rnaking regular blood agar). 'fhi!> pruLt::uurc lilJt::1aLes
NAD fro m cells and inactivates Pn7ymes clestructive to
NAD.
Alternatively, a "feeder" bacterium (c.g., Staphylococcus)
n1<1y be inoculatcd across platcs wherc llaemophilus has
been streaked. ()n otherwisc i.nadequate media, growth
occurs only near the feeder streak, a phenomenon called
sacellitisn1(Fig14.1). lt 1nay be duplicatcu uy Lu111111e1cially
prcpared X and V factor-impn•gn;itpfl filter papers placed
v11 ll1t:: i11oculaled area (fig 14.2) . for thc X und V factor rc-
quirements, see ·rable 14.1.
Biochemical Activities
flae1nopl1ilus and llislopl1ilus of anirnals are uxida:.e a lld
nitratase-positive and ferment c;irhohyclr;ites.
variability
Serotypes rnay differ in pathogcnicity and geograph
prevalence, and determine the specificity of bacter:.r-
induccd irnrnunity. 111ere are thrce serotypes (A-C in th
so-called Page scheme, or I-III in the Kume scheme) of ~
paragallinarum anti dt lt::ast sevelf uf l-1. purasuis. NAD i n~~
pPnc!Pnt ~tra i ns of ff. paragallinarurn have been isolatt:'
fron1 South Africa. Since NAD-dcpcndcncy is used in t
cultural diagnosis ot lnlectious <.~oryz.a, tl1is has becorne
serious issue.
Ecology
Reservoir
Members of the generd Huernupl1ilu~ and Histophilus li\·t:
the uppPr rPspi r;itory tract, or in thc Jov.•er genital tr.:~
Haernophilus parasuis lives in the nasopharynx of nor:-
swine, while H. paragallinanJ111 is more closely associ:
\vith thc rcspiroto(y tract~; of sick or recovered p ou
ffistophilus son111i is found in .normal cattlt' lJutli i1
lower genital tract (prepucc, vaein;i) <IS Wf'IJ as in the Uf''.°
rt'spiratory l1acl. Oddly, isolates fro m these sites do •
see1n to phasp v;iry their LOS (and are tht1s relativcly a•
ulcnt) . Histopllilus so11111i also inhabits thc lowcr gc.r
tract of n ormal shccp.
 Cltapler 14 PusteuH:lluc,eue: Hue1Tiup/1ilus <1111.l Histuphilus 97

f 1G U RE 1 4. 2. Determination of cofactor needs with (left to righV XV, 11, and X factor-
imprcgnatcd filtcr papcr discs. Growth has occurrcd around the XV and X discs (lower left and
right), but not the V disc (top center). The organlsm accordlngly was identified as Haemophilus
haemoglobinophilus, which requires X but not V factor.

,.

Tab l e 14.1. Pathogenic and Common Species of Haemophilus and Histophilus

X V Host Clinical Signifkance

Haemophilus influenzae + Human Me11i11gili1, ;eplic~mid, ulilb, ~¡.iiylullilil,

H. pilrilgilllinilrum
H. parasws
+
+
Fowl
Swine
conjunctivitis, bronchopneumonia
lnfertious coryza of fowl
Secondary pneumonia, Glásser's disease
-m
Hirtophilus somni -· -· Cattle, sheep Septicemia, meningoencephalitis,
pneumonia, abonion, epididymitis
H. fe/is + Cat Conjunctivitis of cats
H. /1demuylubiriupl1ilu> + Dog None (opportunistic)

"Requires <yrt(e)ine, is stimul•ted by thiamine pyrophosphate.

- ·s- ss1on
__:r.1ss1on of hacmophili ancl Hi~tnphi/11~ is prob;.ibly
_ ....._-.-¡e o r by close contact. Indircct transmission is likely
=::::::=:-: cpidemics.
• belonging to t11e IgG2 isotype) binds to lgBPs by their Fe
-
: : :'lCSIS
.1s111.s. Di:it'.ase is usuall y pru<.luced v.1 hen haemophlli
&:::..:~
- - -...,rophilus fin d thcir way to a norm;:illy sterile site (a
e exception is JI. paragalli11aru111). Whether an ü1va-
enotype is triggered by as yct sorne undefined event
-·~ •íhis somni septicen1ia, for ex<implc), or whcthcr it
~appe nstance "contam1nat1on" of a previously
__-,,_:-:-?ised site (e.g., H. parasuis pneumonia following
«r both Is nor known.
- ~T1'ÍC'P mir <fjc;p;:ic;p prnrl11rPrl hy 1-listophilus son1ni,
:aoorganism is resistant to con1plemc11t-n1edialed
lysts (LOS phase variation) and survives within phagocytic
cclls by an unknown mechanism. !ron is acquired by re-
111oval (10111 L1a11sferri11:. uf Lhc hu~t. Effective immune re-
sponses are evaded by means of phas<> variation of the
outcr surface (LOS). In addition, antibody (especially that
portion (antibody bound in such fashion docs not triggcr
complement activation, nor does it serve as an opsonin).
Histophil11s sornni adheres to endothel ial cclls by way of the
high 111ule<.:ul<1r ~veight IgBPs, and trlggers apoptosis of this
cell. 'l'hromhosis occurs upon th<> s11rface of the damaged
cndothelial cell. Intravascular multiplication brings aboul
endotoxcmia (see Fig 8.1).
Dcposition of Histophilus sotn11i or J-1. parasuis into the
lowcr rcspiratory tract stimulates an inflammatory re-
sponse by virtue of their ccll wall lipopolysaccharidc (also
k11uw11 <1s lipooligosaccharldc, or L()S). Iron is acquired
98 PART II Bacteria and Fungi
from transferrin. Immune evaslon occurs beca use of LOS
phase variation, as well as IgBPs sidetracking antibody mP-
dialed opso1lizalio11, a11d con1plcn1cnt activation. llisto-
philus so1nni survives intracellularly within phagocytic
cclls (H. parasuis does not have this property) .
Haeniopl'lilus paraxallinarum is resistant to complement
1nediated lysis and phagocytosis because of its capsule.
Though llttle is kno\vn regardl ng the procluctiun uf clisease
by this microorganism, thP lipopol y~arrha ricle c.ompo-
nent uf thc cc.:11 wall Lrigger:. an inílan1n1alory respo11se.
Pat11nlngy. Ali infections have suppurative components
brought about by the chernistry of thc gram-ncgative cell
;vall, which triggers the release ot prointlamn1atory cy-
tol<incs from macrophages. lnfection of lungs, body cavi-
ties, and 10111ts te11ds to be serofibrinous to fibrinopuru-
lent. Bacteria! colonization of the merlingeal vessels
produces a thro1nbotic vasculitis lc<itliug tu e11cephalilis
and men ingitis. 1-IPmorrhagir nrcrotizing processes are
caused by l-Tistophilus so11111i.
Powl coryza is marked by catarrhal intlammation with
hcterophil cxudates.
Disease Patterns
Cattle. Thrornboembo/ic Meningoencepl1nlitis. TEME CThrom-
l.Jue111\.Julic flteningoe11ccpl<alitis) is a consequence of sep-
ticemia produced by Histop/lilus sornni leading to throm-
botic meningocnccphalitis und infarcts i11 brain and
cerebellum. ·r11e preencephalitic stage is marked b y high
fever. With central nervous system (CNS) involven1ent,
motor and behaviora.1abnormalities develop.
J>neu1nonia. Histophilus son111i occurs in pneumonic proc-
esses, u:.ua1ly \'Vill! oll1e1 agenLs, for exan1plc Pasteurella/
1 ulations.
Mannlleirnia spp., as part of the shipping fever complex.
Septicc111ic Discasc. Histop/lilus son1ni may produce sep-
ticemia (manitcsting as an endotoxemia), sometimes re-
sulting in arthritis, mycoarditis, or abortion.
Jlbortíon. Abortion dueto Histophilus sornni sometirnes oc-
curs. Whether this is secondary to septicemia, or to genital
trat:I tli:.ea~e (i11ilialed by an ascendiJ1g route) is unknown .
Swine. 1'11cu111onia. Hacrnophilus para:;uis can cause bron
chopneumo11ia secondary to virus infections (e.g.1 swine
influenza). Other bacteria (e.g., Paste11rel/a spp. and Myco-
plasma spp.) may also participa te.
Septicernic Disease. In young weaned pig•;, H. parasui<;
al:.u t:au:.e:. Glasser's disease (polyserositis), an acute in-
flammation affecting pleura, pcritoneum, mediastinum,
pcricardium, joints, and meninges. Wcaning, transport,
and management stress are prectisposing causes. 'fhe d1s-
ease strikes sporadically within days of the stressing event.
Morbidity and mortality are often low because uf ~viue­
spread acquired resistance, but they m;iy hP high in previ-
uu:.ly u11t::xvosed herds (e.g. 1 spccific pathogen-free herds).
Oisease manifestations include fcver ancl general malaise,
rcspiratory and abdominal distrcss, lamencss, and para-
Jytic or convulsive signs. Recovery begi.ns in 1 to 2 wecks.
Similar syndron1es are dueto Mycoplas1na h)'orhinis.
Poultry. Jnfectious Coryza. Infectious coryza (causecl hy H.
purugtlllinururr1) i:. ai1 acule contagious upper-respiratory
infection uf chickens. it C1ffect:. l.Jirtl:. uf vraclically ali ages.
ThP ~igns inrlnc!P nasal clisc.harge, swelling of sinuses, fa-
cial ede1na, and conjunctivitis. With air sac and lung in·
vo1ven1ent, rales ruay be <ietected . ln t he uncomplicatec
infection, mortality is low. Loss of productivity is the most
damaging aspect. Superlmposcd lnfections with my-
coplasmas and helminth parasites exacerbate and prolong
UUll.Jreaks. Qf OLhe1 species, 011ly japanese quail are highl~
susceptible.
Sheep. Histophilus so1nni causes respiratory and mamman-
infcctions, epididymitis of immature rams, and occasion-
ally septicemias (see Fig 74.6).
Dogs and Cats. Dogs. flaernuphilu~ huerr1oglobinopllilus1 z
commensal of th.e caninP lowPr genital tract, sometime<
causes cystitis and neo11atal infcctions. Its role in balano-
posth itis and vaginitis, where it is frequently tound, is un-
ccrtain.
Cats. Hae111opf1ilus (e/is is associated with conjunctlvltls
(follo"ving Chla1n)'dophila fe/is and l11fycoplasma spp. in pre-
valence).
Epidemiology
Ali the agents namcd, cxcept for H. paragallinarum, ir.
habit normal mucous membranes of the respiratory o-
genital tract. Sources of infection are therefore often er.-
dogenous to herds or indivlduals. Colonization of pig
with H. parasuis probably occurs vvhile anin1als ar
shlelclecl IJy maternal i111111u1ti1 y. Glasser's disease il1 pigs a.
all agPs orrnrs in previously H. parasuis-free, stressed pop·
Respiratory and septicemic diseases produced b'
Histophilus sornni are similarly related to stress factors, :i
the1r predileetion for feedlots and the fall and winrt-
months suggests.
Haemophilus aru.l Histuphilu~ a1e gene1ally hosl-specifh..
lmmunologic Aspects
Clrculating antibody develops In lnfecteu i11divitlual:; an ....
has a protective funrtion, at IPast in mam mal s. Presence
:.cru111 aulil.Jody cor1elales well with resistance ::
ffistnphilus sornni infection. The role of cell-mediated im-
rounity is unkno•vn.
Immunity develops atter infection . following infec-
tious coryza, it extends to heterologous serotypes.
Immunoprophylaxis is used to control infectio:_
coryza, polyserositis (Glasser's disease), and TF.ME. Raer -
ri11:. preveuLseiious diseasc but not infection owing to h
111ologous serotypes.
Laboratory Diagnosis
Recovcry of the organism from infPctt>cl tissnes or fluid<;
u:.ually requited Lo eslablish a diagnosis, though detecti-
of genus and species-spccific DNA makes this considerab'
casier (however, diagnosis madc in this manner is withcr_
 Chapter 14
sola te, maki ng type and suscepttbility testing difficult,
:'.'.or impossible). Observation of gram-negative rods in
-.:iecimens ¡.aio1 lu cullu11:: 111ay ::.ugg1::::.t Huernuphilus ur His-
-·:ílus infection. Jsolation of such an organism is fol -
"Cd by demonstration of a growth factor requircment.
lrganisms requiring X factor cannot convert delta-
:t.~!.."'!olevulinic acid to urobilinogen and porphyrin. The
:?!'lyrin rest determines this ability and X factor require-
-=nt most reliably. Definitive assignment to a species usu-
~equires addilional Lesls.
--:fectious coryza is diagnosed by culture or hy serologi-
~ ~cans (agglutination, agar gel immunodiffusion, and
""""!agglutination-inhibition tests). ·rhe polymerase chain
c:-!on with prímcrs spccific for H . paragallínarurn (thc
-2 test) is quicker, and less cxpcnsive tha11 classical cul-
- · tf'chniques (NA[).c\ependent and NAD-iI1dependent
---:!.ns react in ét sin1 ilar n1anne1 in lhis Lesl). Ornílhubucle-
rhinotracheale (scc bclow) is a gram-negative rod-
-=""d bactcrium that mimics hacmophili (and other
- ~bers of the family l'asteurellaceae), and is often iso-
~ frorn thc respiratory tracts of bi rds. It is not, however
~-dependcnt (as are the classical strains of H. paragalli-
:.i11. ~f'f' ;ibove, "Variability"). NAD-independent vari-
.c=::- of 1 l. paragallí11arun1 are difficull lo diffe1e11 Liale Iro1u
.11otrachea/e, except with the HP-2 test.
-·::tment and Control
~ animal haemophili are susceptible to penicillin G,
~~" ~ur, a11tl tctracycli11c::.. Histuphilus surnni is susceptible
- :phenicol, peni<:illin G, tilmirosin, ;incl tptr;iryrline.
J!e septicemíc-meningocnccphalitic forro, timeliness
~~ :naintenancc of trcatment are critical.
- : infectious coryza of fowl, crythrornycin or sulfon-
~ ~s can be administered in feed or water, but may also
ntrolled hy el im í nation of carriers and immunization
~ctividuals at risk. Poultry producers may clepopulate
_.:-cd flocks. When breeding stock m ust be preserved,
-'--'-""' a<l<li liu11s are vacci tt<itctl <it 16 wccks, fuur wccks bc-
~in ing the infected flock.
Pasteurellaceae: Haen1ophllus and liistophilus 99
Bactenns are of vaJue in prevention ot 'l'EME but are Iess
effective in other Histopl1iltis son111i infections.
0RN ITHOBACT ER IUM RH INOTRACHEALE
Ornítllobactcriu1n rl1inotrachea/e is a grarn-negative rod
that resembles sorne members of the family Pasteurella-
ceae. It is a facultative anaerobe, whosegrowth is enhanced
by carbon dioxide, is oxidase-positive, attacks carbohy-
dratcs fermentatively, and does not grow on MacConkey
agar. Il req ui1es neilh1::1 purvt1yri11~/l1c1uc (factor X) nor
nicotinamide adenine dinucleotidc• (NAD, NAf)P; f;ictor
V). Tt is important to note, however, that it is not a member
of the fa mi Iy Pasteurellaceae, and in fact is nota n1ember of
any recognized group of microorganisms. Based upon thc
sequence of the DNA en cocling the 165 ribosomal RNA, it
is more closcly related to Ríen1erella and Capnocytophaga.
Thcrc are 12 se10Lype~ (A-L), with A bcing the most
common.
Ornithobacteriu111 rhínotrachcale causes (by unknown
mechanisms) respiratory tract disease (sinusitis, airsacculi-
tis, pneumonia) in birds (domestic and wild). In turkcys
and chickens resplratory tract disease may be relatively
mild, rcsulting in poor performance, or severe '\.\rith in-
creased n1ortality. ·r1ansn1is:>iv11 b l1orizuutal (acrosols,
contaminated fomites) or vertical (it is unc:le;ir wht>thf'r
this is transovarian or cecal contamination of eggs).
With thc appearance of NAD-independent strains of
Haernopl1ilus paragallí11an1m (see above) diffcrcntiation of H.
paragalli11arun1 from O. r/1inorracl1ente is d1fficult by classical
culture techniqucs. A polyn1erase chain reaction- based test
(HP-2, see abuvt:) yuickly anti casily tliffcrentiates the n.vo
organisms.
Most isolates of O. r!zínotracheale are susceptible to an1-
picillin, erythromycin, penicillin, spectinomycin, tetracy-
cline, and tylosin. Vaccination with autogcnous bacterins
reduces morbidity.
 Bordetella
DWIGHT C. HIRSII
Members of tl1e genus Burdetellu are grar11-11cgalive cuc-
cob<t<'illi belonging to the f;imily Alcaligenaceae. There are
eight species described, sorne of which are important
pathogens of humans and other animals (fable 15.1 ). All
but one (B. pctrii) are acrobic bacteria that are parasites of
ciliated respíratory epitheJJum. Boráetella petrii is a faculta-
tive anaerobe that lives in the environmer1t and l1as not
t)een associatcd with disease. Bordelella pertussis, B. puruper-
tussis, and B. bronchiseptira are ve ry rlosely related, and
pro bably belo11g lo tl1e san1e species. Bordetella pertussis
(and, rarely, B. parapertussis) causes whooping cough in
humans. Of veterinary unportancc, and thc main topic of
discussion in this chapter are H. broncl11sept1ca, which has
been implicated in porcine Atrophic Rhinitis, canine ken-
ncl cough, and bronchopneumonla In many species, and
B. avi11rn, the cause of rhinotracheitis of birds (mai.nly
lurkcys).
Descriptive Featu res
Mor pholo9y and Stainin9
Bordetellae are pleomorphic gram-negative coccobacilli,
about 0.5 ¡11n X up to 211m in size.
Structure and composition
1'he cell wall is typical of gram-negative bacteria being
composcd of lipopolysaccharidc and protein . .Bordetella
bronchiseptica produces a capsule. Most 1f not ali members
of the genus produce fimbria] adhesins (pili). Bordetella
bronc/1/septica and B. avium are motile !Jy peritricl1uus
flagella
Cellular Products of Medica! lnterest
With tew exceptions, /J. pertuss1s, li. parapertussis, B. bron-
chiseptica, and B. avium produce (or at least have the genes
to produce) many of the same products iruporta11t i11 tlic
pathogenesls of disease (T;ihlf' 1.'i .7.) .
Adhesíns. Thc role of adhesins, as in other microorgan-
isms, is to allow the bacterium expressiI1g the1n to adhere
to cclls Hning a pürticular nichc, as wcll as to the surface of
so-callcd "target" cells prior to the initiat1on of disease (in
sorne cases, niche and target cells may be the same). The
expresston of adhcsins depends upon various enviru11-
m P nt;i 1 l'llP~
Bordetellae produce a numbcr of structures that are re-
100
ERNS'f L. BIBERSTEIN
sponsible for adl1erencc to host cells. These adhcsins ir -
clude fimbriae (or pili), filamentous haemagglutinin, per-
taetin, cell wall lipopolysaccharidc, tracl1eal colonizatio,..
factor, and pertussis toxin. Ali are posítively regulated tT
the BvgAS regulon (see below, "llegulation of the Cellula:
Products of Medica! Intercst").
l. Fimbriae. Fimbriae are protein structures co.rr-
posed, in part, of subunits (pilins) responsible fe·
adherence to recepturs u11 hosl cells. Bordetella fim-
briae adhere to ri liate<I Ppithelial cells of the respir::
Lory tract. They are encoded by four structural gen--.
fi1112, fi1113, fimX, and fi111A that give rise to at lea
four scrotypcs (scc below, "Variability") .
2. 1:uamentous haemagglutirun. fha (for filamentor.:.
haemagglutinin) is a protein adhesin that has ~
tcast th ree activities assoclatcd with various c!-
mains within the protein: adhcrence to ciliate
eµitl1elial <..:ell:; uf l11t:: 1espi1alory trae l and n1ac.
phages (hy way of an "Arg-Gly-Asp" or RGD x:
quence targeting complement receptor 3 on h
cells); adherence to ciliated epithclial cells ot t.
respiratory tract and phagocytic cells (by way o
"carbohyclrate recognJtlon domain"); and a hw
magglutinil1.
3. l'erlacU11. Pr11(for pertacti11) is an outer membra~
protcin that functions as an adhesin. Like Fha
above), pcrtactin contains an "Arg-Gly-Asp" or IltJ
sequence. Though a particular host target cell .
not been identified for J>rn, it is likely that cilia.
epithelial cclls of the respiratory tract, as wel! _
phagocytic cells are the targets for tl1ls adhesi n
4. Cell vvall lipopolysaccharide. Cell wall lipopol y~
charide of B. aviu111, and by inference B. pertussi
parapert11ssis, and B. bronchiscptica, serves as an _.
hesin by binding to ciliatcd epithelium of the re-.
ratory tract, and as a "shield" to cover the o_
membrane prorecting !t from the actlon of nie-:
brane attack complexes gener;1ted by the rolT':-
1ueul systen1 (see below).
5. Tracheal colonization factor. Tracheal coloniza~
factor (lcf) is a protcin u<lhcsin that bh1ds to cili-
epithelial cells oí thc respiratory tract.
6. Pertussis toxi11. Ptx (for pertussis toxin) is bot~
ADP-ribosylating toxin (see below), andan adhe:-
Following synthesis, a portion nf Ptx is exr-
f 1u1u ll1e bacteria! cell, and a portion remaim
tached to its surface. It is the surface-associate<i ~
tion that serves as thc adhesin. The host cell t~ ..
 Chapter 15 Bordetella 101

Table 15.1. Members ot the Genus Bordetella and Their Usual Source or Associated
Condition

Usual Source or Ass:ociated Concfition

801cletelld pet l~b Whooµiny lough in hu111a11 pdlienb

B. parapertussis Whooping cough-like condition in human patients: uncommon cause of
pneumonia in sheep (sheep strains different from human strains)
B. bronchiseptica Associated with Atrophk Rhinitis in S\vine; respiratory tract disease in a variety
of animals
B. avfum Rhinotracheitis in pouluy (espedally turlceys)
R. hin1ii Normal flora of fowl
B. hofme>ii A>>U<.idle!l wilh upper 1e>pi1dlu1y ltdtl !li>t:d>e, d11!l >eµliu:mid i11 humdll ¡Jdlitmb
B. trematum Associated with ear and wound infections in human patients
8. petrii Environment

Table 15 .2. Distribution of Cellular Products of Medical lnterest Within Selected Members
of the Genus Bordetella

l'roduct O.pertmm O. pon1pcrtu"i' O. b<onchixptica B. ovium

Adenylyl cyclase + + + +
Brk + + +
(d¡J)Ule +
DNT + + + +
Fha + + +
Fimbria + + + +
lron scavenging + + + +
LPS + + + +
Pertactin + + + ?
Ptx
Tri
+
+
-· -· }

Trdtl1edl lyluloxi11 ~
• + + +
Type 111 secretion system :¡. + + +

'Genes encodinq Ptx are present, but thi?"toxin is not produced.

\Vhich Ptx adheres are ciliated epithelial cells of the
respirarory tract, and phagocytic cells.
Cipsule. 'fhe capsule plays many roles, the most impor-
- · of which are lnterference wlth phagocytosis (an-
- "gocytic) and protection of thc outer membrane from
ceposilio11 of 1nen1b1ane a llack con1plexes generaled
~-n,,-ation of thc complemcnt system.
JI Wal/. Thc ccll wall of thc mcmbcrs of this genus is
~·•pical ot gram-negative bacteria being composed ot
-~aydrates, lipids, and protein. Bordetellae possess
r>Irsaccharlde and outer membrane proteins that are
rtant virulence determinants.
Lipopolysaccharldc. The llpopolysaccharide (LPS)
in the outer memhrane is an important virulence
determinant. Not only is the lipid A component
tox.ic (cndotox.in), but the length of the side chain
in the 0 -rcpcat unit hindcrs thc attachmcnt of thc
membrane attack complcx of the complement sys-
tem to t hc outer membrane and imparts resistance
Lo auliu1h.:ru!Jial pruteiHs (uefeHsins) found in respi-
.ratory tract sccrctions and within phagocytic cell
granules. Llpopolysaccharide binds to lipopolysac-
charide-binding protein, w hich transfers it to the
blood-phase of CD14. ·r11e CD14-LPS coiuµ lex. bluds
to ·roll-like receptor proteins (see Chapter 2) on the
surface of macrophage cel Is triggering the release of
proinflammatory cytokines. The composition of
the LPS (presence or absence of the 0-repeat por-
tian, its length, and charge) 1ntluences the extent of
protection against the complement system and an-
liwlcru!Jial pcptltles. LPS cornposition is regulated
by the RvgAS regulan (see below, "Regulation of the
CellulaI Producls of Medica! lnle1esL").
2. Outcr mcmbrane protein. Brk (for Bordetella resist-
ancc to killing) is on outcr mcmbranc p rotcin. Drk im-
parts res1stance to damage by the complement sys-
tem (bestows the serum resistant phenotype). Brk is
regulated by the BvgAS regulan (see below, "Regula-
tion of the Cellular Products of Medical Interest").
Exotoxins. There are four exotoxins produced by thc bor
detellae that play lmportant roles in dlseases associated
102 PART 11 Bacteria an<l Fungí
with IDCf!lbcrs o1 this genus: tracheal cytotoxin, der-
mo~1ecrot1c toxin, adcnylyl cyclase toxin, and pertussis
tox1n. Ali, except trachcal cytotoxln, whlch is a portian of
;,he ceJJ i~1all, are re,gulated by the BvgAS regulon (see below,
Regulallou uf lht: Ct:llular P1uuu<.:L::. uf Mt:Ji<.:al Iutt:re::.t").
l. ·rracheal cytotoxin. ·rracheal cytotoxin is a portian
of the cell "vall, and damages ciliated epithelial cells
lJy iutcrfcriug with DNA :-..ynthe:-..is. It aI:-..o lJinds to
n1acrophagPs triggPring tht> rt>lease of proinflamma-
tory cytokincs.
2. Dermonccrotic toxin. DNT (fo r dermonecrotic
toxin) is a membcr of a family of toxins vvith similar
structures an<l b1ological activity. The other "der-
1no11ccrotic" toxins i nclude Pmt produced by Pasteu-
rella rnultoclda (scc Chapter 12), and CNfl an.d CNF2
produced by f.:scherichia coli (see Chapter 8). These
toxin:; are :;o nan1cd hecause <lern1onecrosis follows
the ir in jection in to the skin, an occurrence tl1at does
not occur nnturnlly. DN·r dcnmi11atcs and transglu-
tamates (preferred) the small GTP-bindíng protein
Rho blocking its G'l'Pasc activity (Le., GTPase activat-
lng protelns are unable to correctly bind to n1odified
Rho). 'fhese modifications result in changes in the
a<.:tin <.:ylu::.k\!l\!luu uf affc<.:lcu <.:cll::. a11u i11hilJiLio11 oí
clifft>rPntiation of ostt>Ol)lasts in honc tissue.
J. i\dcnylyl cyclo:sc toxin. Adcnylyl cycla:;c toxin is a
bitunctional protein \.Yith adenylyl cyctase as weH as
hemolytic activity. Adenylyl cyclase toxinenters the
.host cell, and follo,ving "activation" by calmodulin,
1ncreases the intracellular concentration of cA~P.
Affe<.:ted cell::, lose control of intracellular levels of
cAMP resulting in an inability to regulate ion and
fluid flow in~o ai:d out of the "target" cell (respira-
tory tract cp1thcl 1uml. Loss ot control o1 cAMP lev-
els in phagocytic cells results in reduction of this
cell's ability to phagocytose and kili. T he hemolytic
activity is associated with an RTX motif (repeats in
Loxir1, so callt:d lJe<.:ause of the common feature of
rrpr;its in g lyrinf'-r irh SP<J.11Pnrt>s within tht> pro-
tein, see also J!,scllerichia coli haemolysin in Chapter
8, l'asteurclla/Ma11nhei1nia leukotoxin in Chapter 12,
AcLinobacillus haemolysin in Chapter 13, Moraxel/a
cyt?toxi~ i~J Chaptcr 19). By vir t uc of the RTX-type
lox1n acl1v1ty, adcnylyl cyclase toxin is also a pore
fv1111iug .1.11vtei11 lltal la1gct:; i1eutJ0.1Jl1il:;, iuacrv-
phages, and lymphocytcs.
4. Pertussis t<>xin. Pertussis tox in (Ptx) is an ADP-
ribosylating toxin \-vhich ribosylates "activated" het-
erotrimeric (; proteins (G'fP bound) rendering them
incapablc of rcturning to thc inactivc state (GDP-
bound). "Activatcd" G proteins stimulate adenylyl
LyLla:.t: lt:aui11g lu i11L1t:ased level:; uf i11tracellula1
cAMP. Abnormal\y high concentration of cAMP re-
sults in loss of fluid and ion flow in to and out of the
cell, and it the cell has phagocytic function, interter-
ence \vith uptake and intracellular killing. The role
of Ptx as an adhesin is discussed above.
lron Acq11isilio11. Because iron is an absolute Do-rowth re-
. .
qwrement, 1n1croorganisms must acquire this substance if
they are to exist ~vithin the host. Bordetcllae acquire iron
by secreting a sidcrophorc and utilizing siderophor'2"
ma<le by other species of n1icroorganism. In addition th~
utilize the !ron found in heme and hemoproteins. '
Under conditions of iron starvation (as would occur 1~
Lhc llu:-..t), lJorJctcllac ::,ccretc a 11yJroxawatc-tyµc sü.ler<..-
phorP callPci a/caligin. Rorclt>t<>llae also havc thc ahilin· +
utilizc cnterobactin, a siderophore produccd by membe:
of thc family Enterobacteriaceae. These siderophores (alca:.-
gin and enlerobactin) remove iron from the iron bindir:
pro~eins of tl1e host (transferrin and lactoferrin), making
ava1lable to the bacterium.
~~1ne anti ht:rnoproteins (allJunliu c1I1tl he1uu.1.1exi11) a;-i:
actct1t1ona l so1irrf's of iron. The outer memhrane protei:-
that binds thes~ substanccs, Dhu R (for Dordetella heme 1rp-
take receptor), 1s rcgulatcd by lcvels of available iron b·
mcans of thc cxtracytoplasmic sigma factor Rhu (for reEU-
lat1on of hc1nc uptal<c) and t h e Fur regulon (see Escherichf...
coli, Chapter 8).
Type fil Selrelion Sy:;Le1n. A ·rype III :;eLreLiou svsleru lid:.
h<>t>n idPntifiPct in thc bordctcllac (unde r the control ofth ....
UvgAS regulon, scc bclo"v, "Rcgulation of the Cell uia-
Products of Mcdical lntcrcst"). The Type III secretion $\...,_
tcm consists of nn asscmblagc of protcins (more than 2c
that torm a hollow tube- like structure tl1rougl1 which e.-
t~ctot ?tOtci.ni; are "'H'\\cc.ted" \nto host "tariet" ce\\s. ......
yet, Borderella effecror proteins have not been identifie...
but hnst "t;irzrt" crll~ (t r;ich<'<ll t>pitht>li;if ct>ll.~, phago0-
cells) undergo apoptosis as a result of effector protein "ir-
jection" resulting in loss of epithelial integrity, and e\·...-
sion ~f the immunc systcm of the host (decreased ¡)hagv-
cytos1s, and dccrcascd antigen processing which leads r
reduced imrounc responses).
Regulatio11 of the Cellular Products ofMedical Tnterest. Bo;-
detellae transcriptionally regulate the genes encodin:
protlu<.:ts involved in the protluction of tlisease. Tl1ese ir-
c ludP thE> gt>nPs Pnrocting all of tht> acthesins (firnhriae, fi -
amentous hacmagglutinin, pcrtactin, the character of th~
cell "vall lipopolysaccharide, tracheal colo11ization facto;
and pertussis toxin), pertactin, Brk, toxi ns (ad e nylyl c-
e lase, DNT, pcrlussis toxin), alcaligin, and the Type III s~
cretion system. Ali are regulated by the products of tl'-
gcucs c11cotl<::tl in uvgt\ anti uvgS (fo r BvrJctella virul<::nl.c
s~n~s~ and are rcfcrrcd to as the RvgAS regulon. BvgS is
h1shd1ne kinase that acts as a "sensor" of environment-
cues resulting in autophosphorylation ot one ot its his::.-
dine residues. 'l'his phosphatc is thcn scrially transferred ~
an aspartate residue, then to anothcr llistidinc befo:~
bcing uscd to phosphorylate BvgA. Phosphorylated B'~
i::. a Lrc111::.criµlio11al a<.:tivalor uf tl!e geues e11codiJ1g .:
products
. mentioned above. lt is unclear exactlv , what t.. -
e11VJ.ronmental cues are that are "sensed" by Bordere
Growth at 37"C in low concentrations ot Mg~ü 4 and n:~
tinic acid activates the regulan. Though gn)\-vtl1 at 374 r
compatible "''lth a parasiric state, it is ttnclear what :-
MgSO.¡ and nicotinic acid concentrations play in vivo.
Growth Characteristics
All but onc ol thc species ot Hordetella are strict aerobe5 ~
riving energy from oxidation of amino acids. Bordr_
petrii (the exceptlon) is a facultative anaerobe. Borde.,.

--..d1isepticu and B. uvíurrt grow on 01di11ary lal.i~>ral.ory
--.:.2 (3S-37ºC), including MacConkey agar, under at-
l:i..:5?heric conditions; the former is inconsistently he-
- _'"lic on blood agar.
-':"--~mica( Activities
-.;..:-rella avium and B. bronchiseptíca are catalase and oxi-
,,.,.e positive, ferment no carbohydrates, and utilize citrate
::.:: o rganic carbon source, but only B. bronchiseptica re
-=-.es nitrate and splits urea.
-..- ~ella spp. are klllcd by hcat or disinfectants. 'rhey are
.:rptlble to l)road-spectrum antibiotics and polymyxin,
-!lt to penicillin. 'fheir environmental survival is epi-
-;ologically sig11ifica11t.
=:iility
a~rtme11t of typing schernes has been developed,
-. ~ly for epldc111iological pu1 poses.
- : least four serotypes have been described for bordetel-
~d upon thc Fim protcin (scc above, "Adhesins").
=- mete/la bronchiseptica dissociates into tour phases
--::ng in colonial characteristics, b.emolytic activity, sus
:e::::s:an stability in saline, ease of coloni.zation, a11d. toxic-
:SOme strains appear to be host-specific.
:I'Jee serotypes, l>aseu 011surfdt:e agglutini11s, dre recog-
:;:.;:.rC tn B. avi um.
Some 20 heat-labile (I<) and hcat-stable (O; 120ºC/
.-'n) antigens exist. Many are cornmon to severa! spe-
Others are species- and type-specific.
:ordetella hronchiseptica from pigs appear different from
-= srrains from dogs a11d horses (which are also different
~ each other) .
-__:; o gy
-o:-:voir
,;.a.el/a spp. are mainJy parasites of ciliated respiratoty
~~ tissue. Bordetella bronchiseptica occurs in wild and do-
~-;SJc carnivores, wild and Iaboratory rodents, swine, rab-
- and occasionally horses, other herbivores, primates,
...;;__ turkeys. Altl1uugh prubably not part of the residcnt
_ _,.,,. it is found in tl1e nasopharynx of hPalthy :inimals.
3ordetella aviun1 inhabits thc rcspiratory tract of in-
-ee fowl, principally turkeys.
-....,-:iission
--. malian infections are prin1arily airborne, wl1ile in
- ~ indirect spread via water and litter is common.
~~genesis
·wnis111s. BoJdctc:llae "se11se" va1iou::. e11viro1111u.:11tal
;es :eading to activation of the BvgAS regulon lc~ading to
- ;egulation of the various genes encoding thc products
Chapter 15 Bordetella 103
dist:ussed dl>ove (see atJove, "Cellular Products of J\1edical
Tnterest"). Attachment of hordetellae to c:iliiltf'cl Ppithelial
cells follows expression of adhesins (fimbria e, filan1entous
haemagglutinin, pertactin, cell wall LPS, tracheal colo-
nization factor, pertussls toxin- scc Table 15.2 for listing
of which aclhes1ns are produced by which bordetellae).
Multiplication occurs (iron is scavenged from host iron-
l>iudiug proteins- lactoferrin, transferrln- and. from
hen1e and hPmoproteins; horc!Ptf'llae 011ter membrane is
p.rotected from co1nplement-ge11erated n1en1brane attack
complexes by capsule, LPS, Brk) and infla1nmation is initi-
ated (LPS; death of epithelial cclls duc to "injection" of cf-
fector proteins; tracheal cytotoxin). Infla1nmation and Joss
of the ability to control fluid and ion flow into and out of
trat:hedl epithelial cells (increased production of cAMP
from effects of increasPcl ;:ictivity of <'lcl<"nylyl cyclase) leads
to increased mucus and fluid accumulation in the upper
respiratory tract. Ciliated epithelial cells become ineffec-
tual in clearing secretions (cilial paralysis dueto incrcascd
levcls of cAMP and cytoskeletal changes brought about by
the action of DNl'; death of ciliated cell dueto tracheal cy-
Lotox in, and the unillentified effector protein "injected"
by way of thc~ ·ryf)P 111 sPcrPtion system) . lfnencapsulated
bordetellae (ali but V. bronchiseptica) adhere to inflanm1a-
tory cells (by way of filamcntous haemagglutinin, per-
tactin, and pertussis toxin), and relcasc adcnylyl cyclasc
(interferes with phagocytosis an<.1 killing), pertussis toxin
(i_n terferes '1-Vith phagocytosis and killing), and "injection"
of ali effect.or prutein (interferes with phagocytosis). If
phagocytosis oc:c11rs, horc!Ptell;ie survive within phagoly-
sosomes (nature of their LPS) and also escape into e11do-
cytic compartments that do not lead to lysosomal fusion .
The consequences of Bordctella-induccd changcs in thc
ciliated portions of th.e respíratory tract are varted: depres-
sion of respirat ory clearance 1nechanisms, facilitating sec-
ondary complications (e.g., pneumonla); in pigs, B. bron-
chise¡1tira provides nasal irritation, rendering the
turbinales susceplible lo lhe aclion of Lhe deru1011ecrolit:
toxin (Pmt) of Pasteurella multocida, which has emerged as
the primary agcnt of Atrophic Ilhinitis ("progressive
Atrophic Rhinitis," see Ct1apter 12). IJ P. rnultociaa does not
become involved, DN'f as well as ali of th.e products de-
scribecl above, produces a mild, reversible turbinate hy-
poplasia ("nonprogressive Atrophic Rhinitis"). Pneumo-
11ia, if lhis occurs, is due lo i11flau1111atory responses C111cl
the inability of host phagocytic cells and complement sys-
tcm to easily clear bordetellae .
J>atholo«<:Y· Destruction of ciliated respiratory epitl1e-
Iium is the hallmark of Bordetella initiated diseasc. Thc
process is a suppurative one affecting various portions of
the tract (rhinitis, sinusitis, tcacheitis). Compromise of the
clearance n1echa1tis1us oI tl1e upper respiratory tract may
lead to a suppurative pneumonia ;:in<l ;iirs:icculitis with
Bordetella itself or sorne other microorganism.
Disease Patterns
Swine. Atrophic Rhinitis. 'fhe progressive form of Atropllic
nhinitis is <11 JP to romhinPrl infPction withP. nn1ltocida and
B. bronchiseptica, and was discussed il1 Chapler 12. 1'he
104 PART 11 Bacteria and Fungí
nonprogressive form of tl1is disease follows infection with
B. bronchiseptica alone, and is transient and self-Jimiting.
Pncumonia. Bordetc/la-mediated compromise of the host
defenses of the upper respiratory tract sometimes leads to
secondary pneumonia associated with B. bronchiseptica or
sorne ot her microorganis1n.
Dogs and Cats. Canine Infectious Traclzeobronchitis (l<ennel
Cough). Kennel cough is associated with B. bronchiseptica.
Thc natural discasc is oftcn accompanied by canine para-
influenza virus, canine adenoviruses 1 and 2, and canine
herpes virus. While most dogs recover within a few weeks,
the bacteriurn ca11 persist fur r11u1 1Ll1:-.. Tra11sfe1 lo suscepli-
b le cats has heen demonstrated.
Pneurnonia. Bordetella. bronchiseptici1 is sometimes iso-
Jated from samples collected from pneurnonic lungs, often
from dogs with canine distemper.
Cats. Bordetella bronchiseptica produces a mild upper res-
piratory tract infection in cats, mainly those housed in
colonies (upper respiratory t ract infections in cats are also
associated with herpe~ vin1•;, r;i lirivir11s, Nfyr:nplasrna,
Chlan1ydophila) . Cats, like dogs, carry the organism asymp-
tomatically (up to 19 weeks) following recovery. Transfer to
susceptible dogs has becn dc1nonstratcd.
Poultry. Turkcy Coryza. Turkey poults infected with B.
avium develop tracl1eobronchitis, sinusitis, and airsacculi-
tis. Signs include nasal exudate, conjunctivitis, tracheal
rales, and dyspnea. Morbic.lity is lligh, l.Jut u1ur ta¡iLy, ex-
cept by seconclary infection, is ge ne rally Jow (<Sº;ó). Re-
covery may begin after 2 weeks, aithough sorne illnesses
may Jast 6 weeks.
Laboratory Animals. Rabbit bordetellosis. Bordetella /Jronchisep-
tica infections in rabbits are usually asymptomatic. How-
ever, in association with other infectious ageut:s (e.g.,
Pasteurella mu1tocida) may be involved with broncho-
pneumonia.
Epidemiology
Atrophic Rhinitis affects pigs under 6 \«.reeks o ld , when os-
teogenesls and bone remolleling are rnu:st activt:.. Affected
p igs sprt>acl R. hronr:hiseptir:a. The ultimate source<; are car-
rier sows, in whicl1 carricr rates decline with age.
Canine kennel cough usually affects young, nonim-
munc dogs that acquire it from clinically affected individ
uals, as well as recovered carriers.
Bordetella avium causes disease main ly in young poults.
'fhe contan11nated environment is impurta11t iI1 perpetu-
atin.g the infection.
lmmunologic Aspects
Pathogenic Factors
Depressed cell-mediated responses havc bccn obscrvcd in
experimental H. avium intections. ·rheir relation to natural
disease is undetermined. Bordete!Ja bronchiseptica can para-
sitize dendritic cells, which results in a dccrcasc in im-
1nune responses dueto iI1efticient antigen processing.
Protective Role
Local antibody ls believed to preve11t B. bronchíseptica col-
onizatio11 in dogs. No ot her immune responses 11ave been
sl1uw11 tu !Je vruteclive.
Rordetella avium antíseru1n is ineffective in protecting
turkeys, but maternal in1munization reduces losscs i.r:
challenged progeny. Antibody to acthesins prevents adl1er-
ence to tracl1eal epitbelia.
lmmunization Procedures
Bacterins used on pregnant sows provide sorne colostra·
imn1u11ity tu IJiglets, especially '\'vl1e11 including toxigenic
P. rnultocida strains. Bacterin-toxoid preparations protec:
piglets.
Intratracheal administered live attenuated vaccine ha;;
been beneficial in. kennel cough control.
Of B. aviurn vaccines, those u:sing atte11ualed live orga11-
isms intratracheally have bee11 most effectivP..
Laboratory Diagnosis
Nasal swal.>s (Atrophic Rhiiliti:s), sediJuen Lof Lranstrachea.
washes (c;:ininf' trache.ohronchitis), and tracheal SV·l ah
(coryza of turkeys) are cultured on blood and MacCon~
agars. Routine Jaboratory tests are used for .identificatio::
A polymcrasc chain rcaction bascd nssay u tilizing prime:.
ctesigned to amplify various species-specific segments
DNA is available for determining the presence ofbordete·-
lae as well as for identiflcatlon purposes.
RnrdPtP.lla aviurn rPact~ likf' Alt:aligenes (aecalis in rou tií'-
laboratory tests. Cellular fatty acid analysis diffcrcntiate!
the two. A n1icroagglutination test is used tor serodiagnosis.
Treatment and Control
Thc progressive form of Atrop!1ic Rhinitis is not treatab~"'­
Preventive measures include maintenance of an aged ~
herd with a low carrier rate; thorough d isinfection a- ._
c1eanup of farrowi11g 11uuse:s a11d 11urseries afler eacl1 ~
vaccination (see ahove); prophylactic use of sulfonamice:-
in feed or water; and elimin ation of carrier sows based --
nasal swab culture.
Canine tracheobronchitis responds inconsistently -:-
antibiotics. Vaccination (see above), fumigation of kl:I.
nels, adequate ventilatio.n, and isolation of aff1~cted do::-
are useful preveutiv<:: praclices. l'elracycli11e ren1ain.s ~
dn1g of choice.
Dordetellcz aviu111 is suscepti ble to tetracyclinc, crytl-..: -
n1ycin, and nitrofura11toin , but resistant to penicillii:
streptomycin, and sulfo11a1nides. 1vfass medication ar.._.
vaccination may prevent outbreaks witl1out el!m tn ~-­
the infection.
 Brucella
RICHARD L.
:>i:> i:. a11 i1úcctious bacteria! disease caused by
rs of the gen u-; Bruce/la. Tt is a ciisPasP of '"'orlc1wic1P
-·--~ncc and affects a number of animal species. Bru-
·e oblígate parasites, requiring an animal host for
_ _ , "'lance. Infections tend to locallzc to the reticuloen-
...__.., di system and genital tract with abortions in fe-
rid epididymitis and orchitis in males tl1e most
n clinical n1a11ifeslalions. Cllro11i<.: infcctions are
n.
,-D:-JA hybridization studies show that Drucella is a
---.. pecific genus based on level of overall homology.
- -ditional species names for the different bruceJias
.ie to be used in a biological species concept system
~-'' ~ host range, in addition to the presencc of species-
"1arke1s. 'l'he dassical 110111c1 1<.:laturc is used ir1 this
- :\dditional Brucella "species" havr rrc:ently bPPn
....'CI from marh1e mammals. Thc different Brucella
c:xhibit host preferences and vary in severity of t he
causect. Dye and phagc susccptibility along with
~:nical, cultural, and serologlc characteristics are
d istinguish among species. The six traditional Bru-
:..ie!> are B. afJortus, B. carlis, B. 1nelirensis, B. neoro-
1·i~, and B. suis.
. spccies of Brucella are capable of causing disease
-ans. Infections are chronic and debilitating. The
brucellosis in humans are relatively nonspecific
~ :vidua ls wíth brucellosis are somctimes labeled
_ ,......, o ndriacs hecause of the va¡,rue prcsenting clinical
.t:=: :igy and Stainlng
rs of the genus Brucella are small, gram-negative
-~ cilli measuring 0.6 to 1.5 µm by 0.5 to 0.7 µm in
..s are fa1rly un1form and can casily be mistaken tor
Tlley are typically arranged singly but also occur in
clu~ters. No capsu les, flagella, or spores are pro-
nowever, an PxtPrn;:il f'nvf'lc.ipe has been demon-
ii.:::::3! ny clcctron m icroscopy around R. abortus, B. rneli-
nd B. suis. Brucella stain red with Macchiavello and
_ ,...,, d Ziehl Neelsen stains.
-----~--'"· Structure and Composition
~=J:i._e po<;SP<;<; ¡:¡ typic;:il gram-negative cell \-vall.
-~"1--nt surface antigens are located on thc lipopolysac-
W ALKER
charldc. Specifically, the A and M anrigcns are found in
varying concentrations among the different smooth
Bruce/la species. 'l'l1e oule1 u11::1111;1a11e <.:outaius otl1er
1najor surface antigens. The peptidoglycan !ayer (3 nm to .'i
nm) is more p romincnt tl1an that of Escherichia coli. The
pcriplasmic space varíes tro1n 3 nm to 30 nm. Th e cyto-
plasmic nlcmbrane is a typical thrce layered lipoprotein
membranc. L-form variants are recognized and may play a
role in persistent infections.
·rhe n1ol% G Te of DNA is 58-59. Most Bruc:ella species
have two circular chromosomes. Plasmid DNA ha-; not
bccn dcmonstratcd. Thc cntirc genornes of Drucella suis
and B. 111elitensís have been sequenced and are likely to pro-
vide substantial insight into factors relatcd to virulence
and pathogenesis. The overall fatty acid composition is
sufficiently unique to be useful for identification and tax-
onon1ic pur poses.
Cell ular Products of Medica! lnterest
Cell Wall. The cell >vall of the members of this genus is one
typical of g ram ncgativcs. Thc lipopolysaccharide (LPS) in
the outer membrane is an important virulence determi-
nant. Not only is the lipid A componcnt toxic (endotoxin),
but the Iength of the side chain in the 0 -repeat unit hin-
dcrs the attachment of the membrane attack complex of
l he con1plen1e11Lsyslen1 Lo l11e uuler 111e;:111lJra11e. LPS biI1ds
to lipopolysaccharide-binding protein (a serum p rotein),
which in turn transfcrs it to the blood phase of CD14. The
CLJ14-LPS complex bit1ds to 'loll-like receptor p roteins (see
Chapter 2) on the surface of macrophage cells triggering
the release of proinflammatory cytokines. In addition, the
cell wall lPS of brucellae aid in its survival within ma-
c1ophages.
Jirythritol. Erythritol (a fo11r-carhon alcohol) is one of
severa! "allantoic fluid factors" found in the gravid uterus,
and appcars responsible for the prefercntial Jocalization to
the reproductive tract of the pregnant animals. "Allantoic
flul<t facrors" stimulate the growth ofbrucellae.
0 11tcr Membrane Proteins. Porin protcins in the outer
r111:: r11 bra1H:~ are tl1ought to stlmulatc delayed-type hyper-
sensitivity anrl ac.c:ount fnr the varying susceptibility to
dyes observed for the different species.
Miscellaneous Products. Production of adenine and gua-
nine monophosphate by Bruce/la inhibits phagolysome
fusion and activanon of the myeloperoxidase-halide sys-
tem. Bn1cella are ablc to inhibit apoptosis in infected ma
<.:roµltage!>, thereby preventlng host cell elimination. Sol-
105
106 PAtrf .Il Bacteria and Fungí
uble protein products int1ihit ·rNF-alpha production. The
Vi r (for vir11/r>nrr>) opPron PnroclPs ;i TypP TV Sf'C'rPtion
systen1, which appears to be involved with intramacro-
phage survivaL
Growth Characteristics
On initial isolat ion, colonies are not apparent until 3 to
5 days' incubation. Most colonie:; are lletectell 1.Jy 10 tu 14
days, b11t in somP c.;:ises incuhation for up to 21 days is
required. Grow th is best in an aerobic environment at
37ºC but occurs between 20º C and 40ºC. Optimal pH is
6.6 to 7.4. Brucclla avis and sorne biovars of B. abartus re-
quire an increased co11ce11trati.on of C0 2 . Enriched
media with 59/o serum are required by B. abartus biovar 2
and B. ovis.
Bruce/Ja colonies have a r.h;iractf'ristic hl11ish color
wl1en exan1ined witl1 obliquely transmitted light. Colo-
nies 11ave sn1ooth or nonsmooth morphologies that are
d etcrn1incd by thc prcscncc or abse11ce, respectively, of the
polysaccharide side chain in the lipopolysaccharide.
These morphologic v ariations are the result of sponta-
neous mutarion and are intluenced by speclfic grovvth fac-
tors. Smooth colonies are ivhite, co11vex witl1 an entire
eJge, dilU 11ave a crea111y co11sisle11cy. No11sn1ooth colonies
have intermediate, rough, or mucoid forn1s. Rough colo-
.n ies are dull yellow, opaque, and friable. Thcy are difficult
to suspend in solution and agglutinate spontaneously. The
mucoid colonies are siinilar to the Tough colonies except
for havir1g a glutinous texture.
Resistance
Brucellae survive freezing and tha\.Ying. Under proper e11-
vironmcntal condition!.;, thcy survivc for up to 1 1no11tl1s
in milk, urine, water, and clamp son. Most disinfectants ac-
ti~le against otl1er gram-negative bacteria kill Bruce/la.
Pasteurization effectively kllls Bruce/la in milk.
Diversity
!he Brucella genome appears to be very stable. Strains asso-
ciated with a specific animal host l1ave a unique genon1ic
organization.
Thf' colony morphology of Rrucella varies from rough
to smooth forms (see "Growth Characteristics,'' above).
Brucella a.bortus, B. melitensis, B. suís, and B. neoton1ae a.re
typically isolated in thc smooth form but can ctevelop
rough forms oo subsequent laboratory passage. Brucella
avis is always in a Tough forn1. lsolates of B. canis have a
111 ucvitl appeara11ce. 111 ~e11e1cd, ~iuvvll 1 ~LI ai11~ vf B1 u ccllu
are more virulent than rough strains.
Variations in C0 2 requiremcnt, H 2S production, urcasc
production, susceptibility to differing concentrations oí
ccrtain dycs (thionin and basic fuchsin), and susceptibil-
ity to naturally or mutagen-derived bacteriophages ac-
cou11t for diversity among species and biovars within
species. 1'he different species vf _Brucel/u vary i11 hosl pref-
Prf'n<'f' an<i <if'gr<"f' of virulPnce within and among animal
spcc1es.
Ecology
zoologic and Geographic Reservoirs
As oblígate parasites, Bruce/la require an a11Lmal rese;..,._-
Host prcfercncc is cxhibitcd by the different fh __
species; however, a broad host range has been d.s;;;;:::
strat ed for son1e species. Survival tirne outside th e:.
variable and depends on temperature and m o1üiS<;;:::;;-
Colder weather extends survival time.
Cattle are tl1e prefert:u.lial hosL for B. abortus. O lhc _
imals, including hison, camels, and yaks, are comm L.
infccted. The d ifferent biovars of B. abartus havc dit!c-~
geographic d.i stributions. Biovars 1 a11d 'l have a worlc
distribution., while biovar 3 is predomi.nantly four .:.
India, Egypt, an.d Africa.
Swine are the preferential host for B. suis biovar l_ -
biovar is widely distril.Jutt:J. Bivvar 2 is ÍOLHld ü1 swine __
\"lile! hilrf's (l.epus timidus) , predominately in westerr: _
central Europe. l3rucel/a suis biovar 3 is prcdominatd~
covered from midwester11 North An1erica in the L:- __
States. Biovar 1 affects reiI1deer and caribou in -~~....
Canada, and Siberia.
Brutella rnelitensis infects goats and sheep worldwíc-c
cept for North An1erica, Australia, dJltl New Zt:a1c
Ca1nels, alpacas, and Jh1m;:is arf' ;:¡ lso i nfected.
Brucella avis infections are lin1ited to sh.e ep and D. e;,-:_
infections are limited to dogs. Both have a worldwide dis::
bution. Brucella ncotomac has only been found .in the '"e
rat (Neotoma lepida) with geographic distribution. lim itai -
the Salt Lake Desert of Utah. Recently, B. rnelitensis aru:
suis have become esta1Jlis11ect in cattle ill so1ne M id_
Eastern and South American countries, respectively.
Tlie 11cwly recvg11ized Brucella. species fou11d i11 n1a.........
mammals also show host preferences similar to other E~
cella :>pccic:>. lvfnrinc Brucclla :;pccic:; (propoGcd spcci~
B. cetaceae and B. pinnipediae) have oeen found in a nurr::
of species of sea 1na1nmals and are considered h
adapted and e11zootic in large populations of these a_-
mals. Patl1ology is typically m inilnal and tl1e ma.~
Bruce/lu :species are currenlly considered opportu11ists
seconda.ry pathogens.
Transmission
Bruce/la are disseminated by director indirect contact \•-
infcctcd animals. lngcstion is the most common routt:
entry, although exposure t11rough ttie conjunctival z:::
genital mucosa, skin, and respiratory routes occurs. "'.""
1najor so urce for exposure tu B. uburlus ir 1 cal Lle a 11l'. _
melitensis in. sh<"<"P ;:in<i goats is through aborted fetns..
the placenta, and postabortion utcrinc fluid:;. Abortcd =-
sue and fluids are also a common means for transrnis2 -
of B. suis and B. canis. Genital infections in cat tle rout i.Ge.
clear within 30 days after calving and cows are not cons_
ered infectious for other cattle after that time. Genital -
fections ln swine, in sorne case:;, per:si:sl lo11ger lhan lh
in c;ittlf'.
lngestion of milk from infected cattle and goats .is ;;7".-
other source for infection of calves and kids. Virect t~
fer in utero has also b een documented.

.::.:ections of thc accessory sex glands of males allo\"IS
.:iissemination of organisms through the semen.
.:t1ons can occur in the accessory sex organs Without
.:ular or epididymal lesions being prescnt. Venereal
111i:>:>h>n of B. suis in swine, B. ovis In sheep, and B.
in dogs is common. l Jrinf' is rinothf'r vehicle for dis-
--:J.ating n. ca11is to other dogs.
-.sects may play a minor role in transmission and
- -tenance of infection in a herd. Facc flics have been
-n ro take up and excrete Brucef/a in their feces.
..·-:genes1s
. terference with fetal circulation dueto the ex1sting placen-
•u1is111s. Y:ollowing exposure, IJrucella penetrate intacl
-..osaJ surfaces. In the ali.mentary tract, the epithe1ium
'l:':ing the ileal Peyer's patches are a preferrcd sitc for
-:. After penetrating mucosa! barriers, organisms may
-rigulfed by phagocytic cells. Br11cella are internalized in
- c.:ophages by generalized 111t::111lJra111! ruffling and
::::;;;...:ropinocytosis. Various mechanisms are PmployP<l by
-:ella to allo"" for survival inside phagocytic cells. Son1e
:..'le genes for intracellular survival are actually shared
·-:. plant pathogcns and cndosymbionts. Brucella are ca-
_..,'. e ot surVJving ana mu1np1y1ng ins1de macrophages by
'ifying the phagosome maturation process and creat-
- .i.n inlraceJJulaJ enviro11111c11L ~uili1l>le for multiplica-
;:. :\cidification of the phagosome induc.r.s thf' virR pro-
~¡;r for the Viril complex, an important virulence factor.
--e \ 'irB operon appears to encode a 1'ype IV secretion sys-
- and is likely involved in intraccllulnr trafficking and
- ;.cropinocytosis. Phagosome maturation is arrested at
?S between acidification and phagolysosome fusion.
=..1tri1cellular survival in macrophages and, to a lesser ex-
~. nP11trophi l.<; is f'n h;inrerl by su pprt'.'ssing the myeloper-
dase-11202-halide systen1. Superoxide disn1utase and
;alase production may play a role in defense against ox-
-~vc killíng. Stress proteins have been demonstrated in
-...c.e11a, however their role in virulence is considered mar·
e -.:JI. 'fhese proteins are thought to play a role in protect-
4 organlsms from hyclrolytic enzymes, oxygen radicals,
- , myeloperoxiclase killing systems in the phagolyso-
me. Tbe lipopolysaccl1aride of Brucellu is d ireclly as~oci­
ed with virulence and is thought to play a role in enhanc-
: intraccllular survival. Production of adenine and
:-.-L..1.ine monophosphate by Bruce/la inhibits phagolysome
•on and activation of the myeloperoxidase halide sys-
....,. Bruce/la are able to inhibit apoptosis in infected
'>CTOphages, thereby preventing host cell elimination.
"uble protcin producls il1l1ibil TNF-i11plta proüu1..,1ion. It
::ielieved that the variations in virulPnC'f' oh~erved among
-ce Brucella species may, in part, be related to the greater
:.Litv ot sorne species to avoid host defenses.
Following entry into tl1e host, Brucella organisms, ei-
--;.er free ln the extracellular environment or in phagocytic
·1~. localizc to regional lymph nodes. 'fherc they prolifer-
- :e and infect other cells or a1e killed a11ú Ll 1c i ofection is
·=:minated. Sorne cattle appcar to be innately resistant to
..:::fection. ·rhis rcsistancc is rclated to the macrophages'
,_ 11lty to contain the organisms. from the regional lymph
- .xies, Brucella disseminate hematogenously and localizc
- the rctlculoendorhelial sysrem and reproducnve tract.
Chapter 16 Brucella 107
Thcrr. is preff'rf'ntial loc;:ili7;ition to thP rPprorl11rtivP
tract of prcgnant animaJs. Unknown factors in the gravid
uterus, collectively referred to as al/antoic fluid factors,
stimulate the growth of Bruce/la. Erythritol is considcrcd
to be one of these factors.
Experimental infection studies have demonstrated
lhal, al the cellular levcl, Bruc:ellu locali;::es into the clster-
nac of the rough endoplas1nic rcticulum of trophohl;ists of
thc placcntome. lnfection subsequently spreads to the
fetus. ·rhe exact mechanism of abortíon is unclear; how-
ever, likely possibilities are that abortion results from 1) in-
titis, 2) the direct effect of endotox.in, and/or 3) fetal stress
resulling f10111 tl1t:: i11fia11unatory response In fetal tlssue.
Although less is knovm ahout thf' frictors involved in lo-
culizution of Bruce/la in the reproductive tract of n1ales, the
presence ot growtl1 stimulating compounds may be a fac-
tor. 'fhe prolonged bacteremia observed with sorne of thc
Brucella species accounts for the greater likelihood for ex-
tragcnital manifestations to occur in animals infected with
ll 1o~c ~i>t:cies.
Pathnlngy. ThPre are grossly visible lesions in the pla-
cen ta associated with Bruce/la abo1 lio11s. 111 tercotyle-
donary thickening With a yellow gelatinous fluid is pres-
ent. ·rhe cotyledons are frequently necrotic, ycllo,"1-gray in
color, and covered vvith a thick brown exuda te. The degree
of necrosis varies among the Brucel/a species with B. meli-
tensis infections i11 goats uciug iuost severe. The aborted
fetus is frequen tly edematous. Ahomri~::i 1 contents may be
turbid and have a lem on-yellow color. 'J'he most con1n1011
histologic findings in the fetus are bronchitis and bro11-
chopneumonia with a predominatcly mononuclear ccll
infiltrate. In general, Bn.1cel/a induce a granulomatous-
type inflammatory reaction.
Tu 111ales pali>iil.Jle enlargement of the epidldymis, espe-
cially involving thf' tail portion, is common. Epididymal
lesions are characterized by hyperplasia and hydropic de-
gcncration of tubular epithelium. Rcsulting extravasation
of spcrm lcads to thc formation of a spcrmatic granuloma
(F1g 16. L). Tn bulls with orchítis the scrotum is swollen,
largely due to an inflammation of the tunica and fibrinop-
uruleut exuuate in the tun!ca vaglnalis. ·rhe testicular
parP.nrhymri hPromPs necrotic and is sometirnes replaced
by pus. Pathology of the accessory scx organs includes pro-
statitis in dogs and fibrinopurulent seminal vesiculitis
in bulls .
Extragenital tract pathology includes lymphocytic en-
dophthalmitis in dogs (B. canis), purulent or fibrinopuru-
lent synovltls in swine (B. suis), osteomyelitis in dogs and
swine (B. canis and B. suis), necrotizing and purulent bur-
silis iu liur~cs (B. uburlus), anú hygroma development in
cattle (B. abortus).
Visease Pattcrns. The primary clinical manifestations of
brucellosis are related to the reproduclive tract. In general,
anima!s do not exhibit overt systemic illness. In fcmalcs,
abortlon Is the most common presentation. No premoni-
tory signs are usually apparent. Abortion in cattle com-
monly occurs iJ1 tl1e fiftl! 111outl1 of ges1:ation or later.
Retained placenta is a possihlr. sf'<]11Plri. ~Pm;iles usually
abort only once, presumably duc to acquired inlillunily.
Hrucella melitensis infections in goats and sheep are similar
108 PART JI Bacteria and Fungi
F 1G U RE 1 6. 1 . Swelling and irregular conformation of the tail of the epididymis of a ram
ínfeeted with Brucella ovis (A). Cross-seetion of the tail of the epididymis showing a pocket of
inspissated matefla/ result1ng from extravasation of sperm {IJ).
to H. ahnrtus in cattle except t hat acute mastitis devclops in
goats lnf<::cted with 13. rnelitensis. ·rhe mastltis in goats prc-
scnt·s w ith palpable noclu les in the udder and milk tJ1at is
clottcd and watery. Abortions in swine can occur at any
tlme in gestation and are rclatcd to time of exposure.
Abortions in dogs dueto [{ cnni<> oc:cur <irounci SO ci::iy<> of
gt>!>tatiuu. Brutella uvi:> i11fecliu11!> i11 :.liee.1:1 ouly 1a1ely 1e-
sult in abortions in ewes.
In males, epididymitis and orchitis are thc most com-
mon presenting signs. Lesions are usually unilateral but
n1ay be bilateral. Semen cxaminaUon reveals increased
numbers of neutropl1ils in acute cases. Ne utrophils are few
in number in more chro11ic infcctions. Bruce/la ovis infec-
tiun:s in rau1!> prc<Jo n 1i11ate;:ly afft:cl tl1e epid idyn1is wilh
tPsticular lesions being u ncommon. Palpable lesio11s in the
cpididy1ni:> of mature rams are frequently thc rcsult of in-
fcction ~vith B. avis, i\1hereas epididymitis in yearlings and
ram lamb:s are usually caused by a varicty of other organ-
1sms. In bulls, infection with B. aborn1s frcquently involves
the testicle. Dogs infected with B. canis develop scrotal
swelling a:s a result of fluid acl:u111ulaliou iu U1e lw1ica.
Scrot.il c1C'rn1atitis may develop because of consta11t licking
of thc scrotum. Infections of the male genital tract rcsult in
clccreasetl fertili ty and, in sorne cases, s terility.
Exlragenital m anifestations dcvclop in many an imal
spcc1cs. Swine in fected wit h 13. suls may deveJop art h ritis,
cspecially in the large joints of thc li mbs, or lu mbar
spondylitis. Lesion:s in the lu1111Jar ve;:rlel;1ae res ull i11 Ussue
necro'\i'\ th;it p11ts prP_<\surc on lhe spi nal c:ord and can lead
t() posterior paralysis. Dogs infected ~vith B. canis can dc-
velop mcningoencephalitis, osteomyclitis, discospondyli-
tis, and anterior uveitis. Ocular manifestations in dogs
may be thc 1D1t1al presenting sigo for canine brucellosis.
C:hronic infectio11s v.rith R. abortus in cattle can result
hygromas. Horscs i11fcctcd with B. ubortus devclop "P'
evil" or "fistulous withcrs" and present 1~>ith fistul _
tracts o riginating from thc atlantal o r supraspinous bur~
rt>:.pectively. lnfectio11:. i11 liu1:.e:. u:.ually resull íro111 ce.
t.ict with infPcte.c1 cattlP.
J3ruccllosis i..11 l1un1ans is primarily a disease of the ret
ulocndothelial system. A mild lyrnphadcnopathy, spler
mcgaly, and hcpatomcgaly may be dctected. Onset of si;-
occurs within z to 3 weeks of cxposurc. Clinical signs;:
nonspecific and include alternating fever and chills \\~
night sweats, fatigue, musclc and joint pains, an<.l ba ..
aches. l1epression ancl insomni;;i <irP com1n on . SpC'c-'
cllnlcai n1a nifestations of infections includc a rth r itis,
teomyelitis and endocarditis.
Epidemiology
Humans acquire infections !Jy l1autlliI1g Li!>:.ues co11Lai11
L~r11cella organisms. Rrurt>lla 1nelite11sis is considered t
1nost virulent species for humans followed by B. suis
abortus, and B. canis. Bruce/la avis and B. neotornae do :-:
infcct humans. Common sources for infection are abor
fetuscs, placentas, and postabortion uterine flu ids, a!:
which con tain large n u mbers oí organi s111s. Veterinaria::-
ranchers, an d slaugttterl!uu:>c 1.vurker:. a1e par liculail.
r isk For <icq11iring in fPcti on.~.
The prolonged bactcrcmic phase of B. suis infcction
swine poses a special risk for slaughterhouse vvorkers h..
dling infcctcd tissucs. Tndividuals who participate in -
huntmg of feral swine are also at risk for contracting B.
infections. Relatively few cases of infections in hum·
dueto B. canis have been repurtctl. I11Llividuals al 1isk

~e1 ~vorkers and brccdcrs co1ning in contact with con-
- =-"nated fluids from infected dogs.
.::fected animals also shed organisms in the mill<. Raw
~-or raw milk products of bovine or caprine origin are
.:~ sou1ce:> fui i1úections in hun1ans. Maril1e n1anm1al
- -:.ella species have caused intracerebral granulomas in
~ans .
'"'cddental selt-inoculation with live Bruce/la vaccine
~- 7'S can result in disease. l-luman-to-human transmis-
~ iS rare.
:-.:.~eristics of lnfection inAnimal Populations. Susceptibility to
:.-..:tíon depends on age, sex, breed, and pregnancy sta-
Youngcr animals tend to be more resistant, and fre-
_;1Uy clear iufecliuu~, allho ugh latenL i.nfecUons do
2 r. Less than 3º/Ú of animals infected at birth remain in-
~~d as udults. Scxuully rnaturc unimals are mucl1 inorc
._x¿ptible to intection, regardless of gender. Most ani-
~ infected as adults remain infected for life.
'-{erd size and animal denslty are directly related to
alencc of discase and difficulty in controlling infec-
•• in a populallon. Calving praclices also play a 111ajo1
:n the spread of hruc:ellosis. Separa te c:alvi ng pe ns
. for n1inímizing exposure of uninfected animals.
-ether a herd raises its own reptacement animals or pur-
~es replacement animals affects the potential for intro-
-Ctlon Jnto the herd.
:.'": contrast to cattle, where bulls play a relatively minor
boars are more llkely to be a source for introuuclng
'1' 1/n into a swine h errl Roth vPnPrP::il tranc;mission ;:inrl
-,,,.lurc to abortcd fctu:sc:s and fetal n~cmbranes are in~-
•
-.ant for maintaining infections in a herd. Confinement
o:-t:>eding swine in common pens or lots provides the
...::- setting for spread1ng infection. Management practices
_ -::eted at climinating infected boars and minimizing ex-
..:re to aborted tlssuc grcatly reduce the inciuence of uis-
".2:X' in commercial swine operations. Feral swine serve as a
~ voi1 fo1 13. su is and a1e n1orc con1n1only infected tban
_ .:::mercial swinc in sorne countries. Tn sorne European
.:ntrics whcrc 8. suis biovar 2 is found, thc Europcan harc
- as a source tor intection in swine.
::>issemination of n. 01 is in shccp occurs during the
1
-eed1ng season. Olctcr raros are more llkely to be infected
- '1 yearlings. lntrodt1ction of an in fected ram during tl1e
_CIJi ug seasu u ca11 lea u tu ra µid sµreau uf iufecliuu
·ri n thc flock . 'l'r::insmission occurs when an uninfected
- b rccd:; u cwc rcccntly brcd by un inf<:ctcd rum . Thc c-,,vc
~ aiainly as a mechanical vector tor transmitting infec-
=>n.. Homosexual activity of rams is another means of
-eadlng lnfcctlon among rams.
"">gs io suburban areas are least likely to be infected
~ - B. can is. Prcvalcncc of infcction is grcatcst il1 eco110111-
.......:-- depressed arcas. Closc confincment settings such as
":::cls incrcasc thc likclihood for transmitting infections.
-:nunologic Aspects
- - :ine Mechanisms in Pathogenesis
dencc indicates that antibodies against Bruce/la play
::i a protectlve and detrlmental role. IgM antibodies,
Chapter 16 Bruce/la 109
which appear initially after infection, and low Jevels of lgG
will cause complement-mediatcd lysis of Brucella. Elcvatcd
levels of IgG antlbodlcs, howcvcr, appcar to actas blocklng
antibodies that m odulate the ability of the complement
n1e111brane attack con1plcx to lyse cclls. This n1ay account
for resistance to complement-mediated lysis in the tace of
high spccific antibody lcvcls and thc lack of corrclation bc-
tween protection and high antibody titers. ·rhe blocking
antibodies are opsonizing and promote uptake by phago-
cytes where Brucella have developed mechanisms for sur-
vival and proliferation.
Phagocytic cclls unablc to elin1i11ate Brucella play a role
in dissemination of organisms to other parts of tbe body
and in pcrsistcncc of infcction.
lgA autoantibody has been demonstrated in dogs in-
fected with B. can is and may explain sorne of the observed
effect on fertll!ty.
Mechanisms of Resistance and Recovery
Effcctivc immunity is primarily ccllulur in oature. Specifi-
cally sensitized ·r lymphocytes release cytokines that acti-
vate macrophages, which in turn control Brucella by reactive
oxygen intermediates. Bn1cella can actívate NK cells by in-
duci ne antigen-prcsenting cells to secrete IL-12. NK cells can
then kili infected target cells. A n1orc cffeclive ü11111u11ily de-
velops "''hen animals are infccted prior to sexual maturity.
Artificial lmmunization
canle are 1mmun1zed w1th e1ther nonVlable (B. abortus
45/ 20) or attenuated live (B. abortus strains 19 and RB51)
vaccines. These p1oducts p1ovide p1otection úo111 abo1-
tion, the major mode of dissemination, but not from infec-
tion. A single dose at 3 to 7 and 4 to 12 months of age is
required with B. aborh's strain 19 and strain RBSl, respec-
tively. Two doses 6 weeks apart in animals over 6 months
of age are requ1red w1th B. abortus 45/20. Adult cow vacci-
nation is sometimes performed as a regulatory effort to
control inft!ction In a hen.l. Strain 19 is uccasionally sl1eu
in thP mi lk ;:incl c;:in c;:i11SP (lhortions in c:attlP. Aclult vac:c:i-
nation with D. abortus strain RD51 only rarely causes abor-
tion. Bulls should not be vaccinatcd becausc orchitis can
dcvclop. Thc typc of vnccinc uscd is gcncrnlly cstablishcd
by the particular country's regulatory agency in charge ot
brucellosis control. Recently, in a number of regulatory
programs 13. atJorrus straln RB.51 has replaced stra1n 19 as
the approved calfhood vaccinc because it does not inter-
fere with serologic evaluation.
Bruce/la melitensis Rev 1, a partially attenuated vaccine,
is uscd to control bruccllosis in goats and shccp causcd by
H. melitensis. A killed product, Hn,cella melitensis H38, is
also available.
Bacterins for control of B. ovis are availablc, but their ef-
ficacy is limited. Bruce/la n1elitensis Rev l has been used to
prolecl againsl B. ovis infeclions in sheep; however, il cau-
not be used in countrics free of B. tnelitensis because the an-
tibody litcrs intcrfcrc with serologic evaluations for D.
n-1elitensis intection.
Vaccination is not practiced for control of disease
caused by B. suis or B. canis.
110 PARr 11 Bacteria and Fungi
Laboratory Diagnosis
Specimens
Great care should be employed when working with in-
fected tissues and cultures in the laboratory. Ali Brucella
cultures should be handled following biosafety leve\ 3
pra1...tices l.Jecause uf tl1t: 9utt:ut.ial fur laborátory infecLion.
All lahoratory proc:edures should he performed in a man-
ner that prevents aerosolizatio11, and all work should be
conducted in a biological safety cabinet.
Appropriate san1ples for diagnosis ofbrucellosis depe11d
on the aniJnal species affected, species of Brucella involved,
and clinical presentation.. Abscess material, semen, and
vag1r1al flulus assuclateu wiLll rece11L auv1 liuu::. a10:: u::.o::ful
for rt>covt>ring organisms anten1ortem. Milk samples from
cattlc and goats are use<l in antemortem isolation attcmpts
and fo r immunodiagnostic evaluation. ln dogs, blood cul-
tures are useful for isolation of B. canis beca use of the pro-
longed bacterenlla that occurs. Serum is ltsed for serologic
eval uatio11.
Samples collected at necropsy shoul<.l include splee11,
liver, udder, and multiple lymph nodes, inch1cling the su-
prau1a111n1ary, relropl1ary11geal, interna! iliac, lumbar, and
mesenteric lymph nodes. The supramammary lymph
node is superior to other lyn1ph nodcs for isolating Brucclla
from dairy cattle. AbomasaJ fluid and lungs of the aborted
fetus and the placenta are the preferred specimens i11 the
case of abortion. In males the epididy1nis, testlcle, and ac-
cessory sex organs are examined.
Direct Examination
Gram stains of fetal stomach contents from an aborted
fetus and the placenta revea! largc nun1bcrs of gram-
negative coccobacilli. Using carbol fuchsin rather than
safranin as a counter-stain in the gram-staining procedure
makes organisms more easily detectable. Modified Ziehl-
Neelsen and Macchiavello stains are also used to demon-
slrale Brucella. Orga11isn1s can be detected ü1 sen1e11 but are
usually in low numbers. Brucella is difficult to detect by di-
rect examlnati.on in othcr samplcs, cspccially from chron-
icall.y intected animals.
lsotation
'fissues are cultured directly on solid media. Milk cultures
are performed by centrifuging milk at 5900 to 7700 x g for
15 n1inules or by allowin.g for gravity crean1 separation to
occur overni.ght. Both the cream layer and sediment, if the
ccntrifugation tcchniquc is uscd, should be platcd on so lid
media. Commonly used media il1clude serum dextrose,
tryptose, and brucella (Albin1i) agars.
If contarnlnatlun ls llkely tul.Je a prul.Jlt:Ju, J:;ulaliu11 al-
tt>mpts shonlcl he made using media containing actidione
(30 mg/L), bacitracin (7500 U/L), and polymyxin B (1800
U/L). Seiective inedia are used hoth with and without the
incorporation of cthyl violct (1:800,000) (Fig 1.6.2).
Cultures should be incubated at 37ºC in 10% C02 for a
mínimum of 10 days and up to 21 days in highly suspi-
cious cases.
F 1G U R E 1 6 . 2 . Culture pfate from the supramammary lymph
node from a cow that reacted positive for serum antibodies to
llrucell a abortus. Numerous Brucella colonies are growing on the
selective Brucella media with ethy/ vio/et. Four large co/onies of
onother bacteria ore o/so present.
Animal inoculation is the mostsensitive method for d.:-
tection of Brucella and is sometimes necessary when V€:"""
low nun1t>ers of organisms are prese11t. Guinea pigs are th:
most sensitive laboratory animals for this pnrpose. T''
guinea pigs are il1oculated a11d at 3 and 6 weeks postinoc-
ltlation an animal is sacrificed. Serum is examined for a:.-
tibodies and tissues are culturcd for organisms.
tdentification
Presumptive identification of Brucella species requircs
<.leu1u11:>tratiI1g culu1lies u( gra111-11egali ve coccobacilli ll=
are nonhen1olytic, catalase positive, and oxidase positi ~
(except for D. ovis and sorne strains of B. abortus). Mos:
species, except B. avis, are strongly urease positive. Glucos..:
and lactose are not ferme11ted by any of the specie:;
Agglutination in unadsorbed antismooth Bruce/fa sen•;.;
helps in presumptive identification of smooth strains_
Dt:fi11itive ide11lificalio11 is usually periorn1ed by •
Bruce/la reference laboratory. A fluorescent antibody test -
used for rapid identification. Urcasc production, C0 2 :-:-
quirement, H 2 S production, oxidation of metabolic su.:-
strates, agglutination in monospecific antisera, growth..::
the presence of varying concentratlon of thionin, a.i:..:.
basic fuchsin and phage typing are used to determ ir-
svecies aud biovars wilhi11 species (Fig 16.3). Brucella ab. ~­
tus strain 19 can be differentiated from field strains of ~
abortus by its lack of rcquircmcnt for C02 for growth a.G.:.
inhibition by 5 mg/ml penicillin or l mg/ml of erythrito..
St rain RBSl can be differentiated from field strains ar:.
srra1n 19 by úemonstratlng lt:; rt::;i:;tauct: Lu 1ifau1pi11 (ZC
pg/m l), st<"lining with c:rystal violet, and aggiutinatic=..
with acriflavin.
Polymerase cl1ain reaction (PCR) methods have bee=
describcd for diffcrcntiation of Brucella species. Pe;.
restriction fragment length polymorphism, particularly
the omp2 gene, has proven particularly useful. A deletio-
in the ery locus can be used to dlfferentiate Strain 19 fro=.

: G U RE 1 6. 3. Lysis of a confluent lawn of Brucella abortus
th increasing tenfold dilutions of Tbilisi phogc (B. abortus specific
Jidge). The "rout ine test dilution" used is the hlghest dllut/on
• '10winq confluent ~vsis where phage is applied. At t he 10-5 dilution.
-dividua/ plaques are becoming apparent.
~ strains of B. abortus and the presence ot an insertion
...ence in the i.vboA gene, which encades a glycotrans
e, can be used to differentiate Straln RBSl from field
.,~
-- .nodiagnosis
=:.bOdy dctection is commonly used for diagnosing bru-
<.is and in control program:;. Somplc:; tcstcd includc
'-"~.:1. milk, and occasionally semen. A number of iJn-
r¡odiagnostic tests have been dcvclopcd for cattle.
tests detect <lifferent classes and types of antibodies
--. vary in t h<>i r ~<>nsi tivity ;in<! specificity. In dividual
d samplcs can be tested by tube aggluti n atio11, plale
_ utination, rose bengal platc, or card tests. ()ther tests
.-de thc buffcrcd platc agglutination assay, rivanol ag-
. rtat1on, complement ft xation, and cnzyme-linked im-
so rbent assay (ELISA).
·t:\lucutly, llighly sensitive but less speclfic tests are
--"'""' ~or ser<'<' O ing p11rpos<>s ;:inri ;:i rf' followed by more spe-
·ots for confirmation purposes. A sin1ilar approacl1 Lo
used in cattle is employed when testing goats ancJ
_,..,
•
fo r B. mclitcnsis. Sera are screcned with a test such as
- .se bengal test and results contirmed with a more spe-
·est.
... k 1:. s1.:ree11cd wltll the Bruce/la mili< r1ng test, Whicn
,fies specific antihocii<>s in mi 1k. ·rhP test is p erformed
•.uk tan k Tn ilk samples as a means of scrce11ing dairy
- .)tained Bruce/la an tigcn is added to milk . If an tibod-
-e prescnt, agglutinated antigcn is buoycd to thc top
'" rising cream and a purple r1ng develops at the top ot
be (Fig 16.4).
Iogit. Le:;L:; i:lrc 1.:ornrnonly used to Identlfy infected
~--- - he rds and monitor h<>rcJ <>tilh1<: Thcse tests are less
;:;;:::i:::;_;e when testing individual pigs bccause son1e iu-
Chaprer 16 Bn1ce/la 111
F 1G U RE 16. 4 . Brucella milk ring test for detection of
antibodies in milk. In a negative sample, the added, st ained Brucel la
antlgen remains dispersed in t he milk port1on and the cream /ayer is
white (left). In a positive sample, the antibodieI reart with ~tainerl
antigen and thc complcx riscs to thc top in the cream /ayer. The
cream /ayer appears purp/e (rlght).
fected swine do n ot have detectable antibody titers. Hercis
can be scrccncd by thc bruccllosis card test. Tests such as ri-
vanol agglutination and 2-rncrcaptoet han ol agglu tination
are used for confirmation .
Rams are tested for antibodies to B. ovis u sin g either a
complement fixation test or ELTSA.
Fo1 <.:anille uruccllosis, screenlng Is performed with a
rapid slide agglutination tP.~t (R~A1') . The RSAT is sensitive
but not vcry 3pecific, therefore positivc rc.sult.s .should be
confirmed with additional tests that are more specific. An
agar gel immunodiffusion test using cytoplasmic antigcn
is more specific but not as sens1t1ve as the RSAT and is used
as a confirmatory test.
Nonculture Detection Methods
A number of nonc11lture methods, including PCR, im-
m unopcroxida sc staining, Dl'lJ\ probcs, nnd co ngglutinu-
tion, have been described for dctection of Hrucella in tis-
sues and fluids.
Treatment
As a general rule, trcatmcnt of infected livestock is not at-
tempted because ot the h igh treatrn en t tailure rate, cost.
and potential problem s related to maintainin g infected
i:luilnals in the tace of ongoing eradicatio n programs.
Tetracycline ;:in<! clihydrostrcptomycin have b een used to
treat D. ovis infections in ran1s \'Vilh va1iable resu!L:;. OnLc i:I
palpable epididymal lesion is present, a11tibiotic treatment
wiH not be beneficia!. l'hc prcscncc of abscesses and fibro-
sis in tissues of the accessory sex organs makes penetration
"1-Vith antibiotics to these areas difficult.
·rreatment of dogs with brucellosis requires a prolongcd
112 PART 11 13acteria and Pungí
course of antitJiotic t11erapy. The cuu1uil1aliu1J of dihy-
drostrPptomycin and tetracyclinc or minocycline for a 2-
to-4-week period is commonly used. fluoroquinolones
may also be uscful; however, only Iimited inforn1ation
about thcir effectiveness is available. ·rreatment failurcs are
common. Treatment should also consist of neutering af-
fected animals. Treatment is not recommendcd in canine
breedlng colonles. In this case, inft:cted dug:s shuulu IJc
culled .
Control and Preventi on
Approaches at control and prcvention of brucellosis de-
pend on the animal species Involved, Brucella specie;),
managcment practices, and availability anrl pfficacy of
vacciue:.. AJJvruacht::s used lo control brucellosis includc
1) immunization alone. 2) testing and re1noval of infectcd
animals in conjunction with an immunization program,
and 3) testing and removaJ of infected an1mals without
immunization.
Control by lmmunization Alone
Tmmuni7..ation by itself reduces the number of abortions
and, thereby, reduces potcntiul for cxposure . .By itself, im
rnunization will not rcsult in eradication ot the intection
in a herd. ln1munization alone should be considered as a
means of controlling the leve! of disease only.
lmmunization Followed by Test and Slaughter
Control of bovine brucellosis routinely employs a combi-
nation of vaccination of females and a test and slaughter
program. Cattle are vaccinated at a young age and evalu
ated vv1th rmmunodiagnostic tests when they rcach sexual
mat urity and vaccination titers havc di1ninishf'ci. Vaccinil-
liu11 wiLl1 slrain 19 is approxi111ately 70% effective on an
inciivirlual hasis h11t more effective when evaluated on a
herd basis. ln experimental challcnge, vaccination with
strain Rl351 providcd protection similar to vaccination
with strain 19. Any animals identified as infected a re
cullcd fron1 the herd and slaughtered. Routine testing Is
done by the milk ring test in dairy cattle o r blood tests
from beef <.:attle at ::.laug11ter. A ~i11dlar v1og1a1n is Jollowed
with R. m1ilit<?nsis in fPctions in sheep and goats.
Test and Slaughter Without lmmunization
lmmunization control programs are not used for swin.:-
brucellosis. ·rhe most successful method of control is de-
population of the entlre herd and restucki11g wil11 u11in-
fected replacement anima Is. MPthods other than depopi.:-
lalion, such as removing only adult animals and retainin~
weanlings, are less successful. Re1noval of only the serolou:
ical rcactors will not control infection in the herd.
Conl.incment operations and closed herds make establish-
ing and maintaining a swine herd free of brucellosis read-
ily achievable. In sorne instances, u1::puvulalio11 is vraL-
ticed with B. m elitensis infPctions in sheep and goats.
Control Methods for Bruce/la ovis
Removing inlected rams and prevent1ng new infections in
raros are the main means of controlling B. ovis infection ir:.
a flock. Practices that allow for intruuu<.:tiu11 uf i11ft:Llt'L
rams into a flock, such as loaning of raros, should tK
avoided. Yearling rams should be maintained separatelc
from mature raros. Ali rams should be palpated tor cpi-
didymal lesions at lcast t wicc a ycar before breeding seasor..
and rams with palpable lesions culled. Serologic tests
(ELISA, C:F) are used to identify infected raros without le-
sions . .Serologic testlng should be perfuru1cu a 111i11i111u1n
of two times before ra1ns are turned in to brPPci PWPS.
Vacclnation ca11lJ1::1::111vluy1::LI bul ils effi cacy is lin1 ited and
it intPrfPrPs with scrologic interpretation. No effort is
~1ade to ~ontrol infections in ewes. Ewes, although play-
1ng a role 111 transmission of infection at breeding, are only
tran sicntly infccted and 11aturally eliminate the infectioi1
by the next breeding season.
Control Methods for Bruce/la canis
Prc:vc:n.tio11 of canine brucellosis involvc3 :;crologic tc:iting
of dogs p rior to breeding. Males are aJso evaluated by paJ-
pati n~ for epididymal and testicular lesions. In breeding
coton1es w1th brucellosis, 1nfected animals identified b\'
serologic tests are removed. Repeat serologic testing is per-
formed to identlfy prevlously undetectt:u iuf<::<.:tt:u a11i-
mals lJntil at IPa~t thrPP nPgative test results are obtained.
a kennel should not be considered free of bruccllosis.
Kennel areas should be thoroughly disin!ected with qua-
ternory ammonium compounds or iodophors.
 Burkholderia mallei and
Burkholderia pseudomallei
DWIGHT C. H IRSH
"llbers of t he genus Burkholderia are gram-negative, aer-
c rods. As of Lhis wriling, Lhert: art: 24 ~ ¡;ecies l.Jelonging
::his genus, most of which live in soil and/or produc:P.
.easc in p lants. Members of the B. cepacia complex pro-
~ disease in compromised human patients (e.g., those
·"- cystic fibrosis; chronic granulomatous discasc). Two
::?.Jbers of this genus, B. mallei (the cause of "g1anders"
- -i::irily in equids) and B. pseudomallei (the cause of "me-
dosis" in a varieLy of species) a1t: i111vur1.<111t in veteri-
--~ medicine, and are the topics of discussion in this
- ~ptcr. Both produce pyogranulomatous disease.
E- ~K HOLDERIA MALLE/
- vzoldcrla mallet Is a gram-negatlve, aerobic rod that
c;.pc; "gla nders," once a widespread disease of Equidae,
~é remains in1porta11t 011ly u1 Asia (Mongolia and China)
.:.'l pockets of activity in India, Iraq, ·rurkey, and the
- .ilippines. Glanders is a systemic pyogranulomatous dis-
~ varying in acuteness and severity. It also affects mem-
.;..-s of the cat family and occasionally dogs, goats, camels,
- eep, and humans.
;~scri ptive Features
:rphology and Staining
_..:rklroldt:ria 111allr:i are gran1-negali vi:: 1ods 0.5 µu1 wiut: d11u
-.dable in lcngth .
: ··ucture and Cornposition
_..rt/1olderia mallei produces a carbohydrate capsule. rhe
cJ wall is typical o f gram-negative bacteria composed of
-. opv ly~d CCharic.le dIHl protein. Being nonmotlle, flagella
-1' not produced (differentiating it from B. pseudomallei
nich is n1otilc).
~ lular Products of Medica! lnterest
.::ipsula. Thc only dcmonstrablc function thc capsule plnys
.:: dlsease produced by B. mallei is to protect the outer
ERNST L. B IBERSTEIN
membrane from membrane attack complexes generat ed
followlng actlvation of the complement system (V\rhich is
m ::t ni ff'~t ;is a serum -resist ant phenotype). Mutants gener-
ated by insertional il1acllvalion of Lhe ge11t::. cucuuiug tl1t:
capsule are avirulent.
Cell Wall. The cell wall of B. tna/lei is typical of gran1-
negative bacteria. The lipopolysaccharide (LPS) in the
o ute r membrane is an important virulence determinant.
Not only Is the lipid A component toXic (endotox1n),
h11t thf' lPneth of the side chain in the 0-repeat unit hin-
ders the attachn1ent of t11e n1en1b1a11e aLLack cuu1plex uf
the complement system to the outer memhrane. T.ipo-
polysaccharidc binds to lipopolysaccharide-binding pro-
t ein (a serum protein), which transfers it to the blood-
·phase of C Dl4. The CD14 LPS complex binds to Toll-like
receptor protein s (see Chapter 2) on the surface of ma-
crophage cells triggering the releasc of proinflammatory
cytokines.
Miscellaneous Product5. Burkholderia n1al/ei possP.ss a cluc;-
ter of genes encoding a Type TII secrction system (an as-
semblage of more than 20 proteins that form a hollow
tu be li ke structure t hrough which cffector p rotcins are
"injected" ínto host "target" cells). Effector proteins have
not been characterized, however, nor has a "target" cell
IJc~JJ iuf:!ntifit:c.L
Proteases, lipases, and a phospholipase C have b een
den1onstrated u1 lhe cullure fluids of B. rrtullei. Nu11e:: uf
these products have b een shown to play a significant role
in discasc.
Growth Characteristics
Tlit: ur8a11isrn gru\vs best on media contalning glycerol
or blood. Nonhemolytic colonif'~ r!Pvelop in 48 hours or
more at 20ºC to 41 ºC. They range from n1ucoid to rough in
tive possible forms. Confluent growth is common. Burk-
holderia mallci <loes not grow on MacConkcy agar or at
42ºC.
Biochemical Characteristics
RurkholdPria mallei is aerobic and oxidase and catalase-
po3itivc; it reduces nitratc~ a nd hyd1olyLe5 u11::.:i. GluLu:>t: i:.
attacked oXidatively.
113
114 PART 11 Bacteria and Fungí
Resistance
l{esistance is unremarkabJe, aJthough in dark, damp, and
cool environmc11ts the agent can survive for months. Ami-
noglycosldes, cl1loramphenlcol, fluoroqu1n.olones, macro-
lides, sulfonan1ides, and tetra(yclines in hi.bit .R. mallei i.n
vitro.
Ecology
Reservoir
Tnfected Equidar?<ire the reservoir.
Transn1ission
Exposure occurs via contaminated feed, water, and fo-
m ites, and sometimes through inhalation and wounds.
Infectious material originates mostly in the resplratory
tractor skin lesions.
Pathogenesis
Pathology. The basic nodular lesion is made up initially of
ncutrophils, fibrin, and red cells. 'fhe neutrophils degener-
ate and the central necrotic area becornes surrouuueu uy
l"pithetioid ancl gi;int cells ;ind h.y lymphocytes emhedded
in gra11ulatio11 tissue. Near epithclial surfaces, ulceration is
common. Strair1 variations determine tl1e suppurative vs.
granulomatous predo1ninance in lesions.
Mechanisrns. Although toxins are suspected in patho-
ge11esis, the mechanisn1s are uncertain. Primary lesions
fon11 al tl1t IJOi11t uf tutry- tlit µ!Iary11x, (or txa1u¡.¡lt.
Infection spreads along lymphatics, producing nodular le-
sions on the vvay to lymph nodes and the bloodstream,
which dissemil1ates the agent. Metastatic lesions form in
tl1e lungs or other orga11s, such as spleen, liver, a11d skin,
producing cutaneous glanders ("farcy"). Lesions in tl1e
nasal septum may be prin1ary, hematoge11ous, or second-
ary to a pulmonary focus.
Disease Patterns
(~!anders. A.c ute infecti.ons are characterized by fevcr, nasa1
discharge, a11d lympl1adenitis of bead and neck, with
swelli11g along thc uppcr rcsp.i ratory tract They tend to
end fatally in <1bout two weeks a11d predon1inate in don-
keys and felids, less so in n1ules.
In horscs, protractcd chronic and subclinical infcctions
are typical; signs, if present, include occasional fever, per-
sistent respiratory problems, skin abscesses ("farcy buds"),
a11d nodular lnduratlo11 of cranlaI lympb nodes.
Human exposures are t raced to acutely ill horsf's <incl
n1ay lcad to acute or cl1ro11ic infections. Ali acutt infec-
tions and 50°/o of cbro11ic ones were fatal prior to the ad-
vcnt of c.ffcctivc antin1i.crobials.
Epidemiotogy
1'l1e persiste11ce of glanders depends on an infected horse
population. Susceptible noncquids acquirc glandcrs from
infecled 11orses or 11orsen1eat, and appear to be dead -eo:::
11osts.
lmmunologic Aspects
Humoral and cell-mediated responses occur.
Appare11l rec.overy fro111 gla11de1s, i11cludiug Joss of da:-
mal hypersensitivity, has been observed under natural oon--
ditions, but with.out .incrcascd rcsistancc to rcinfcction.
No met hod of immunization is knOWil.
Laboratory Diagnosis
Nodular contents are cultured on blood or glycerol a~:.::
1'hey may be examü1ed for gram-ncgativc rods and b~- i::::­
munotluorescence.
Guinea pigs and hamsters are highly susceptible to fa;¡_
infectlon with virulent strains.
Any suspect isolates should be submittecl to a qua Hf>~­
reference laboratory. Differe11tiation from B. pseudo1n~
is important.
Serologically, glanders is diagnosed by complc.m ent ~­
ation tests employing aqueous bacteria! extracts as an::-
gen. The intradermo palpebral n1allein test detects C--=
mediated hypersensitivity, which indica tes infection ¿:i...:.
has served as a basis for glanders eradication. Mallein is_
heat extract of old B. mallei broth Lultures.
A polymerase chain reaction- based assay tl1at utili--
primers specific for D. rnallei is available for detection ar;.:.
identification.
Treatment and Control
Although glanders is treatable by many antimicrob~c:
(see above), treatment is inappropriate in countrie.
con1n1illed Lo glanders eradicalion. Equi11e in1porls (ro:;:;
endemic areas are mallein-tested, and reactors are ~­
stroyed.
BURKHOLDERIA PSEUDOMALLEI
Burktiultleriu pseutlornullei are aerobic gran1-11egalive, fac_
tative intracellular rods that cause a pyogranulomat<Y.:!..
disease called "melioidosis," a disease superficially rese--
bling glanders. Important distinctions are that 1) melic
dosis affects a wide host range and 2) the agent is a sap:
phyte (an endosymbiont of amoebae living in r.~­
environment), whose prevalence is unaffected by eli.rni!~
lio11 of i11fected a11i1nal:;.
Descriptive Features
Morphology and Staining
Burkholderia pseudornallei are gram-negative rods 0.5 ;::=
widc and variable in lcngth.
 Chapter 17
: ··.icture and Composition
-rkllolderia pseudornallei produces a carbohydrate capsule.
e cell wall Is typlcal of gram-negatlve bacteria composed
'ipopol y~;:iccharide
a11d protein . Reing motile, flagella
--. produced (differentiating it fro111 B. rnallei wl1ich is
"lffiOtile).
~ ular Products of Medica! lnterest
esin. Burkholdcria pseudo111allei adheres to amoebic
~hozoites prior to uptake (and by infcrcncc adhcrcncc
..,hagocytic cells). Adherence is by means of the flagellar
tein Fli (for flage/lin).
~.1p:>ule. 'fhe ur1ly c.lemunstrable funcrlon the capsule
been shown to play in R. pseudnn1allei is to protPrt th <-'
er men1 brane fron1 n1en1brane attack con1plexes gene1-
.... following activation of the complement system
...ch is 1n;n1ifcst as a scrun1-rcsistant phcnotypc). Mu-
•s generated by iI1sertional inactivation of the genes
Jding the capsule are avirulent.
..:JI Wull. Tl1e cell wall uf B. p:>eudurnallei is typical of
...,-negative bacteria. The lipopotysarrhari<lP (l.PS) in
utcr membrane is an important virulcnce determi-
- . ot only is the lipid A component toxic (endo-
__..... , but thc lcngth of thc sidc chain in thc 0-rcpeat
!11nders the attachment of the membrane attack
p!ex of the complement system to the outer mem-
t:. Li puµoly s<1ccharic.1e bind:; to l!popolysaccharide-
~;ng prot·c>in (;.i sernm protPin), which transfers it to
-Jood-phase of CD14. The CD14-LPS con1plex bi.11ds
·1-like recepto r p roteins on the surface of macro-
- "' cclls triggcring t hc rclcasc of proinflammatory
'les.
cellnneous Products. The chromosome of B. pseudo-
possess at Ieast one Pathogeniclty Tsland (a cluster of
encoding virulence determinant(s), an integrase
~::e... •. a :.pccific i11:;ertiu11 :;itl!, a11J 111ul.Jility) er1cucling a
JI secretion system (an assemhlage of more than 20
'.1S that form a hollow tube-like structure through
el tcctor proteins are "injected" into host "target"
However, effector protcins havc not bcen character
-ior has their "effect" bcen delincated. A "target" cell
t been identified.
,eases, liµa:;e:;, C1.11d a µl1u:.phuliµase C have been
'"1Strated in the culture fluid~ of M. pseudomallei
~~ n v itro. None of these products have been shown
a significant role in disease production.
_ __. · - Characteristics
_ B. rnallei, B. pseudornallei grows on MacConkey agar,
nresence of 2% sodium chloridc, and at 42°C.
---f'e
=--~- '
--'--~,.,:-ria pseudomallPi is kil1Pc1 hy and
c1i~infectants
• survive chilling and freezing in biologic speci-
;s generally susceptible in vitro to fluoroquino-
ctracyclines, chloramphcnicol, trimcthoprim-
_z=:~ hoxa2ole, and novobiocin.
Burkholderia rnallei and Burkholderia pseudornallei 11 S
Ecology
Reservo ir
Burkholderia pseudoniallci is considered a soil a11d water
dwc!Jer (most likely an endosymbiont of arnoebae).
Although most p revaient between 20º northern and
southern latin1de, extra tropical foci do exist, for example,
in France, Tran, China, and North America.
Transmission
Lngestion, wound infection. and possibly arthropod bites
introduce infection. In humans, consumption of infcctcd
animal products and airborne 1nfect1on maybe signiiicant.
Pathogenesis
Meclla11is1ns. ileing an endosyn1biont of an1oebae, B.
pseudornallei is adapted to survive within phagocytic cells
of the host. Microorganisms are taken up by a proccss of
"coll!ng" phagocytosis and surv1ve w1thin the cell by
being resistant to lysosomal contents (e.g., defensins), and
by cscaping phagosu111e:. a11d pllagolysosumes. Actin-
bascd motility has been observcd within phagorytir rPll<>
(see Chapters 11 and 33), and actin-associated ubudding"
occurs from affected to nonaffected cells. Host cells in-
-
fected with B. pscudoniallci rclcar.c proinflammutory cy-
tokines, and undergo apoptosis.
Pnthology. The lesions are primarily pyogranulo1natous.
S111all ¡jlJ:;ccsses tenll tu Cü(;llt'sc:e, tlevelop!ng into largcr ...J
suppurative foci or gr;.in11 l oma~.
D
Disease Patterns
Melioidosis. This d1sease is typically systemic. Manifesta-
-
tions dcpcnd on the extent and distribution of lesions.
Thc equlne disease may mimic glanders. In cattle, acute
and chronic infections can localize in Iung, joints, and
uterus. A1·thritis and lyn1 phadcn il is occur iJ1 sl1eep. Goals
suffer loss of condition, respiratory and central nervous
systcm disturbanccs, arthritis, and 1nastitis. Si1nilar signs
are seen in swine, along ~vith abortions and diarrhea. Uogs
dcvclop a febrile disease with localizing suppurative foci.
Epidemiology
Clinical disease is usually sporadic. The host rangc> in
mammals is virtually unlirnited, and avian cases are rc-
ported. Human intections range trom the rapidly fatal to
the subclinical. .A. wet environment, such as a swampy ter-
raln or rice paddies, is related to exposure.
lmmunologic Aspects
Complement-fi:xing and indircct hemagglutinating anti-
bodies are produced duri11g i11fcctiur1s. Cell-mediated
hypersensitivity has been demonstratPcl in infPrtP<l goats
Succcssful vaccination of horscs and zoo anin1als is re-
portcd.
116 PART 11 Bacteria and Fungí

Laboratory Diagnosis Treatment and Control

Tht> mi>tho<is for isolating an<i i<ii>ntifying R. mallei apply Antirnic:rohia 1susc:eptihi lities should he verified by labora-
to B. pseudon1allei. Chilling and freezing of specimens tory tests. Fluoroquinolones and the tetracyclines are con-
should be avoided. Motility, growth on citrate, growth at sidered drugs of choice. Vaccines are not commcrcially
42°C, and rcduction of nitratcs to gascous nitrogen distin- available.
guish H. pseudomallei from B. mallei. A polymerase chail1
reaction-based assay utilizing primers specific for B.
pseudomallei is available for detectlon and ldentlflcatlon.
 Francisella tularensis
ÜWIGI IT C.
fe1nbe1s uf tlie ge11u::. Frur1Lisellu élrc gra111-I1egative, aero-
c. rods. ·rhere are two species: F. tularensis, anct F. philnmi-
•.;gia. Thcrc are four subspecies of F. tularcnsis: tularensis
~reviously known as b.iotype A, oras subspecies nearctica);
iartica (previously known as biotype .B, or as subspecies
ul.iearct/ca); novtcida; and rnediasiatíca. Francisella tularen-
<;11 h-.pecies tularensis and subspecies holartica are facul-
,., ·ively intraccllular pall1oge11s of hu111a11s a111.l, ur1tler
~ited epidemiologic conditions, occasionally infec.t do-
- estic nnimals (shccp, horses, swine, carnivores) produc-
~ thc díscase tularem.ia. Francisella pl1ilorniragia and F. tu-
•1<11sis subspecies novicida and subspecies 1nediasiatica are
uncertaln animal pathogenicity.
:: escri ptive Featu res
: rph ology and Staining
~.;•1cisella
tularensis is a gram-negative coccobacillus,
-easuring les~ than 1 µ1n in any dimcnsion. 'l'hey can pass
·1:r memhranes of 600 nm porosity. In older cultures,
- ·;e pleomorphisn1 develops. Gicmsa stain is preferred.
:~ :.llar Anatomy and Composition
-.cisella tularensis proctuces a caps11 IP composed of lipids
- J- -<:>0A1), arnino ac.ids, and c.arbob.ydLales. ll possesse::; a
.:-an1-negative cell wall, whose lipopolysaccharide portian
ndotoxin) has substuntially lcss toxic activity than the
iX>POlysacct1aride foun<1 in the cell walls ol other g ram-
::egative microorganisms.
:~llul ar Products of Medical lnterest
-.1ps11le. Capsules protect the outer memhrane from the
:icmbranc attack complex of the complement cascade.
.-.."lee capsules ot oth.er microorganisms inhibit attach-
....,ent to, and ingestion by, phagocytic host cells, it is un
. ear what the role (aside from protccting the outer mem-
-ranP. from complPm ent compone11ts) of a capsule is for F.
:;.larensis, an intracellular parasite (see below).
Ce// Wall. l'he cell wall of the 1nembers of this genus is
ne fairly typical of gram-ncgntive bnctcria. Thc lipopoly-
..í:Charide (LPS) in the outer mcmbrane is an important
.:ulence determinant. Not only is the lipid A component
-.i<: (cutlutoxin), but the length of the slde chain tn the
-repeat 11nit hin<IPrs the attachment of the membrane at-
1..k complex ofthe complen1enJ systcn1 to the outer iue1u-
HIRSH ERNS'f L. B IBERSTEIN
branc. Llpopotysaccharide binds to lipopolysacchande·
binding protein, 't<\•hich transfers it to the blood-phase of
CD14. The CD14-LPS con1plex. J;ir 1tls lo Tull-like receptor
proteins on the surface of macrophage cells triggP.ring the
rclcnse oí prointlam m.atory cytokines. As mentioned above
("t: cllular Anatomy and <.:omposition"), the potency of
lipopolysaccharide of F. tularensis, as compared to othcr
gram-negarlvc microorganisms, is much less. Neither thc
rPa~on nor the role for this is known.
Acid Phosphatase (Acp). Tlit: At:p (fur acitl phosphatase)
of 1:. tularensis suppresscs the respiratory hurst of phago-
cytic cells, cspccially ncutrophils.
Products of the lntracel/ular <.Jrowt/1 Locus (Jgl). The 23 kDa
protein, termed lgl (for intracellular growth locus), is asso
<:iate<1 wlth the prevention of fusion of phagosomes (con-
t::iin ing P. tularensis) with lysosomes. However, acidifica-
tion of t hc pl1agoson1e still 01.:1.:ur~, fé!<:ili tatir1g irun
acquisition from intracellular iron stores. How thesf'
cvcnts occur is not known. lgl also prevents secretion of
proinftammatory c:y"tokines by intected macrophages.
Macrophage Growth Locus (Mgl). 'l'he Mgl (for tnacro-
phage growth tocus) proteins are associated with preven-
tion of the fusion of phagosomes (containing F. tu/arensis)
with lysosur11es. Huvvever, ai.:itliflcatlon uf the phagosome
occurs, facilitating iron ac.q11isition from intracellular iron
stores. TTow these events occur is not known.
Growth Characteristics
Francisella tularensis is a fastidious aerobe. 'f'he p referred
medlum Is glucose-cysteine-blood-agar. Two to four days
of incubation (35- 37ºC) produce grayish, viscous, oxidase-
11egative colonies 1 Lo 4 r11u1 i11 tlia111eter. 1e:-'ts for acillifi-
cation of certa.in carbohydrates anct c:itr11lline 11rPi<lase ac-
tivity permit subdivision of the F. tu/arensis into biotypes A
(now designated subspecies tularensis) and B (subspecies
holnrtica), which differ in geographic distribution, host
speclflC!ty, and V.irulcnce for humans (see "Epidemiology,"
below) .
FtufltiSt'. l/u lulc111:roi:, :.u1 vi ve:. <:~ilLI 1e1uµeratures fur
months in water, soil, and animal tissues.
Ecology
Reservoir
The reservoirs of F. tularensis are infectcd lagomorphs
(llares, rabbits; subspecies rularensls) and rodents (sub-
117
118 PART II nacteria and fungi- Graxn-Negative Rods
~pecies hulurlica) . Sorne t1ave suggested that F. tularensis is
<in t>n<losymhiont of frff-living <imofhil.
Transmission
Tl1e n1ore virulent subspecies tularensis (predomi11a11t in
Nortl1 America) is trans1nitted largely by ticks and hemo-
phagous insects. Surface waters co11ta1nlnated by rodents
are sources of infectio11 witl1 tl1e less virulent subspecies
holartica (predon1inant in Eurasia). h1gcstio11 of i11fected
pcey spreads F. tularensis to the predator. Sheep on the
~vc:;tcrn rangc:; of ]'¡orth An1crica are infcct cd by way of
ticks. Transmission by animal bite has been documented.
Humans are commonly infected by contact (percuta-
ncous, co11junctival, inhalation, in.gestion).
Pathogenesis
Pollo•ving the infectious eve11t (bite, inhalation, in.gcs-
tion), F. tularensis comes in contact with tissue fluids (com-
plement protcins) followcd by initiation of an inflamma-
tory response (chemistry of cell \~•all). <.::omplement
protcins (cspccially the membrane attack complex) are in-
cttectual dueto the presencc of the capsule. Early arriving
neutropl1ils do not easily eliminate the organism (suppres-
sion of the respiratory burst by Acp; it is unclear whetl1er
the capsule plays a role at this st:1gf). Tntr<ic:ellu l<ir growth
occurs following pl1agocytosis by macrophages (made pos-
síble by a suppressed respiratory burst by Acp, lack of fu-
sion of phagosomcs and Iysosomcs by lgl and Mgl pro-
teins, and iron acquisition fron1 iron-binding proteins
'1-Vithin the phagosome under acid conditions). lntracellu-
la.c growth of F. tularensis results in apoptotic deatb of the
macrophage. Liberation of the microorganism is followf>rl
by an0Ll1er "rou11d" of pl1agocyto.sis. Endotoxen1ia (see Fig
8.1) follows if the infectious process reaches the dissemi-
natcd pbasc.
Epidemiology
Tnl<irf>mi;i is ;i northern disease, its southern limits being
Mexico and Mediterrancan Africa. Seasonal peaks reflect
vector activlly and co11tact with the reservoir. Waterbom ;:
i n fections predo111inate in fall and winter. Francisella tu-
larensis subspecies tularensis is predon1inant in Norti:
America, and F. tularensis subspecies holartica is predorn!-
nant in Europc and Asia.
lmmunologic Aspects
Solid, largcly cell-n1ediated ilnn1u11ity follows recovery ir;.
h umans. Anin1als in endemic areas carry antibody. Its re1a-
ti on to immu nity i::; unrclatcd. A livc attenuated vaccin-e
(e.g., LSV for live strain vaccine) is used in hum.ans at ri.sk.
Laboratory Diagnosis
Demonst rat ion of t he organism 1n exudates is by
Romanovsky-typc stains (Wright's or Giemsa), immun o
fluorescence, and culture. Jsolation is facilita ted by injec-
tion of suspect n1aterial into m ice o r guinea pigs.
Rising serum t ube agglutination t iters (1 :>80) are evi-
dence of infection.
DNA primers are available for the <liag11u:;i:, a 11d ideuli-
fication o f Francisella by the polymer<ist> ch<i in rt~action .
See "G1owll1 Characteristics" earlier in this chapter for
isolation mediurn.
Treatment and Control
Aminoglycosides (especially streptomycin) are effecth·E
drug:; fur tre<1 ting 11uu1a11 tulare1nia, bul due lo toxicit;
ot hf>r <intimic:rohials are preferred. The fluoroquinolones.
have been uscd successfully, and are a safe alternative to
the aminoglycosides. Tetracycline has been ettective in an -
i1uals. Other antimicrobials (1nacrolides, beta lactams..
Chloramphenicol) are less effect1ve. Conrrol measures are
ai m ed at limiting tick exposure and access to c:ont<imi-
naled feed a11d waler.
 Moraxella
DWIGIIT C.
\.fpmhPr<: of the genus Moraxella are gram-negative rods
m d cocci belonging to tl1e Ia111Hy Mo1u.xelluleue. Tl1e ger1us
.foraxella is subdivided into two subgenera, Moraxella
containing thc rod-shapcd mcmbcrs of the genus) and
rubgenus Branl1amella (the coccoid members). 'l'here are 14
;;iembers of thi.~ genus (see 'Table 19.1), most of wl1ich are
~ul.'.la ted with <.liseases of human patlents. From a veteri-
~ perspPrtivP, Moraxe/la subgenus Moraxella bovis (here-
_-:er rcferred to as Moraxella bovis) is the 1nost i111purta11t
-~:nber of the group. Moraxella bovis is the cause of infec-
u.s bovinc kcratoconjunctivitis (IBK), thc most common
-cu1ar d1scasc of cattle.
: :scriptive Features
··phology and Staining
~ raxcllac are short, plump gram-negative rods, 11.5 µm
:..:> to 2.5 mm, and are often arrangecl in pairs ("diplo-
;cil!i") o.r short chains (Fig 19.1).
: ·-. cture and Composition
- e:: cell wall is typical of gram-negatlve bacteria being
;:iposed of Hpopolysaccharide and protein. The lipo-
·: :>accl1ariue uf 1noraxellae uue:> not contain 0 -repeat
-=-~t:> . in contrast to many other gram-negative micro-
;-:;inisms (c.g., mcmbers of the family Enterobacteriaceae).
• he fimbria! adhesins (pili) ot M. bovis are virulence de-
:minants and can be lost in subculture (see below,
.a1ablllty"). Capsules may be present on fresh isolates.
::. !.llar Products of Medical lnterest
. dases, peptidases, and proteases. Thcre is little evidence
"1esins. The role of adhesins, as in othPr mirroorganisms,
:o allow the bacterium expressing them to adhere to
~s lining a particular niche, as well as to the surface of
;;..called "target" cells prior to the initiation of discasc (in
:ne cases, niche and target ceUs may be the same).
~raxelln bovis produces a type 4 pilus (fimbria) that ad-
·eres Lo con j u11clival auu coru..:al t!pitht!lial cells. This
- ·us is similar to those of Pseudomonas aP.r11ginosa, Neis-
~-na gonorrhoeac, Dicholobacter nodosus, Pa.steurella rnulto-
-.ta, and Vibrio cholerae. Mutants unable to produce this
-.!.'iesin are avirulent.
....Jpsule. The capsule plays many roles, the most impor-
l)f which are interference with phagocytosis (an-
:::;:=:¿:ocytic), and proLecliou uf Llte;: oute;:r rnembrane from
dcposition of membrane attack romplPxPs generated
~ ... vation of the complement system.
HIRSI-T ERNS1' L. BIBERSTEIN
Ce// Tl\lall. The cell wall of the members of this gcnus is
one typlcal of gram-negative bacteria (except for the ab-
<:PnrP of the 0 -repeat unit). The lipopolysaccharide (LPS) in
the outer n1en1bra11e is an i1u¡.iucla11t virulence determ1-
nant. LPS binds to lipopolysaccharide-hinding protPin (a
serum protcin), which transfcrs it to the blood-phase of
CD14. Th e CD14-LP.S complex binds to Toll-like receptor
proteins (see Chapter 2) on the surface of macropl1agc cclls
tr!ggertng the release of proinflammatory cytoldnes.
l\xntoxin~. The most noteworthy toxin produced by M.
bovis, is an RTX (repeats in to.xi n, so called becau:se;: uf tlt..:
common feature of repeats in glycine-rich sequences
within the protein) typc of cytotoxin (scc also Escherichia
coli haemolysin, Chapter 8, Pasteurella/Mannheimia leuko-
toxin, Chapter 12, Actinobacillus haen1olysin, Chapter 13,
a<.h.:uylyl cyclase toxin of Bordetella, Chapter 15). This
cytoxin, somPtimPs referred to as "hemolysin" dueto its
bchavior on blood agar p lates, has been tern1ed Mbx (for
J\foraxella bovis toxin). Mbx is a porc-forn1ing toxin •vith
spccificity for conjunctival and cornea! epithelíal cells,
and neutrophils. Mutants unable to produce Mbx are
avirulent.
Tron Acquisition. Because iron ts an absolute growth re-
quirement, microorganísms must acquire this substance if
they are to exist \-Vitl1in tl1e hos1:. Mo raxellae acqulre lron
from the iron-hindingprotpin<: ofthP hn-.t (tran~fprrin anit
Jactoferrin) by expressing 'lbp and Lbp (for transferrin-
and /actoterrin-binding proteins, respectively) on their
surface. Tbp and Lbp bind their respective proteins giving
moraxellae access to iron .
Miscellaneous Prod11cts. A numbcr of proteins with toxic
acLivilies are p1oduced i11 vilro lJy 'NI. buvis. 'I"hese inclu<.le
complement-degrading protp;i~P<:, lip;ises, phosphoami-
showing that any of these play a ro le in vivo.
Growth Characteristics
Moraxella bovis grows hPst at .'~.<;º\.in the presence of serum
and blood. No growth occurs on MacConkey aga1 01
anaerobically. In 48 hours, fresh isolates produce flat, he-
molytic, friable colonics, about 1 mm in size that corrode
the agar and autoagglutinate when suspended in satine.
Biochemical Activities
Moraxel/a bovis is oxidase-positive, nontermenting, and
catalase-variable. Nitrates and urea are not attacked, but
p10Le;:iu:> are lligested.
119
120 PARr 11 Dacteria a11d Fungi

Table 19.1. Mernbers of the Genus Moraxe//a and Their Usual Source or Assotiated
Condition

Specles Usual Sóurce or Assodafed (:omlition

Moraxel/a atlantae {COC Group M-3) Septicemia in human patients

M. boevrei Respiratory tráct ot normal goats
M. bovis lnfectious kerato~onjunctivitis in cattle{IBI<)
M. cuniculi (Neisseria runicµ/i) Respiratory tract of raliblts
M. canis Respiratory tracts of normal dogs.and cats
M. caprae R~1µir<1tu1y traüs el 11ormal goal5 and sheep
M. catarrhalís (N. catarrh¡¡/is) Middle ear infectíons in d'lildren; upper respiratory tract infections
in human patients
M. caviae (N, caviae) Respir.atory traét ot g'\.linea p(gs
M. Jacunata Conjunctivitis~nd keratitis io human ,patients
M. lincolnii Res¡iiratocy tract of .tiuman patieots
M. nonliquefaciens Respiratory tract of 11ormal human patients; blood, cerebr.al spinal
fluid, and lungs of compromised human patíents
M. ov/s (N o\/ís) Keratoconjandiliitis in sheep
M. osloesis Nematodes; varíous conditions in human patients
M. phenylp.vruvíca Respiratory tract of normal human patients; blood stream of
compromisca humaff patients

F 1G U RE 1 9 . 1 . Moruxcllu bovis in thc cornco:i of o:in cxpcrimcnto:i//y infcctcd calf.

There Is evidence of digestion of cornea/ substance around the bacteria/ ce/Is. scanning
electron micrograph, 22,000X. (Photograph courtesy of Dr. G. Kagonyera.)

Resistance
Resistance to phy<;ical an<l chemical agents is not remark-
able. It is usually susceptible to con1n1only used a11Lil.Jiutics.
Variability
In culture, M. hovis undcrgoes colonial dissociation (phase
varialiuH) ¡.irutlucing smooth butyrous colonies composed
of cells, which Jack pili (<1111> to invcrsion of the pilin en-
coding gene) and infcctivity, and are lcss auloagglutinal>le.
Pili are irnmunogen ically di verse, and this trait is responsi-
ble for a classification schcmc bascd on scrological sirnilar-
ities. Nonhemolyt1c var1ants are nonpathogenic.
Ecology
Reservo ir
.\lfnraxt'lla hovis occurs worldwidc on the bovine conjunc-
tiva and upper respiraL01 y 1nuco:.a1 uf ten without clinical
:nanifestations.
-ransm1ss1on
. .
::n:.semination is by dlrect and indirect contact, including
:':ying ino;ects and possibly other airborne transmission.
::thogenesis
!«hanisn1s. Disease produccd by M. bovis is closely linked
~:::cytoxin (Mbx) and pili. Attachmcnt (pili) to conjuncti-
-al epithclium is followed by dcstruction (Mbx) ot con-
~'1ctival and cornea! cells. Growth of M . hovis in the con
-z.ctival and corneal Ieslons Ieads to inflammation
~m -negativc ccll wall). Mbx-mediated lysis of neu-
phils a1nplifies i11fl<!111u1atiuI1 auc.I tissue destruction.
:nvironn1ental factor<; impliratP<l include ultraviolet ir-
~~iati on, flies, dust, and woody pasture p lanls, all of
'"'.ich contrit>ute to irritation of the target tissues. Con-
---rent infections with viruses, such as bovinc hcrpcsvirus
_ ·nfectious bovine rhinotracheitis virus) a11d adenovirus,
-·.·coplasma (1\tfycopl asma hovoculi), bacteria (Listeria
'.'nocytogenes), and ncmatodcs (Tflelasia), may com plicate
-!? disease.
: .sease Pattern and Pathology
-:tectious Bovine Keraroconjunctivitis (TBK). IBK begins with
.::vasion of conjunctiva and cornea by M. bovis, rcsulting
.n eden1a anda predominantly neutrophilic 1nflammatory
:6ponse. Jt may progress from mild epiphora and cornea!
.:.ouctiug lu ¡.iru<.luctiun uf severe edema, corneal opacities,
-ascularization, ulccration, an<l n1ph1ri> IP;uiing to uveal
:--olapse and panophthalmitis (see Fig 72.2). Healing of
Chapter 19 Moraxella 121
the ulcers proceeds from the pPriphPry and requires sev-
era! wecks. Central scarring may persist for 111011lhs.
Though a selt-limiting disease, losscs occur becausevision-
impaired animals do not forage and lose condition .
Epidemiology
IBK is a highly infectious <li<;Pa'>i>, mostly of beef cattle.
Young animals are prefercntially affected, probably Llut: tu
lack of acquired immunity. Lack of eyelid pigmentation
and prominent placement of eycs are apparcnt predispos-
ing factors, as is vitamin A deficicncy.
Prevalence is greatest during summer and early fall,
whe11 envi1u1111 1t:11tal stresses are maximal.
lmmunologic Aspects
Antlbodies of all isotypes are produced during infection,
with secretory lgA predominating locally. Temporary re-
sista11ce lo 1t:i11fcctiun fullows rccovery. T11e relative roles
in immunity ancl rc>rovPry of general vs. local responses
and humoral vs. ccll-mediated rcspo11ses are u11:.ettletl.
Experimental bacterins and fimbria! antigens stimulatP
resistance, optimally to homologous challenge. Appar-
ently, fimbria! proteins, Mbx, and proteolytic enzymes
l1ave protection-inducing activity. Fimbrial vaccines are
con1me1cially availa!Jle.
Laboratory Diagnosis
The agcnt may be demonstrated in smears of exudate,
most convincingly by immunofluorescence (by using anti-
IJutly sveciflc for M. bovts antlgens). Exudate is cultured on
hloocl agar an<l Mora.'Xella are identified by colonial charac-
teristics, oxidase acLiviLy, lle111uly:.is, ¡.iroteol ysis, and fall-
ure to fermcnt carbohydrates. Spc>c.ific flnorescent anti-
body conjugatcs can be applied dircctly to suspect colonies
on p lates tor idcnlilication even o f dissociant colonies
(epifluorescence). Poly1nerase chain reaction-based assays
utilizing M. bovis-spccific primers are available for detec-
tion and idcntification.
Treatment and Control
Affected animals should be placed in a dark stall, free from
dust and fl1es. Topical corticosteroids may relieve the in-
flamrnation, while antimicrobial drugs, given topically or
systemically, may be beneficia!. Long-ac.'ting tetracycline
or florfPnicol ar(' considered the drugs of choice.
Fimbrial vaccines a1e Lile 111osl ¡.irulllisir1g specific pro-
phylactics.
 Pseudomonas
DwrGH'f C. HrRsH
Members of the genus Pseudornonas are gram-negat ive, aer-
obic rods. Of the rnany recognized species of I'seudomonas,
only P. aeru,'{inosa is of veterinary importance. Previously
named pseud.omonads of veterinary importance, P. mallei
and P. pseudornallei, have been moved to the genus Burk-
holderia (see Cl1apter 17). · quirement for ali living tl1ings. Ps.eudomonas ar~ruginosa
Pseudornunus ueruginosu is very rarely involved wilh pri-
mary di.sease, although it is extremely important in clini-
cal medicine. Most strains are resistant to the commonly
used anti.luicrobial agents and are therefore sometimes
dLfficult to eliminate when they contaminate a compro
mísed site.
Descriptive Features
Morphology and Staining
The organisms are gram-negative rods, O.Sto 1.0 ¡1m hy 1.S
to 5 .() prn .
Cellular Anatomy and Composition
Pseudomonads produce a typical gram-negative cell wall,
surrounded by a carbohydrale-conlai1ú11g cavsule. All
members of t he genus are motile by n1eans of polar fla-
gella. Pili (fimbria! adhesins) are produced.
Cellular Products of Medica! lnterest
Adhesíns. Pseudomonas aer11gínosa produces several prod-
u<.:l;:, ll.1at ;:,erve a:> adl1t:siu;:,. Tlit:st:. iucludt: a JlJulJrial ad-
hesin that has affinity for certain glycoproteins on epithe-
lial cells. In addition, t here are non-fimbria ad.hesins, an
outer memhrane protein witl1 affinity for mucin, and an-
other, the lipopolysacchatide of the cell wall that has
affinity for chloride channeJ proteins.
Capsule. The capsule prot ects the outer membrane from
the membrane attack complex of the complement cas-
cade. The capsule also inhibits attachment to, and inges-
tion by, phagocytic host cells.
Cell Wall. The cPll wall of the memhers of this genus is
011e typical of g.ran1-negative bacteria. The lipopolysaccha-
ridc (LPS) in the outer 111embra11e is an important viru-
lcncc dctcrminant. Not ouly is the lipid A compone11t
toxic (endotoxin), but the length of the side chain in the
0 -repeat unit h inders the attachment of the me.m brane at-
tack complex of the complement syste111 tu lile ouLer
membran<!. Lipopolysarrharicle h incls to lipopolysaccha-
122
ride-binding protein, which transfers it to the blood-phase
of CD14. Thc CD14-LPS complcx binds to 'foll-likc recepto:
proteins (see Chapter Z) on the surface of macrophage cells
triggering the release of proinflammatory cytokines.
lron-Acquiring Systems. Irun is a11 alJ:>ulute gruvvtli rt>-
produces tl1e iro11-acquiri11g siderophores p}'Ochelin and
pyoverdin, as '-vell as using the siderophores produced by
other bacteria living in its cnvironmcnt (c.g., cnterobactin
and aerobactiI1). 'fhese products are used to remove iron
from host iron binding proteins.
Exotoxins. Pseudomonas aeruginosa produces a number
of protein exotoxins: exotoxin A, exotoxin S, exotoxin T,
exutoxin U, exutuxin Y, ela:sta:>e, a11J ¡1 uu1ube1 of oll1er
proteins with hiologic.al activity (proteases, phospholi-
pases) . .E.xotoxins S, 1', U , and Y art~ "injected" into host
cells by way of a Type 111 secretion apparatus (an assem-
blagc of protcins- morc than 20- tl1at form a holloi;.v
tube-like structure through which effector proteins are
"injected" into host "target" cells):
l. Exotoxtn A. Exotoxin A i.nhibits protein synthes1s
hy rihosylation of Plongation fartor-2 (F.F-2) follow-
h1g receplor-111edialed endocylosis.
2. Exotoxins S and T. Exotoxins S and T ribosylate
host cell G1'f'-binding proteins, intcrrupting cell
íun.ctions relying on the actin cytoskeleton, e.g.,
phagocytosis.
3. Exotoxin U . Exotoxin U is cytotoxic, but the mech-
anism is undefined.
4. Exoloxh1 Y. Exoloxin Y is a11 adc11ylyl cyclase that
raises tbe amount of intracellular cAMP to damag-
i11g Jcvcls.
Miscellaneous Products. Pseudomonas aeruginosa pro-
duces bactcriocins (pyocins) and pigments (pyocyanins).
Pyocins are useful epidemiologically for tracing epidemics
within the hospital environn1ent. Pyocyanin has toxic ac-
ti\1ity and is used asan aid in the laburatury iJl:'.11LificalioL
of P. aeruginosa. Pyocyanin rearts with oxygen to forro re-
active oxygen rad icals t hat are toxic to eukaryotic and
prokaryotic organisms. Pseudomonas aeruginosa protects i~­
self from thc toxic cffccts of pyocyanin by increasing syr..
thesis of catalase and superoxide dismutase.
Product Regulation. Regulation of the expression and e...:-
cretio11 of cellular proclucts invulveJ i11 t!JI:'. palhoge11esh
of disease producen hy P. aeruginnsa is complex. Secretio::
of products that are secreted by way of the 'fype III secre-
tion apparatus (exotoxins S, 1', U, and Y) is initiated iollo•"-

·,g hacteria-host cell interaction. 'fhe remaining bacteria]
cell products are under the control of thc "quorum scns-
.ng" system of P. aeruginosa. ·rhe genes that encode these
!J'FOducts are expressed when concentrations of bacterially
?IOduced homoserine lactones reach a threshold leve! (a
-~uo.rum"). All P. aeruginosa cells excrete homoserine lac-
.011t::., IJuL Liie couce11 L1alion is Loo low lo lrigger virule11ce
-zene expression until a critica! number of bacteria] cells is
:eached. Finally, exotox.in A and a11 c11doprotcasc are also
:eguJated by levels ot pyoverdin. When free iron concen-
:ration.s are low (as wo1lld be the case in vivo), these two
protei ns are expresse<.1 and excreted.
<:1owth Characteristics
?:;eudurnunus uerusínu~u isobligaLe aerobe, derivi11g e11-
a11
ergy from the oxidation of organic materials and using oxy-
gen as a terminal clectron acccptor. It grows on all common
media overa wide range of temperatures: 4 ºC: to 41 ºC.
Ecology
~eservoir
~ lost members of the genus Pseudornonas live in soil and
,·ater. Pseudon1onas aerugínosa may also be found ln the
:eces of n.ormal an.imals, but notas a mernber of t11e nor-
!!1al flU['1 (i.e., l11ey are lra11sienls).
:ransm1ss1on
:Uvironmental or endogenous exposure is constant, and
most infections are secondary to compromised host de-
-
:cuses.
?athogenesis
~fechanísrns. Pseudornonas aeruginosa contaminates areas
..::: the body that possess reduced numbers of 11or1nal flor'1.
Disruption of the norm;"Jl flor;'! is almost always dueto an-
:!microbial agents. Since P. aeruginosa is resistant to most
~mmo nly used antimicrobial agents, it will replace t he
- ormal flora. Tf the si te colonized is compro1niscd or con-
Liguous to a compromised site, there is risk ot infection oí
~e site. Tissue destruction follows initiation of inflarnma-
:ion (cell wall), and liberation of exotoxin(s) and py-
ocyanin.
Pseudornonas aeruginosa is also isolated fron1 certain sites
of animals that have no history of antimicrobial therapy.
:>isease Patterns
.9og ((:at). Conditions in which P. aeruginosa is associated
; nclude otitis externa, lower urinary tract infection, and
pyoderma.
Horse. Conditions in which P. aeruginosa is associated
!nclude iuetritis (vagirlitis) sccondary lo prolo11ged Lreal-
:nent with antimicrobial agents, keratitis, and conjunc-
tivitis followiJ1g trcatment of corncal ulcers with topical
steroi.d-antibiotic mixtures.
Chapter 20 Pse.udo1no11as 123
Bovine. Conditions in which P. aeruginosa is associated
include mastitis (uncommon).
MiScellane.ous. l'seudornonas ae.ruginosa is an uncommon
cause of septicemia in immunocompromised animals, but
a frequent cause of bacteremla in human belngs wlth
burns, leukemia, or cystic fib.rosis .
Epiderniology
'lhe organism is ubiquitous in the enVironment. lJisease
determi11a11ts therefore lie largely with the hosts and their
immediate environment. In a veterinary hospital, how-
ever, a number of situations favor selection of this organ-
i:s1u. P:seudurnunas aeruginu::;u t11rive:; in wet, poorly aeratecJ
environments within the hospital, espe.cially in surgery
areas within support bags that have not been properly
dried, in 11oses on anesthetic machines that have not been
cleaned and dried properly, or in disinfectant solutions
that have not been changed frequently. These situations
result in an increase in the nu1nber of pseudomonads in
the e11vi1011111ent of tl1e con1pron1ised anin1al (sile),
thereby increasing the risk of infection (contamination).
lmmunologic Aspects
Speciftc immunf' responses <lo not seem to play much of a
role in pathogenesis or resistance, though artificial protec-
tion has been shov.rn to occur in anirnals vaccinated with
cxtracts of the organism or cxotoxin A. Thc most impor-
tant consideration is to decrease tl1e risk of infection by re-
ducing the concentration of the organism in the environ-
ment of thc patlent, in addition to reducing the extent of
compromisc, for example, by rlf'<lning ;"Jnd drying an in-
fecLed ear.
Laboratory Diagnosis
Pseudomonas aerugínosa grows well on blood agar :m.edl um.
The colonies are somewhat large, >1 mm in diameter, gray
(gunn1eLal), rough, usually with a zone of hen1olysis. A
plate containing P. aeruginosa has a characteristic odor,
reminiscent of corn tortillas. Desides being oxidase-
positive, a trait that sets it apart from members of the fam-
ily Entcrobactcriaccac, it turn:; triple :;ugur iron ugur :iligbtly
alkaline (without gas), utilizes glucose oxidatively, gro'"'s
at 42ºC, and forms a blue-green, chloroform-soluble pig-
ment, pyocyanin. Resistance to sorne antl1n1crobials is due
to permeability barr.ier of the Pse11don1onas cell wa 11, and to
others because of inactivation dueto products encoded by
plasmid-based genes (R plasmids).
Treatment and Control
·rreatment involves corre<.."tion of compromise and, if nec-
essary, Lhe use of au a11ti111icru!Jial agt11t. P::;eudun1unus
aeruginosa is usually susceptible to gentamicin, tohran1y-
cin, amikacin, carbenicillin, ciprofloxacin, and ticarcillin-
clavulanic acid, and these agents are used for the treat-
124 l'ART 11 Hacteria and t<ungi
.me11t of soft tissue infections. In the canine urinary tract,
tetracycline achieves concc11trations sufficicnt to kili most
isolates. Most pseudon1onads are susceptible to levels
achieved by antimicrobial agents in otic preparations: en-
rofloxacin, neomycin, polymyxin, chloramphenicol, and
gentamicin. Tt shoul<l he note<l that there are no in vitrf'
tests that predict susceptibility/resistance of a11 isolate
from infectious processes that will be treated topicaU:-
(e.g., the ear).
 Taylorella equigenitalis
ERNST L. B IDERSTEIN
embers of thc gcnus Taylorclla are grani.-negative, facul-
_:.J\'ely anaerobic rods. ·rhe genus contains two species, T.
;.¡igenitalis, the cause of contagious equinc metritis
_f.:\f), and T. aslntgenitalis, an inhabitant of the genital
::.a of cl inically normal male donkeys. Because of its clin-
- and economic in1por lance, T. equigenitafis wlll be diS·
'.\ed in detaiL J3ecause of its phenotypic similarity to T.
;enitalis, T. asinigcnítalis will be briefly described.
-• . ORELLA EQU/GENITALIS
.llu equigenitalis causes an acute, suppurative, self-
---'"'&ciisease of the uterus of mares calJed contagious
-.: meL1ilis (CEM). TI1e c.lisease results in temporary
~-, is high ly comm11nicable, and is followed by Iong-
.,.....-i asymptomatic carriage of Lhe agenl. Slallio11s de-
~o signs of illness but rnay remain carriers of thP
~-- .:idefinitcly. 'fhc discasc is geographically límited at
..-.=:··ptive Features
• ·; ogy and Staining
4 equígenitalis is a gram negative coccobacillus,
S µm by S to 6 µm in size.
- _·: and Composition
__..¡ equige11italis produces a carbohydrate capsule
~e·1 \vall that is typical of gram-negative bacteria
"t:::::::: .::::nposed of lipopolysaccharide and p rotein. It is in-
::ntly piliated. It shares sorne antigens with Haemo-
~asteurella, and Bruce/la.
-=............ - oducts of Medica! lnterest
:he capsule protects the outer membrane from
-~brane attack complcx of thc complement cas-
.._,::. --=.e capsule also inbibits attachment to and inges-
"agocytic host cells.
a!l. The ccll wall of T. equigenit.alis is typtcal of
_ _~ -- sative b acteria. The IipopolysacchariciP (T.PS) in
:=. mcmbranc is an important virulence detern1i-
DWIGHT C. 1lTRSH
nant. Not only Is the lipid A compon ent to.xlc (endotoxin),
h11t the length of the side chain in the 0 -repeat unit hin-
ders the attach111e11L uf the membrane auack complex of
the complement system to thP 011tPr membrane. Lipopoly-
saccharidc binds to lipopolysaccharide-binding µroteir1 (a
serum protein), which transfcrs it to the blood-phasP of
CD14. The CD14-LPS complex binds to Toll-like receptor
proteins (see Chapter 2) on the surface ot macrophage ccHs
triggering the release of proinflammatory cytokincs.
Growth Characteristics
Taylorella equigenitalis is a fac11ftative anaerobe that grows
optimally at 37ºC under 5% to 10% carbon dioxide 011 cl10-
colate agar (see Chapter 14) in a rich base (Columhia,
Eugon ). After 48 hours of incubation, colonies have a di-
ameter of l mm and may enlarge turther upon Jonger in-
cubation. They are shiny, smooth, grayish white, waxy,
aud sometimes pleomorphlc.
The organi~m is oxidase, catalase, and phosphatase-
positive and produces no acid f101n car1Jol1yurates.
The agent is unrelated to other gram-ncgative hac.tP.ria .
Resistan ce
·rhe resistance ot T. equigenitalis is not remarkable. Samples
on swabs can be shipped in transport medium undcr rc-
frlgeration an<l successfully cultured w1th1n 48 hours of
c:o llection.
Most anlibloLics exceµL slrt:pto1nycin inhtbit T. equigen-
italis, although they are strain-s11scP.ptihlP to this antibi-
otíc. Trimethoprim-sulfamethoxazole, linco1nycil1, and
clindamycin are sufficiently well tolerated to have been in-
cluded in selective isolation media.
Variability
Apart from harboring streptomycin-susceptible variants,
Lhe specie~ aµpears to be antigenically homogeneous,
thongh sorne strains (as defined by DNA analysis, sce
below) appear n1ore virule11L L11a11 otl1ers as measured by
entry into cells in vitro.
Molecular methods such as Random Amplification of
Polymorphic DNA (RAPD) and pulsea tield gel electropho-
resis of restriction endonuclease-digested DNA have iden
Lified differeut strains of T. equtgenitalis.
125
126 PART 11 Bacteria and Fungi
Ecology
Reservo ir
Taylorella equigenitalis is a parasite exclusively of the equine
genital tract. Antibodies to thc agent but no infections
have been found in cattle and humans.
The re:;ervolr 1!5 in E.urope, t>ut the ageut ha:; l>1::e11 r1::t:uv-
PrPcl from ;inim;il~ \.Vith C:F.M in J;ip;in, Austr;ili;i, ;ind
North America.
Transmission
1'a)'lorella equigenitalis is scxually transmitted, although its
occurrencc in ncwbor11 and vlrgln anin1als suggests alter-
native means of transmission.
Pathogenesis
Witl1in a ícw days of cxposure, a purulent endometritis de-
velops with variable amounts of exudatc. ·rhc main dam-
age is to uterine epithelium (exclusive of glands), wl1ich
becomcs covcrcd by ncutrophilic exudate. 'fhe cellular
infiltratc in the endomerrial stroma is predominantly
mononuclcar. Epithclium is eroded or undergoes severe
ucgc11crativc t:l1<111gc:.. Tl1c ulc1 iue iufeclion usually sub-
sidcs spontaneously within several weeks. Endometrial re-
pair is complete and there is no lasting impairmcnt of
breeding performance. lntection has been demonstrated
in placentas and newborn foals, but abortions havc been
few.
1·here is no fever or other sign of illness. 'fhe 011ly effect
rnay lJe f<tilurc uf t:o111.:c¡;tiuu. Altcru(ltivcly, there 111ay lle a
m11coici to mucopur11lent vulvar ciischarge of variahle
abundancc sorne days after scrvicc; interna! examination
reveals a cervicovaginal exudate of uterine origin. Externa!
signs of disease disappear within 2 weeks, although the
uterine infection may persist longer.
Epidemiology
1I1a¡;¡;arcut ¡;cr:;btcnt:c uf T. 1.:t1ui¿;1.:niluli:; i~ frey_ue11l i11 re-
covered mares and in stallions in endemic areas. 'fhe foci
of carriage in mares are the clitoridal fossa and sinuses; iI1
stallions, the foci are the prepuce, urethra, and the ure-
thral fossa and sinus.
Dissemination of infection is tied to breeding opera-
tiuns and the movement and use of infected animals.
lmmunologic Aspects
Recovered animals show increased resistancc for severa!
months, manifested by milder signs and lower numbers of
bacteria when reinfected. The mechanism of rcsistance is
u11t:e1 lai11.
Complement-fixing scrum antibody appears late in the
first vvcck and riscs for 3 wccks aftcr experimental cxpo-
sure. Uuration ot titers in mares varies, but tests are cons1s-
tcntly positive during the third to the seventh week post-
1nicct1on. T1ters are apparently unrelated to the carrier
status. Antibody is present in vaginal mucus, but its rela-
tion to infection is unknown.
Bacterins do not prevent infection, but do reduce it::
severity.
Laboratory Diagnosis
Diagno:;is of CEM and carrier identification require de-
monstration of T equigenitalis in the ge11ital tract. In clini-
cal cases, the agent can be dcmonstrated in the uterine e."-
udate by Gram stain.
Definitive identification of an infected animal requires
rc¡;Ht:atc t:ulLur1:::. uf avp1op1iale specime11s (see under
"Treatrnent and Control," below) on chocolate Eugon aga;
(prcparcd with horsc blood) with and without addcd strcp-
tomycin. A third medium incorporating clindamycin anc
trimethoprim permits growth of ali strains. Plates are e,.,_.
amined daily from the sccond ro the seventh days of
incubation (sce "Growth Characteristics," above). Gram-
11egative, uxiua:.1::-vu~i live, and calalase-positive organisms
from s11spect colon ies must be confirmed serologically. An:-
identification of T. equigenitalis should be considercd tcnta-
tive until confirmed by a competent reference laboratory.
A polymcrasc chain rcaction (PCR) based assay has
been developed and found to be more sensitive tl1a11 tradi-
tional culture techniques uscd to identify affected animals
and to ldentlfy Jsolates.
Serological testing (CF) may be done on snspect m;ire<;
belween Lhe CifLeenlh and fortietl1 day postbreeding.
Treatment and Control
Uterine infusions of disinfcctants or antimicrobics and
systcmic antibiotic tccatmcnt are uscd in attempts to r~
duce the sevcrity and dura ti.on of illness and perhaps abort
tl1c cacrier state. Their benefit has been questioned.
Topicat treatment of affected mares conslsts of a cleans-
ing of the c.:litoridal fossa with 2o/o chlorl1exidine followed
by liberal applicaLloi1 of nilrofurazo11c ointn1ent (0.2<M>).
·rhe prepuce, urethral sinus, and fossa glandis of stallions
importcd in to thc Unitcd Statcs of Amcrica from couotcies
in which CEM is known to exist are cultured. The stallions
are then bred to two uninfcctcd rnares, which subse-
quently are sampled threc rimes for culture of t:liturida'.
fossa and clitoridal sinuscs. In mares to hP imported, earh
of Ll1ese si.Les is cullurcd rcpcatedly throughout 011e estr us.
Sorne countries require surgical obliteration of the cli-
toridal sinuscs in importcd mares.
In CEM-endemic countr1es, attempts at control have in-
cluded mandatory veterinary examinations, negative cul-
tures of ali anlmals lntcnded for breeding, ar1u :;upe::rvi.:;iuu
of the movement of horscs.
TAYLORELLA ASINIGENITALIS
Taylorella asinigenitalis is a gram-negative, facultativel y
anaeroblc rod that Is phenotypically similar to T. equigeni-
 Chapter 21 Taylorella l!quigenitalis 127

At the p resent t ime the only ~"ªY the two microorgan-
can l>e <.liffere11tiatc<.l is l>y a polyu1erase cltaili rea<.:-
- - _hat utilizes especially designed primers.
-::~vlorella asinigenitalís appears to reside in the genital
- ...Jo. of donkey jacks. It can be transmitted to mares by
--=al service, but not by artificia l means. Depending
~ the strain of T asinigeniralis that is used, sorne af-
-ed mares develop clinical disease that is similar to
CE1'4. Ho~.vever, mares do test positive \~•hen t he C(Hnpl e -
111e11t fixation test is use<.l to i<.lentify anirnals infecte<.J with
T equigenitalis. This, coupled with t he fact that isolati>s of
T. asinígenitalis and T equigenitalis are similar phenotypi-
cally, makes identification of horses with T. equigenitalis
(an economically dcvastating discasc) difficult. At this
time, the signtficance (other tha11 making control of CEM
more difficult) of T. asinigenitalis is not known.
 Spiral-Curved
Organisllls 1: Borrelia
RANCE B. LEFEBVRE
Borreliae are spirochetes transmitted a11d r11aintained pri-
marily by ticks. 'l'he infections they cause have blood-
borne phases acco1npanied or followed by general and Jo-
calized manifestations.
Animal pathogens include B. anserina, the fowl :;piro-
cl1etosis agent; B. theíleri, o mild pathogi>n m;iin ly of c.;itth~;
and B. burgdo1ferÍ serlsu lato, con1prised of three geno-
species, the cause of Lyme disease in dogs, horses, cattle,
a11d humans. Tick-bor11e relapsing fcvcr borrcliac of hu-
mans (B. hermsii, B. parkeri, B. turicata) occur asymptomati-
cally in. feral man1mals, birds, and reptiles.
Descriptive Features
Morphology and Staining
Borreliae are gram-negative but stain poorly. for demon-
stration by light microscopy, silver, or Romanovsky-type
strains (Gien1sa, Wright's) are best (Fig 22.1 ) . Dark field ex-
amination reveaJs spirals and motility.
Cellular Anaton1y and Cornposition
ilorreliae have a structure like other spirochetes, with an
outer sheath encasing the axial fibrils consisting of 15 to
20 endoflagella (dependlng on specles).
Members of the genus .Borrelia are u.n ique am.ong pro-
karyotes in. that they have a linear double-stranded chro-
mosome of approximately 900 kbp and a multiplicity of
linear and circular plas1nids that may in fact constitutc
components of the genon1e.
Cellular Products of Medica! lnterest
(;el/ Wall. Memhers of the genus Rorellia have a gram-
negative cell wall. The lipopolysaccharjde (LPS) in the
outer me1nbrane is an important virulence determinant.
Not only is the lipid A con1po11ent toxic (endotoxin), but
the length of thc side ch.ain in the 0 -repeat unit hinders
the attachrnent of the n1e1nbrane attack complex of the
complement systerr1 to tl1e outer u1eu11Jrane. LPS 1Jir1Ll~ lu
lipopolysac.charidf'-hinding rrotein (a .serum protein),
which in turr1 transfers it to the blood phase of CD14. The
CD14-LPS complex binds to Toll-like receptor proteins (see
128
Chapter 2) on the surtace of macrophage cells triggertn.~
t he release of proinflamn1atory cytokines.
T-Temolysin. Hemolysin activity has becn associated \-Vit t
B. burgdorferi.
Growth Characteristics
Of the borreliae pathogenic for animals, B. anserina is
propagated in embryonated hen's eggs, and B. burgdorfer
sensu lato is cultivated at 33ºC on modified Kelly's
medium (BSK), an enriched serun1 broth made selective b:-
inclusion of kanamycin and 5-fluorouracil.
The organisrr1s are :o;low-growing 1uicruaeru¡Jllile:s (duu-
bling times of 17. to 18 ho11rs). 'l'hf'y ferment gluc.ose anG
possibly otlJer carbohydrates.
Survival of borreliae is about a week at roon1 tempera-
turc in b lood clots, severa! months at 4°C, and indcfinitc a:
Jess t han 20•<.:.
Variability
A numher of genes enc.oding outer surface proteins ha,-.,
been identified in severa! Dorrelia species. The antigenic
variability available to tl1c sp.irochetes is utilized for im-
mune evasion in the relapsing fever borreliae. Lyme dis-
ease spirochetes are also equipped to express a wide rang1:
of outer surface proteins. Though not utilized as with the
relap:siI1g fever uurrel.iae, au iu11uu11e evasion funclion v-
these antigens may still be involved in their maintenanct
and expression.
Ecology
Reservoír and Transmission
Pathogenic borreliae of anin1als are vcctorcd by ticks
Ticks become infected at sorne stage in their life cycle b:
feeding on infected anilnals. Son1e vertical (tr~u1sovarian
maternal) trans1nission occurs. ()ther artllropods ma•
serve as short-term vectors. Infection occurs by woun.:
co11la.111i11aliou, usually du1i11g feeding by infected ticks.
Passage via placenta, 1nilk, and urine has been docu-
mented. With birds, infection may occur by coprophagi.=
and cannibalism.

- =__,R E 2 2 . 1 . Section of liver from il horse showing presence
- :..::rrella. Steiner Sí/ver stain, TOOOX.
•
..
• though not routinely treated, animals respond to tetracy-
·-~gen es is
-= ~toxin is incriminated in tl1e pathogenesis of relaps-
- ~~-er and probably other bor.r elioses. Hemolysin activ -
-_,s becn described in Ly1ue clisease spirocl1t:Lt:s.
- MA L BORRELIOSES
: : ;~fl/A ANSER/NA
~..'.!a anserina causes fowl spirochetosis in chickens,
• h~"S, gccsc, ducks, phcasants, p igcons, canarics, and
.....e specics of wild birds. Onset of disease is marked by
:-- depression, and anorexia. Affected birds are cyanotic
.:. jevelop a greenish d iarrhea. Later signs may include
'":Lysis and anemia. Mortality ranges from lOo/o to al-
1001X>. Necrnpsy reveals splenon1egaly a11cl ~vicle­
eEd hemorrhages. The enlarged liver may contain
.=otic foci. Peripheral blood is often sterile.
-.T.fa11 spirochetosis occurs on ali continents and in all
i2!!'.-~ ofbirds. The young suffer high mortality rates and die
c:-lier, septicemic stages o f infection. FoJJowing an out-
-=-'· the agen.t generally disappears fr.om the flock
...;L_n 30 days.
-:'lf' lt>:irling vf'rtnr, Arga.~ pr~rsir:us, c'an rpmain infer.ted
O'·er ayear and can pass the agent transovarially.
~::::nporary immunity, apparently antibody mediated,
J,,-s recovery. Ant i.sera confcr protcction for severa!
~ - Inactivated vaccines m.ade fro1n infected blood or
--pro pagated B. anst>rina are beneficial.
'pirocheleS a1e detllO!lSlrable ill blOUU lJy uarkfielu IIIÍ-
>i...-Opy, stained smear, or immunotluorescence. Suspec.t
- -eria l (i.e., blood, spleen, or Ji ver suspension) maybe in-
_ ated into t11e yolk sac of S-to-6- day em bryonated eggs.
:och etes \-vill appear within 2 or 3 days. Antigen or an-
- .e.y m ay be demonstrated in agar gel diffusion tests.
=:vrrelia anserina is susceptible to penicillin G, tetracy-
-=:::, chloran1phenicol, slre¡JL0111yci11, ka11a1uycin, arH.l ty-
- Im1nune serum has protective potc~ntial ano h:ir-
-.,...,..;--~·1s produce long-lasting immunity.
...ontrol ot ectoparasites is essential.
Chapter 22 Spiral-Curved Organisms l: Borrelia 129
BO RRELIA THEILERI
Borrelia. theileri causes a lnild febrile anemia, most often in
African and Australian cattle, and occasionally in sheep
and horses. Tl1e di.sease is associated with severa! species of
ixod.id Licl<.s. Tl1e µa ll1uge1llc iuecha1Ji:sn1(:s) i:s r1ot urH.ler-
stood. Most information comes from fiel<i c.ases, whic.h
may be complicated by other tick-bor11e infcclio11s. Al-
cline. Tick control is advisablc.
LYME BORRELIOSIS
Lyme disease is caused by D. burgdor(erí sensu lato, a patl1-
ogenic spirochete. Genetic analysis now defines three
genospecies, B. burgdorferi scnsu stricto, B. garínii, and n.
atzelii. Borrelia burgdor{eri sensu stricto is the predominant
pathogen of North America.
Dlstrlbutlon and Transmlssion
Endemic areas in.elude the North American Ati anti e states.
as well as Minnesota and Wisconsin¡ parts of thc North
American South and far West; most of continental Europe
and Britain¡ Russia; Asia; Japan; and parts of New South
Wales ln Australia. May through Octobcr is the time of
peak p rPva lt>nrP. Tnrrt>:ising spread of the disease is attrib-
uted to increased deer populations, i11creasin.g hun1an
movement into ru ral areas, and t he dissemination of in-
fectcd ticks by migratory birds. 'rhe agent is harborcd by
several ixodid ticks (.primarily Jxodes scapularis and J. paci-
ficus in North America). The tick has a 2-year life cycle
comprlsed of a larval, nymph, and adult stage, requiring a
blood meal at each molt. Deer mice, white-footed mice,
and oll1er sn1all rodenls serve as reservoirs for Lile spiro-
chete. Borrelia burgdorferi has been isolated from the u rine
of dogs and cows as well as milk from infected cows, poten-
tially serving as aJternate routes for exposure and infec-
tion.
Hu man Lyme borreliosis, caused by B. burgdorferi, typi-
cally begins with a skin lesion (erythema migrans) often
followed vveeks or u1011ll1s laler !Jy ut:ural, cartliac, anu
:irthritic complications. F.ndotoxin, hemolysin, immune
complexes, and in1n1u11osuppressio11 n1ay be i11volved in
pathogenesis.
In othcr animals, dogs are most <>ftcn affected, witl1
manifestations of polyarthritis, fever, and anorexia the
most coxnmon signs. 1vfalaise, lymphadenopathy, carditis,
auu rt:11al c.lisease lklVt: also lJeen noted in dogs. S11n1lar
sih'Tls have also heen rt>pnrtf'rl in clomesti.c cats, thougl1 fe-
line b orreliosis is rare.
Borreliosis also occurs in horses and cattle . T11 horses,
polyart hritis, ocular a11d ncural involvcmcnt, and foal
mortality have been reported.
Diagnosis
Diag11osis involves den1onstratlo11 of Lhe agenL in lissues
and fluids (darkfield, immunofluorescence microscopy),
130 PART 11 Bacteria and fungí
antibody in serum or other fluids (indirect immunofluo-
rescent test, enzyme linked immunosorbcnt assay, ELISA),
or DNA amplification of tissue or fluid samples using
genus specific DNA primers and the polyrnerase chain re~
a.:lion.
Culture is laborious and often unrewarding. However,
culturing cnr punch biopsies from infected dogs and mice
has proven to be reliable. Culture of synovial fluid from af-
fected joints is also possiblc. 13SK is n goo<l isolation
medium. The sp1rochctcs grow best at 33ºC.
Treatment and Control
'l'etracycline, doxycycline, enrofloxacin, erythromycin,
and pcnicillin e; urc gcncrally cffcctivc, although not in-
variably so. ·nck control is vital.
lmmunologic Factors
A humoral iminunc response appcars essential for protec-
tion against B. burgdorferi infectio11. Most animals appear
to selt-immunizc with no apparent clinical manifestations
subscquent to exposure to the spirochete.
Artificial lmmunization
Antibodies produced in response to vaccination \\"'!~
hurgdor(erl have proveo effective in preventing iniect: ~~
J;ihnr;itnry ;inim;:il~ ThP~P nh~Prv;itinn~ h;ivP Jpd to the
velopmcnt o f a commerciaHy available whole cell bdl..~...=.~
for use in canines. Subunit vaccines comprised of ,,,,~,,_
surfacc protcin A (OspA) are also commercially aYai_.~~a,,;;;
Protective immunity tor these vaccines is not : ~
Protective in1munity also appcars to be of short dura::.
ano llrnltetl tn range, prolJalJJy due ro cons1deralJlehe:::::.
gent>ity in th f' OspA antigf'n. Tt has hPi>n <lemonstr.o
that thc OspA antigen is synthesized by B. burgdorfe ri
while in thc 1nid-gut of the tick vector. Its synthes:
turncd off during the first 24 hours of a blood meal as --
spirochetes migrate toward tl1e tick's mouth and su...
quc nt inoculation into the ma1nmalian capillary. ~­
(./spA subuntt vacctnes runcnon 111 a cwo-step proce-
Vacciníltf'<'I mf!mmals st>r vi> ílS antihndy fílctoriPs to Os~
which in turn must be delíverect to the midgut of a feedi-_ _
tick to neutralize the spirochetes at this site.
 Spiral-Curved
Organisins II:
Brachyspira (Serpulina)
DWIGI-IT C.
..embers of lhe geuus Bruchyspíru are gra111-uegative,
-:-;al-shaped ohligately <in<ii>robic b;icteria belonging to
:::= family Spirochaetaceae. Brachyspira hyodysenteriae is the
~sative agent of swine dysentery, a disease of actively
=:t1'ving pjgs. Brachyspira pilosicoli (Anguillina coli) is asso
~:ffl \\1ith intestinal spirochetosis of pigs in the post-
'"!!aning period, dogs, birds, and humans (usually those
;.;:;a~ are i111u 1u11oco.1I1JJruu1ised). Bruchyspiru uulbur;.;i i~ a
-:...-e cause of human spirochetosis. Other hrachyspiras
-~ uncertain pathogenic potential include n. intennedia
z::~ B. murdochii. Brachyspira innocens, found in teces of
-:::pt omatic as well as asymptomatic pigs, has little if any
-~-.:iogenic potential. "Brachyspira canis" is often tound in
: intestinal content of sympto1natic as well as asympto-
~~tic dogs.
: ?scriptive Features
~ ·phology and Staining
c;¡r!ryspira hyodyse11terh¡e and D. pílosicoli an~ loosely coiled
~ _:ochetes. 6 to 11 µm long by 0.25 to 0.35 µm in width
- :: 23.1). Another very similar brachyspira, B. innocens
- eso-called small spirochete), is often found in the teces
- ?igs with signs of dysentery as well as in normal feces .
hyspira innucens rneasures 5 tu 7 µrn by 0.2 µrn tightly
:::!led .
• ne brachyspiras are gram-negative, but this character-
- is not used to identify or detect them. Romanovsky-
- ~ stains (e.g., Wright's, Giemsa) are more useft.tl in
...r--0:'lstrating these organisn1s in smears.
:.· Anatomy and Composition
-~ne typical of spirochctcs. Thc axial filament of D. hy-
--;-,eriae is made up of 8 to 1;¿ flagella inserted at either
---=·:: i.nnocens has 10 to 13 flagella, and B. pilosicoli 1 to 6.
- • ~ · Products of Medica! lnterest
- : JJ. T he hrac.hyspir<is possess a gram-negative cell
~-~-:e lipopolysaccharide (LPS) il1 the outer iuerulJrane
HIRSH
is ;u1 irnpurtant virulence determinant . Not only is the
lipid A component toxic (endotoxin), but the length of the
side cbain il1 tl1e 0 -repeat unil l1inders the atla1.:l11ueut uf
the membrane attack complex of the complement system
to the outer membranc. Lipopolysacchnridc binds to thc
plasma protein Jipopolysaccharide-binding protein,
which then binds to CD14. The CD14-LPS complex binds
tu a 'full-like receptor (see Chapter 2) on the surface of
macrophage c.ells, triggi>ring thP rPlP<iSt> of proinfl;im1na-
tory cytokines.
Cytoxin/Hemolysin. 1'he protein, Tly (for cytoxin/he-
molysin) is responsible for the strong beta-hemolysis dis-
played by B. hyodysenteriae in vitro (the degree of hemoly-
sis in vitro is often used to differentiate S. h)'Dd}'senteriae,
whtch is strongly beta-l1emolytic, from B. innocens and B.
pilosicoli) . This protein is a virulence determinant in vivo.
Muta11ts that are unable to produce Tly are less virulent.
Tly is a pore-for ming c.ytotoxin affec.ting host target c.ells
(goblet cells and colonic epitl1elial cells) .
Hemolysin. A.nother hemolysin, Hly (for hemolysin) has
been described, but its relation to virulcncc is unclear.
Flageua. Flagella, though present on virulentas well as
avirulent br:achyspiras, appear necessary for virulence.
This Lrail is related Lo 111ove111en L Lhrough Lhe inleslinal
mucus to gain access to target cells in the large intestine. It
has also been shown that virulent strains have an affinity
for intestinal mucus.
Growth Characteristics
.to.U u11::1111Jers uf t l1e genus Brachyspira. are obligate ana-
erobes.
Brachyspira hyodysenteriae is strongly beta-hen1olylic, a
trait that has been used by sorne to differentiate it from B.
innocens and B. pilosicoli, vvhich are weakly bctn-h cm o lytic
(Fig 23.2).
Rrachyspira hyodysenteriae and B. pilosicoli are resistan t
to high concenlralions of sµecti1101nycin, a characteristic
useful in isolating these organisms f rom fi>ces. Brachyspi ra
hyodyscntcríae and presumably D. pilosicoli ren1ain infective
for long periods it enclosed within organic n1aterial in
temperatures of SºC to 2SºC. They do not withstan.d dry-
ing or direct sunlight.
131
132 PAKr 11 Bacteria and Fun<ri
b

F 1G U RE 2 3 . 1 . Brachyspira hyodysenteriae in a co/oníc scrapping of a píg with swine

dysentery Victoria Dlue 4R stain, 1000X.

rlíiíl!lba1 ;r w
•

.@fil O '~
'

Ecology
FIGURE 23.2. Brachyspira hyodysenteriae growing on a
b/ood ;:ig;:ir p/;:itc.
Reservoir and Transmission
'fhe reservoir for B. hyodysenteriae is the gastrointesr'~--:­
tract of pigs, especially asympto1natic carrlers of the Of82~
ism (animals recovered from tl1e disease). The agen t :=-.
bee11 isolated fron1 tl1e feces of dogs, rat:s, a11d n1ice li>'~
on farms where the disease exists. Transmission is thro~~
thc fecal-oral routc.
.Hrachyspira pilosicoli has been isolated from dogs, bi~=­
and hu1nans. ·rhere is evidence that humans 1nay acq~·--..,.
B. pilosicolí from affected dogs.

Variability
There are at least twelve serotypes of B. hyodys<'nt<!riae.
Fi.Hgerpri rr Li 11g isolales by 1neans of reslriclion-le11glh
polymorphisn1s of whole-cell DNA, DNA. encoding riboso-
mal llNA, L)NA encoding specific genes (c.g., flagcllin),
and n1ultiiocus enzyn1e electrophoresis has den1onstrated
the hetcrogcneity of lnen1bers of this species as well as the
otl1ers (B. innocens and B. pilosicoli).
Pathogenesis
Brachyspira hyodyscntcriac multiplics and produces di<e..,
ín the colon (swine dysentery). Tt appears that B. hyo<T
teriae alo11e will not produce disease. Other bacteria -
mally found in the colon of pigs, such a:; Bacteruides l~
tus, B. fragilis, Fusohacterium n<!r.rnphnrum, (~ampyloi- -
coli, a Clostridiu.111 sp., and Listeria denitrific.111s, have t-=
s~1ovvn to be involved in this supporting role. Superficia.
agulation necrosis witl1 cpithelial cell erosion is obse: ..
Edema, hypere1nia, .b emorrhage, and influx of pol~-::­
phonuclear neutrophil leukocytes (PMNs) into the m·,_.·'--'-
and submucosa are seen. There is failure uf <.:ulu11ic abs-·-
tion. Tnfla.mm;1tion, b ro11ght ahout hy c.ytotoxin-mE><i" --
destr uction of colonic target cells (goblet <:ells ini. :...
t hen enterocytes), may induce a secretory diarrhea. .-..
scqucnccs cncoding known enterotoxins have no<_""'~
found in B. hyodysenteriae.
The signs of disease are rat her typical. Affected PÍ0
void gray to strawberry-colored fece~, be<.:u111c Jeh) d:" -
 Chapter 23
:. iJ ~ t; l '?.. '?.. e.tacn·r:.?ita ?ilosi<:.o\\ in the intestinal lt é!Ll <JÍ d
; Nith 1ntest1nal spirochetosis. Transmission electron micrograph,
_::Jt 40,000X.
~. 1n the extre1ne, be acidotic and hyperkalemic. Tem-
~ture generally re1nai ns normal. Morbidity ratcs in sus-
-:ible pigs will he close to 90o/o, with mortality in un-
~Pd hcrds of approximatcly 20% to 40%. Duration of
ess ra11ges f10111 few uay:. to several weeks. Survivors
be permanently stunt·r<1 anrl remain asymptomatic
rlders. 'fhere is no easy way to detect such ani111als.
ntestinal spirochetosis of pigs (post-wcaning period),
:s.. birds, and humans is associatcd with B. pilosicoli.
clisease is characterizcd by a milct, persistent diarrhea
... low n1ortality. Biopsies of affected colon show large
:L:Jps of spirochetes adhering "en d on" to the intestinal
-hpJium (fig 23.3).
- '"'lUnologic Aspects
- e is known about the immunologic facto rs of thcse
-c2ses. Plgs recovered fro1TI swinc clysentery are resistant
-.-infection. llacterins have mct with sorne success in re-
_"1g the seve1 iLy of Lhe ui:.casc iu affeLted sw1ne.
...:_ oratory Diagnosis
-: e Collection
_ samples from affected animals showing signs of thP
_...J5e are uscd to dctcct B. l1yodysenteriae and .TJ. pilosicoli.
~r: Examination
of fpc;,l material are stained with a Romanovsky-
-c....;<;
-:-c ~tain (e.g., Wright's, Gie111sa) or carlJul fuchsin . Oh-
Spiral-Curved Organisms ll: Brachyspira (Serpulina) 133
servation of large, loosely coiled spirochctcs in diarrheal
\ete'.) \'.) pre~umpt\ve ev\oence o\ \n\ett1on with 13.
hyodysenteriae (swine dysentery) or B. pilosicoli (intestinal
spirucl 1closis) (see Fig 23.1 ). Brachyspira innoccns may be
present in s:implPs from pigs w ith swine dysentery, hut
t hése will be smaller and have lighle1 Ct>il:., a uistiuctlon
that is somctimes difficul t to make.
lsoliiti on/ Detection
Isolatiuu uf B. hyodysenteriae and B. pilosicoli from fecal
san1ples is acco m pli~hPd by inoculation onto blood agar
plates containing spectinon1ycit1 (400 µ~/1111). 1'11e plates
are incubated 24 to 48 hours in an anaerohic environn1Pnt
containing 10% carbon dioxidc. Colonies of D. hyodysente-
riae will be small and strongly beta-hemolytic; thosc of B.
pilosicoli will not be as strongly hemolytic (see Fig 23.2).
A 111ulliµlt!x polyn1crase chain reaction (PCR) assay
using prim<'rs <lPsigned to dctect the DNA of common di-
arrhea-associatcd n1ic1oor~a11i:;1us (B. hyodysenteriae, Law-
sonia intracel/ularis, and Sa/nu111P.lla) has been described.
ldcntificotion
Bruchyspiru hyodysenteriae must he differentiated from R.
innocens. This is hf'_"t <1onP by gas chromatographic analy-
sis of volatile fatty acids or by DNA ¡J1uui11g/a11alysls.
Unfortunately these techniques do not Iend themselves to
performance in a busy diagnostic laboratory. Therefore,
observing thc strengt h of the beta-hemolysis. fructose fer-
mentation (R. innocens will be positive), and índole pro-
lluLtion (B. l1yodysentertae wlll be positive) are traits used to
make thP <li<:tincti.on. Though the hemolysis trait seems to
be relatively stablc, the 0Lhe1 LesL:. <irt: ~0111ewhat variable
and misidentifications are possihle. Rrar.hyspira pilosiroli
hydrolyzcs hippurate¡ JJ. innocens <loes not. l3rachyspira pi-
losicoli is ditterentiated from "B. canis" by molecular
meaos (sequence of the gene encodi.ng thc 16$ rRNA com-
bined with multilocus enzyme electrophorcsis).
Treatment, Control, and Prevention
L>rugs shown to be cffective in treating swine dysentery
and intestinal spirochetosis in swinc include o rganic ar-
senicals, tylosin, gentamicin, nitroturazone, virgini-
amycin, and lincomycin. These have been used at low pro
phylaclic lt::vcls, lJut it should be kept in mind that drugs
used routinely to prPvPnt the disease will ultimately lose
their effectivcness. Metronidazole is Lile 11:<.:u1111111;:11ucu
treatment for dogs with intestinal spirochetosis.
 Spiral-Curved
Organism.s III:
Campylobacter, Arcobacter,
Lawsonia
D WIGHT
Mcmbcrs of the genera Ca111pylobacter, Arcobactcr, and
Lawsonia are gram-negative, curved rods. ·rhey are associ-
atcd with diseases of the reproductive and intestinal tracts.
Taxonom1cally, Campylobacrer and Arcobacter (previously
classified within the genus Canzpylobncter) are members of
the farnily CampylubaLlmaLt:ut:. Luw;,uniu Llves nol appea1
tn hf' rf'latf'd phylogenetically to any other species of path-
ogenic bacteria.
Descriptive Features
Morphology and Staining
Members of the genera c;ampylobacter, Arcobacter, and Law-
sonia are gram-negative, slender, curved rods that measure
0.2 to 0.5 µrn !Jy 0.5 to 5 µm. Whe11 twv vr 1nore l.Ja<...terial
Cf'lls arf' placed together, they form Sor gu llwinged shapes
Lhal n1ay appear as "spirals" (Flg 24.1).
Cellular Anatomy and Composition
Members of the genera Campylobncter, Arcobacter, and
Lawsuniu have typicaJ gra.111-111-:galivt: ccll wall:>, cavsules,
::inc1 fl;igplla.
(AM PYLOBACTER
Members of the genus Campylobactc'r are implicated in en-
tcric and reproductive diseases of animals. ·rhough tl1c
genus contains 15 specles, only a few have bee11 implicated
in <.lisea::.t::. Tht:::> t:' incluut: C. {elu;,, C. jejuni, C. coli, C. con-
cisus, C. he/veticus, C. hyointestinalis, C. tnucosalis, C. lari,
and C. upsaliensis.
Two species of Can1pylobacter are important in regard to
the genital tract and reproductive performance: C. fetus
and C. jejuni. Can1pylobacter (etus has t~vo sulJ:>pecies: verze-
realis and fet1.1s. The disease produced by these organisms is
::.01111-:tiiuc:> rcft:rrt:Ll lu a::. vihr iu;,b bt::cause lhe agents wcrc
134
c. HIElSH
once classified as members of the genus Vibrio. 'fhey are
not.
Can1pylobacter jejuni and C. co!i are major causes of gas-
troenter1t1s ln people and nonhu1na11 µriiuatt:::., a11d ha\c;
been found in fecal s::implf's from dogs and cats with diar-
rl"1ea. Carnpylobacter jeju11i is the more commonly isolatec.
of the two. Though C. coli occurs in high numbers in swine
dysentery and was once thought to be the causative agen r
the disease is caused by Hrachyspíra (Serpulina) hyodysente-
riae (see Cl1apter 23). Both C. coli and C. jejuni may occur ir.
feces of normal animals.
Cnmpylobacter concisus has been associated with gas-
lrviI1lt::>lil1al ili::.t:a::.t: vf hu111ans.
Can1pylobacter helveticus has been recovered from th
feces of dogs and cats with diarrhca.
c..;ampylobacter nyo1ntest1na11s and <..,. mucosat1s were once
implicated as contributors to the swine proliferative en-
teritis complex. Thls dlsease Is caused by Lawsuniu inlracel-
!1.1lnris (see below), a microorganism that produces this dis·
1-:a:.1-: i 11 cv11ve11tio11al pigs Ül pu re cullure, son1cth in::
neither C. hyointestinalis nor C. nzucosalís will do.
Ca111pylobactcr lari, i:;olutcd from thc fcccs of osympto
mat1c gulls (from which it gets its nan1e), has also been iso-
Jated from the feces of various hosts, including dogs, birds
and horses. Tts role in tlisease i:> uuccrtaiu.
Carnpylobacter sputon1111 is sometí mes isolatf'c1 from tli.-
prepuce of bulls or vagina of cattle, but are not considere....
significant.
Carnpylobacter upsa/icnsis has bccn isolated from feces
dogs and cats \.Yith diarrhea. Entenc d1sease and abortio::-
have been associated with this microorganism in human
Descriptive Features
Cellular Products of Medical lnterest
Adhesin. Can1pylobacter jejuni involved with f'nteric <lisf'a
µrvLluct: a 1uaiu1ose-resislan l adhesin that binds to
fucose-cont::iining rf'cPptor on the target cell (epithelia.
cells of the small intestinc).
 Chapter 24 Spiral-Curved Organisms III: Catnp)'IObacter, Arcobacter, La1vsonia
= G U RE 2 4 . 1 . Cd111µylol>atl er felu ~ ~ulJ~peUes fetus in the
;-omach fluid of an aborted /amb. Gram stain, 1000X.
...1psule. 'fhe glycoprotein capsule protccts thc outer
-brane from tl1e mcmbrane attack complex ot the
plement cascade. Thc capsule also inhibits attach
~to, and ingesuon by, phagocyrtc host cells.
!l Wall. The cell wall of the members of this genus is
:··pi cal uf grau1-11cgati ve bacteria. The l!popolysaccha-
LPS) in the outC'r memhr;ine is an impnrt;int viru-
~ determina11t. Not only is the lipid A con1po11ent
-:: endotoxin). but the length of the side chain in the
~at unit hinders the attachment of the membranc at-
.:omplex of the complement systcm to the outer
...,rane. Lipopolysaccharide binds to the serum pro-
.:popolysaccharldc-binding protein, "vhtch transfers
thP hloorl-phase of CD14. 'fhe CL)14-LPS complex
'--'• to Toll-like receptor protcins (sce Chaplc1 2) on Llie
..e of macrophage cells triggering the rclease of proin-
...._.-:1atory cytokincs.
_Tocoxin. campylobacter ¡e¡un1 1nvo1vcd with enteric
e secrete a toxin "vith similar activity to cholera toxin
.1e heat-lauilc tuxin (LT) uf Eschericl1ia coli (see
•er 8) by increasing intr;ic.ellul;ir IPvPls of cAMP and
~ · ... letal rearrangements. Iloth toxins are in1n1unologi-
: Eolated and bind to the sanie ganglioside (GMl ) on
_:face of tl1e target cell. Can1pylobactcr coli and C. lari
-·'-<=e uncharactcrized substances w 1th cytotonic and
'.:ÍC activi ty.
-·coxins. Curnpylubacter jejuni involvcd wlth enteric
""produce a numher of prnteinc; '""ith rytotoxic activ-
.:sc cytotoxins includc a heat and trypsin labil.e pro-
-u küa in size which is neutralizcd by antibody to
_,.._ .ke toxin (scc Chapter 11); a 73 kDa protcin, tcrmcd
: Campylobacter 1nvasion antigen) that is inserted
~e target epithelial cell allowing subscquent invasion
..t:l1 (a TyIJe III sccn:uon sysrem may be 1nvo1vea, see
a protein ac.tivP on Vi>ro tissue culture cells; a pro-
135
tein that increascs intraccllular cAMP follo,.ved by cell
death (a cytolethal dlstcnding toxin, see Chapter 8); a pro-
tcin that has hemolytic activity (hemolysin); anda protci n
tltat was shuwn tu Induce hepatitis in mice (hepatotoxin).
All of these toxic compounds have a tenuous association
wilh Lhe disease 111ucess lu l1Ulllaus and other ani:mals.
Miscellaneous Products. Campylnhartt>r jejuni posscss a
Typc TTT sccrction system (an assemblage of proteins-
more tnan LU-tnat torm a hollow tu bc-like st ructure
th rough which effector proteins are "injected" lnto host
"target" cells). ·rhe proteins involved wlth this syste1n1 Cía
(for Campylnh;i1tPr inv;ision antigens) are responsiblc for
triggering lhe uplake uf C. jejuni after adherence to intes-
tinal epithelial cells.
Growth Characteristics
Can1pylobacter spp. a re m icroaerophilic (with the excep-
tion of C. hormnis which appears to be obligatcly anaero
bic), re4ulriug an atmosphere contaln!ng 3o/o to 15º/o oxy-
gen and 3% to :'l% 1arbon dioxide fo r growth. Sorne, such
as C. ¡ejuni, grow at 42°C, a charactc1islic Lhal i~ useful fur
its selectivity in isolation from intestinal sources. Unlike
mcmbers of the fam ily Entcrobactcriaccac, thcy are oxidase-
positive. They do not fer1nent or oxidize carbohydrates,
generating cnergy from oxidation of amino acids or tricar
boxylic acitl iuterrnetliares through the respiratory path-
way. Though thPy po<;<;Pss catalasc and superoxide dismu-
tase, these enzymes are over~vl1cln1ed by lhe excess uf
hydrogen pcroxide and superoxide anions formed \.Yhen
they are grown in thc prcscn cc of atmospheric concentra-
tions ot oxygen.
Variability
Campylobacter ferus subspecies fetus contains nvo serovars,
A-'?. and B, based o n heat-stable surfacc antigens. Strains
with both antigcns occui. Curnpylu/Jctt ler (elus su!Jspe(;ies
venerealis has two serov;irs, A-1 ;incl A-<:11b l. The serovars
differ not only with rcspcct to heat-stable surface anligen
(A) but also culturally and biocheniically. (,'arnpylobacter je-
juni possesses one serovar C, bascd on a hcnt-stablc surfacc
antige11. Carnpylobacter jejuni has twenty-two serovars as
dctcrmined by analysis of extractable heat-labile antigcns
(Lior scheme) 01 lwt:r1ty-tluee serovars by utilizing ex-
tractable heat-stable antigens (PennPr <;<hPmi>).
Ecology
Reservo ir
Catnpylobacter fetu s subspedes venerealis. ·rhe reservoir of C.
fet11s ssp. veneren/is is thc preputiai crypts of the bull (main)
a11d Liie vagina uf carrier animals (rare after one to two
breeding seasons "l<\rithnnt re-exposure).
Ca111pylobacter fetus subspccies fetus. 'fhe reservoir fur C.
fetus ssp. fetus is the intestinal trac.t of infP<ted (r.ecovcred)
shccp (pcrhaps through contamination fron1 a colo11iz.cd
ga11n1aaaer).
Catnpylobacter je¡uni. The rcservoir for C. jcjuni is thc
136 l'ART 11 Hacteria and Fung¡
intestinal tract of normal animals (cspccially young rumi-
nants, various birds, asy1npto1natic d<>gs and cats) or ani
rnals that have had the disease.
Other Can1pylobacters. The sources of C. Inri, C. helveti-
cus, and C. upsalie11sis are not known but are presumed tu
be the intestinal tract of infected individuals. FP<PS from
healthy puppies aud kittt:H::. have been sl1own to contain
C. 11psaliensis.
Transrnission
H.eproductive Disease. Acquisition of carnpylobacters affect-
ing the reproductive tract occurs either venereally (C. frt11~
ssp. venerealis, cattle) or by inge::.tiu11 (C. (elus ssp. fetus and
C. ;e;uni, sheep and goats, rarPly cattle).
Enti;rit Dbcuse. Acquisltion of campylobacters associ-
:itPcl with cnteric disease (C. jejuni, C. coli) occurs by the
fecal-oral route, dircct or indircct, n nd is probably the
rnain mode of spread.
Pathogenesis
Reproduaive Diseu~e (Guille). The agent (;. (etus ssp. venere-
alis is introd11<Prl into a susceptible fen1ale by a11 iI1fected
bull al coltus. The organisn1s remain at thc ccrvicovagi.nal
junction until the end of estrus. 'l'his is probably a conse-
qucncc of thc incrcnsed blood supply and active polymor-
phonuclear neutrophil leukocytcs (PMNs) seen in tl1e re-
pro<luctive tract during this time. The organisms m11ltiply
at this site and, when conilitio11::. ¡¡re suitable, move into
the uten1s. 1'\1rther m11ltiplication and perhaps active in-
va:.ion resull in inflammatio11 of the uterus v.¡ith rcsultant
endometritis and cessation of pregnancy. "ll1c animal wilJ
return to cstrus. 'f!1is process continues u n til t he fe1nale
makes an iinmune response sufficicn1 to elimir1ate the
agent from thc uterus. Subsequcntly, the endomctriti.;
subsides, and the animal couceivt:~ é!llU p1egna11cy goes to
term. Sporadic abortions somctimes occur.
Cli1lically, there are signs of repeat breeding and ex-
tended cycles (10-to-60-day cycles; 21 days is normal),
which are usually mnnifcsted in an infectcd herd as pro-
longed calving intcrvals anc.l extended calving periods. In
addition, the herd bull will show loss o f conditio11. ThP
herd, if it remalns closed, develoµ::. i111111u11ily, a11d calving
intervals gradually r<>htrn toward normal. This process
take::. yea1s, howcvcr, and the economic consequcnccs o f
thr_disease are disastrous.
Reproductive Visease (Shccp and Goats). These species are
infected following ingestion of C. fetus ssp. (etus or c. je-
¡uni. Following ingestion, the organism son1Phow ga i ns
entry into the bloodstr1::a111 a11d localizes iJ.1 the prcgnant
uterus, especially in thP latter stages of pregnancy.
h1cubalion may be as long as 2 months. A placcntitis de
velops along with infection oí thc tettts (amniot1c fluid)
nnd abortion results. The placenta, fluids, and fetus con-
tain Iarge numbers of thc organism an<l actas a sou1ce uf
infpction fo r susc:cptihle animals. Abortions, when they
occur, are usually in "storms" occurring in grcat numbcrs
relatively suddenly touowing a few scattered abornons.
Cattle are rarely infected '.vith C. (etus ssp . fetus and, when
they are, sporadlc abortlons are observed.
Enteric Visease. c.;ampylobacter ¡ejuni adheres to cells of
the srnall intcstine, cspecially thc distal segments. 'l'hc or-
ganism rnultlplies and invades the target epitln.:lial c~ll
(Cia), with resultant inflamm;itinn . lt is 11nc:crtain whether
ll 1e toxiI1s elabo rated by C. jejuní are rcsponsible for thc dis-
ease. However, the LT-like toxin is thought to deregulate lhe
adenylyl cyclase systcm as dcscribcd for enterotoxigenic E.
coli (see C~hapter 8). At the same time, the cytotoxln c.le-
stroys the mucosa! cpithelium. Thc inflammatory response
elicited by Campylobacterinteractlon with tl1e tCJrget t:¡.>ilhe-
Iial cells, and the c.:hemistry of thf' ce ll wall lead~ to the de-
velop111c111 uf tliairl1ea. Diarrhea follows prostaglandin sy11-
thesis hy the recruitcd PMNs (and perhaps by the altccted
host ce lis), as well as activation of vnrious inositol signaling
path>vays within atlccted host cells. The net result is the se-
crction of chloride ions and water. The bactericidal effects
of serun1 <.lestroy C. jejuni that escape into the ly111phalics
a11d into the systemic circulation l)i;:irrheal feces contain-
lng cell debris aud 111ucus are produced, and pro<lucts of thc
infl;imm11tory response are scen in direct smears. Mucus
and blood are sometimes secn grossly.
Epidemiology
Reproductive Visease (Cattle). Thc disease is seen mainly in
beef cattle, slnce the ager1t i~ e ffectively killed by tcch-
niques used to prep;irP ;inci store semen for insemination
il1 llie dairy industry.
Reproductive Disease (Sheep and Goats). ·rhe gallbladder
of sheep, and prcsumably gonts, may become colonized
witb Can1pylobacter. lt this occurs, t hese animals bccome a
source of infectious organisrns for susceptible stock.
Enteric Visease. In acldition to proc.lucing dist:a)t'. of the
reproductive tract of animals, mc->mhPrs of the genus
Campylobuc/er are a sig11ifican 1 cause of human c.lisease
l,antpylnfJacter jejuni is onc of the leading causes ot gas-
troenteritis in human belngs. Human beings in devcloped
societics acquire e;. jejuni from symptornatic or asympto-
matic companion anin1als (dogs and cats) and from food
such as raw milk, water, and poultry products. Mu::.L :>!Jl."
radie cases probably arise from c:ons11n1ption of irnprop-
erly 11a11dlctl poullry or contact witb infected pcts
whercas large outbreaks rnost often occur from contaC'
with raw milk or contaminatcd water. The feccs of approx-
imately 10% ol asyrnptomatic dogs and approximately 5
of asymptomatic cats are found to contaln C. jejuni Thí:.
pcrcentage may be higher l.11 animal::. ac4uüed fron1 an.-
n1al shelters. "I'he ceca of approximately SOo/o of chicker
sa1npletl cu11lé!i11 C. jejuni. At slaughter, these organism
contaminate the e11vironment and, as a consequence, ai-
111ost as n1any chicken carcasses found in stores will b-
contaminated. From 2°10 to 100% of cattle may uc liealll,
sheddcrs of C. jejuni, a circumstance that may expl;iin on--
breaJ<s of campylobacter-induccd liia1 Lheal disease follo\'·
ing ingestion of 11np;istPurizcd rnilk.
lmmunologic Aspects
Reproductive Disense (Caltlt~).
l)PVPloprnent of an active im·
llll111e response results in the uterus being cleared of the o·-
 (;hapter 24 Spiral-Cu rved O rganisms III: Campylobacter, Arcobactcr, Lawsonia
:a_'lism. Sp0riñr c;pn1m IgG and IgM function by initiating
-ne complement cascadc (IgG and I~M) auu uy actlng as
pson.i zing agents (lgG). In the latter case, these antihodi ~.c;
'"'.nd to the antigenic determinants composing the capsular
_,ttgens, resulting in the phagocytosis and subsequent de-
11ction of the agent. Secretory IgA, IgG, and IgM specific
r surface ~Lructures uind a11d prevent the adherence of
"le organ ism to the s~1.rface of the cpithelium. All isotypes,
~ specific for the flagellar a11lige11s, µreveu t movement of
~ore organisms from the vagina. Animals clf'ílr tht> PntirP
-act of the agent if they are not rcinfcctcd. Clearance rarely
...ies more than one year. The mechanism responsible for
ra ring is unknown but is p robably due to the fact that tl1c
~:npylobacle1s have tu deal with an immune response as
·¡as the normal flora of thP vagina.
Thc disease can be controlled by vaccil1atlng heifers or
\"'S vvith bacterins o r by eliminating carrier an imals, in-
iding the bull. Bulls do not carry the organism effi-
::::!tly untll they are older than about tive years. The most
'nmonly held explanation is that the preputial crypts of
_:er bulls a1e dee¡;cr and more hospitablc than the shal-
-er crypts found in yo11ngt>r h11 ll~
Reproductivc Discase (Sheep and Goats). Shccp aud guats
= illlmune following abortion. The basis for this irnmu-
- is mainly antibody of the IgM and IgC types. These
....bollit!S btnd to the surface of the agent while it is in t he
:'.ldstrpam, resulting in removal by phagocytic cells in
-= '.~ ,-er and spleen. A11Liliud y lJound to the surface also
~tes the complement c.asraclf' leading to lysis of the
--=:::r. Thc imrnune response is also effeclive i11 killing or-
- .sms that had reachcd the placenta but hacl not yet
~uced enough damagc to tcrminate pregnan cy.
:':iertc Dlsease. Circulating antibody develops as a result
-íection. The disease is self-limiting due to the com-
-=K~ effe::cts uf secretory antibody and the normal flora .
...:: ::ratory Diagnosis
-- ¿ Collection
d11ctivc Disease (Catl'le). Samplcs for culture or obser-
-~~ are best taken trom the prepuce ofthe hull. SmPgma
.:eted by aspiration into thc tip of an insemination
-...,-""'- If the female is to be cultured, samples are col-
-- from the anterior vagina. With either sex, 101Jfi of
= d 01 20 arürnals (whlchever is greater) are sampled
-;nostic testi ng.
~-.iuctive Dísease (Shecp a11d Gouts). Sa111µl~s from the
a.::a thc abomasum of the aborted fetus are most rP-
-~-." ~- The placenta and fluids of thc abortus are usually
. :amlnated.
:;:;;r;_ Disease. Fecal samples are taken for thc diagnosis
..111i infecliou~.
~-~ ::.... amination
-'-'--·i·e Disease (Cattle). T11t: luw numbers of campy-
::;;;:~;. together with the high n11mbers of organisms of
- ~al flora m akc it diffi.cult to observe Lhe carnpy-
--=:::::¡:¡~~ small curved rods) in stained s1nears (Gram or
137
Romanovsky). Fluorcsccnt antibody-stained prepa1alio11s
have preven useful in detection. Campylobacters exhibit a
characteristic "tumbling" motility when observed in wct
n1ounls of affectcd rnaterial.
Reproductive Dí~P.ase (Sheep and Goats). Gram (carbol
fuchsin as counterstain) or Ro1nauuv:.ky-~1:ained prepara-
tions of stomach contents from ahortPcl fPtuses often
demo11stratc thc age11t (see Pig 24.1). Such fi11Lliug:;, in
conj unction with doughnut-shaped necrotic foci somc-
times found on the liver, help support thc diagnosis (scc
Fig 74.5). Exau1ination of wet mounts of affected material
is sometimes uscf111.
Rnteric Disease. Stained (G1a1n sLaiu with carbol fuchsin
as counterstain; Romanovsky-type stain) ~mears of fecal
material will revea! numcrous slcnder, curved rods in n1osl
cases ot diarrhea produced by C. jejuni.
lsolation
Reprod11ctive Dísease (Cutlle). Smegma, vaginal fluid, or
stomach contents are plateo onto media that contain an-
timicrobial agcnt:; (vonco rnycin, polyn1y:ti11 B V! e, a1IU
trimerhopri1n are common Jy added to decreasc t he
growth of noncampylobacters; amphoterici n Bis included
in son1e forwulatlons to lnhibit growth of fungí). ·rhe
plate is incubated at ~7°C in an atmosphere containing 6%
oxygcn and 5% to IO'Xi carbon dioxiue. Piares are exam-
ined in 48 hours.
Reproductive Díscasc (Sheep and Goats). Abomasal con-
tenrs and liver (aborted fetus) are plated onto blood agar
plates (with o r without antimicrobials, dcpcndin g upon
lhe de::gree uf contamlnation). The plates are incubated at
37ºC in an atmo-;phere containing 6% oxygen and SO/o to
10% carbon dioxide. Plales a1t! exa1nin~d In 48 hours.
Enteric Disease. Campylobacter jejuni and C. coli are best
isolated from affccted intestinal samples on selecl.ivt!
m edia containing antimicrobial agents (e.g., Campy-CVA
containing cefoperazone, vancomycin, and ampl1otericin
B). 'fhe plates are Incubated at 37ºC, or at 4zvc when isola-
tion of r.. jeiuni or C. coli from feces is attempted , in an at
mosphere of 6% oxygeu aud So/o to 10o/o carbon dioxide.
ldentification
Tsolation of a gram-ncgativc, curved rod that is oxidase-
positive is presumptive evidcnce that a rnember of the
genus Campylohacter (or "Arcobacter," see bclow) h as been
isolalt:Ll. Tl1uugh thcre are a number of fermentation reac-
tions that havP hl'Pn rlP~rrihed far the identification of
Carnpylobacter, thesc are tedious and Li111e consumlng.
Methods based on detection of specific DNA sequc~nces
may be used for identification (e.g., gencration of fJag-
ments of a certain s1ze following amplification of specific
DNA sequences by polymerase chain reaction, or dctcrmin-
ing the seque::11<.:t! of the gene encoding ribosomal KNA) .
Likewise, analyses of rPll wall fatty acids have been useful.
Serodiagnosis
Serodlagnosis is sometimes used in the diagnosis of repro-
ductive disease involving Campylohactcr-associated disease
138 PART IT Bacteria an.d f.ungi
in cattle. In particular, the disease can be diagnosed by
tests of cervicovaginal mucus for antibody to campylobac-
tcr antigens. For collection, a tampon is placed in tl1e
v<1giua 111::<1r t11e <.:l:!rvix. Fulluwing rt:uiuval, tl1e u1u<.:u:; i~
coll ec:tc~d and diluted serially. Agglutinins appear in the
cervicovaginal 1n.ucus in 3 to 80 days of infection and per-
sist for approximately 7 n1onths. Samples are collected 4 to
5 days aftcr cstrus or 1to 2 days beforc. Samplcs tal<en a tes-
trus are too d1lute due to tl1e excess mucus. The presen.ce of
blood invaHdates t h e test. Antibody to C. fetus can also be
detectcd by using an enzyme-linke<.I in1 munosorbent assay
(F.T JSA). Rr>portt>clly, this tf'~t ran hf' nin on samples c:ol-
Jected during estrus and will be positive vvithin 18 to 40
days after infection.
Treatme nt
Reproductive Disease (Cattle). Bulls can be treateci parenter-
ally 1'\'ilh slrepton1ycin. For topical treatn;ent, a n aqueous
soiution of penicilli.11 and strepto1nycin is instilled into the
prcpucc. Vnccinatio11 of bulls has been reported to clear
the carricr state.
·rhe disease is best co11trolled by preven tion. Sound h us-
bandry practices reduce tl1c chances of introducing the cn-
ganism into the herd. T he use of young b u lls t hat h ave
te.stt:d ueg<1Llve 1·vltt:11 l.Jrt:d to a vi1 gi 11J1eifer, aud barringof
replac:ernent heife rs or cows originating fron1 herds with
unk11own history, are means to keep out campylobacters.
Once t he agent is in the herd, a nun1ber of a lternatives a re
available. T·he least acceptable to t he p roducer is to rest the
herd for one breeclinK season and cull the bu lls. Tl1is elim-
inates the organis1n from the females a11d removes a source
uf tl1t: org<uli.s1u vvl1t:11 1Jret:úiI1g re.su1ue.s. Artifi<.:i<1l iu.st:1ui-
nation is a very effective '"'ªY to control and elirn ina te t he
di::;ea:;e from a herd. Antibiotic treatrnent of females is un-
rewarding, but bacterins are used to prevent t he disease ín
herds in which it is endemic. Vaccination is performed
yearly.
Reproductive Disease (Sheep and Goats). Ad mi11istering
<111til>ioti<.:.s <.:ar1 :;top al.Jortiuu :;turru.s. 'fl1t: Iuu.st effe<.:ti ve b
penicillin G . Aho rting evves should he isolated fron1 t hose
that are apparently unaffected.
Administering a bacterin prior to breeding can prevent
the d isease.
Enteric Disease. The enteritis produced by C. jejuni is
most often self-limiting. Macrolide antibiotics (clar-
ithromycin; eryt hrorny<.:in, tylusin) re1nai11 the Jrug~ u f
choice for treatment of (~. jeju11i díarrhea. Tetracyc1ines
are cffcctive when 111acrolides cannot be used, alt hough
tetracyc1ine-resistant strains exist (R p lasmid- based). Most
can1pylobacters frorn animals are susceptible to fluoro-
quinolone antit)iotics. Ho,·vever, due to the 11igh rate of
1nutational resistance can1pylo bacters l1ave t o this group
of antitJiotics, a resistance that souH:>tilue.s u<.:cur:; wlüle a11-
imals i'lrf' heing trea ted, fluoroguinolones a re not the drugs
of cl1oice. ln additioJ1, d.iarrhea dueto C. ¡e¡uni most often
occurs in younger ani1nals, most too young to be sately
treated wi t h this group of drugs.
(:ontrol in t he veterinary hospital and kennel req uires
n1eticulous adh.erence to hygienic measures, such as har-"-
washing, cleaning, and disinfection protocols.
1'here are no im1nunizing prepara tions that will prew::::.-
t:r1tt:ritis proJu<.:t:u l>y C. jejuni.
ARCOBACTER
Me1nbers of tlle genus Arcobacter are associated with d1a:-
rheal conditio11s (calves), 1.n astitis in cattle, and reprodu~­
tlve dlsease of livestock, especialiy .swint:. Tlrt: geni.:.
Arcohar.ter (at onf' ti n1f' cit>signated as Campylobacter-l ikeo--
ganis111s) contains t h ree species '"'itl1 pathogenic poten tu.
for humans and other animals: A. cryaerophilus, A. butzler
and A . skirrowii.
Descriptive Features
Cellular Products of Medica! lnterest
Virtually nothing is known about the cellular products C'
Arcobacter, aside fron1 a typical cell wall (see abo•·é
" Campytobacter").
Growth Characteristics
Men1bers of the genus Arcobacter are aerutulen.111t, a trai:
that separates them from Campylobacter. Thcy gro\>V over z
widc tcmpcraturc rangc, and sorne strains of A. butzlcr
grow at 42.ºC.
Ecology
Reservo ir
The reservoirs of 1lrcobacter are presumably the ir1testina:
tracts and the imn1ediate e11viro11ment of affectcd animals
(calves vvith diarrhea, cattle with mastitis, aburted p ig fe-
t uses). A:;y1 uptu111atic :;wi11e, callle, aud pou!Lry n1ay servt:
as an important reservoir for humans.
Transmission
It is unknown how anima1s acquire arcobacters, tl1o ugb
lI1gestiOll or entry through other mucosal surfaces are
li kel y vossib ililies.
Pathogenesis
Little is known regarding the interactions o f Arcobacter
with the host.
Epidemiology
Very little is known about thc cpidcmiology of thc discases
associated with Arcobacter infection. ·r11e organ1sm is prob-
ably an inhabitant of t he intestinal tract of asym p to1natic
swine, cattle, and poultr y.
 Chapter 24 Spiral-C:urvPd ()rganisms TTI: Ca1npylobacter, Arcobacter, Lawsonia
11munologic Aspects
':e immune response of affected animals to Arcobacter has
r been clarillcd.
_:boratory Diagnosis
:.:-,ple Collection
·~obacters have bcen isolated from stomach contents,
j 1Py, and placenta of aborted swine fetuses. Fecal sam-
es are takeJ1 for the diag11osis of A1cub11c..le1-assuciateu
~rrhea .
: •i.'ct Examination
".3m (carbol fuchsin as counterstain) 01 Ro1nanovsky-lype
;::.. Wright's, Gicmsa) stained preparation of stomach
-tents, kidncy impressions, or placenta! fluids often
".":10nstrate the agent in affected swine fetuscs. Little is
·~-n regarding the usefulness of direct smcars in thc di-
- .vsis ufArcobacter-assoclatcd diarrhea. An important dis-
CTion is diffPrPntiating Arcohacter from Campylobacter-
_1cult without specific antibody (e.g., fluort::sct::ully
led antibody).
.
· ;:ion
same n1cdia uscd lo isolale Curr1pytubulLt:r is successful
• -.rcobacter. Arcobacter does not requir<' thc> s::i mP ::itmos-
.ric environment as do campylobactcrs. They wilJ, how-
~ grow in a microaerophiliic atmospherc.
_tion of a gram-negative, curved rod that is oxidase-
tive is presumptive evidence that a member of the
"='"'J.S Carnpylobaclcr (sce abovc) or Arcobacter has been iso-
_._ Though thcrc are a number ot termentation reac-
-s that have becn dcscribed for the identificat ion of
bacrer, these are tedious and time consum lng.
riods based on detection of spccific !)NA sequences
e prove11 useful fo1 idenliflcaliu11 (c.g., generation of
.::".1ents of a certain size following amplification of spe-
- D~A scquences by polymerase chain reaction, or de-
""'"'-:~ng the scqucnce of the gene encoding ribosomal
. Likewise, analyses of cell wall fatty acids havc bccn
_;;;:¡:{_ ! .
. ;cobacters, A. butzleri and A. cryacropllilus, are resist-
~ the macrolidc antibiotics but susceptible to the
===~-clines and fluoroquinoloncs. Whether these mi-
r-,,anisms havc the same resistancc problems as with
...o.mpyloh::icters is unknown.
-catment of arcobaclcr-associaleu cv11uitiu11s in swine
rly documented. Use of autologo11s h::ictPrins has
_ , . "!' promisc.
139
LAWSONIA
'l'he genus La~vsonia contains only onc spccies, L. intraccl-
/u/aris, previously known as ílea/ symbiont intracellularis. lt
is an obliga te intracellular microorganism causing prolifcr-
ative enter1tls of swine. rhe microorganism is also associ-
ated with a similar condition in birds (emus, ostriches),
l.Jlue fux, <.leer, <.logs, horses, rabblts, and rodenrs ("wet tail"
of hamst.ers).
Descriptive Features
Cellular Products of Medica! lnterest
Adhesin~. T.aw~nnia intrace1l11/aris exprcsses a surface pro-
tcin, LsaA (for Lawsonia surface anlige11) that is responsihle
for the attacl1ment to and entry into target cells (the ;ipical
surface of in1mature intestinal epithelial cells).
c·eu Wall. ·rhc cell wall of the mcmbers of this gcnus is
one typical of gram negative bacteria. 1'he lipopolysaccha-
rtue (LPS) tn rhc ourer memorane 1s an 1mportant viru-
lencP c1PtPrminant ot only is thc lipid A component
toxic (cndotoxin), but tl1e lc11glh of L11t: sille cl1<:1in in the
0-repeat unit hinders the attachment ofthe m emhr::inP at-
tack complex of the complcn1cnt system to thc outer
membrane. Lipopolysaccharide binds to the serum pro-
tein, lipopolysaccharide-binding protcin, which transfers
it to the blood-phase of CD14. The CD14-LPS complex
hinds to Toll-like receptor proteins (see Chapter 2) on the
surface of macrophage cells l1iggt:ri11g LI 1c relea se uf proin-
flammatory cytokines.
Growth Characteristics
Lawsonta inlracellutaris has not been grown in lifeless
media.
Ecology
Reservoir
The reservoirs of L. intracellularis are the intestinal tracts
and the immediate environment of affected animals .
Transmission
lnfcction occurs following the ingcstion of an anin1al
product contaminatcd with infected feccs.
Pathogenesis
Followi11g i11gt::.liuu, L. intracellutaris associates with and is
intcrnalized (endocytosis involving actin polyrnerization)
by immature (crypt) cclls of the distal sn1al1 u1lesli11e (duc
in part to Lsa). After internalization, the microorganisms
escape from the vacuole tnto thc ccll cytoplasm where
multiplication and spread to adjaccnt cells occurs. ·rhere is
as yet n o evidence to suggest that L. intracel/ularis utilizes
aclin volyu1erization (see Chapters 11 and 33) as a means
140 PART 11 Bacteria and fungi
of propulsion anll spreall througl1 tl1e cell cytoplasIIL
Affec.ted c.ells proliferate resulting in the c.harac.teristic. le-
sio11s associated \'Vith this disease. An inflammatory re-
sponse in most cases is mínima!. ~wine proliterative enteri-
tis is a disease complex involv ing a number of intestinal
abnormalities: intestinal adenomatosis, necrotic enteritis,
regional ileitis, and proliferative 'hemorrhagic enteropa-
tl1y. Thicke1lin g of Ll1e ileal wall, bul including, on occa-
sion, segments on either side, characterize the disease.
Epidemiology
Lawsonia intracellularis is w idcly distributed among swine
herds worldwide (sorne estimat es run as high. as 509/o of
herds are infected). 'fhe disease is most often apparent as
poor performance. Affected pigs ("•veaners" and gro\-ving
pigs), infecled w hile nursing or sl1ortly tl1ereafter, do not
make target weights. Overt manifestations of disease (diar-
rhea, often bloody) are not a common prcscntation, a11d
are often brought about by stressful situahons. However,
infectio11 of mature pigs commonly results in clinical
disease.
lmmunologic Aspects
Both humoral and cellular immune responses are gener-
ated following infection of susceptible species. There is
sorne suggesti0n that an immunosuppressive pheno1ne-
non may account for the progression of tl1e disease. A di-
1nínished inflammatory response thélt is corn1norlly olJ-
served supports this notion.
Laboratory Diagnosis
Sample Collection
Scrapings of affected intestinc are uscd to diagnosc prolif-
erative enteritis. Lil<ewise, tonnalin-fixed tissues acquired
from affected sites are also used.
Direct Examlnatlon
Impression smears of the intestine of swine with prolitera-
tivc enteritis contain small curved rods '1-Vithin the cells
Jining the area. The silver stains, such as Warthin-Starry, or
a IIlOllifieu acid-félst :stélill (u:se 0.5°/o acelic acid for 3 0 :sc~­
onds for decolorization) are someti1nes used to dem<rr
strate organisms in this site. Immunol1istochemical meú:-
ods are used to detect the presence ot t11e m1croorgan1s-
in fixed tissue.
lsolation
Lawsonia intracellularis has not been grown in lifeles:
Inedia. Howcvcr, DNA mcthods (DNA pro bes, or t he use ....
UNA primers to amplify La\vsonia-specific sequences i r
using the polymerase chain reaction) have been develope:.
that enable the detection of this microorganis1n in tisst.::
or feces.
tdentification
The nature of the lesion, together with t he results of dire~
smear examination, is presum·ptive evidence that L. in u-J-
ce/lularis is involved. Methods utilizing DNA. analysls a:::
quick, easy, and specific.
A multiplex po1y1uera:se cl1ai n reaclion (PCR) a5-s¿
using p.rimers designt>d to detect the com1non diarrh e-
associated 111icroorganisms affecting swine (B. hyodysr
teriae, Lawsonia intracellularis, and Salrnonella) has bee;:
dcscribcd.
Treatment
llave show11 U1at tl1e leUacyclines a re~
Cli11ica.I (ria.Is
effective in treating disease produced by L. intracell.!.-
Alternate drugs include tia1nulin and tylosin.
Excluding the organism trom swine herds is at p'5" -
the most desirable method of control. 'rhere is a rel<r.:
sl1ip between berd size and occurrence of the d..Y=::::~
(smaller t he herd, Iess disease) . Tl1us, a small "ali -
out" J¡erd type n1anagen1e11L systen1 see.u1s the mos-
cient way to prevent this disease.
 Spiral-Curved
Organisllls IV:
Helicobacter The Spiral
Shaped Microorganisllls
of the Gastrointestinal
Tract and Liver
JAMES G. Fox
.istrie spiral shaped microorganisms havc bcen noted in
-mans and other arumals for more than a ccntury. After
t: discovery of Helicobacter pylori in diseased gastric tissue
:1umans Jn 1982, hellcobacters have bcen cultured from
~tom;ichs of ferrets, non-human primates, dogs, cats,
- 3msters, cl1eelahs1 dolphi11s, wl1ale~, autl harp seals.
-1ese gram-negative, m icroaerophilic c:urvrcl to spir;il-
~pcd bacteria isolated from gastric rnucosa of hun1ans
-d othcr animals during the last 20 years have created a
-..at dcal of interest because of their causal role in gastric
..easc. The type spec1es1 H. pytori colonízes the stomach
:!O to 95o/o of adult human populations worldwide. This
.. rourgauisrn causes perslstent, active, chronic gastritis
-1 pPptic u leer di sea se in h·umans and al so has been
-;.;ed to the develop1ncnl of gaslric ade11ucae<..:ir101na anti
_.:. trie mucosal- associated lymphoma. In addition to gas-
' hclicobactcrs, an incrcasing number of species of
•1cobacter have been isolated trom the lower gastroin-
tinal tract of mammals and birds. At least 27 membcrs
.he gt:11u~ lielicubacter have heen identlfted and named
ahli> 2S. l ). Members of the genus Helicobacter are taxo-
mically distinct fron1 lhc genus Curnpylu/Jucler, which
-1.SO havc spiral rr1orphology a11d grow un<l<~ r m i-
-;;-aero phílic condition:;, u:; wcll reside in thc gnstroin-
-jtJnal tract. The con1plete genomes of both 11. pytori and
·repnticus have been sequenced. ·
:: escriptive Features
::•phology and Staining
t:mbcrs of the genus Helicobacter have a vast array of mor-
~ologies, ranging from spirals (c.g., H. pylori, 0.5-l.O by
: "'-5 µm), to slightly bent rods (e.g., H. mustelae, 0.5 by 2
pm). Ali express flagella uiffl:'.ri11g in number and distribu-
tion (Table 25.2), and except for H. pullonun and H. roden-
tiun1, thc flagella are shcathed.
Cellular Products of Medica! lnterest
·r11c cellular products of medica! intercst listed below are
for H. pylori (the n1ost studicd of thc hclicobacters), unless
otherWise stated. It is assumed that other helicobacters
will have similar traits and characteristics.
Adhesins. f\dheslns are proteins that mediate adherencc
to t;irgi>t cells in the gastrointestinal tract and to cells com-
prising the niche for thc strain. Helitubuc.ler pyluri expresses
at lcast two adhesins with spt>c:ifirity for eiistric epithelia:
l. Sialic acid-blnding adhesin (SabA). SabA (for sialic
acid-úi11ui 1tg udhesin) bin<.15 to slalylated glycopro-
teins on the surfarf' of g;istric epithelial cells .
2. Blood group nntigen-binding adhcsin (Ba.bA). BctlJA
(for blood group antígen-binding adhesin) binds to
fucosylated blood group antigens found on gastric
epithelial cells (Lcwis blood group).
Ce// Wall. The Hpopolysaccharidc (LPS) in the 0111·pr
mcmbrane is an important virulcncc dctcrmi nnnt. l'~ot
only Is the lipid A component toxic (cndotoxin), but the
IPngth of the side chain in the 0-rcpcat unit hinders the
attachn1enl of lht: 1111:1111.Jrane attack complex of the com-
plement system to thc 011ti>r mi>mbrane. LPS binds to
lipopolysaccharide-binding protein (a seru111 pr0Ltd11),
which in turn transfers it to the blood phase of CD14. The
CDl4-LPS complex binds to Toll-likc receptor proteins (see
Chapter 2) on thc surface of macrophage cells triggering
thc release of proinflammatory cytokines
cag Pulftu8eriic1y lsland. cag (for cytotoxin-associated
gene product) Pathogf'nicity Island (a cluster of genes en-
141
142 PART 11 Bacteria and Fungi

Table 25 .1. Habitats of Helicobacrer species

Helícobacter taxon Source(s) Primary Site Secondary Site

HUMAN
H. bízzozeron/F' Human, cat, dog, cheetah, Stomach
primates. wild rats
H. canís Human, cat, dog lntestine liver {dog)
H. canadensis Human. wild geese lntestine
H. cinaedi Human, hall15ter, macaquc, lntC$tinc Blood, brain, joint (human)
dog
H. fennel/iae Human lntestine
H. pullorom Human. dlicken lntestine Liver (ú1itke11)
H. pylori Human, m;;raque. cat Stomach
Flexispitil (Helkoúdth!r) Human, dog, sheep, mouse lntestine Placentalfetus (sheep)
taxon gb Blood (humans)
H. winghamensis Human lntc1tinc

NONHUMAN
H. aurati Hamster Stomach. intestine
H. acinonychis Cheetah Stomach
H. bilis Mouse, dog, rat cat lntestine liver {mouse)
H. cetorom Dolphins, whales Stomach
H. cholecystus Hamster Gallbladder
H. fe/is Cat. dog Stomach
H. ganmani Mouse lntestine
H. hepatictl( Mouse lntestine
H. llldllllUld~ Cat woodchuck lntestine Liver/woodchuck
H. mesocricetorum Hamster lntestine
N. muridarum Mouse, rat lntcstinc
H. mustelae Ferret, mirik Stomach
/-/. pametensis Birds, swine lntestine
H. rodentium Mouse lntestine
H. salomonis Dog Stomarh
H. 5UÍ~ Piy~ Stomach
H. typhlonius Mouse lntestine
H. trogontum Rat lntcstinc

• t ikely the same as •H. hei/maMií.• "flelicobaeter heilmannii" (form¡rly Gastrospirillum hominis) has the same phenotype as
listed here for H. biuozeronii. Only a single "H. heilmannii" strain has been isolate<l by cultu¡e, and thus it has not been in
duded in Table 1.
°Formerly regarded as· Flexispira rappini"-now has been subgrouped 1nto ten taxa.

coding virulencf' df'tt>rminant[s]. an integrase protein, a
sp~cifi<.; i11scrtio11 si Le, and n1obilily) encades the Cag pro-
tein (sce below), and a 'f ype lV sccrction system, which
mcdiates translocation of Cag in to host cclls.
Cytoletl'lal lJistending 'Jbxin . A cytolethal distend1ng
toxin is produced by H. hepaticus, H. bilis, H. rnarmotae, H.
pullorun1, and fT. cinaedi. ·rhis proteln produces significaut
in vitro cytopathic effects in cell lines <1nd ca11si>s ri>ll c.yc.le
arn.:sl. !L i:; 110L known whal cffcct thi:; cyt otoxin has in
vivo.
Cytotoxin Associated Gene l'roduct (Cax). Cag (for cyto-
toxin-associated gene product) is a protein encoded in the
cag Pathogenicity lsland (see above). Cag is secreted, and
subsequcntly phosphorylated by gastrlc epithelial cells.
'l'he phosphorylated product leads to polymeriz;ition of
cellular actil1 resulliug iu dis1uplion of cellular functíon.
Flagella. Motility is vital for H. pylori to tind its '"'ª
through mucus in order to adhcrc to gastric cpithclial ccll
Urease. Urease hydrolyzcs urea (secreted by gastric cp-
ithel ial cells) forming ammonium ions. Ammonium ion
neutralizc stomach acid, thereby allowing the 111icruur-
ganism to live in the gastric envirnnmi>nt. l Jrease al ~
:;li1uulale:> lh.e p1oduclion of inflammation.
Vacuolating Cytotoxin (Vac). Yac (for vacuolating cyto-
toxin) is responsiblc for disruption of the epithelial cell
barrier stimulating an inJ'lammatory response.
Miscellaneous Products. Gastric hclicobacters colonize
the mucosa and induce an inflammatory response. Leve!~
of reactive oxygen species arC' t hPrPhy inrri>ased. Helico-
fJuc.let p ylor i inininlizes oxidative damage by producing su-
peroxide dismutase and catalase. -i·hc recA gene products
play a role in repair of damagcd DNA.
 Chap ter 2S Spiral-C:urvPrl Organisms IV: Helicobacter- The Spiral Shaped Microorganisms 143

-~ble 25.2. Characteristíc$ That Diffcrcntiotc Hclicobactcr Spccicsª

Growth Reslstance to":

lndoxyl
Helicobactel catalase Nitratl! Alkaline Aa!tate "f'(ilutamyl At With1% Nalidixk
taxon Production Reduction Phosphatase Urease Hydrolysis Transferase 42( Glydne Acid Cephalothin flagella

HUMAN
H. bizzozeroníf + + + + + ... + R s Bipolar
H. canis + + + + s 1 Bipolar
H. canadensis + +/- + t + R R Mono/Bipolar
1.¡_ cínaedi ... + + s l Oipolar
H. tennelliae t + t + s s Bipolar
- pullnn1m + + Nod + R s Monopolar
~ pylori + + + + R s Monopolar
: exispira +/- + t + R R llípolar
Hclicobiletar) toxon se
- '"mghamensis + ND + R R Bipolar

-.O'tHUMAN
- acinonychis + + + R s Bipolar
- aurati
- :iilís + + + + + R n Sipolar
- chotecysrus + + + + + 1 R Monopolar
-
1
P/i~ + + + + ... + R s Bipolar
- dUI 111.Í + + + + + s R Bipolar
- repatícus + t + + + R R Bipolar
- rnarmotae + + + + R R Bipolar
- rr;e50Cficetorum t t t ND + s R Bipolar
- "luridarum + + + + R R Bipolar
- -uste/ae t + t + + t + s R Peritrichous
- ::iametensis + + + + + s s Bipolar
- "Odentium t + + + K R Bipolar
- ¡¿Jomonís + + + + + + NO R s Bipolar
-agontum + + + + t NO R R Bipolar

~. wba<te species are oxid~ive and lack the ability to oxídíze or ferment carbohyd<ates in rovtine reactions.
,,-,ce is determined by disk dfffuslon. lsolates are lncubated fOf severa! days at 3rc on blOO!kontaining rnedium contaimng ~o µg anttbtotic dísts. Miaoaerophllte conditions are typically
= aaQ 111wbation times vary between organisms. Resistance (R) is defined as the complete absence of an inhlbition zone, whereas intermediate (I; zones usually <15 mm) and susceptible
.isually >20 mm in diameter) isolates have visible inhibition zones of variotJS sizes.
-¿ s.:me as •H. heílmannii.' • Helicobacter heilmannii" (formerly Ga1tro<;pírilf11m hnmíní<) h>< th• .omP pheootype as listed here for H. bízzozeronií. Only •single •H. heilmam:iii' strain has
~ by culture, and thus it ha< not been indudQd in this table.
~:ermined.
·~;¡¡¡raed as "Ffexisptra rappini"-now has been subgrouped inro ten taxa.

:::=a- - Cllaracteristics high humidity with microaProphilic conditions (10%

C02, 80<}6 Nz, 10% II2).
~z:~•><" helicobacters are fastidious, selective media are
~e ::Or isolation. A con1111ercially avdllal.Jle medium con-
of brucella agar with lOºAi horse hloorl as well ;i<; varlabllity
... mycin (10 mg/l), polymyxin B (2500 units/l) and
-erhoprim (5 mg/J). Fresh media are recommended for Hclicobacter pylori exhibits a high d0.gri>f' of genomic hct-
'Tial growth. Specific 1-falicobacter sp. also may have dif erogcnicity, duc in part to nucleotidc substitutions an1011g
• anliuio tic susceptibilltles; therefore, selection of a11- strains. However, H. pylori Iacks a SOS mutagenesis path-
:cs in culture merlia may determine the success of way, and the large number of nuclcotidc substitutions are
--!"non. The organisms do not grow w1der aerouic or probably ctue to mcchanisms such as replication tictelity
~~:obic conditions and achieve optimum growth in ;i dcficiencies or mismatch repair dcfects.
144 PART 11 Bacteria and Fungi
Ecology
Reservoirs
Gastric hclicobacters reside in the gastric mucus layer of a
variety of n1am1nals. Enterohepatic 11elicobacters' ecologi-
c<tl 1Iicl1es <1re tl1e crypts of tl1e colon arH.l cecum, and in
son1c~ cases, the organisrn also coloni7.es t h e h ile c;:in;:iliculi
of tl1e liver. Animals maintained in closed colonies-
reared in pou11ds or kennels, for example- often have pre-
valence rutes of gastric helicobacters approacl1ing 100o/o.
Sorne l1el icobacters colon ize specific hosts wh ile others are
t:apable of infecting a number of different animal species.
Mamn1als, and birc.!s in sorne cases, n1ay b e reservoirs for
zoonotic transmission to humans (see "7.oonotic Poten-
tlal," below).
Sit1ce the original observation of helicobacters in mice
in thc 1990:;, it is now known that Hclicobactcr spp. are
prevalent in 1nany roden t colonies both commercial and
academic, throughout the world . 1'hey are also ü1creas-
ingly isolated fron1 the intestines of huma11s, other mam-
mals, and birds. 'fhough the epizootiology of these enteric
infections is unk11ow11, lhe bacteria appare11lly persist iJ1
the intestine for the life of mice, other rodents, a11d per-
haps other animals as well.
Transmission
Both oral-oral and fecal-oral routes are probably operable
in transn1ission of gastric helicobacters. Transmission of
intestinal helicobacters is via the fecal oral route. 1'here is
cu11 Li11 ued cu11 truversy wbether vla\Jle, \Jut uo11-cultur-
able, coccoid forros of helicohacter exist in the environ-
ment , and if present, whether they are important in trans-
mission to susceptible hosts.
Pathogenesis
Although it is now knowo that both urease and flagella are
necessary to sustain colonization of Helicohacter spp. il1 tl1e
gaslric inucus, n1echanis1ns are being aclively explored Lu
explain the chronic inflammation induced by some heli-
cobactcrs. Thcsc include several putative bacterial viru-
lence tactors, (adhesins, Cag, LP:>, Vac, and orease, to-
gether with certain proteins exp.ressed by genes located on
the cag Pathogenicity Island), which ln tl1e persiste11tly in-
fected host, initiate sustalned productlon of proilúlan1111a-
ll)ry cyloki11es.
Pathology
Helicobacter fe/is and "H. bizzozeronii" ("H. heilmannii")
llave been associated vvith gastric h istopathology in labo-
ratory-reared beagle dogs. When these b acteria were ob-
~erveu il1 lovv 110111\Jers e.g., i11 lhe fundus o f lhe slo1nach,
the organisn1 was considered innocuous; ho,.vever, in large
numbers, as seen in the cardia and fundic pyloric junctio11,
thc organisn1 1nay induce ly1nphoreticular hyperp lasia
and n1ay cause premature se11escence of parietal cells. The
presence of gastric Helicobacter-l i ke organisms (GI-ILOs) is
often accompanied by reduction in m ucus content of sur-
face epithelia, occasional intracpit hclial lcukoc;-
son1e degenerating glands. Of the glandular epitl:1e .:
only t h e parietal cells are markedly altered ...\b- -.
findings include vacuolation, enlarged size, and :- ~---­
degeneration consisting of hoth karyolysis ancl - ¿~­
rhexis. The presence of large nun1bers of !l. pyl~
gastric n1ucosa of commercially reared cats is ass. ~~
with a lymphofollicular gastritis, characterizcd tr
phoid aggregates and diffuse int1ammation in th.:
1n.ucosa and lamina propria.
Helicobacter pylori colonizes the gastrointestinal ="'-
gnotohiotic ciogs o rally rhallenged with this mirrr>----
ism. Such dogs are colonized with JI pylori in all pa.-
tl1e stomach examined: cardia, fu11dus, antrum, an:.
loric ant rum; thc fundus is tl1e 1nost heavHy colon ..
Focal to diffuse Iymphoplasmacytic infiltrates with f.,,-~-­
formation a11d focal infil tration of neutrophils
eoslnophils ln the gastric lamina propria are olJ5e:~"'"~·
Also, gnotobiotir ciogs orally inoculated with H. fe/i.; -
the organisn1 recovered from all areas of the stomacl1,
colonization being 11eaviest in the body and an~
Occasionally, H . fe/is is obscrvcd vvithi11 the canalicu..
gastric p ariet al cells.
Ex peri m entally, "H. bizzozeronii" ("H. heílrnan--;
causes m ucosa-associated Iymphoma in rnice and is ü :1,
with the saine d iseases in h u mans.
The hisLopalhological changes occurrh1g in ll1e sl, _
ach closely coincided in topography with the presenet
H . 1nustclae. A superficial gastritis p resent in the bod;·
t he ston1ach shows tha t H. rnustelae is located on the sl.4-
face of the mucosa b ut 11ot in the crypts. In the dw._
antru m, infla1n mation occupies t he full thickness of t::,,
mucosa, t he so-called d iffuse antral gastritis described ~
humans. In t his locatio n H . mustelae is seen at the surfaC>.:
in the pits, and on the superficial portion of the glancis. ·-
tl1e proxin1al an trum and thc transitional mucosa, a prt-
cancerous lesion, focal gland ular atropl1y, and regenera-
tio11 is present, in addition to those lesio11s seen 111 the d is-
tal antru1n .
The liver lesion present in naturally H. hepaticus-
ir1fecteu rnice prugressively ir1<..:reases in severity. It is <tn ir.-
flam matory and ne.crotizi ng lesion, wh ich involves the h i:-
p atic parenchyma, the portal triads, and importantly, the
small in tralobular hepatic venu les. 'fhe \.videspread, multi-
focal hepatitis and single cell to coalescing hepatocellula:
necrosis appears to be random in distribution. Within
both the parenchymal perivascular leslo11 and the affected
portal triads, varia\Jlt: uegrees uf oval, Ilo, a11cl Kupffer cell
hyperplasia a re present. There is an age-associated increase
in cell proliferation in infected animals, whlch is not seen
in uninfected control mice. Liver celJ proliteration is more
pronounced in male mice than in age-matched female
inice. Tl1e increased leveJs o f hepatocyte proliferation in-
dices in H. hepaticus-infectecl male mice are consistent
willr ll1e ul.Jserv<1liu11 oI iJJC.reased hepalo1nas and 11epalo-
cellular carcinom as ohserved in H. hepaticus-il1fccted
aged A/JCr male mice. More recently, it has bccn dcmon-
strated that the B6C3Fl and B6AF1 hybrids are susceptible
to H. hepaticus-lI1duced liver tumors, indicating liver
tumor for1nation susceptibility is a <.1ominant
. trait. Helico-
hacter canis has also been observed in a dog liver with n1ul-
 Chapter 25 Spiral-Curved Organisms IV: Heficobacter-The Spiral Shaped
tifocal hepatitis, in a liver of a rhesus monkey i.v1th hepati-
::is, a11d in woodch ucks with hepatitis. Helicobncter hepati-
i:u:. C111u H. bili~ i11fecliu11 C1rc C1:>:.uciatcu with iI1flarr1matory
bowel disease in immunodeficient micr íln<l only infrf'-
quently in !l. hcpaticus infcctcd immunocompetent mice.
r he immune response to H. hepaticus infection appears to
be T helper 1 n1ediated since splenocytcs produccd largc
3mounts of interferon ( when stimulated witl1 H. hepaticus
.mtigens. No rrnally, l1umans and other animals remain
·,ypo1e:.po11:.i ve:: ur lulera11 l tu ti 1ci r uw11 e11teric flora. In
:1atients with inflammatory howel disease, toleran ce is clis-
:uptcd as individuals become "hyper-responsive" to en-
.:ogenous cnteric antigens. IL-10 is a T helper-2 cytokine
i th anti inflammatory and T helper 1 suppressivc cffccts,
.lnd the lack of IL-10 manifests predictably as an unop-
:-osed ·r helper-1 im1nune response. 'fhe IL- IQ·/· mice
;eared conven Lionally prese11l wi ll 1 t: 11Lerucolitis, !Jut sutJ-
.equent rcaring in specific pathogen-free (SPF) fac ili ties re-
-:rictcd thc inflammation to the colon, whereas in germ-
:::ee TL-10-/· there is no Iower bowcl inflammation. Thus,
_1teric bacteria! antigens including H. hepaticus con-
::ibutc to the induction of colitis and more recently 1nduc-
'"'n of colon cancer in rag mice and TGF~ 1 deficient mice.
: sease Features
-:.-:>rets. Helicobacter mustelae is routinely present in fer-
~ (}..1usLela putorius furo) with gastritis and pcptic ulccr:;.
.:rets lnfected with. H. mustelae, are recognized clinically
\"Omiting, 1nelena, chronic weight Ioss, and lowered
i:.uiilucrit. Acutc episodes of gastric bleedtng can also be
·ted on occílsion. H'elir.nhar.ter rnn~telru• híls also been
.....:nonstratcd within the pyloric mucosa of ferrets with
•oric adenocarcinoma and mucosa-associated lym-
~ma of lhc stomach.
_ -¡¡, Swine with gastric disease have a higher prevalence
H. suis (closely related to "H. heílmnnnii" type 1) associ-
\vi.th gastric ulcers located in the parsoesophagea.
"Uman Primates. H'elicobactcr pylori has bcen isolated
-, nonhuman primates, p<1rticula rly macaques. 'rhe
=-ence of H. pylori in gastric mucosa ot intected monkeys
:'ten accompan ied by a lymphocytic plasmacytic gastri
-!lat appa rently persists. c;linical slgns h avc not been
~ht>rl Primates are also commo nly colonized with
_e astric spiral "H. heilrnannii"-like 01ga11isn1s. Mosl re-
0 11egative enteric helicobactcr lsolated from wild bird and
. y enteric h elicobacter havc bccn isolated from
=::it'.¡qucs and cotton top tamarins with idiopathic colitis.
;;.;s and Cats. Clínica! signs in pet animals, attributable to
b"cter-assoclated gastritis mayor may not be present.
ht1rtr1r pylori has been identified in lOOo/o of the stom-
....__, exan1ined fron1 a group of specific p<1Ll1ugeu free,
~toma tic
•
cats, obtained frorn a commercial vendcH-.
.obactcr carris has bccn isolatcd from feces of norm al
- - • arrheic dogs and normal cats. ·rhis microorganism
o been isolated from the liver of a puppy diagnosed
11g an active, multifocal hepat1t1s. Helicobacter canis
- · - ,... hf'Pn cultured from the feces o f c hildren and adult
-----:s suffering fron1 gaslioente1 ilis.
Microor~anisms 145
H el1cobacter n1arrnotae, originally isolated from the dis-
eased livcr of woodchucks, has a lso been isolatcd and char
acterlzed from the teces of cats, as has been H. bilis.
Helicol1acter cinaedi and H . fennelliae, have been linked
to procti lis and colitis in in1n1u1101.:u111pronlised hu1uar1~,
and septicemia in n eonates. ()f interest is the recent isolíl-
tion, bascd on cellular fatty acid analysís, of JI. cinaedi
from the teces ot dogs, a cat, and rhesus monkeys.
Helicobacter (ennelliae has also been identified in the fece:;
of a dog and a macaque.
Sheep. "Flcxispira rappini"was fi1 st isolaleu fru1u a 11u1r1a11
patient with diarrhea. ln the same householcl, thf' hílc-
tcrium was isolated from the feces of a young asympto-
matic dog. A similar bacterium has been cultured from
aborted ovine fetuses and was givcn the provisional namc
"Flexisptra rappíni." Apparently "F. rappini" crosses the pla-
cf'nt;.i in pregnant sl1eep, induces abortions, and causes
acute hepatic neo:osis i11 ll1e fe Lus. l:'.xperi 111e11tally, "l:'. rap-
pini" causes abortions and necrotic hepatitis in guinPíl
pigs. Annlysis of t h c DNA cncoding the 16S rRNA shows
that this microorganism belongs in the genus Helicobacter.
Helicobacters of Rodents. To date, 12 spccies of Helicobacter
have been isolatcd from the intestinal tract and/ or livers of
iodenls; Lwo uf l11e:se::, H. heputic:us and H. bilis have been
isolated fron1 the ceca, colons, íln<l livPr.:: of mice.
Hclicobactcr 1nuridaru111 colonizes the lo,ver intestinal
tract of rats and n1ice, and undcr certain circumstances
colonizes the gastric tissue of mice and induces a gastritis.
"Flextspira rappini" colonizcs the colons an d ceca of
mi ce.
Helicobacter cinuedi a11d H. rr11::>uc..ric..1:Lurum color1izes tl1e
intestinal tract of asymptomatic hamstcrs .
Helicobacter aura ti is isolated from hamstcrs with severe
gastritis.
Helicobacter trogontunz and H. bilis havc bccn isolatcd
from the colons of rats, H. rodentiu111, H. typhlonius, and H.
gan111a11i from the colons and ceca of mice a11d H. cholecys-
lus l 1a::. 1Jec11 cultured frorn diseased gallbladders of harn-
sters. Clínica! signs in infrctt>il rorl Pnt<: h;ive n.ot been
noted, except in im n1unocon1promised n1ice infected with.
H. h epaticus or H . bilis wherc diarrhea and/or rectal pro-
Iapse may be present.
Helicobacters in Birds. Helicobacter parnetensis is a urease-
porcinc> fc>cP<; .
Ilelicobacter pullorurn has been isolated f1on1 ceca uf
asymptomatic chickens, the livers and intestinal content~
of chickcns w ith hepatitis, and fcccs of humans with gas-
troentent1s. There is speculation that H. pu./forunz may be
the microbial agent responsible for the syndrorne termed
avían vibrtontc hepaCiLts describcd in both chicken s and
hJ rkPy.-;. 'Though Campylobacler jejuni has been suggested as
an ctiological agent iJ1 tl1is disease, stuuic:; co11ducted to
ascertain w hether C. jejuni (derived from humans or ch ick-
ens) produces hcpatopathy have failed. Most rccently, wild
geese have been identitied as rcservoirs for H . canadensis, a
hclicobacter first identified in diarrheic humans and mis-
classlfled as H. pullorun1.
146 PART ll Bacteria and Fungi
Zoonotic Potential
Helicubacter pylori-ínfected cats have been scree.ned by cul-
ture anct polymerase chain reaction (PCR) for the presence
of JI. pylori in salivary secretions, gastric juice, gastric tis-
sue, a11d feces. Helicobacter pylori ;vas cultured from sali-
vary secretions in 6 of 12 (50%) cats and from gastric fluid
samples in 11 of 12 (91 o/o) cats. Isolation of H. pylori from fe-
line mucosal secretions suggest a zoonotic risk from expo-
sure to personnel ha11dling H. pylori-lnfected cats ln vi-
varia . However, to date, there is 110 indication based 011
several epiden1iological studies tl1at pets pose il1creased
risk of H. pylori zoonotic transmission to humans.
Because "H. heilrnannii" ("H. bizzozeronii") and to a
lesser extent H. felis colonize a smatt percentage of humans
with gastritis, and no environrnental source for these bac-
teria b.as been recogntzed, pets in severa! published reports
have been iinplicated in zoonotic transmission of these
n1icroorganisn1s. l!1 Gern1any a rece11L survey of 125 i11di-
1
viduals infected with GHLOs provided information in a
questionnaire regarding animal contact. Of thcsc paticnts,
70% had contact with one or more anilnals (as compared
with 37o/o in the "normal" population). More than a three-
fold preponderance of males over female patie11ts witl1
GHLOs was recorded.
SiI1ce H. c;ínuedi ha:s l!t:e11 i:sulateu fru111 l1u111a11:s a11u tl1t
normal intestine flora of hamsters, it has heen suggested
that pet harnsters serve as a reservoir for transmission to
humans. Also given that H. canis, Flexispira (He/icobacter)
rappini, H. pullorum, and H. canadensis, have been isolated
from animals and diarrheic humans, the probability that
these helicobacters are also zoonotic agents exists.
lmmunologic Aspects
A variety of serolo!,>ic tests have been used to n1easure the
increased anti-H. pylori- specific IgG and Ig/\ antibody
found in humans with different types of gast ritis a11d duo-
denal or gastric ulcers. The rnost popular tests have been
an enzyrne-linked irnmunosorbant assay (ELISA) uslng
glycine-ext ractecl antigen or \.Vhole-cell sonicates of the
bacteria. The severity of gastritis in terms of int1amn1atory
Tesponse does not correlate with levels of antibody.
Specific serum IgG a11tibodies to gastric 11elicobacters in
animals have also been used to diagnose those both natu-
rally and experimen tally infected with Helícobt1cter spp.
Analyses of serum and rnucosal secretions by ELISA ln H.
pylnri nat11rall y infecteci c;:its reve;ils ;in T-T. pylori-spPcific
IgG response, and elevated IgA anti-11. pylori antibody lev-
els in salivary and local gastric secretions. Like humans.
though helpful in diagnosis, neither secretory nor seru1n
antibody responses are protectíve.
Helicobacter hepaticus has appare11tly developed strate-
gies to evade host immune responses silnilar to those of
gastric helicobacters. H<'.licol1acter hepaticus i nfected A/JCr
mice with hepatitis have a persistent IgG anti body re-
sponse to this rnicroorganism, which does not confer pro-
tection. Younger mice, whose intestinal crypts are colo-
nized with H. hepaticus, but without appreciable hepatitis,
do not have elevated IgG antibody to H . hepaticus.
Laboratory Diagnosis
Direct Exarnination
In addition to using a Gram stain on homogenized gastric
tissue for a rapid, presumptive diagnosis, the urease activ -
1ty of tl1ese gastrlc bacteria is utllized. A dlagnostlc t est fo:
urease is commercially available tl1at detects urease activ-
ily in gaslric Lissue willlin 15 n1i11utes to 3 11ours. Tl1us, a
gastric biopsy can be minced and placed directly in to urea
broth anda posi.tivc rcaction obtaincd in 1 hour.
Gastric brushing cytology can be performed during
routine endoscopy. Cells and mucus that adhere to the
brush are applied to a glass sllde, air-dried and stained with
a Giemsa stain. Por visualizatio11 of gastric belicobacters,
oil in1n1ersio11 n1ag11ilicatio11 (lOOX) is used. Phase micro-
scopy is also helpful in establishing a tentative diagnosis.
Jsolation and Jdentification
Helicobacters can now be isolated routü1ely fron1 the gas-
tric tissue of infected humans, ferrets, nonhuman pri-
mates, and increasingly from other laboratory, domestic,
and wild animals. It is more difficult to isolate H. felis from
Lio~s a11d cats, a11d unlil very recenlly H . bizzozerorzii ("H.
heilmannii"), the large gastric spiral, was uncultivable.
I-Iowever, this organism has now been isolated fro1n both
dogs and humans. 1-/e/icobacter salornonis has been isolated
from dog stornachs as well. When attempting to grow H.
fe/is, H. bizzozeronii, and H. salomonis, it is very important
to use moist plates, which are incubated lid uppermost.
These species do not form distinct colonies, but rather bac-
t eria ! gro'\o\rth consists of a very fine spreacling film, which
could easily be dismissed as water stains. Another limiting
factor in isolation of gastric Helicobacter spp. from animals
and humans is tl1e neccssity of obtaining gastrlc blopsies.
The organisms are not routinely cultured from gastric juice
or feces. Enterohepatic helicobacters can be isolated on
t he same selective antibtotic media u sed to isolate gastric
helicob;icters. Also, the use of O 45 JI or 0.65 p fil ters to se-
lectively filter feces helps 111inimize contan1ination from
other enteric organisms during primary culture on selec-
tive agar. Higl1er hydroge11 levels in the gas n1ixture- i.e. 1
10°16- enhances recovery of the enterohepatic helicobac-
ters. It is now recognized that helicobacters and campy-
lobacters can be a mixed infection and isolated from tbe
sanie fe<:es ofboth animals a11d huma ns. PolymerasP chain
reaction (PCR)- based assays n1ay therefore be required in
many cases to verify whether mixed infections exist in the
samehost.
Fig11re 25.1 and Table 25.2 outline tbe various pheno-
typic characteristics of various helicobacters that are used
to isolale and idenlify helicobaclers fron1 gaslric biopsies.
Molecular Methods
DNA prilners specific for genes encodlng various proteins
specific for helicobacters, as well as primers specific for seg-
ments of DNA encoding the 165 rRNA, have been de
scribed and used for demonstration in samples and identi-
fication by the use of the polymerase chain reaction.
 Chapcer 25 Spiral-Curved Organisms IV: Helicobacter-The Spiral Shaped Microorganisms 147

1 Figure 25 . 1 . lsolati on of Helicobacter from gastric biopsies.

BIOPSY ] - Free2e in 20% glycerol

[ Store at -20C
~-------'

M«..1oos~1 tiss1.1e 0.5 m i of 20% glucosé Sélacted ti&Guet:

Formalín fiKed
~r.r~pino 4r. 11nlil innr:1 1l~tinn prQp;ired for EM

Fx::1m;Mwrfh
-------- TISSUe Gtinder
phase microscopy

lnoculoto ti33uO homogenate Gram stain lnoculoto in o. cclcctivc incdio

into uroo broth (counter stain with (Brucolla agar w ith 10% blood)
carbofuchsin)
· Vancomycin 10 mg/L
•Polymyxin B 2500 ug
•TrimAthn()ñm !i. mg/l

Uro:ieo • G rom ncg.:itivc l ncub.,te 37C "nd 42C

wtthin 1 hour mo:phok>gy dependont S-..1 doy.) in ge.-s mixture
(up t.o 24 hrs) on spociq_s is;.obt9d ( 5% co,. 5% ~· !)()'!(. N,
or
10'll. CO,, 10% H,_ 80% N,)

!)•/o u , (m1croaerob1c)

Gram negatlve organlsm

Mvtilily T
Oxidase 'T
Urease •
Cotolesc +

-~: at ment and Control
_-obncter priori. A triple-therapy regimen consisting of
'-h.:ill iu au<l 1uctru11i<l¡¡zulc1 ur tctr¡¡cycline an<l
_;:;o nida7.olP., in comhination w ith hismuth s11hs;ilic.y-
given for 2 to J weeks has proven to be cfficicnt in
dication of rf. priori. Indeed, this antimicrobial regi-
- .;n. plus ranitidine, has p roven successful in treating pa-
- ¡s with utcer disease. In studies comparing th1s treat-
- .:r. t to those patients receiving ranitidine alone, ulcers
011Iy Jicalcu raster, uut tlte recurrt::nct:: uf ulcers was sig-
cantly less than the antihiotic-trr.atP<I group whf'rf' H.
~..¡ had been eradicated. Recently, thcrapy regimens
_ ..,g proton pump inhibitors (e.g., omepra7.ole) in combi-
-ion with othcr antibiotics also havc shown consider-
e e!flcacy 1n erad1canng H. pylon. ~everal reports de-
-:bing antimicrobial therapy in domestic dogs and cats
• ..i1 gastriti:. ltave faileu to erauicdte the large gastrlc splral
:.:anisms from the sto1nach of infr.c.tr.cl ;i nim:-ils.
- ;er Helicobacters. Antimicrobial susccptibility testing of
- d naeái indicatcs that tetracycline, chloramphcnicol,
A.:.d various aminoglycosides should be effcctivc in trcat-
-=- - infections with this miaoorgan1sm. Apparcnt relapses
i·I. cinnedi bacteremia have occurred in pcople treated
:1 ciµruíloxaci n uespite its previous use to treat H.
dnacdi infcction succcssfully. Thc occurrcncc of in vitro re-
sistance of isolates of H. cinaedi to ciprolloxacin suggests
that fluoroquinolones should be used with caution.
"Flexispria (He/fcobacrer) rappiní"-llke organisms have
been isolatcd from imn11.111ocon1petent and immunoin-
con1petent (X-linkcd aga111n1aglobuline1 nia) childreu.
'J'hese isolates have had variable susceptibility to a number
of antimicrobials. However, most isolates have sho~vn sus-
ceptibílity to imipenem, metronidazole, minocycline, and
rifampin, and intermcdiate sensitivity to doxycycline.
Studles In ferrets indicate that thc triple therapy con-
sisting o f amoxicillin (30 mg/ kg), metronida2ole (20
mg/kg), and bisn1ulh sulJ:,alicylatc (17.5 1ng/ kg) (Pepto-
Rismol original form11lil, Proctor & Gamble) three times a
day for 3 to 4 weeks has successfully c1 adica led H. rnuslelue.
L>osages ot clarithromycin and ranitidine bismuth citrate
tl1at successfully eradicat ed H. n1ustclac wcrc 12.5 and 24
n1g/kg of body \"7eight per os every 8 h rs, respectívely for a
14-day period.
HelicolJu¡,ler flepalic;us- irife.cte.d mlce recclving triple
thcrapy consisting of amoxicillin, mPtronirlazole, and bis-
muth 3x daily for 2 weeks by gastric intubation cradicaled
thc microorganism. Use of antibiotic impregnated dietary
wafers has not been succcssful in cradicating this micro-
organism.
 Spiral-Curved
Organisills V: Leptospira
RANCE B. LEFEBVRF.
l.PptospiraP a rP spirochi>ti>s tha t arP morphologically a11d
physiologically uniform but serologically and epide11.1 io-
logically diverse. Domestic animals most coinn1only af-
fcctcd are dogs, cattle, swine, and horses. Canine leptospi-
rosis tn.anitestations are septicemic, hepatic, and renal
disease. ln cattle and swü1e, septicemic illness is largely
confined to the young, whilc abortion is tl1e priI1cipal
1nanifestatio11 iI1 adults. Abortion and recurrent uveitis
( l llVVU l>lii lUUI::>:>, Vi JJt:liVUÍL vpl1ll1al1uia) ale lht' lUU:>l
common manifestations in horses. California sea lions are
susceptible to acutc, scpticcn1ic le.ptospiral infections.
Other host species. though susceptible to infection, de-
velop clinical signs less frequently. Leptospirosis in hu-
mans is typically an acute febri le disease.
'l'axonomy studies, based on DNA analyses, have led to
the description of eight pathogenic species: Leptospira
horgpetersenii, f .. inarlar., T.. interrogans sensu stricto, L.
kirschneri, L. 111eyeri, L. noguchii, L. sa11tarosac, and L. weilee.
Notwithstanding, leptospires are classified and referred to
by their antigenic composition. Thcy are currcntly sortcd
into 23 serogroups . Lept ospire tsolates placed within
these scrogroups are further characterized and ide11tified
a11tige1lically as unique serovarietics (serovars), of which
there are more than 200. RPferenci> to t hese organisms
is typically by genus and serovar in clinical setth1gs, (l.e.,
Leptospira serovar canicola is the accept ed designation of
the most common canine pathogcn found in North
An1erica. Other serovars important i.n Nortl1 i\merica
and tl1eir principal hosts and clinical hosts (in parenthc-
ses) are:
icterohaemorrhagiae: rodents (dogs, horses, cattle, swine)
grippotyphosa: rodents (dogs, cattle, swine)
canícula: dogs (swine, cattle)
pornona: cattle, sv.rini> (horsPs, shPi>p, s~;i lions)
hardío: cattle
bratislava: swine (horses, sea lions)
Descriptive Features
Morphology and Staining
Leptospirae (from the Greek leptos, meaning "thin") are
tightly coiled spiral organisms. Because tl1ey stain poorly,
they require darkfield or pl1asc contrast mlcroscopy for vi-
sualization. The spirals are bcst demonstrated by electron
148
microscopy. Typical cells have a hook at each end makir::
tl1e111 S- or C-sl1aped. Wct n1ounts revea! ll1cn1 Lo !:>::
motile.
Leptospirae are gram-negative b ut stain poorly ar.:.
thus, unrecognizable in routinely fixed, stainecl smea!"'"
'fhey can be den1onstrated by fluore.scent antibody or sL
ver impregnation (Fig 26.l).
Cellular Anatomy and Composition
Leptospiral cells consist of an outer sheat h, axial fib~
("endoflageJJa"), and a cytoplasrnic cylinder. '!he oute:
sheath combines features of a capsule and outer m ee:
brane. A cell mernt)rane and t he peptidoglycan !ayer of tt..::
cell \vall cover the cytoplasm ic <..)'lü1der.
Cellular Products of Medical lnterest
Cell Wall. Members of the genus Leptospira have a graT:"-
ncgative cell wall. 'l'he lipopolysaccharide (LPS) in th"
oule1 1nec uura11e is au iluµortaut viruleuce determinar;-
Not only is the lipid A component toxic (endotoxin), h1 -
the length of t he side chail1 in tl1e 0 -repeat unit h inde.:
the attachment of the 1nembrane attack complex of tt=
complement system to the outer membrane. LPS binds::
lipopolysaccharide-binding prot ein (a serun1 proteir:.
which in turn. tra11sfers it to the blood phase of CD14. Tt.<:
CD14-LPS co111plex !Jiud~ Lu Tull-like re<.:eµtur µroteiI1s (se=
Chapter 2) on the su rface of macrophage cells tri ggeri~­
thc rclcasc of proinfla1nmatory cytokines
Hemolysin. ·rhe hemolysin (a cytotoxin) produced t;c-
some serovars Leptospira is a spl1ingomyelinase C .
Growth Characteristics
Leptospirae are oblígate aerobes, which grow optimally a-
29ºC to 30°C. Generation tin1e averages about 12 hou--
No growth occurs on bloo<.l agar or other routine mect¡,.:_
'fraditional mPdia ari> i>ssentially rahhit sPn1m (10ºAi), in sr...-
lutions of peptones, vitamins, electrolytes, anct bufft:.!'
Sorne newer media have substituted polysorbates a~
bovine albumin substituted for rabbit serum. Protein is r. -
required. Unlikc most prokaryotes, lcptospi rae are not ab•.::
to synthesize pyrimidines and th us 5-fluorouracil is addE:-
to growth media as an inhibitory agent to contarrlinati::~
bacteria and fungí .
 Chapter 26
F 1G U RE 2 6. 1 . Leptospira interroga ns, serovar pomona in the renal tubules of a pig.
Levaditi si/ver stain, 1000X.
~-íost media are fluid or semisolid (0.1 % agar). In fluid
__ dia , little turl.Jillity Llevelups. Iu seruisulid u1edia,
:::::;:::-,\· th is concentrated in a disc- called a "dinger zone"-
.....:cut 0 .5 cm below the surface.
· .;{hemical Reactions
'"""-ptospirae are oxidase and catalase-positive; many have
.:tase activity. Sorne produce u rease. Identification be-
-o:Jd genus is based on serology. Species-specific L)NA pri-
_ ers in conjunclion vvilh Lhe polyn1erase chain reaclion
:CR) have also heen developed for a more accurate char-
1CTerization of pathogenic leptospirae.
;:>istance
:_¿ptospirae are killed by drying, freezing, heat (SOºC for 10
°!liJ1uLe:s), soav, bile salLs, deLeigenLs, acidic environrnenls,
.!..:d putrefaction. They persist in a moist, temperate envi-
:;Jnment at neutral to slightly alkaline pH (see "Epidemiol-
::-gy," below).
.ari¡ibility
_.!o re Lha11 200 servvars o í varasi lic leIJLOSIJirae exi:sL Tl1t:y
<UY in host and geographic distribution, and in pat ho-
.::enicity.
::cology
:te~ervoir
~1em.bers of the genus Leptospira inhabit the tubules
o f mammalian kidneys. Tl1ey have been isolat ed from
bin.ls, reptiles, amphibians, and invertebrates, but the epi-
Spiral-Curved Organisms V: Leptospira 149
demiologic significance of such associations is not es-
tablished.
Roclrnts arr thr most frequent leptospiral carriers, with
wild carnivores ranking second. No n1a11.1n1al ca11 be ex-
cluded as a possible host. Typically reservoir hosts show
mínima!, if any, signs of discnsc.
Transmission
Exposure is through contact of n1ucous n1embranes or
skin with urine-contaminated wat er, fomites, or feed .
Othcr sourccs are milk from infcctcd cows and genital sc-
cretions from cattle and swine of either sex.
Pathogenesis
Clínica! a11d pathological manifestations suggest toxic
1ncchanisms. Filtratcs of tissuc fluids from cxpcrirncn-
tally infected animals contain cyt otoxic factors that p ro-
duce vascular lesions similar to those seen with lep-
tospirosis.
The spirochetes enter the bloodstream subsequent to
n-iucous n1embra1-ie or reproductive inoculatio11, colon.iz-
ing particularly liver and kidney, where they produce de-
gcncrativc changcs. Othcr affcctcd organs may be muscles,
eyes, and meninges, where a nonsuppurative meningitis
may develop. Hemorrhages result from damaged vascular
endothelium. Ali serovars produce these cbanges to vary-
ing degrees. Leptosj>ira serovar pomona in cattle causes
intravascular ben1olysis due Lo a hen1olylic exoloxin. Au-
toimmune phenomena may also contribute to this con-
dition. Sccondary changes include icterus due to liver
damage an.d blood destruction, and acute, subacute, or
chronic nephritis dueto renal tubular injury. 'l'he cellular
exudates contain predominantly lymphocytes and plasma
150 PA!tT 11 Bacteria and Fu11gi
cells. In surviving ani rnals, leptospirae are ren1oved from
circulation with the appearance of antibo<ly but persist in
the kidneys for many weeks (see Fig 26.l).
Disease Patterns
Most leptospiraJ infcctions run an inapparent course prob-
ably due to infection of the animal by a host-adapted
scrovar. Clinicnl infcctions manifcsting overt signs are pri-
martly due to non-host-adaptcd scrovar infeetions. These
occur 1nainly in dogs, ca ltle, and s'<Vine; increasingly in sea
líons; occaslonally in horses, goats, ancl sheep; and excep-
tionally in cats.
Canine. Leading serovars involved are icterohaemorrhagiae
and canicola, with thc lattcr thc n1on.: common. lncrcasing
i1umbcrs ot dogs with acute renal failure d ue to serovars
grippotrphosn, pomona, and bratislava infections are being
rcported.
The n1ost acute form affccts young pups preferf'nti;:illy,
p1oducing rever 'vilhout Jocalizing signs, and is con1-
monly fatal within days. Hcmorrl1ages are often apparent
antemortem on mucous mcmbrancs and skin, or mani-
fested hy epistaxis or by bloodslained teces anú vomitus.
Jcterus is absent.
Tl1e icteric type runs a slower course, and hemorrhages
are less conspicuous. lctcrus is prominent. Renal localiza-
tio11 cau::.c::. Jlitrugc11 rt:lt:11liu11, l'vhile 1enal casls and leuko-
cytcs appcar in thc urine.
Thc urcmic typc, centered in the kidneys, results subse-
quent to either of the types of infections described above
or may develop in their absence. It may be acute and rap
iclly fatal with signs of gastrointestinal upsets, uremic
breath, and ulcerations in the anterior aJirnentary tract; or
it u1ay rull a ::.low cour::.t: will1 lit:layt:u un::.t:l. Tltt: rt:latiu11-
ship of leptospirosis to ch ro nic interstitial nephritis lead-
ing to uremic death is controversia!.
Cattle. 'fh e predominant manifestation of bovine lep
tospirosis is abortion, usually late term, but may occur at
any time following infection. J\bortion is d ueto primary
fetal Ul..!<ttlJ rc1tlicr thG111 t.Jlacc11tal i11fecliu11. Fetal re teuti uu
with progressive autolysis is common. Abortions due to
serovar ilardjo, the host-adapted serovar for cattle, are pri-
n1arily a prohlem ot heiters in dairies due to management
practices that differ between beef cattJe and dairy opera-
tions. Leptospira serovar 11arrtjo infections affect calves in
utero, lea<ling either to abortion or "weak-calf syndrome."
'fhe::.e iufeclions a1e often subclinical or n1ay be 111arked by
"milk-rtrop synrtromf'," reproductive failure, and infertil-
ity. Chronic infcction of the kidneys and the shedding of
leptospirae in urine are common.
Acute leptospirosis dueto serovar po1nona affects mostly
calves and sometimes adult cattle. lt is n1arked by fever,
hemoglobinuria, icterus, anemia, anda fatality rate of 5%
to 15o/o.
Swine. ·rhc scrovars implicated in porcine leptospirosis in-
clude serovars po1nona, icterof1ae1norrhagiae, canicola,
tarassovi, /Jrntisla~·a, and tnuenchen. As in bovine Jeptospiro-
s1s1 sept1cem1a witt1 icterus and hemorrhages occurs, espe-
cially in piglets, while abortion and infertility are the man-
ifestations in sows.
Horses. Equinc lcptospirosis is due most often to serovars
pon1ona, grippotyphosa, and icterohae1norrhagiae. Signs in
natural infections have been fever, mild icterus, and abor-
tion. Leptospirosis is involved in equine recurre11t uvcitis
(periodlc ophthalmla, muun IJ!ir1dncs:s; :scc "ll1uuu11ulugiL
F;:ictor.~." hi>low). l .Pptn~pira sprovar pnrnnna and an as yet
unidentified leptospira havc bccn cuJtured from the aque-
ous humor of horses with clinical signs of this disease.
Miscellaneous Species. In small ruminants, Jeptospirosis,
usually dueto serovar pornona, resembles that seen in cat-
tle. Tnfectlons wlth seruv<1r:s hurclju a11u ;srippulyphusu alsu
occur. EpiciPmirs rl11P to sProv;ir pnrnnna h ave caused high
n1ortality an1ong California sea Jions periodically since the
1940s.
Humans. Humans a re susceptible to all serovars with no
host-adaptcd strains i<lentified. Jnfcctions cause fever,
icterus, muscular pains, ra:shes, a11d i1011:suppurati ve 1nen-
ingitis, manifestations v;irying somc.what with the
serovars involvcd. A 111aligr1ant forrn, most oftcn associ-
ated with serovar icterohaen1orrhagiae, can cause fatal liver
or renal diseasc.
Epidemiology
Thc many tolcrant hosts and the protracted shedder state
perpetua te leptu::.piru::.i::.. Iudi rct.:L eXJ?OSu1e depends on
1nilrt anrt '-VC't condilions, which favor environmental sur-
vival o f lcptospirac. More direct transfer occurs by urine
aerosols in milking barns and cattle sheds or by canine
courting habits, which may explain the male bias of ca
nine leptospirosis.
Contaminated bodies of water are i n1portant sources of
infectiuu tu liv~::.tock, a4uati1.: u1a1uu1al::., a1.1d hu111a1 1~.
Anima l hanrt lcrs, scwcr workers, field hands, n1iners, and
vctcrínarians are at íncrcased risk of exposure.
lmmunologic Factors
lmmune Mechanlsms of Disease
Immunologic mechanisms may relate to sorne features or
lcpto5pi ral discasc:
1. The hen1o lytic anemia characteristic of septicemiL
leptospirosis duc to servar pon1ona in ruminants !
associatcd with thc prcscncc of cold h emagglu-
tinins, suggesting an autoimmune process. The re'-
ative roles of this and the bacteria! hen1olysin a:L
uncertaln.
2. Canine chronic interstitial nephritis is con.<>icif'r~
by 111any a poslleptospiral lesion. A leptospiral etiol-
ogy is suggested by a frequcnt history of leptosp.-
rosis and thc prcscncc of leptospiral antibod-
particularly in urine. An immune-basecl etiology •
attractive, as antirenal antibody has been demo:--
strated in lnfected dogs.
 Chapter 26
3. Evidence of a leptospiraJ basis for equ1ne recurrent
uveitis (periodic ophthalmia, moon blindness) rests
i11 parL un leptu~piral a11tiliuuy a11d its relative titers
in serum and aqueous humor. The condition has
bccn rcproduccd in horscs and dogs cxperimentally
by using leptospiraJ antigens.
le(hanisms of Resistance and Recovery
ecovery from acute leptospirosis c:oincic1f'.~ with thi> ci>ss;i-
::>n of septicemia and the appcarancc of circulating anti-
- ...<Hes, usually during the second week of infection.
--otective antibodies are of lgM and TgG isotypcs and are
_ rected mainly at the outer sheath anti gens.
Agglutinating antibodies, which persist aftcr recov-
.~..')', i~ 110L ar a i11<..licatiu11 uf iuuuuuity IIOr uf the shedder
ratc, which may exist in the presence or ahsenc:i> of <inti-
!>ody.
lmmunity toUowing recovery is generally solid and
4:rovar-specific. Repeated abortions due to serovar hardjo
-.ave been reported in co,\fs, probably dueto weak in1mune
-.-..ponse to this hovine-adapted serovar.
- 7ficial lmmunization
_.:cination with bacterins is used in dogs (bivalent con-
-üing serovars icterohaen·1orrhagiae and canica/a). /\ biva-
dlt bactcrin containing serovars grippotyphosa a11d
··nona is also commercially available. 1'hese bacterins are
u1prlsed of killed, whole-cell leptosplrcs. Toxlc compo-
- nt~ of thi>.~P proc111rts, ~nrh ;:is endotoxin, may be re-
-:-onsible for the adverse reactions reported in dogs.
ln North America, cattle and swine are vaccinated with
pentavalcnt bactcrin containing thc most common
:Jrth American serovars (fzardjo, grippotypfzosa, pomona,
-.aohae1norrhagiae, and canica/a), with thc addition of
~:o\·ar bratislava anda second scrovar f1ardjo component
.:: sorne vaccines. Humans who are at risk may opt for vac-
.naliuu as we;:IL ProLecLiu11 i::. ::.eruvctr-:.pecifi<.: auu teu1po-
-::ry, requiring at least annual hoosters. Vaccination pre-
~nts overt disease but not necessarily infection.
_aboratory Diagnosis
iagnosis of leptospirosis must be established by labora-
"•Y co11fi1111aLio11.
!:mple Collection
.,. uu1 1iviI1g sulJjects, t;lood, urin<::, cercbrosplnal fluid,
.. ~eri ne f111 ic1.~, 11nc1 p l11ri>nt;:i I cotylc~dons are examined.
~lood is usually negative after the first febri le pl1ase. Milk
destructive to leptospirae and nota pron1ising source for
_-.iltures. Urine should always be testcd.
From cadavers, including aborted fetuscs, kidneys are
±e most likely organs to harbor leptospi rae. In septic fatal-
..:t::. (i11<.:ludi11g al.lurtions) many organs, espccia1ly the
_;;er, spleen, l11ng, hr11in, ;:inc1 i>yP, may contain the agent.
Culturing is done promptly after san1plc colleclion.
Spiral-Curved Organisms V: Leptospira 151
Direct Examination
MPthoc1' of c1irPct vis11;il demonstration are wet mounts,
cxamined by darkfield (or phasc) niícroscopy, in1111u110Ilu-
orescent stains, and silver impregnation of fixed tissue.
Routine darkfield micrüscopy should be limitcd to
urine. Other body fluids conta1n art1facts morphologicall y
similar to leptospirae. Brief, low-speed centrifugation
clea1s LI 1e;: :.µeci.iue11 uf i11t~rf~ring particles but will not
sediment leptospirae. Methods 11.~ing formlllini7.Pcl 11rinp
have been described, but they destroy motility, 'l\ hich aids
1
in thc idcntitication of leptospirae. Negative results of di-
rect examinations do not rule out leptospirosis.
Fluorcscent antibody has been uscd on fluids, tissue
sections, homogenates, organ impressions, and, most ef-
feL:li ve;:ly, llll al.lurtell l>uvine f~tuses, where examlnation of
kidney is most rewardi ng. Silvt>r-imprt>gnated sections
must be interpreted with. caution, because argyropl1ilic tis-
suc 1ibrils mimic leptospirae.
DNA amplification using the polymcrasc chain rcac-
tion togcthcrwith specific DNA primcrs has become an ex-
cellent diagnostic tool for detecting the presence of lep-
luSiJiiae in ct11irual tissues and flulds.
lsolation and ldentification
The nlcdiun1 of Ellinghauser1, McCulluugh, Johnson, and
Harris (EMJH medium) is a good isolation medi11m, i>spi>-
cially for serovar hardjo, the slowest growing of the
com rnon scrovars. Replicate inoculations are 1nade into
EMJH medium with and without sclcctivc inhibitors
(5-fluorouracil, neomycin, cyclohex1m1de). Cultures are
exam ined microscopically at intervals during incubation
fui up lo :.everal 1noutl1s.
Animal inoculation (hamsti>r' or gninP;:i pigs) elimi-
nates minor contaminants from the prirnary inoculun1,
which is injccted intraperitoneally. Blood is drawn period-
ically for culture starting a fcw days aftcr inoculation. Aftcr
3 to 4 weeks, the animals are killed and their kidneys ex-
amined and cultured for leptospirae. Jf irlfected with lep-
to.splrae, they will have dcvclopcd antlbody. Any isolate
ri>rovi>ri>c1 hy thi>si> methods can bC' identified morphologi-
cally as a 1nen1be1 of Lhe ge11u::. Lcplu:;piru. Defi1litive ider1-
tification is carried out hy refercnc:e J;ihoratories.
Serology
Dircct examination is often unreli;ihlP 11nc1 r11lt11re l11bori-
ous, cxpensive, and slo'"'· Serology is the most common
diagnostic method. ·rhe microscopic agglutination test
employing live antigen is most widely uscd. Othcrs in-
clude macroscopic plate and tube agglutination tests,
complemcnt fixation tests, and enzyme-linked antibody
a:-.say:.. Paircll sa1nples are preferred: onc collected at first
presentation and one 2 wi>Pk.~ lllti>r. Tf leptospirosis was
the problern, a fourfüld or greater rise in titer should have
occurrcd in the interval. In bovine abortion, these rela-
tions may not hold. This is duc to thc fact that scrovar
/1ardjo infections ot cattle clicit a very weak immune re-
sponse that is probably dueto their adaptation to this an
hual species.
152 PA1rr TI R<'lC'tPria ¡¡nrJ !=11ngi
Treatment and Control
Leptospirae are susceptible to penicillin G, fluoroquino-
lones, tetracycUnc, chloramphenicol, streptomycin, and
erythromycin. Treatment, to be of benefit, must be insti
tuted early, possibly cvcn prophylactically in cases of
known exposure. Doxycycline is used to treat humans pro-
phylactically. Howevcr, cvidcncc of leptospiral infection in
the kidneys and reproductive tracts of cattle subsequent to
antibiotic treatment is not uncommon.
Vaccinalion ge11erally preve11ls disease. ll does r1ol p1e-
vent infection nor shedding, although it does reduce its
cxtcnt.
 Staphylococcus
DWIGHT c. HIRSH
...-:-.,hylococci are spherica\ gram-positive bacteria that di-
.:.1: in scveral planes to form irregular clusters. ·rhey are
- -sent in thc upper respiratory tract and on other epithe-
- ~urfaces of ali warrJJ-lJluuucu aulu1als. Four of sorne 20
?e(:ies are of veterinary importanc:r.: S. a11re1LS, S. inter-
Jius, S. hyicus, and S. schleiferi subspccies coagulans. Sta-
lococcus aureus is a common pyogenic agent in humans
-d severa! animal species. Staphylococcus intcrrncdius is
·e leading pus-formlng bacterium in dogs. Staphy/ococcus
.:us, which is found in severa! species, causes exudative
..:1:1111lc.lls of s~v111e anu sometlmes, bov1ne mastitís. sca-
ococcus schleiferi suhspPcipc; rnag11/n11s (a long with S. in-
"'ledius) is sometimes associated with otitis externa of
::s. Staphylococcus sciuri and S. x.vlosus (and rarely S. epi-
~nidis) are univcrsally prcscnt on skin and sorne mucous
-.:nbranes, but are ra rely pathogenic. Pathogenic staphy-
.. .cci usually produce the enzyme coagulase (see below).
: : scriptive Features
• ·::hology and Staining
~!"lylococci are 0.5 to 1.5 µm in diamctcr and gcncrally
;i strongly gram-posit1ve. In exudates they form clus-
pairs, or short chains (Fig 27.1). Spores and flagella are
.11. E11<.:a¡.>:.ulatiu11 i:s varJalJlc.
_:ture and Composition
.. ell wall consists of proteins and polysarrharirlt>s.
~ protein ("clumping factor," "bound coagulase") is
y present in S. aureus and S. interrnedius. Clumping
- intcracts in vitro with fibrinogcn to produce an
--.,,...-~-nation-lil<e reaction. Another, Protcin A, produces
--o..~ation by combining with the Fe fragment of immu
uli11:.. Tht: prt:LioIDiI1ant poly:sacchar1de is teJchoic
'lked to peptidoglycan. Tts alc:ohol moiPty is rihitol in
-~:.s, and glycerol in S. epidennidis and S. íntennedius.
---enoid pigments in the cell rnernbrane can imparta
.:n" (Latin: "aureus") color to colonics of S. aurcus. A
--__,,... e Is sometimes produced by S. aureus, and often a
'.:>capsule," a loosely associated carbol1ydrate struc-
p1oclucec.1 lJy :strains <.:au:siI1g lJovlne mast1tis.
- : • Products of Medica! lnterest
: \'1.·hat follows has been determined for S. aureus, the
.1lt:11:.ivcly studied specles of staphylococcus.
ERNST L. BIBERS"fEIN
I'resumably, the othcr species have sin1ilar trails and cha1-
acteristics that make them potentially pathogenic.
Adl1esí11s. Staphylococci produce a number of surface
proteins that bind to a var1ety of extracel lular matrix pro- ,.
teins of the host (fibronectin, fibrinogen , collagen, vitro
neclin, Ia11 1i11iu) . 1'he:;e "adheslns" have been termed
MSCRAMi\lfs (rnicrohia 1 s11 rfacP r.omponents recognizing
adhesive 1natrix rnolecules). Staphylococci produce a 11uu1-
ber of different adhesins, giving ~orne .strains afñnity for a
ccrtain tissuc typcs (bone, kidney, bladdcr) .
Capsule. ::,tap11y1ococcus aureus produces 11 serologically
distinct polysaccharide capsules. The genes encoding cap
:,ult: µrotluction are located on a Staphylococcal Cassette
w
Chro1nosome Gt>nPtic F.lt>mf'nt (SCC.cnp). SCCs satisfy the <(
characteristics outlined for defining a Patl1ogeniciLy
Islancl: a cluster of genes encoding virulence determi-
nant(s), an integrase protcin, a spccific insertion site, and
rnobility. The capsule is thought to act in preventing
phagocytosis.
Ce// Wall. The cell '"'ªll of the members of this genus is 1
one typical of gram-posi tive bacteria. ·rhe lipoteichoic
acids and peplidoglyca11 uf lhc gra111-posit1ve cell wall in- 1
teract with macrophage cell~ rPc;11lting in thf' relPase of
proinflammatory cytokines.
Enterotoxins/Pyrogenic Toxin Superantigens. Coagulase-
positive staphylococci produce n group of cxotoxins of
similar slze and three-dimensional shapc. These include 11
cntcrotoxins (SE, for staphylococcal enterotoxin) A-M
(Lhcre is no f 1101 J), and ll1e Lvxi<.: sl1uck sy11Liro1ue toxin
(fSST-1 ). The genes encoding these toxins are located on
Pathogcnicity lslands (SEI3, SEC, SEl<-M, TSST-1), pro-
phagcs (SEA, SEE), or plasmids (SED). Ali are small proteins
(20,000 to 30,000 in molecular wcight) and dissimilar in
pr1mary amino acid sequence, but remarkably similar in
shape. They are resistant to heat and digestive enzymcs.
1"hc SEs, ~vhicli are mually i11ge:sted as a preformed toxln,
act by binding toan undefi ned recPptor in the wall of the
intestinal tract triggering reflcx stimulation of the von1it-
ing ccnter. Both SEs and TSST-1 are superantigens, and
part of the systemic symptomntology mny be rclatcd to the
cytoklne "storm" that results from the interaction o1 1'
lymphocyte receptors, macrophages, and t hese toxins
wl1icli are n::leased in vivo (TSST-1 crosses mucus men1-
branes, the SEs do not).
Exfolíatíve Toxins. Stapl1ylococc11s aureus produces lwo
cxtoliative toxins (sETA, El"B), and S. hyicus produces three
antigenically differcnt cxfoliativc toxins (shETA-C). The
genes encod1ng the Efs of S. aureus are Jocated either on a
153
154 PART II Bacteria and fungí

FI G URE 27.1 . Staphylococcus aurc us in urinary sediment from a cat. Gram stain,
7000X.

. .~
""-~· )-
,..,.. ·.
•
• ••
• ..
4
....
. • -
• •••• •
•

•
..
-· •
..... •

prophage (sETA) or plasmid (ETB). The exfoliative toxins agar at 37ºC, a partial hemolysis ("water-stain" ar-
are atypical glutan1atc-specific serine proteases. ·rhey tar- peara11ce) that occurs an<.l goes to co1upletiou u=
get the intercellular adhesion protein, desmoglein (a cad- furtl1er incubation at lowt>r tP.mper<iturP.s (Fig 27.2
l1eri11), found only in lile epidernlis. Whelhe1 lhe exfolia- Its role iI1 vivo is unclear, but damage to host CeL
tive toxins are superantigens, is unsettled. membranes is a reasonable assumption.
T-Ten10/ytic Toxins. 1'he re are four hemolytic toxins 3. Gamma toxln. Gamma toxin is a bicomponcr·
(alpha, beta, gamma, and delta), so-called because of their toxiI1 composed ot two proteins that combine •
action on erythrocytes in vitro. Heinolysis is not a prop forman active moiety. This toxin stimulates degraL
erty ot)served duriI1g the disease process. The l1emolytic ulation of pl1agocytic cells, thereby intensifying ~-
tox.ins are ex.pressed siogly, in combin.at ion, or not at all.
TJ1ey tlifíer a11lige11 it.:ally, l.Jivclie111it.:ally, a11tl i11 Llieir
effect on the erythrocytes of various species. The genes
F1G U RE 2 7 . 2 . Staphylococcus beta toxin activity on bovine
encoding the hemolytic toxins are located on the chro-
b/ood agar. For explanation, see text.
mosome:
l . Alpha toxin ..A.lpha toxin acts on membrane lipids,
is hemolytic in vit ro, is mitogenic, is lethal to rab-
bits follo•ving intravenous injectio11, and is r1ecro-
tizing u pon intradermal injectic>n. The n1ajor effect
in vivo is related to its insertion into membranes.
The toxin is excreted as Inonome.rs, vvhich con1e to-
getl1er in tl1e l1ost ccll n1en1bra11e to forn1 a c..yli11de.r
througl1 wl1ich io11s flow. Loss of membrane in-
tegrity in a variety of ccll typcs rcsults in .u11toward
ettects on the host. At high concentrations alpha
toxin initiates target-cell death by necrosis (ion and
ArP depletion). In lower concentrations, apoptosis
is triggcred . I.n certain instances, coagu1;1s1~-posi ti vt>
staphylococci are internalized by nonprofessional
phagocytes (endothelial cells, sorne epithelial cells),
but c:;cupc th.c cn.do:>oroc to multiply within t hc cy
toplasm. Endoso1nal escape is thought to be associ-
ated vvith alpha hemolysin-mediated lysis of the en-
dosomal membrane.
2 . Beta tox.i11. Beta toxi11, a phospholipase C is prt>va-
lent i11 a11in1al strains. Beta toxin p roduces broad
zones of "hot-cold lysis" on sheep or cattle blood

flammatory responses and tissue damage. Gamma
toxin is not observed surrounding colonies growing
0111.JlouLl agar plate::; ::;iuce il i::; i11hibitecl l.Jy agar, l.Jut
viTtually ali strains of coagulase-positive staphylo-
cocci produce the toxin.
_ Delta toxin. Delta toxin lyses cells of various species
by a detergent like action but is inh ibited by seru·m.
Like gamma tox.in, almost ali strains of coagu lase-
positive straios produce this toxin. Its role in dis-
ease, however, is undefined.
Acquisition. Staphylococci with pathogenic poten-
- ;;n
~e., coahTUlase-positive strains) grow bett er in iron-
~~-":cted conditions (as would occur in vivo), 0.5 compo.rcd
;!!ose staphylococci with less potential (coagulase-
.:-"ive strains) . lTnder iron-limiting conditions, the
~gu:lase-positive strains produce siderophores, auroche-
- ;nd staphyloferrin, which are responsible for iron ac-
-.Si:tion from cxtraccllular sourccs (transfcrrin, lactofer-
- Staphylococci also utilize the siderophores produced
_-~er bacteria, specifically enterobactin and aerobactin
-:= gram-negatlve organisms .
. niknr.idin. Like gamma 11emolysin, leukocidin, also
-n as the Panton-VaJenti11e toxin, is a bico111ponenl
.......__._ In fact, sorne of t he proteins encoded at the locus
-z..c.~""ining the genes for gamma hcmolysin, forma part of
e: .:>Cid í n . Leukocidin stimulates the degranu.l ation ot
:::ocytic cells, thereby intensifying inflammatory re-
-"5e5 and tissue damage.
¡,TTF (M11ltiple Peptide R1Jsistance Factor). MprF bestows
~ice Lo Ll1e aclio11 uf clefe11::;in:; tl1ruugl1 tl1<:: ly:>iI1yla-
=:= of phospholipids in the cell memhranes of coagu-
-N)Sitive staphylococci. Not only <loes this permit sur-
- \\l'ithin defensin-containing phagolysosomes (this
¡¡:¡,s :'!Ot protect against oxygen-dependent killing, how-
it allows existente within niches that are bathed i11
:'::lSin s (upper respiratory tract, intestinal tract, genital
iü:P.llaneous Products. Staphylococci produce a
~:a of other products that n1ay or n1ay not have a role
-"6lse production. 'l'hese products include lipases, ser-
_::oteases, thiol proteascs, metaloprotcascs (aurc-
-~~: , esterases, deoxyribonucleases, staphylokinase (a
• :=t'nogen activator), hyaluronidase, and phospholi-
-.::::.C5.. Urease, an enzyme produced by coagulase-posítive
"""'-- ~:lococci is ;:issoci;:ited with the production of uroliths
--=e canine bladder. Coagulase, an enzyn1e t hat causes
""""'~-ia coagulation in vitro and aids in the identification
~~e patl1ogcnic spccics: S. aurcus, S. intermedius, S.
~...rm ssp. coagutans, and sorne S. hyicus, probably has
i.f any role in vivo.
-:."JUlalion o(the Cellular Products o(Medical Interese. 1·he
.,_._-_·ction of m;:iny of the products involved with patho-
=~~15 of staphylococcal disease is regulated by way of a
-..=n-sensing, global-regulatory system. Aside from the.
r::::; ;egu latcd products, most if not all of t11e products of
~~~-::sr are regulated by the aKr-sar (for accessory Kene reg-
-= ~ staphylococcal accessory gene regulator, respec-
:sy,)tern. 1'his system ts comprlsed of a series of genes
.,_-J. ano sarA) whose products "sense" and respond to
- "'lactone-contain ing pl1eron1011e (A u loinducing
Chapter 27 Staphylococcus 155
Peptide Pheromone or AIP, wbich is the modified product
encoded by the agrD gene) produced by other staphylo-
cucci iI1 tl1e irrnne<liate er1vironment. Each staphylococcal
cell produces a hasal lPvPl of AIP, ;incl will produce more,
when a certain environmental concentration of AIP is at-
tained (quorum sensing). AIP induces the formation of
RNATTJ (not its translatcd product) t hat rcgulat cs thc tran-
scription of many of the genes involved with st aphylococ-
cal disease (global regulation). Thus, when tl1e numbers of
::;tapl1ylucucci iI1crea:;e tu a certain rnass, th.e increased
amount of AIP directs the up regulation (by way of RNATII)
of certain genes (those e1icoding 11e111olysi11s, enlerolox-
ins, exfoliative toxins, leukotoxins, lipases, serine est erase,
dcoxyribonuclca:;c, :;taphylokinnsc, hynluronidasc, phos-
pholipase, and capsule productio11), while down regulat-
ing those gene products that are made 1o\7hen AIP is made
iu les:;er aruuuut:; (aclhe:;ins).
Growth Characteristics
Staphylococci grow overnight on common laboratory
media, over a Wide temperature range, and atmospheric
conditions producing on agar smooth opaque colonies
over 1 u 1111 iI1 clíarueter.
Biochemical Characterization
Staphylococci are catalasc-positivc, facultative anaerobes
that attack carbohydrates oxidatively and tennentat ively.
Resistance
Staphylococci withstand drying (especially in exudates)
for weeks, heating up to 60ºC for 30 minutes, pH fluctua-
tíons frorn 4.0 to 9.5, and salt conccntrations of 7.51J1l,
which are used in selective media for isolation of staphylo-
cocci.
Staphylococci are inhibited by bacteriostatic dyes (e.g.,
crystal víolet), bile salts, disinfectants like chlorhexidine,
and n1any ai1ti111icrobial drugs.
Variability
Colonies vary from smootl1 (S) to rough (R). G ("gonidial")
and L (wall-less) variant s reflect progressive cell wall loss
produced by unfavorable environmental conditions, such
as a11libiolic LreaL111enL.
Isolates can be "typed" hy their <;usceptihilities to hacte.-
riophage lysis. Phage-typing sets have been developed for
hun1an, bovine, and avían S. aureus.
Resistance to beta -lactam antimicrobics is dueto posses-
SiOn of a plasmid-encoded penicillinase (beta-lactamase),
or the presence of a Staphylococcal Cassette Chromo-
son1e Ge111::ti.c Elt::111er1t cur1taíning the gene that encodes a
pPnic.i llin-resistant penicillin-bi11ding protein (SCCrnec).
Tolerance, a rarer forn1 of penicilli11 resislance, is allribuled
to failure of autolytic celJ wall enzymes. Intrinsic penicillin
resistance may be due to ch.auges in pcnicillin-binding
proteins (enzymes responsible for cell synthesis, see
Chapter 4).
Re:;í:;ta11ce to other antimicroblcs is common.
 156 PAI~T 11 Bacteria and Fungi
Ecology
Reservoir
C~oagulase-positive species S. aureus and S. interrnedius in-
habit thc dist<1l nasal passages, externa] nares, and skin, es-
pecially near mucocuta11eous borders such as the per-
i neum, external genitalia, and bovine udder. They also
occur as transie11ts il1 the gastrointestinal tract.
Coagulase-11egative staphylococci, especialJy S. scíuri
and S. xylosus (and rarely S. epidermidis) are predominant
amo11g the resident skin flora but also colonize the upper
re:;¡;iralvry lract. 111 :.1.vi11e, lh.is geueralizalion applies Lo
S. hyicus, a species potentially pathogenic, especially for
piglets.
Staphylococci are found worldi~1 ide in warm-blooded
anin1als. Interspecies spread (e.g., humans to cows, dogs to
l1un1ans) appears to be limited.
Transmission
Staphylococci sprea<.l lJy <.lirect cu1d i.11dirt:ct cvulacl. Man y
anirn;i 1infi>ctions ;irp proh;ihly enct og~nous, that is, caused
by a resident strain.
Pathogenesis
lvtcchanis1ns. The deposition of staphylococcal cells into a
norrnally steríle s1te is the first step in the infectious
p ro<.:ess. Presumably, the i1un1bers of staphylococci at this
stage i-vould be fcw, and thc amour1t of AIP (see above,
"Ri>gnliltion of thP Cellnlilr Products of Meliical Interest"),
loiv. 'fhus, expression of adhesins (MSCRAMMs) results in
adherence to extracellular matrix proteins. Inflammation
is initiated by cell wall constituents (peptidoglycans and
lipoteichoic acic.1s), and by deposition oí complement pro-
teins on the bacteria! cell surface leading to the generati.on
of splil producls Lhal are cl'Je1nolaclic a11d a11a¡;ltylvtoxl<.:.
At this stage, the infecting strair1 may be elin1inated, or
not, depe11ding u pon bacteria! factors (inoculun1 size, vir-
ulence of the straiI1) and host factors (strength of the in-
nate ilnmune systen1, underlying diseasc or dcfccts such ns
the extent of tissue damage). Tf the rnfecting strain is not
controlled and eliminated, their numbers increase so that
tt1e concentration of AIP rises lea<.ling to the <.lo•vn regula-
tion of adhesi11s, and the up regulation of capsule and tox-
ii1s. Siderophore produclio11 aids i11 lhe acquisilion of iro11.
'l'he capsule prevents further phagocytosis; tl1e toxins ag-
gravate tissuc damage by dcstroying recruitcd inflamma-
tory cells (n1embrane active toxins, and triggering degran-
ulation). ·rhe predominant pattern of staphylococcal
pathogenesls is suppuratlon and abscess formatlon. Syste-
n1ic effects inay be the resul t of sup<~rantigen activity.
Ccl1-111cdiatcd in1n1u11e pl1e110111e11a inte11sify inflan1-
matory responses in sorne stapl1ylococcal ir1fections whi.le
spatially confining them. In so1nc forms of ca11il1c pyo-
dcrrna (juven ile pyoderrna, tolliculitis), cell- and antiboc!y-
mediated hypersensitivity may be induced.
'!'he ent erotoXin-induced diseases (food polso11ing) are
not prominent in animals (tl1ougl1 so-called "garbage can
er1le1ilis" n1ay be stapl1yloc.occal cntcroto.xin-iJ1duced in
ctogs). -roxic shock syndrome cloes not commonly occur ~­
at all) in the veterinary species.
l'l1e e.xfolialive loxü1s n1ay play a role ü1 exud;1tive det-
matitis of pigs. A conditio11 rese1nbling staphylococca
scaldcd-skin syndromc has hccn dcscribcd in dogs.
vatho1ogy. J'he typ1ca1 1es1on is tne atJscess, an innam-
matory focus in which participating cells have been de-
stroyecl by the combine<.! effects of hctcterial au<.l iuflaII1ma-
tory cell activity. 1'his confrontation between leukocyt6
and n1icroorganisn1s produces pus, a n1ixture of host ce...
debris and bacteria, living and dead. In an abscess, ptis .-
surrounded by intact leukocytes and fibrin strands. Unles-
thc pus is drained, a tibrous capsule will gradually bE:
formed . In chronic, ulcerative staphylococcal ..,,vound in-
fections ("botryomycosis"), fibrous ele1ne11ts predo.tfu-
nate, interspersed vvith pockets of suppuration.
Disease Patterns
Although all warm-blooded animals can be clinically a:-
fected by coagulase-positive. staphylococci, the prevalen~
and form of such interactions vary a1nong host spectes
'fhe n1ore com1no11 presentations are listed, a11d it shotLC
lJt: kept in rnin<.l that sta¡.il1ylvcocci can affecl any organ
tiSSllP.
Dog and Cat. 1'he term canine pyoderma covers many cJin_-
cal pictures, all of which include sorne degree of pyoger:!.
skin inflammation associated vvith bacteria! infectior.
Staphylococcus inl'ermedius is the chief bacteriuin ilnpL-
cated. Its contribution and the degree of suppuration a:c
va riable. Tn chronic and recurrent forms, cell-mediated h\-..
'
persensitiviLy and iu1111uue cornplexes are Lhoug11l lo bt:
involved. Host aspects, incJuding genetic, endocrine.. and
imrnunological factors, may plny an importar1t pnrt.
Osteomyelitís (especially discospo11dylitis) and arthritiS
are frequently associated with S. intermedius.
Mastitis is trequently assoclated witl1 S. interrnedius.
Otitis extPrna is usually a n-1ixture o f ye;:ist (Malassr.zía,
see Chapler 45) aud b<tcleria. T!Je bacteria frequenlly asso-
ciated with this condition are S. intermedius and S. schleiferi
ssp. coa,r:ulans.
!'ripie Phosphate (struvite, apatite) urolithiasis is a l-
most always infectious, with S. intermedius as the etiologic
agent. Other bacteria frequently found 1nclude 1~seudo­
monas (see Chapter 20), Proteus (see Chapter 7), and Entero-
COl:cus (see Cha¡;ler 28).
Ruminant. t\s a leading cause ofbovinc mastitis, S. aurcus ri-
vals !:>treptococcus agalactiac (see Chapter 28). lnfection oc-
curs vía the teat canal, and tl1e course of the infectious
'
process varies from subclinícal to acute suppurative, gan-
grenous, or chronic, depending on the infecting str;iin, in-
fecling dose, a11d hosl resista11ce. Bovine n1astitis is son1e-
tin1es caused by coagulase-11egative staphylococci, notabty
S. epiderrnidis, S. hyicus, S. xylosus, and S. sciuri.
Tick pyemia ot lam bs, resulting from inoculation of in -
digenous skin S. aureus by tick bites, may be acute '"'ith tox-
emic deatl1, or chronlc wlth dlssen1inate<.1 abscess forrna-
tion. lt is often linked with tick-borne fevi>r (c;iusect by the
rickellsia.l agenl Anaplasrna phagocytophila, Chapter 43).

;oscess disease of sheep, resembling caseous lymphade-
~ w hich is caused by Corynebacterium pseudotuberculosis,
.J:...;?~er 31) is caused by an anaerobic subspecies of S. aureus.
:. · re. Mastitis i11 111ares is frequently associated vvitl1 S.
~-
:'ectoral abscesses are sometimes associated with S. au-
Coryriebacteriurn pseuaotuberculosis is tar more com-
-=- ~- however).
S::>ermatic
•
cord abscesses after castration are sometimes
ociated with S. aureus.
- • - ~'E!.Exudative epiderrnitis (greasy pig disease) dueto S.
--US affecting young pigs (7 weeks). lt is often systemic
- rapidly tatal, attecting the lungs, lymph nodes, kid-
-~. and brain. Skin lesions are characterized by a thick,
:. 1: iSl1-brown exudate (Fig 27.3), especially around the
-- ;in d ea.rs.
- ---~ "Bumblefoot" of gallinaceous birds is a chronic
---granulomntous proccss in thc subcutnncous tissucs of
- ~ foot resulting in thick-waUed swellings on one or more
~t:s. This condition is associated with trauma together
_'! S. aureus.
(\taphylococcosis in turkeys, a bacteremia locaJizing ü1
~ and tendon sl1eaths, is often produced by S. aureus.
::...:em iology
~?hylococcal diseases (e.g., pyoderma, otitis externa,
_-nary tract and wound infections) often arise endoge-
- :usly. Studies on humans suggest widespread staphylo-
'-'-<tl culu11izatiu11 witlliu 11uurs oI birtlt. Cliui1..al i11fe1..-
~s appear to he decisively detennined hy host factors.
F1G U RE 2 7 . 3. Porcine exudative epidermitis (greasy pig disease). The skin is covered and
thP hri~tfP~ are matted hy ah11ndant hrownish ex11date (arrow) made 11p of epidermal debris and
inflammatory components. (Photograph courtesy of Dr. Hurvey O!under.)
Chapter 27 Staphylococcus 157
ln bovine mastitis, staphylococci may enter the gland
during milkir1g. Manageznent practices and milking hy-
giene influence prevalence significantly .
·1ransmission of S. aureus between animals and humans
occurs infrequenlly.
Prolonged environmental survival of staphylococci
pennits their indirect tra.nsmission.
lmmunologic Aspects
Possible in1n1une n1ecl1anisn1s in palhogenesis l1ave al-
ready been cited.
Recovery and Resistance
Clearance of staphylococci depends chiefly on phagocyto-
sis. Humoral factors are apparently important because
agan1n1aglobulinen1ic individuals suffer frequenl infec-
tions. Cell-mediated factors contribute to localization ancl
rcsolution of Iesions.
J{ecovery trom stapl1ylococcal infection confers no last-
ing resistance.
Artificial lmmunization
The benefits of vacciI1ation are doubtful. Commercial or
autogcnous wholc-culturc prcparations, toxoids plus bac-
terins, are used prophylacticaily on dairy cattle and some-
times in small animal dermatology t o treat persistent
Infections. Although successes have been report.ed, coo-
trolled evaluations are lacking.
U se of :.La¡..>hylucoccal ¡..>l1age lysate:. a1H.1 11011svecific
stimulants of cell-mediated imrnunity in cases of non.re-
158 PAnT JI Bacteria and Fungí
sponsive pyoderma awaits :;upport by adequate clillical or
experimental evidence.
Laboratory Diagnosis
Sample Collection
Aspira tes from unopened lesion s, in sterile syringes or ster-
ile containers, are preferred. 5wabs in transport media are
acceptable. Milk is collected into containers under sterile
precautiou:.. l3lootl a11ú urir11:• culture routines are appro-
priatc for sta phylococca 1 isolation.
Direct Examination
011 gram-staincd fllms, stapl1ylococci appear as gran1-
positívc cocci in pairs, clusters, or short chains (see Fig
27. l) . Tu S(J<.:CllJ1e11~ frvu1 ~k l11 J.JU:.Lule;::., Ll1e::y 1uay !Je;: :>JJar:se;:.
lsolation and ldentification
Bovinc blood agar is bcst for thc dctcction of bet a toxin
("water stain" appearance), which is diagnostic for coagu-
lase-positive staph ylococci (S. aureus, S. intermedius, and S.
schlei{eri ssp. coagulans; see Fig 27.2). Colonial appearance
is as described . Biochemical tests are tised to identify
slaphylococcal isolales. Coo101ercial kiLs are available.
Treatment and Control
Absccsses are dra1oed of pus. For the most supertidaJ forrn.s
of pyoderma, topical application of mild antiseptics (3Q
hexacltlorophene) may be aúequate.
F.xtPn'\iVP, inaccPs'\ihlP, anc1 c1issPminatPc1 proC"PSSPS re-
quire systemic treatment. Staphylococci are commonl·
resistant to penicillin G, streptomycin, and tetracyclinÉ
Usually cffcctivc antim icrobics includc pcnicillinase-
res1stant pcn1ctlhns, fluoroqumoloncs, chloramphcn1co!
cephalosporins (first generation), vancomycin, lincosa.
mides (llncomycln and clindamycin), the macToliúes (err-
thromycin, azithromycin, clarithromycin), anc1 trimethÓ-
prim-sulfas. Clavulanic acid inactivates the beta-lactamase
produced by S. aureus and S. interrnedius, therefore cell war
antibiotics containing this substancc are protected (e.g.
clavulanlc acid/amoxicillin). Th.e penicillin ase-resistan;
penicillin cloxacillin is effective in treating staphylococ~
1.11aslilis , C::~JJt:C ially i11 dry cows .
For staphylococcal cystitis, penicillins remain cffectiYe
because of their high urinary conce11trations. Cloxacillli:
is used topically and systemically on exudative epidcrnlif~
duc to S. hyicus.
A controversia! approach to prevention of staphylococ-
cal infections in infants utilizes "bacterial interference~
the implantatton of a nonvirulent strain to preclude colo-
ni7::itif'ln hy vir11IPnt <:t::iphylf'lrf'lrri ThP mPthf'lrl <:ho~'
p romise for control o f staphylococcosis in turkeys.
 Streptococcus and
Enterococcus
D WTGHT C. H IRSH
STRE PTO COCCUS
Streptococci are gram.-positive cocci occurring in p airs and
chalns; they show considerable ecologic, physiologíc,
sProloglc, and gen etic diversity. There are 55 recognized
specics wi thin ll1e genus, bul only a l 1a11<.lf ul is regularly as-
sociated with diseases of veterinary import;incP. 'íhPsP,
along with thc 11istoricaUy important streptococci mainly
affecting primates are shü\.\Tll in 1able 28.1.
The streptococci are supcrficially "grouped" by how
they grow on blood agar plates. Effects on sheep or bovine
hlood agar are used to divide streptococci into three types:
Alpha-lternolytic streplococ.ci (u) úu 11ul lyse erythrocytes
but produce a zone of green disc:olor;ition around the
colonies (change the hemoglobin to methemoglobin).
Yrost commensal streptococci of animals are alpha-he-
molytic. Strcptococci that do this are sometimes referred
to as "viridans streptococci." .rhey are not hemolytic in the
true sense of the word.
Beta-l1en1olytic streptococci (B) lyse erythrocytes and pro-
duce a "clear" zone arou11d thc colonies. Most pathogenic
ate l.lt::la-llo::u1ulyliL.
L)' lJC:>
Ga1n1na streptococd ('y) are nonhemolytic. Most are non-
pathogcn ic.
Descriptive Features
'-'orphology and Staining
~trcptococci vary from sphcrical to short bacillary celJs,
about 1µm in diameter. lJivision occurs in one plane, pro-
::iucing pairs and chains. Chain formation is variable,
:hough sorne species (e.g., S. cqui subspecics equi) are con-
i ~cnt chain formers (Figs 28.1, 28.2).
Young cultures are g1an1-posili vt:. I 11o::xu<.lates aucl ulc.Jer
cultures (> 18 houis) organisms often stain grarn-n PgativP.
Structure and Composition
~eptococci havP ;i typic<1l gram -positive cell wall com-
"'."Osed of proteins and polysaccharidcs. Capsules are 1uau1::
-· sorne species. Cell wall polysaccharides (C-substance)
..:e somctimcs uscd in strcptococcal idcntification (see
.elo,v, "Variability").
ERNST L. BIBERSTEIN
Cellular Products and Activities of Medica! lnterest
Adhcsins. Streptococci produce a nun1ber of surface pro-
tcins that bincl to a varicty of extracellular matrix proteins
of the host (fibronectin, fibri11ogcn, collagcn, vitronectin,
lamín in). These "adhesins" have been termed MSCAAMMs
(microbial surface components recognizing adhesive ma
LI ix rnulecules). Sorne MSCRAMMs, specifically those that
bind fihrinogen (M protPin, othPr«, <;ee below, "Cell Wall"),
impartan antiphagocytic property to the streplococcal pa1-
ticle. "Coating" of strcptococcal ceUs with this host pro-
tein, is thought to result in the "covcring" of sitcs for con1-
plc1nent act ivation (and thus decrease opsonization) as
well as those recognized by serum proteins (collectins/
íicullír1s) that opsonize foreign partlcles. The hyaluronic
acicl c;ips11IP of S. pyog(ln<~~ is an ac.lhcsin (as well as impart-
ing antiphagocytic effects, see below), will1 affi11ily fu1
human epithelial ce lis via c:044, a hyaluron ic ;icicl-
binding glycoproteill. Whether the hyaluronic acid cap-
sules ot streptococci ot veterinary importance also act as
adhesins is unclear. Other adhesins are responsible for billd-
tng of srreptococci to host cells. Psa (for pneumococcal sur- -
face adhesin) is a lipoprotein found on S. pneumoniae,
S. equi ssp. equi, ai1d S. equi ssp. 1.ooepicJe111icus au<.l is rcspor1-
sible for adherence to cells lining the upper and low0r
airways).
Capsule. ~orne species ot strcptococci p roduce a capsule.
Streptococcal capsules are composcd of hyaluronic acid.
Hyaluronic acid, also a constituent of mammalian connec-
tivc tissue, is poorly antigenic and does not readily bind
con1plen1enl con1ponelll:. (allú i~ thus, antiphagocytic).
Hyaluronic acid capsules may also serve asan adhPsin («PP
abovc).
c·e11 Wa/I. 1·he gram-positive cell wall contains proteins
and polysaccharides that are of medica] intcrcst. Thc
llpotelchoic acids and peptidoglycan of the Riam-positive
cell wall interact ivith macrophage cells resulting i11 the re-
lease of µrui 11ílauuuatury cytuklnes. A flbrillar surface pro-
tein, termed th e 'tvf protpin (a MSCRAMM) imparts an-
tiphagocytic properties by binding fibrinogen (see above).
Pyrogenic Toxin Supcrantigens. Most of what is known
about the streptococcal pyrogcnic cxotoxins (SPEs) in-
volves those produced by Strcptococcus pyoxenes (a liroup A,
beta-hemolytic streptococcus affccting people, see Table
28.1 a11<.l lJelow, "Variability"). Strepcococcus pyogencs pro-
duces sevpr;il SPF.~: SPF.A, C, G-1, J, and Z (SPEF has bcen
159
160 PART 11 Bacteria and Fungí

Table 28.1. Streptococci of Veterina1y Jnterest

Species \411$!ill.e.l!i_G1'.9UR Ho~tSJl!!~Íes,;_~ff&tedª f,ri,~cipa!.Q¡sea~~s Remarks

Strep,tococcus. pyogenes A Humans, rodents (dairy cattle) Pharyngotonsillitis, pyoderma, erysipelas, Poststreptococcal immune
puerperal tever, rheumatic fever, sequelae. Morethan 50
glomerutonephrjtis (mastitis) serotypes (B)b
5. agafactiae B Oairy cattlee (sheep, goats) Mastitis S serotypes (o:, B, ¡)
humans' kats, dogs) Neonatal ínfectíons
·5.dysgalacti<w ssp equisimHis e Swine, horses (hum¡¡ns, dogs) Miscell<Jneous 5uppur<Jtivli conditions .g scrotypcs (B)
S. dysgalactiae ssp. dysga/actiae e Dairy cattle Mastitis 3 ~erofypes (o. 1)
S. equi ssp. equí e Horses "Strangles" 1 .serotype (13)
5. equl ssp.zooepldemlcus e liorses (towl, dogs, rumlnants, Secondary pneumonl~ rniscelJaneous 15 seroty¡¡~s (B)
lab animals. humans) suppurative conditions. genital and
neonatal conditions
Enterococcus spp. d D All species Many opportunistic infections, canine urinary Normál gut flóra (n, y)
tract infections, chicken septicemi~
S. porcinus E Swine Jowl abscesses o.serotypes (B)
S. canis G Carnivores Feline 1ymphadenítis, miscellaneous pyogenic (B)
co~tlltions ot'Clogs and cats, humans"
S. suis R (:type- 7.) SwinP (hum;m~-R) !iJP.onatai infections. septicemias. pneumonia React with.group D
s (=lyp~ 1) (humans- se¡Jticemia, arthritis, meningitis) antiserum (ex, y)
RS
T
unj:jroupable
S, uberis Cattle Mastitis (u;, y)
). pneumoniae Primates (lab animafs, cattle} Pneumoniá, septicemia (mastitis, calf More than 80 serotypes (a)
septicemia, meningitis)

• Parentheses indicate that species is affe<ted, but only rarely.

b Typ.e of hemo~¡sis on sheep blood agar.
'<:attle and hu!l1<ln strains are diíferent.
0ThlS'genus has been proposed based on nudetc acid studies. Not ali group Dstreptococci belong to it (e.9., S. bovfs, S. equlnus).
"lanme ana tiuman stram> appear d1tterent.

shown to be a DNase). Sorne streptococci of veterinary in1-
portance, S. equi ssp. equi for exan1ple, have been shown to
produce SPEs as well (see below). 1·he genes encoding the
SPEs are chro1noso1nally located. 'fhe SPEs are supcranti-
gens, and part of the systcmic symptomatology may be re-
lated to the cytokine "storm" that results fro1u the interac-
lluu uf 'f lyu1pllucyle cell receplu1s, 111ucrupl1uge::;, u!Hl
these toxins.
Miscellancous f'roducts. The streptococci produce a num-
ber oí proteins in vitro with "toxic" activity. 'l'hese so-
called toxins rnay or may not play a role in discase produc-
tion. and include a peptldase that degrades CSa (Scp for
streptococcal cysteine protease), the hernolysins responsi-
!Jle [ur l!ela-!1e111oly::.i~ 011 ~Jieep !1lo(JU agur piule:. (!>Lrep -
tolysins () anc1 S), hy;iluroniclilSP, DNilSPS (P.g ., SPEF, which
is a DNase), NADases, proteases (e.g., SPED which is a cys-
teine protease), and streptokinases (a fibrinolysin) . Strep-
tolysins O and S are 1nembra11e active substa11ces a11d rnay
serve in vivo to damage cell membranes (tl1ey are tnade
in vivo since antibodies are produced to tl1ese substances
by patients that have streptococcal disease). Streptolysin
<)and suilysin O (produced by S. suis) are cholt>sterol-
binding cy+.olysins (see for exan1ple, listerial listeriolysin
O, C:hapter 33; clostridial pertringolysin O, Chapter 36;
and arcanobacterial pyolysin, Chapter 29). Tl1ese toxins
bind to cholesterol-containing rafts in the eukaryotic cell
n1en1brane, forn1ing a pore, resulting i11 death of the cell.
Regulation of the Cellular Products of Medica! lnterest. Ex-
prcssion of virulcnce-rclated genes in S. pyogenes is regu-
Jated by at Jeast three global systen1s (whether these sys,
te1ns occur in other streptococci is unkno..,vn). 'fhe first is a
"growLl1 -µ1ta~t: relatt:u ::;ig11ul" Ll1aL i!> u1Hlefi11ell, !JuL cer-
tain genes (including those encoding streptolysin S and
DNases) are up regulated during stationary pl1asc, while
others (including those encodiI1g capsule, streptoki.nase..
streptolysin O, ru1d a protein called rnultige11e regulator in
group A streptococci or Mga) are up regulatcd during late
exponential phase of growtl1. The second syste1n involves
regululio11 by Mga. Mga ilself is regulaled by growll1 pl1ase
;is wPll ;is othPr 11nc1Pfínec1 enví ron mental cues. Mga up reg-
ula tes the ge11es encoding the M protein, and Sep. Pinally, a
third system involves the protein Cov (control of virulencel.
Cov down regulates all of the genes that were up regulatee
by growtl1 phase, except Mga. The gene encoding c:ov, likf:
Mga, responds to undeflned environmental cues.
Growth Characteristics
::>treptococci have tairly exacting growth needs best satiS-
fied by media co11taining blood or serun1.
 Chapter 28 Streptococcus and Entcrococcus 161

F 1G U RE 2 8. 1 . Streptococcus e4ui lUbsp. equi in pus from ceNica/ lymph node of a

horse. Gram stain, 1OOOX.

IL!S-~

F 1G U R E 2 8. 2. Strcptococcus equ i subsp. zooepidemicus in ce1Vica/ exuc.ldLt: fio111 ¡¡

111d1e. Gr¡¡rn stain, IOOOX.
, -
..
I

. •f
•
•

. "

.\fter overnlght incubation at 37QC, streptococci pro-
_ce clP<'IT colonies, usually less than 1 mm in diameter.
-~psulated forn1s, such a;) S. e4ui ::.::.p. cqui, µru<.Juce
_er, mucoid colonies.
'.lathogenic species grow bcst at 37ºC.
::..-emical Activities
-=ptococci are> cat;:il;:ise-negative facultativc anaerobes,
-·ing energy fron1 ferrueuli:ltiou.
tolerate these conditions (see below). Virídans strPptorocci
vary >Vith respcct to hcat and bile resistancc. Only S. pncu-
moniae 1s blle-soluble. ~treptococci tolerate U.U2o/o sodium
azide, which is used in streptococcal isolation media.
Palhogeuic :;treµtucucci are usually susceptible to pen1-
cillins, cephalosporins, m;:icrolides, chloramphenícol, and
trim cthoprim-sulfonamides¡ they a1e ofle11 resistant to
aminoglycosides. fluoroquinolones, and tetracyclinP.
Variability

= itance Rebecca Lansfield developed the serological method used

--hemolytic streptococci can survive in drícd pus for
~- TI1ey are 1<.llle<.1 ar .s.s·c ro 6o·c in 30 m inutes, and
-t>ited by 6 ..'i% sorli11m rhlori.de and 4()<l{i bíle (except S.
....<..rtiae), 0.1 % methylene bluc, and low (lOºC) and hlgh
- """ temperatures. Members of the genu~ P.ntf'cror.ocrus
to group ~trPptococci based upon serologic similarities of
cell wall polysaccl1aiide. Tliis rnethod Is called lansfield
serologic grouping. Groups are designatert hy r;ipital let-
ters (A to V) . Serologic subdivisions exist in most strepto-
coccal species except S. equi ssp. equi (see Table 28.1 ). Strep-
tococcus pyogenes has sorne 70 immunotypcs, bascd on M
162 PAitT II Bacteria and Fungi
and other proteins, while s. pneumoniae has over 80 capsu-
lar types. In S. pyogenes, change from rough (matte) to
:;111uull 1 acco111pa11ies loss of M protein a11d virule11ce. Cell
wall deficient forms (L forms) also occur.
Ecology
Reservo ir
Most streptococci of veterinary interest live commensally in
the upper respiratory, alimentary, and lowcr genital tract.
Transmission
'l'hose streptococci that <lH' ront;igious (S. equi ssp. equi, S.
porcinus, a11d S. agalactiae) are transmitted by inhalation or
ingcstion . sexually, congenitally, or indirectly via hands
and fomitcs.
Pathogenesis
Mechanisms. 1'he relation of streptococcal p roducts to
pathogenesis is Jargely spcculatlve, with the followiI1g
exceptions. The capsule of S pnP11moniaP. i.~ a proven viru-
le111.:e factor. M proleh1 is ar1 in1portant virulence determi-
nant, and antibody to it is protective. Other antiphago-
cytic cell constituents and cytotoxins of streptococci are
probable virulence factors.
Streptococci trigger inflarnmatory processes tha t lead to
suppuratio11 and abscess formation .
JJathology. Th.e basic p;ithologic proress rf'sf'mhle.s that
of :>la¡;hylucuccal iufeclion, i.e., ll1e typlcal les.ion is an ab-
scess, an inflammatory focus in which participating cells
have been destroyed by the combined cffccts of bacteria!
and intlammatory cell activity. ·rrus confrontatíon be-
tween Jeukocytes and microorganisms produces pus, a
mixture of host cell debris and bacteria, living and dead. In
an abscess, pus is surrounded by intact leukocytes and fib-
ri u :>lra11ú:.. U11Jess Lhe p us is drained, a fibrous capsule will
gra<lually be formed.
Disease Patterns
Equlne. 5trangles is a highly contaglous rhinopharyngitis
caused by S. equi su bspecies eq11i (hereafter referrecl to ;is S.
equl). Aft1.:r ue¡;osilion on lhe 111ucous n1embranes of the
uppe r respiratory tract, S. equi adheres to epithelial cells by
way of MSCRAMMs (M protein, fibronccti n -binding pro-
tein, tibrinogen-binding prote1n, l'sa) ana nya1uron1c ac1a
capsule. Adherence triggers internalization and subse-
quent localization in the :;ulJcµitlJelial :;paces. Celi wail
constituents as \.vell as pyrogenic exotoxins (SPFH ;incl
SPEI) iiüli.ale an acule inflan1n1atory response. Capsule, M
protei n, and Scp protect S. equi from opsonization and
phagocytosis. Streptolysins may act to dcstroy host cclls by
damaging their membranes. Systemic symptomatology
may be due to the superantigen effects of pyrogenic exo-
toxins (SPEH and SPEI). Other "toxlns" may contribute tu
the proccss by digesting DNA (DNases), fibrin (streptoki-
nas1::), a11u 11yaluro11ic acid (hyaluronidase). The disease is
marked by a serous or purulent 11a:;al uis1.:l1arge, tl!:- ~­
temperature risf', lor;il p;iin, cough, and anorexia_ -
gional Iymph nodes, abscesses develop, which ty-¡:-:•....._
rupture and drain within two weeks. Recovery follO\•-
ovcrDll mortality rate is under 2%. Complications L'1._
pyemic disse1nination to meninges, lungs, pericarG..
and abdominal viscera, or extension to the gut<_.._
pouches. Purpura hemorrhagk;a, a Ty¡;t: IIJ hypt'.r:.e1_
ity manifested by subcutaneous swellings, mucosa! ~
orrhages, and fever, may follow thc acutc discasc by_
three weeks.
Bacteria! pneumonia/pyothorax in horses is almo
ways associated with a beta-hemolytic streptococcus
S. equi ssp. zooepidemia1s (hereafter referred to ;is S. 7
dtmzicu:>) lJ1::iI1g tl 1e 1nosL con1n1011ly isolated. In addié
gram-negative microorg;inism (Actinobacillus is the :-
co1nn1on, see Chapter 13) is frequen tly found along,,-:-
zooepidemicus. 1'he infectious p rocess is endogenous.
microorganisms involved are part of the normal flon
the upper respiratory tract, which then contamin2-
compromised lung (e.g., following an episode of -
pneumonia). Streptococcus zuuepidernit us (plus lhe ol.
are deposited in the lune ;incl initiatf' or amplify a pree.-
i11g i11ilau1malory proccss (cell wall constituents, p:-
genic exotoxins). The intensity of the inflammatory -
sponse is not as extreme as with the response initiated
S. equi, nor are the constitutional signs as severe. Hov•e'
it is presumed that tl1e mechanisms involved in the pat;..
genesis are similar. Pyothorax !s probably an extenslor.
the pneumonic process just desr rihPrt 1.ikP pn<'11mo ni~
t.uvepiderrlicu.s is Lhe n1osl co111111on isolate, combined "~­
a gram-negative species. Unlike pneumonia, an obli~­
anaerobe (Bacteroides or Fusobacteriu1n are the most co=
mon, see Chapter 35) is frequently found as weli.
Genital tract diseases in horses are associated with
zooepidemicus, which is frequently associated with cerv11...
tis and metritis. Infections in newborn foals, which a.;
often umbilical infe<.tion:; (11av1::l 111, IJyoseplicen1ia, jo.
ill, poly;irthritis) disse.minate via the bloodstream to joir.-
and the renal cortex. The microorganis1ns originate fro:-
the genital tract of the dam (they are a part ot .her norm:..
flora).
Beta-hemolytic streptococci (S. zooepídemtcus Is th.
most common) are associated with a variety of misrf'll--
111.:ou:> l.:011uillu11s i11 h orses il1cluding osteomyelin
arthritis, ahscesses, and wounds. All are endogenous, wil: -
the infccting strain arising from the normal flora .
Swine. Cervical lymphadenitls of swine (jowl abscess) is -
contagious aisease affccrtng swtne. 1't11:; couu1uu11 1:. a:.:>o-
ciated with S. porr.in11s (prP.viously known as "Group !:.
Streptococcus"). The diseasc is analogous to strangles bu:
el in ically less drama tic and frequently not diagnosed un ti
slaughter. Its most damaging aspcct is carcass condem
nation.
Secondary pneumonias in swine are sometimes assoc!-
ated with S. dysgalactiae ssp. equisimilis.
Streptococcus suis, S. dysgalactiae ssp. P.q11i~imilis ;ind
:>tre¡;lucocci belo11gh1g lo Groups L and U cause neonata.
septicemia, pneumonia, arthritis, and meningitis. The
exotoxin, suilysin, a cholesterol-binding cytolysin (see

=bove, "Cellular Products and Activities of Medica)
m terest"), is produced by s. suis. lt has been proposed that
~is cytotoxin is active in vivo and may account for sorne
of the tissue damage associated with this disease.
, uminants. ThP Jp;:i<1ing ;:igpnt of strPptoc:occ;:i l m;:istitis is
S. agalactiae. Less frequent causes are S. dysgalactiae ssp.
ivsgalactiae (hereafter referred to as S. dysgalactiae) and
S. uberis.
)ogs and Cats . Secondary pneumonias affecting dogs and
_at:; are :;u1netiII1e:; a:;:;uciateJ with S. cuni~.
T..ahoratory c.rits oc.c.risionri lly experienc.e c.ervic.a l lym-
:-hadenitis fro1n which S. canis is isolated (sec fig 67.5). ·rhe
:.-ondition probably arises endogenously being precipi-
tated by an unkr1own event.
Streptococcus canis is associated w ith septicemia in new-
born puppies.
Streptococcus canis has been associated wiLh a Loxic
shock- like syndrome a11d necrotizing fasciitis in dogs. No
·irulence-associated traits have been defined as they have
.:or similar conditions seen in humans affected w ith S. pyo-
genes.
'rimates. Streptococcus pneumon iae is a leading cause of
!Jne un1onia, septicenlia, and 111eningitis i11 pri111ales.
i>neumococcal pneumonia in monkeys runs an acute
course with high mortality rates. '!'he lesions are those of a
!lbrinous p leuropneumonia. Recent shipment and viral
.ilfection are common antecedents.
\Uscellaneous Species. Cervical lymphadenitis in guinea pigs
:s caused by S. zooepídemicus ssp. zooepidemícus.
Septicen1ic disease in fish from frPshw;:itPr !H}11ac11lh1rf'
farn1s and fron1 salt-\'Vater environn1ents has been asso-
ciated w.itl1 S. iníae, a beta-hemolytic streptococcus with-
out a Lansfield designation. People handling (cleaning/
:iecropsy) affected fish are at risl< ot developü1g celJuli-
tis, er1docarditls, or arthrit is presumably following self-
!!10culation.
Septicemic clisease in seals is associated '"'ith S. phocae, a
Jela-hen1oly Lic slreplococcus tha t reacls lo Lansfield
zroup a11tisera C and F.
Septicemic disease and dermatitis in opossums is associ-
ated with S. didelphis, a beta-hemolytic streptococcus with-
out affiliation ivith kt1own Lansfield groups.
: pidemiology
Healthy individuals n1ay carry all the streptococci dis-
cusscd, and many infections are probably endogenous and
stress-related. Neo.natal iníections are commonly mater-
::!al in origin.
Strangles and porcine lymphadenitis are cont agious
diseases preferably affecting young ani1.nals (past infancy) .
Streptococcus equi and S. porcinas are spread by co11ta111i-
nated food, drinking water, or utensils and by recovered
animals, which may remain clinically healt hy shedders for
:nonths. Mili<ing equipment, unskilled attempts at intra-
m.ammary .m edication, and unsanitary milking practices
often spread Streptococcus agalacriae among dairy CO\-VS.
Chapter 28 Streptococcus and J:,nterococcus 163
Animal streptococci have limited public health signifi-
cance. The Group B streptococci that cause disease in
human infants are apparently distinct froni. bovine st rains,
but infections vvith S. zooepidernicus have been traced to in-
fected mHk, and S. suis (Type 2) has caused serlous infec-
tions in swinP h;:indlers. The Gro up G streptococci affect-
ing dogs (S. canis) are apparently different fron1 the Group
G streptococci affecting human patients.
lmmunologic Aspects
lmmune Mechanisms of Disease
Human poststr.eptococcal diseases (rheumatic fever, acute
glomerulonephritiS) are attributed to immunopathogenic
mechanisms. Similarly, equine purpura hemorrhagica fol-
lowil1g strangles is probably inHnune co111plex- 111ec.liateJ.
Recovery and Resistance
"!'he main defenses against streptococcal infections are
phagocytic, and the antiphagocytic M protein elicits pro-
tective antibodies. Animals recovered from strangles and
cervical lyn1phadenitis are at leasl Leu1porarily iJ11111uue tu
reinfection.
Polysacchar,idc capsules of S. agalactiae and S. prieu"
moniae evoke the forn1ation ot opsonizing antibody. In
streptococcaJ pneumonia, their appeara11ce determines re
covery from infection. In bovine inastit is, no useful in1mu-
nity develops : Cows, unless treated, remain infected.
Ex11eriu1eulal eviúe11ce :sugge:st:; Ll1at anticap:;ular lgGz.-
type antibody is protective.
Ali immunity is serotype-specific.
Artificial lmmunization
A whole-cell bacterin an.d an Jvf protein vaccine are avail-
able for vaccination against strangles. Neither is uniformly
pffpctivf', anrl oftPn Plicits loc;:il rP<"lrtions ;:it t hP injection
site. An intranasal avirulent live vaccine that stin1ulates es-
sential local antibody responses appears promising.
Feeding live avirulent cultures has produccd immunity to
porcine jowl abscesses.
Laboratory Diagnosis
Sample Collection
Aspirates from unopPnf'd IPsions, in stPrilP syringPs or ster-
ile containers, are preferred . S'l>vabs in transport n1edia are
acceptable. Mili< is collected into containers under sterile
precautions.
Direct Examination
Smf.ars of exu<1atPs o r sediments of suspect fluid s are fixed
and gram stained. Streptococci appear as gran1-posilive
cocci in pairs, short chains, and in sorne instances very
long chains (typically sccn in pus aspirated from cervi-
cal lymph nodes of horses i11fected with S. equi- see Fig
164 PART 11 llacteria and f.ungi

28.1). SlrevLococci l1ave a teuueucy to lose their gram-
positivity and sometimes stain wt>akly gram-positive or.
gram-negative.
Culture
Exudales, 111ilk, lissue, urlut:, Lra11:;tra<.J1eal a:spirate:s, auu
cerebrospinal fluid are cultured directly on co•v or sheer
blood agar. Jncubation at 37ºC in 3% to 5% C0 2 is prefer-
able_ Streptococcal colonies, smooth or mucoid, will ap-
sheep or bovine blood agar plate. At righr ~ _
this line, and approximately 0.5 cm from ~
pect S. agalactiae is inoculaled. Afler i11culJa-<~;,,,.-­
molysis by CAMP-positive bacteria v.ii: --
hanccd in thc bcta-toxin zonc (fig 2 8..3
combined action of these two toxins on sh:::---
bovine blood agar 1)roduces Iarger and cleare:: ~
of l1ernolysis than eitl1er agent alone (see Fi; :_
This reaction has diagnostic value.
2 . Bacitraci11 se11sitivily. Bacitrac.u1 disks (O.Ch ~:::

pear 111 18 to 48 l1ours. It is sometimes difficult to distin-
guish alpha frorn beta hemolysis. Intact erythrocytes
remai11 adjace11t to alpha- but not to beta-hemolytic
culu11it:s. Beta-hernolytic :strain:s consister1tly lyse rec.l cells
in hloocl hroth; alpha-hPmolytic strains of animal origin
ge11erally do not.
Identification relies on a combination of classical tech-
niques (detcrmination of Lansfield grouplng and bio-
chemical tests), and molecular techn..iques (e.g., determL-
nation of the sequence of DNA encoding tbe 16S
ribosomal DNA, or using species specific prin1ers in the
polymerase ch.ain reaction). Commercial klts are avai.lable
for both purposes. Other useful diagnostic tests includc
the following:
l . 1"hc CAMP phcnomcnon (namcd aftcr Christic,
Atkins, and Munch-Fetersen) reflects hemolytic
synergisn1 between staphylococcal beta toxin and a
S. agalactiae toxin (CAMP protein sometimes re-
ferred to as cocytolysh1). A beta toxin-producing
stapl 1ylucuccus is iI1oculateJ a cross the equator of a
inhibit growth of S. pyogenes oo blood agar. __
action is not cntircly co.n sistcnt or specific.
3. Hile esculin agar tests the ability of 4Uo/o bi.'..-.-
tolerant bacteria to hydrolyze esc'Ulin, a char-..,..--
tic of those belonging to Lansfield Group D.
4 . Optochin sensitivity. Growth of S. pne11111oni..<=
11ol oll1e1 alpl1a-l1e111olytic slreplococci, is int>-:.
around disks impregnated witb optochin (eth.
drocuprcin hydroch loridc).
Treatment and Control
Loca!iL:ed SUJJJJUralive co11diLio1.1s are drai11ed of JJU:S.
For systemic treatment, penicillin G and ampicillb
cffcctivc on most bcta-hcn1olytic and virida11s streE- -
cocei. Cephatosporins, chloramphenicol, and trimec_
prim sulfas are alternatives. Streptococcal endocarditis
treated with combined penicillin and gentamici.n. Susc_-
tibility to fluoroquinolones is unpredictable. Streptococ:::::..
toxic shock a11d necrotlzing fasciitis are treated with pe;:.c

F1G U RE 2 8. 3 . CAMP phenomenon on bovine blood agar. The dark Jine across the
field is growth of Staphylococcus aureus surrounded by a zone of beta-toxin activity.
Growing in fines at right ang/es to it are, left to right: a viridans streptococcus, Streptococcus
agalactiae, another viridans streptococcus, Streptococcus equi subsp. zooepidemicus, and
Streptococcus agalactiae. See text for discussion of CAMP phPnnmPnon. (Photograph courtesy
of Dr. Rich<ird W<ilker.)

d llin G and clindamycin (clindamycin dccrcascs toxin
¡>roduction, and p en1cill1n Gis bactericida!).
Penicillins (intrarnammary) are cffectlve for treating
.11<1:.liti:; <.lue to S. asalactiae and most othcr streptococci.
~o r th<' many avail;ihlP ;i ltPrnatives, a specialty text should
be consulted. lmportant aspects of 111astilis conL1ol lit: iu
~he arca of sa11itation and herd managcment.
For strangles, it is most beneficia! to trcat cxposed and
affected animals prior to absccss for1nation and to con-
-::inue treatmcnt past the febrile stage. lnappropriate or in-
.ldequale Ll 1eraµy uf :;tr;1ngles is blamec.l for prolonging the
lness and causing "hast;i rcl strangl p~" (wic.lesprcad abscess
ormation vvith systcmic manifestations). Populalions al
- sk may be vaccinatcd. Affected or suspccted horses
hould be rigorously isolated.
f NTER OCOCCUS
..nterococci were once classified as Group D streptococci.
.. 111ike 1nost true members of the genus Streptococcus,
·nese mic:roorgani~m<; possess phenotypic traits (resist-
:.nce to salt, bile, methylene blue, and giowlli al increa:;etl
-emperatures) tl1at set them apart. Molecular genetic:
..nalysis showed that thcy are uniquc, and prompted the
c:-.tab lishment of a n cw genus, Hnterococcus. 'l'here are 28
pecics in the genus, most of which Iive in the intestinal
~ac.:ts uf rnammals and blrds. ·rhey are mainly oppor-
- nists that infect compron1ised sites to produce disease.
~e1e are su111e (E. durans, E. hirae, and E. vfllorun1) that
.'.!Ve heen assoriatP<I with intestinal disease in neonatal
-nimals (piglets, foals, calves, puppies, killcns) as well a::.
_.:ult dogs and cats (though only the isolates from p iglets
lve heen subjcctcd to thc gcnctic analyscs needed to
~fl nltivc1y determine the 1dent1ty or the 1solates). Ente-
- .cocci are either alpha- or gamma-hemolytic on sheep or
.... le re<.l !Jloo<.l cell-contain ing blood agar plates (see
'lve, "StrPptororr11c;").
:escriptive Features
: ·phology and Staining
-rerococci vary from spherical to short bacillary cells,
ut 1 µm in diameter. Division occuIS in one plane, pro-
'"'ng pairs and chains.
• ~cture and Cornposition
-erococci have a typical gram-positive ccll wall com-
~d of proteins and polysaccharides. Polysaccharidc
ules are made by sorne spectes. Most enterococci pos-
• .ancefield Group D carbohydrate.
== Jfar Products and Activities of Medica! lnterest
~.::h ot what is known regarding cellular products and ac-
ctes of medical intetest comes from study of E. faecalis
.. E. (aecium, tt1e most common enterococci producing
~c;p in human patients. Presumably, sorne (if not all) of
Chaptcr 28 . Strcptococcus and Enterococcus 165
what is k11own about these tvvo species also applie~ Lu ll1e
other m embers of the genus.
Ag,gregation Substancc. Aggrcgation substance is a surface
protein that promotcs adherence ot enterococci to each
other (to form aggrcgates) and to epithelial surfaces (an
a<.lhesin).
r.ap.<:ull?. Snm<' cnterococci produce a polysaccharide
capsule. l'he capsule discourages assu<.:i<1liuu witl1 pha-
gocytic cells by interfering with complement d eposi-
tion and in1parting n relat ivc hyd rophilicity to the mi-
crobe's surface. It is unlikely that the capsule is made
while enterococci associate with enterocytes in the intes-
Li ual tract.
r.ell \l\fal/. F.ntPrncocci posses a typical gram-positive cell
wall. Cell wall peptidoglycan and liIJolt:i<.:l10ic acic.ls init1-
ate an inflammatory response follo~ving their interartinn
with macrophages.
Cytolysin. Cytolysí n is a cytotoxin whose mechanism of
cell destruction is not understood. Jt is also a hemolytic
Loxi11, lysir1g human and horse red blood cells (but not
sheep or hovi nP rP<I hlood cells, red blood cells most often
used in blood agar platcs). Cytoly:.i11 protluciion is under
the control of a quorum-sensing system .
Extracellular Supcroxidc. Sorne cnterococci secrete a su-
peroxide that appears to affo rd sorne protection against
killing by phagocytic cells.
Clclulinuse. Sorne enterococcl produce a gelatinase that
is marginally relatPrl tn virule11ce.
!ron Acquisitio11. Enlerocucci protluce a hydroxamate
type of siderophore in response to low IPvels of iron.
Growth Characteristics
Enterococci prod uce clear to gray colonies 1- L. mm in di-
ami>ti>r (either alpha- or gamma-hcmolytic) after over-
n ight incubatio11 al 37ºC, Lhougl1 ll1ey will grow between
10 to 45ºC. Entcrococci will grow in 6 ..'iºAi soclium chloride,
40% bilc, and in 0 .1 % methylene blue.
Biochemical Activities
F.nterococci are catalase-negative facu ltativc anaerobes,
deriving e11ergy í1on1 feriue1rl<1tiuu.
Resistance
Enterococci are hardy microorganisms ablc to survive in
the environmcnt for extended pcriods of time. ·rhey are
able to grovv in 6.So/o sodium chloride and 40% bile, O.lºA>
n1cthylene IJlue, <111tl luw (lOºC) and hlgh (45.C) tempera-
tures. 1'hey are in tri nsic.a lly rPsio;t;in t to the beta-lactan1 an-
tibiotics (includi ng cephalosporins and pen icil li11a:.t:-
rcsistant penicillins), aminoglycosidcs, clindam ycin, fluo-
roquinolones, and the trimcthoprim-sulfonamides (they
are effectlve scavengers ot thymidine found in exudates,
thereby bypassing the effect of the trimethoprim-sulfon-
arnic.les). They are able to acquire resistance to high levels
of hPta-lactan1s, high levels of aminoglycosides, the gly-
copeptides (va11co111 yci11), Letracycline, erythromycirl, flu-
o roquinolones, rifampin. and c:h lora mphPnicol SPli>rtion
of vancomycin-rcsistant strains by the gro,'\'th promoter
166 PAKT 11 Bacteria and Fungi
avoparcin is discussed below (see below, "Epidemiology,"
and Chapter 4).
Ecology
Reservoir
Enterococci live in the intestil1al tra1.:1. uf 111a1nrnals and
hirds as part of the normal flora of these species. Whether
the enterococci associated with "primary" disease, H. du-
rans, E. hirae, E. víllorurn, as opposed to opportunistic-type
discase, are inembers of the normal flora, is unknoiv11.
Transmission
Enterococci that are associated vvith opportunistic disease
are part of tl1e norn1al flora of the 11ost. It is unknown
whether this is true for E. durans, E. hirae, and E. villorum.
Pathogenesis
Except for E. durans, E. hirae, and E. villorum- associated dis-
ease, endogenous enterococci infect a comprornised site
(e.g., urinary bladder, moist externa! ear canal, catl1eter).
The cell wall peptidoglycan and Upoteichoic acids initlate
an infla1nmatory response. Capsule, cytolysin, and super-
oxide puteut<1te the i11íla111111alory processes.
Enterococcus durans, E. hírae, and E. villorum associate
with the villi (fronJ tip to crypt) of thc :;n1all intcstinc of
affected anin1als (see below). Associated diarrl1ea does not
appear to be due to an enterotoxin or epitl1elial cell
damage.
Oisease Patterns
Dogs and Cats. Otitis externa is usually the result of infec-
tion (bacteria anú a yea:;t, Mulusseziu, ~ee Cl1apler 45) oí a
con1pron1i$ed t=>xternal ear canal. 'fhe bacteria involved are
usually an environmental species (e.g., Pseudon1011as and
Proteu_s, see Chapters 20 and 7, rcspectively} ora member ot
the patient's normal flora (e.g., Enterococcus, Staph}'lococcus
interrnedius, see Chapter 27).
Enterococcus is a common isolate frorn do8S with lower
uri11ary tract infections (see Fig 74.2B).
F.ntf!ror:ot:t:11s spp. (P. durans, E. hirae, E. villorum) are asso-
ciated with diarrhea in puppies, kittens, and adult dogs
and cats.
Aln1ost any compro1niscd site may be contaminated
\-vith an enterococcus.
Horse. Enterococcus spp. (H. durans, E. hirae, E. villorun1) are
associated with diarrhea in foals.
Entcrococcus spp. should be expected in any co11dltloa
th.at resutts from co11tamination of a compronused s1re
witl1 fecal material (e.g., street nail/sole abscess, ~.vound) .
Cattle. Enterococc;us spp. (E. durans, E. hirae, J::. villorurn} are
associated with diarrhea in calves.
Enterococcus spp. should be expecteú in any conditlon
that results from contamination of a comprornised site
with fecal material (e.g., wuu1H.l).
Swine. cnterococcus spp. (E. durans, E. fzirae, E. villon1m
associated with diarrhea in piglets.
Enterococcus spp. should be expected in any 1.:un<ls
that results fron1 contamin;:ition of ;:¡ compromised
with fecal n1aterial (e.g., wound).
Miscellaneous Species. Enterococcus spp. (E. durans, E. hirlk -
villorum) are associated with diarrhea in infant rats.
Epidemiology
Most of the infectious processes fro1n whic·h enteroo......
are isolated are üue tu cu11ta1ni11alio11 w ilh n1en1bers o~
normal flor;i. ln hospital settings, nosocon1ial spread (e._
fonlites, hands of care giver~, soles of shoes) to coro¡:;~ -
.m ised si tes is a11 important issue. 'fhe epiden1iology ot ·-
diarrhea-associated enterococci (.li. durans, E. hirae, E. iil -
rurn) is unk11own.
Vancomycin-resistant strains of er1terococcl are a s.e-
ous problem in huu1a11rucdicj11e, beca use n1en1bers of t;....
genus (f~sper.iall y /?. faecalis, a11d E. faecium) are major co-
trlbutors to nosocomially acquired disease. Entcrococd.;....
a group are very resista11t to antimicrobial agcnts SP--
abovc, "Ilcsistancc"). Vancomycin (a glycopeptide anr~
otic) is one ot the few effective drugs for treatment of su"-'-
infectious processes. Vancomyci n-resistant strains of ef.!~,,,­
rococci arose in Europe after the initiatitn1 uf feeüiug -
another glycopeptide avoparcin (;l growth prornoterJ -
fuu<.1-¡;r oduci ng ani1nals. ·rhough at first the vancomyc:í:--
rcsistant strains were limited to the intestinal tract of a.::..-
mals fed this antibiotic, thcy soon spread, as did the gene
e11coding vancomycin resistance lvanA), to the human ::-.-
testinal tract. In the United States (where avoparcin \ \-a-
not allowed), injlH.1icious use of vancomycin in hum"-::
hospitals resulted in the same effect, i.e., an increase in se-
le1.:tive pressure rcsulliug i11 a11 increase in coloniz:atlon h-
van co m yci n -resistan t enterococci (especially in h osp i tals
Laboratory Diagnosis
Sample Collection
Aspirates from unopened lesions, in sterile syringes or st~­
ile containers, are preferred. ~wabs in transport media a:-::
acceptable. Urine samples are obtained by antepubic cysro-
centesls (bladder tap), catt1eri.z.atiuu, ur u1idslrea1n calch.
Direct Examination
Stained (C~ram's or Romanovsky-type such as Wright's o:
G icn1sa) sn1ears of exudates are examined. Urine can be ex-
an1ined unstained or stained ((~ram's or l{omanovsb.1--
typc). Histopathologic sections of small intestine obtaineC.
from an1ma1s w1th associateCl enterococcal enterltls are
needed to demonstrate characteristic adhcrence of cocc;f
forms tu villi.
Culture
Samples arP. streaked onto the surface of blood agar plates
and incubated at 37ºC overnigl1t in air (bccausc entero-

~e facultative anaerobes, any atmosphere will suf-
?aterococci produce clear to gray colonies 1- 2 mm in
ili&I:::c':er (cithcr alpha- or gamma-hc1nolytic). Prcliminary
...::.::-..:ication entails t esting for the production of catalase
::;::~~=-e), and ability to grow in 6.5% sodium cl1loride
-=- b1le. Most tsolates can be speclated by uslng com-
y Treatment options of d1arrheal disease associated wlth
!IE=-;.·y ;:ivailable kits, seqnencing the gf>ne f>ncoding the
-;;-osomal RNA, ora combination of both.
-:~ent and Control
=-"QO of the underlying condition is t he most impor-
"" .iect of t reatment of most situations from which en-
"'::"::::.m· are isolated. ln son1e il1sta11ces, re111oval of tl1e
~,_.::.:nise is enough to in itiate host-elimination of en-
:;"";"'.'=:.~ (becausc thcy do not havc potcnt virulcncc dc-
-..:nts). This is an important concept because entero-
~d to be quite resistant to antimicrobials (see
~~-- -Resislance"). ·roo, in lhose infecLious ]Jrocesses
~ i; a polymicrohic etiology (along vvith t he compro-
":.!ccess can be achieved by correction of the com -
::::::=::.::e 2nd antimicrobic therapy aimed at the other (usu-
::e susceptible) microorganisms.
--ococci isolated from the Iower urinary tract of
~ usually susceptible to urine concentratiol1S of
.e::::: · >: 'ln-clavulanale, chlora111]Jhenicol, a11d Lelra<.:y-
==~ ..:.~ though most urinary strains of enterococci test
iC!i::;::i::'b!e to trimethoprim-sulfonaroides, care should be
¡¡....,;:=. - interpreting in vitro test results because of the
--~.:: -:::_e-savaging ability of this group of microorgal1isms.
----ococci associated with otitis externa in dogs are
Chaptcr 28 Streptococcus and Enterococcus 167
dealt with by correcting the underlying compromise and
use of any of the otic preparations tl1at contain an antimi-
crobial (topical concentrations of most i11corporated an -
timicrob ics usually far exceed the ininimal inhibitory con-
centration needed to inhibit gro,AJth of enterococcí).
F.. d11rans, F.. híra(!, or F.. villorurn i'lre undefi.ned .
ABIOTROPHIA AND GRA NULICATELLA (NUTRITIONALLY
VARIANT STREPTOCOCCI)
Members of the genera Abiotrophia and Granulicatella "l>vere
discovered as sn1all colonies g iowing as saLelliLes arou1 1Ll
other bacteria] coloni.es when samples obtained from nor-
mal human m ucosa! surfaccs (eycs, genital tract, mouth,
respiratory tract) were inoculated onto blood agar plates.
'fhe bacteria comprising t hese colo11ies were gram-
]JU:;itiv«::, <.:atalase-r1egative cocci requiring vitamin B 6 for
growth (satisfif'rl hy t hf' ;irlrlition of O.OO?ºA:i pyridoxal hy-
drochloride to media) . These microorganisn1s were provi-
sionally tertned "nutritionally variant streptococci," but
later placed into the genera Abiotrophia and Granulicatclla
based u pon the sequence of the gene encoding the 165 rí-
bosomal RNA.
Cliui<.:ally, u1eu11.Jers uf tl1ese genera have been isolated
frorn the hloodstrP.am, ;ihsr.P.SSf'S, dP.n t;il pl;:iq11i>, joints,
corneal ulcers, and cardiac valve vegetations of huma11 pa-
tients. They have been isolated from the genital tracts, res-
piratory tracts, absccsscs, and cycs ofhorscs and ruminants.
•
 Arcanobacterium
DWIGHT C. HIRSH
Me111bers of lile ge11us Arcunobucleriurrt art:: µl1:::u111u.rµhic,
non- spore-forming, nonmotile gram-positive hacilli. In
shape, 1ne1nbers of this genus are "dipht heroids" (see
Chapter 31 ), an·ct many were at one t i1ne inclu ded within
the genus Corynebacterium (e.g., C. pyogenes) as well as the
genus Actinomyces (e.g., Actinomyces pyogenes). Sequence
analysis of the gene encoding t he 16S ribosomal RNA re-
vealed that the ge11us Arcunubucleriurn is tl1e aµµruµriatt:
plac.c> for t h ese m icroorgan isms.
Of the five n1embers of the genus, Arca11obacteriu1n pyo-
genes is the main species of veteriI1ary importance, being
involvcd in suppurativc proccsscs of rurninants and swinc.
Otl1er arcanobacteria of note include A. phocae, A. pluraní-
malii11n, al1d A . hippocoleae isolated from the respiratory
tract of seals, the spleen of a harbor porpoise and the lung
of a deer, and from an equlne va gin.al. exudate, respectively.
ARCANOBACTER/UM PYOGENES
Descriptive Features
Morphology and Staining
Arcanobacterium pyogenes is a small, gram-positive, pleo-
morphic rod (0.5 µIn by up to 2 µm; Fig 29.1) that lacks
capsules and metacl1romatic granules. The instability of
the gram-positive reaction is comparable to that of strep
tOCOCCi.
Structure and Composition
Its cell v.1all peptiduglyca11 coutaius lysiue, rl1arnr1ose, anll
gluc.ost>. l Jn li kt> mt>m ht>rs of tht> ge.nt>r<i l.nrynehar.teri11m
and Rhodococcus, it does not contain n1ycolic acids.
Cellular Products of Medica! lnterest
Cell Wall. The lipoteichoic acids and peptidoglycan of the
gram-positive cell wall interact with macrophage cells, re-
sulting in the release of proinflammatory cytokines.
Exuluxins. Arcunobucleriu.rn pyugenes prolluces a 11u1nlJer
of prot eins with toxic. propt>rties:
l. Pyolysin O. PLC) (for pyolysin O) is produced by ali
straiI1s uf A . pyugi:ru::> (see alsu, streµtucuccal streµ -
tolysi n O, \.hapter 28; listerial listeriolysin O,
Ch.apter 33; an.d clostridial perfringolysin O,
168
ERNST L. BIBERSTEIN
Cl1aµtt::r 36). Jt is a µore-for1uing, cholestC'
dependent cytolysin that is a major virulence ee
minant for this species (mut ants deficient i:: --
protein have reduced virulence; antibodies to'.:
protective). PLO binds to cholesterol-conta!:-~-·
rafts in the eukaryotic cell membrane, form:::_
pore, resultlng in death of the cell. PLO is a.S-
sµo11sible Cor lhe he1nolysis observed whe.n -~- _
genes is grown on blood containing media. --""-,_
PLO acts in vivo is not known, but it is assumed _-....
it damages host cell membranes.
2. Ncuraminidascs. Arcanobactcrium progenes proC-
at least two neuraminidases, Nans (for N-acet\~
raminyl hydrolases). Hovv the Nans work .in '~
not clear, but they appear to be involved i.n a:...
sion of the rnicroorganisms to host cell.s.
3. Miscella11eous products. Severa! proteins witl-__
teolytic activity in vitro have been demonstrarr-=..
well as a DNase. What roles, if any, these othe:: •
ins" play in the pathogenesis of disease a:c:
k11own.
Growth Characteristics
Arcanobacteriu.m pyogenes is a facultative anaerobe :e::~
ing cnrichcd media and up to 40 hours to produce
cernible colonies on blood agar. Growth occurs opu._--....
·_
at 37ºC and produces transluce11t, he1nolytic colon-=<'
to 1 mm in size.
Biochemical Activities
Arcanobactcrium pyogcncs is catalasc-ncgativc, fcrmcz_
tose, and digests protein (gelatin, casein, coagulated se;_
Resistan ce
Arcanobacteriurn pyogenes is susceptible to dryinÉ
(60ºC), d isinfectants, and beta-lactam antibiotics. ~
sistant to sulfonamidcs, and increasingly so to tetrae"':
Ecology
Reservo ir
Arcunubucleriurrt pyu¿)t:rtes is found on rn ucous rneu... _
(including the rumen and stomach) and skin of Sl.::-'.l~
ble species.

: , GURE 29.1 . Arcanobacterium
=yogenes in pus from sheep lung. Gram
~in, 1000X.
--=nsmission
_-ist infections are
probably endogenous. In "summer mas-
:..~s." cow-to-cow spread, aided by flies, is thought to occur.
-=:.-ogenes1s.
-~iuinisms. Arcanobacteriurn pyogenes causes suppuratlve
- _:cesses, usually complicatecl by other potentially pa tho-
~.Jc con1111ensals, especially non-spore-forn1ing anaer-
-.e-; ( Rar.tr.rnidr.s, Pusnhar.teriurn, Porphyrnrnonas, Prevotella,
-.ostreptococcus, see Chapter 35). Pyolysin O (PLO) is a
~ or virulence determinant (supported by the finding
-~- antibodies to PLO are protective, and mutants defi-
--:t in PLO are avirulent). Deposition of A. pyogenes (fro1n
~ ;itiguous surface or from the iin1nediate environn1ent)
.;._,- a normally sterile ~ite initiate~ au iuflauunatury re-
--~,p (arlhPrPnc.P., hy way of Nans, cell wall constituents,
::;,on of PLO). The exudate (pus) consists of bacteria, neu-
... ~il s (live or dead), and host cell debris.
Patl1olo3y. The lesions are abscesses, empyemas, or pyo-
---.-_uJomas. Abscesses are often heavily enea psulated.
~ offensive odors are contributions of anaerobic parti-
~t:; .
- -:-;se Patterns
.=-'E. Arcanobacteriun1 pyogenes is involved in most puru
- 'nfections of traumatic or opportunistic origins;
-~~-.:i may be local, regional, or metastatic. Common lo-
__,.___~· ~iu11~ CJre the luug, pericCJrtliurn, entlocCJrtliurn,
___,_:ti. p eritoneum, liver, joints, uterus, renal cortex,
=::::.. bones, and subcutaneous tissues. In other suscepti-
;pecies (sheep, goats, wild rurninants, S>vine) similar le-
.:;s may be found.
--~mobacterium pyogenes causes abortion and mastitis in.
_ ::.e. "Summer mastitis" is a communicable disease
'--""'ng pdsture<.l dairy cattle <.luring their dry peri o<.!. lt often
...M"" a ilPstn1ctivP conrsP, ca11sing ahsrPsS formation anrl
Chapter ?.9 .Arcanobacteri11m 169
J l: .
•
sloughing. Bacteria implicated in its etiology include A . pyo-
genes, Streptococcus dysgalactiae ssp. ctysgalactiae (see Chapter
28), ancl non- sporP-forming ;inat>robes (set> C haptt>r 35).
Epidemiology
Because ,4. pyogenes is a permanent resident of susceptible
species, disease prevalence is sporadic and governed by
precipilali11g slress or trau1na.
"Sum1nf~r mastitis" is most prevalent in northern
Europe. Jt .is spread by flies attracted to traumatized teats.
lmmunologic Aspects
Itnmune r~~sponses to A. pyogenes are not '"'ell understood .
Infection or vaccination confers no useful resistance. The
usefulness of pyolysin as a vaccine remains to be demon
strated, but toxoids produced from PLO are protective in
laboratory anima1s.
Laboratory Diagnosis
Gram-stained smears fro1n tissues or exudates reveal gram-
µo:;ili ve µleu111orpl!ic ru<.l~ (~ee Fig 29.1) ufte11 uüxe<.l w itl1
other bacteria. Material suspected of containing A . pyo-
genes is cultured on blood agar. Routine methods are used
to identify this microorganism. Primers designed to am-
plify A. pyogenes specific DNJ\ by the polymerase chain re-
action have been described.
Treatment and Control
Incision and drainage of abscesses are essential. Although
many antibiotics, including all penicillins, are active in
vitro, medical trean11ent alone is usually dlsappolntlng
cine to inadequate clrug delivery to the lesions.
 Bacillus
DWIGHT C. HIRSH
Members of the genus Badllus are gram positive, sporc-
forming, facultatively anaerobic rods that typically in-
habit soi 1and "''a ter. So me species cause diseases of insects.
·r111:: u11ly Lu11::.i:;Le1n JJC1tl1uge11 uf vertet>rates, 1ncludlng
humans, is B. anthracís, the causativf> agf>n t of anthrax
Bacillus anthracis is a close relative of D. cereus that causes
caninc and human food poisoning.
8 ACILLUS ANTHRACIS
Descriptive Features
Morphology and Staining
Cclls of B. anthracis are grarn-positive, nonmotile, roughty
rectangular rods with squarc cnds (about l µm by 3 to 5
µ111). Cliai11:; uf ruds are common. Spores within the cell
cause no swC'll i ng. A caps11 le is forroed in vivo, o r l.1nder ap-
propriate condition in vitro.
Cellular Composition
Thc capsule consists of a v-glutamyl polypeptide. Tl1e cell
wall is largely polysaccharide. Covering the cell wall is a
proteinaccous paracrystalline structurc (S-laycr). When B.
anthracls produces a capsule, the S-layer lies betwee11 it and
the rest of the ccll wall. No virulencc propcrties have been
ascrlbed lo lhe S-Iayt:r ur it:; ¡.¡rute1n:;.
Cellular Products of Medica! lnterest
Capsule. ·rhe vegelalivc furru uf B. unthrulis produces a cap-
sule composed of a o-glutamyl polypf'pti<1f>. Thf> genes en-
coding this structure are on a plasmid called pXOZ. The ex-
pression ol the genes encoding the capsule are under
control of two regulatory gene products, AtxA and AcpJ\
(for anthlax roxin activator and a11thrax capsule activator,
respcctlvely) which respond to cnvironmer1tal cues (see
bclow, "Ilegulalion of Celluldr Pruuu<.:t:; uf Mellical Inter-
cst"). 'l'he capsule enables thc vegetativf> form to avoi<I
phagocytosis. 'fhe genes encodiog AtxA and AcpA are on
the plasmids, pXOl (see below, "Lcthal Toxin") and pXOZ,
respectively.
1oxins. A number of toXins are made by B. ant/1racis. The
most important of th.ese in the production of disease are
ll1e lelhal loxi11 aud Lhe euema tuxil1:
170
ERNST L. IlIBERSTEIN
l. Lcthal torjn. The gen es e ncoding the lethai ---~­
(LeTx for lethal toxin) are found on a pla'-..
termed pXOl. LeTx is a binary toxin compo:;e.:
"protective anttgen" (PA) and "lethal factor
PA is responsible for binding of LeTx to ~a;
cells," wl1ile Lf is responsible fo1 ils lo:x.icaclivit
and LF associate only after PA has been oligo......,..-
izcd on thc "target" cell surface (see below). t.:
zinc metalloprotease that inactivates mito __-
activated protein kinase kinases (MJ\PK kinases
sulting in the disruption of signaling path' _
within the affected cell (macrophages appear t
the principal cell that LT affects, even though it
tf'rs scveral others). PA binds to cell surface rece~
011 a variety of cell lypes. IL is aclivaled by furill.)
the cell surface resulting in o ligomerizat ion, "'· h
is followed by internalization of LT (as well asede-
lactor, see below). At low concentrations, the Le~
macrophage interaction results in a cytok..::.
"storro" leading to muttiorgan dysfunctlon, sim
to what occurs during endotoxemia (see Fig S -
Thal i::., u1u.Ier tl1e üúlul:'IH.:e uf LeTx, macropha~­
become hyperresponsivP an<1 pro<111cf> lar _
amounts of tumor necrosis factor (fNF), interleuL
(lL)-1, and IL-6 resulting in increased vascular pt:
meability CfNF on endotheliu1n), incrcascd i:-
travascular clotting (TNF on endotheliun1), attra
tion of lnflammatory cells (TNF/IL-1 signalir.
e11doll1eliu111 lo vruc..luLc lL-8 ar1d macropha e
chemotactic factor), release of acutc phase protpi-
(IL-6 on livcr), cndogenous pyrogen (TNf, IL-1, lL-<
on brain), an<.1 somnogenic ('l'Nf, lL-1 on brain) . .
higher concentrations, LcTx produces apoptoL.
death of affeaed mac rophages. Producrton of LeT·
is under control of the rcgulatory gene produc<
ALXA (for u11lh1ax lu.A:i11 uclivatur), whi<.:11 rl:!:;pund
to as yet unknown environmental cues (see belo\
"Rcgulation of Ccllular Products of Medica! Inter-
est"). AtxA is also encocted on pOXl.
2. Ede1na toxin. 'fhe genes encoding the ede1na toxir
(Ed'fx for edema roxln) are found on a plasmic.
termed pXOl. EdTx is a binary toxi n composcd o;
"protecllve a11tigen" (PA) and "ede1na faclo1" (EF
PA is responsible for binding of EdTx to "targe-
cclls," whilc Ef is rcsponsiblc for its toxic aetivity. P.~
and E.F associate only atter PA has been oligomer-
i2ed on the "target" cell surface (see below). EF is a
calmodulin-dependent adenylyl cyclase that in-

creases levels of c1\MP witlún the atfccted ccll caus-
i ng <'lec:trolyte and fl11id loss. PA hind~ tocPll surface
rcccptors on a variety of cell types. It is activated by
furins on thc ccll surface resulting in oligomeriz.a-
tion, which is followcd by intcrn<llization of EF (as
well as lethal factor, sce above). l'roduction of Ed.l'x
is under control of the rcgulatory gene product,
AtxA (for anthrax toxin activator), whlch responds
to as yet un known envi ronm Pnta 1 c11Ps (.~f'f' below,
"Regulation of CelluJar Products of Medica! li1Ler-
est"). AtxA is also encoded on pOXl.
3. Misccllancous "toxins." Scvcral homologues of pro-
teins snown to be important 1n v1ru1ence ot other
crucroorganisms have been discovercd in the L)NA
~1::4ucr1ce of the genume uf B. anthracis. These in-
clude genes encoding proteins important in s11r-
vival within phagolysosomes and on mucosa! sur-
taccs (lnh and Mprr), escape from phagolysosomes,
and phagocyti.c cells (anthrolysins), and iron ac-
qutr!ng products (Dlp).
a. lnh and MprF. The genomc of B. anthracis con-
Laius Lite ge11es e11cutli11g tl1c hurnulogues ofinh
(for immune inhibitor protei n) in Rar.illus r:rTl'.11~,
and MprF (for 1nultiple peptide resistancc factor)
in St.aphytococcus (see Chapter 27). which have
been shown to bestow resistance to defensins
(found within the phagolysosome, and in se-
cretions bathing mucosal surfaces) by lysiny-
lal iun uf pl1uspl1ulipids in the bacterial ccll
mc~mhrilnPs.
b. l\nthrolysins. ·rhe genon1e of B. anthracis con-
tains the genes encoding anthrolysins (also re-
fcrrcd to as anthralysins), which are homologues
of severa! phospl1olipase C and chotesterol-
binding cytolysins (anthrolysin O) in other
pathogenic bacteria (see also clostridial per-
fringolysin O, Chapter 36; streptococcal strep-
lul ysi11 O, CltavLer 28; li~lt:rial listcriulysin O,
Chapter 33; and arcanohacterial pyolysin,
Chapter 29). ·rhe phospholipases are pore-fonu-
ing cytolysins, and anthrolysin ü. also a pore-
forming toxi n, binds to cholcstcrol containing
rafts in the eukaryot1c cell membranes. Though a
definitive role has yet to be determincd, there is
strong evitlence that the anthrolystns are re-
-~ronsihlP for lysis of phagoly~o~nme containing
n. a11thraces followed by the liberation of the n1i-
croorganism into the cytoplasm of the affected
cell, as wcll as Jysis of the cell membrane leading
to llberation into the extracellular enVironment.
Note too, that the anthrolysins have hemolytic
aclivily. ·rhis acli vi Ly is tleu1u11str<IIJle untler con-
ditions not used in the clinical lahoratory for iso-
lation and identification of D. anthracis (it is typ-
ically "nonhemolytic" under clinical laboratory
conditions).
c. Dls. ·rhe genome of B. anthracis contains the
genes encoding Dlp (for Dps like protein) which
is an iron-bindil1g vrult:ill 11uruulugue of that
found in Escherichia coli calleci Dps (for DNA pro-
tccting protein under starvcd conditions).
Chaplc.'T 30 Bacillus 171
4. Regulation of the Cellular Products of Medica!
lnterest. ·rhere are n.vo proteins, AtxA and AcpA (for
11nlh1ax toxi11 uclivalor a11tl u11thrax i·apsule activa-
tor, rcspectively) that are produc:eci in rPsponsP to
cnvironmental cues. What thcsc cucs are in vivo is
urtknown. Under t he appropriate conditions, how-
ever, AtxA ir1creases the production of Lc'J'x, Ed'rx,
and proteins involved in the "escape" from phago-
lysosomes and macrophagcs (presumably the an-
LIIrulysiI1s). AcpA production Is amplified under in-
creaseci amounts of C0 2 (5% or greater). Atx.A acts
syncrgistically with AcpA in this rcgard.
Growth Characteristics
Rncillus anthracis is a facultative anaerobe and grows on
con1n1on 111edia beLwee11 l5nC auu 40nC. Colonies reach a
diametcr of 2 mm or greater in 24 hours at 17ºC:. <~olonies
grown in air havc a dull surface and wavy margin forn1ed
by strands ot bacteria! chains ("medusa-hcad"). Cells are
nonencapsulated. Colonies grown in greatcr tl1an So/o car-
bon dloxlde on serum agar conta1n1ng 0.7o/o bicarbonate
are mucoid and consist of encapsulated bacteria (Fig 30.1).
Sporulalion occurs untler <IIJu11tlant oxygen, but not
while inside the animal. Organisms in infPc:tPc1 tissuP or
fluids cxposcd to air sporulate after severa! hours.
Biochemical Activities
Reactions differentiating B . anthracis fron1 R. ccr<:us, its
nearest relative and a frequent contam1nant, are summa-
rized in ·rabie :~0.1.
Resistance
Vegetative cells in unopened carca<;Se><; may s11rvivf' for 11p
to 1 to 2 weeks, but spores can persist for decades in a sta-
ble, dry environment. Spores are killed by autoclaving
(121 ºC/15 min) and dry hcat (lSOºC/60 min), but not by
boiling (lOOºC) for less than 10 minutes. ·rhey are not
highly susceptible to phenolic, alcoholic, and quaternary
ammonlum dlsinfectants. Aldehydes, oxtdtz.ing and chlo-
rinating disínfectants, beta-propiolactone, and ethylene
oxide are n1ore use ful. Heal fixaliu11 uf s111e<Irs tlues r1ut kill
spores.
Variability
Colon1al and patho~enic variability may be caused by en-
vironmental manipulation or spontaneous mutations.
U11tler natural conditlons the bacterlum is antigenically
uniform .
Ecology
Reservoir
·rhe soll IS the source of anthrax 1nfectlon for herbivores.
Other species, including humans, are exposcd via infected
ani111als aud a1li1ual prutlucts.
172 PART JI Bacteria and Fungi

F1G U R E 3 O. 1 . Bacillus anthracis grown on ca/cium carbonate

agar under 20% carbon díoxíde for capsule productíon. Mcradyean
capsule stain, 7OOOX.

Table 30.1. Differentiation of Bacillus anthracis from Bacillus
cereusª
8. ililthr<idS"
Motility
Hemolysis (sheep blood agar) :!:
Reduction of methylene blue ± +++
Fermentation of salicin
Growth at 45ºC
Mucoid coJonies on bicarbonate ao_ar + cd'J'x produces severe tluid and electrolyte ünbala11ces, IE-
under 20% COz
susceptible to gamma phage + sule tliscourages further association of tlle vegetative forc:
• Mod.ified after Burdon !';L RaP.id isolatio!l'and identification of tJafilfus anthraCts.
Preseílted at the Symposium on Anthrax in Man, Philadelpnja, October 1954.
b Elífferent stralns exist, with most being moti!e.
Transmission
The endospore is the "infcctious unit." lnfection takes
place by ingestion of contaminatcd feed or water or via
\'>'Ound infection and artl1ropod bites.
Human infections occur via skin wounds (malignant
carbuncle), inhalation (e.g., "v.rool-sorter's dlsease") and,
exceptionally, ingestion, which is also the likely exposure
for predatnrs.
Pathogenesis
Mechanisms. Spores are acquired from the environment
(e.g., soil, animal products, see above, "Transmlssion").
They are phagocytosed by macrophages (polymorpho11u-
clt>ar nt>ntrophil lt>ncocytt>s cio not appt>ar to play a role in
the disease process). The spores germinate within the
phagolysoson1e con1partn1ent. Vegetative bacteria, re-
sponding to cues, produce the regulatory protcil1s AtxA
and AcpA, whích in turn up regulate the productio11 of
LeTx, Ed1'x, capsule, a11d other putative toxins (an-
throlysins, iron-acqulring products, defensin-resistance
factors). Inh and MprF aid in the surv.ival of B. anthrax
while it is in the phagolysosome. Anthro1ysins permit es.-
cape from first the phagolysoson1e 1 and then t he
+++ m.acrophar,e itself. Production of LeTx triggers the hyp~­
productio11 of cytokines leadil1g to n1ultiple organ dys-
+ functio11, shock, and depletion of clotting factors (see FiE
;- 8 .1), as well as apoptotic death of affected macrophag~
sulting in localized edema and systemic shock. The cap-
with phagocytic ct>lls.
Pat11ology. In lissue, spores genninale and lhe vegetali\-.
form prolifera tes, prodúcing gelatinous edema. Inflarruna-
tory rcactions are minimal. lnfection disseminates to retic-
utoenc1othelial sites. When these are saturated, a termina.
bacteremia occurs, with enormous numbers of organism...
in circulation. Postmorten1 findings are widespread llem-
orrhage.s; a hlack, e.ngorgecl, friahle spleen; tarry, noncloc-
ting blood¡ and abse11ce of rigor n1ortis. Bleedi11g at bod
orífices is common.
Disease Pattern
Ruminants. ·rhe process described above is typical for rfit:
n1ost susceptible species- cattle and sl1eep. 'fhe coUJ:k.
following an incubation period of 1 to 5 days, ranges fro!"'"
a few hours to 2 days. Sorne animals d ie without overt cTI:-
ical signs. Othcrs dcvclop high fcvcr, agalactia, and t b ....
n1ay abort. ·rhere is congestion of mucous men1bran~
hematuria, hemorrhagic diarrhea, and often-regior_
edema. 'l'hese forms are regularly fatal. Occasio11al arli.ma...
show just localized edema oran ulcerative sk.Ln lesionar;.~
recover.
Horses. Horscs dcvclop colic and diarrhea¡ edema also e~­
curs, particularly of dependent parts and at the point of w-

"ection (e.g., the intestine or thc throat) where it may
cause death by asphyxiation. Altern;:itively, thP course m;:iy
-.e sepliccn1ic, as in run1inants.
5.vine. In swinc, localization in phuryngcal tissucs is typi-
::al. An u lcerative lesion at the portal of entry is associated
ith regional lymphadenitis. Obstructive edema may
cause death. ülcerative he1norrl1agtc enteritis and 1nesen-
·.-ric lymph;idenitis sometimP~ ncc11r.
:arnivores. Carnivores (induding mink) are rarely affected;
·hcn thcy are, thc discasc pattcrn is similar to that in
~\·ine, although massive exposure throu,gh tainted meat
-
~ay trigger septicemia.
-
-iumans. In h11m;ins, percutilnf>o11 .~ introc1urtion of sporf>s
..:auses "malignant carbuncle" (pustule), a local ulcerative
~--iflammatory lesio11 covered by a black scab (escl1ar). Pos-
s_ble complication s are subcutaneous edema and sep-
·~cemia. ·rhe case fatality rate is 10% to 200/o. A shock-like
!>~ate precedes death. Inhalation anthrax (e.g., "wool-
<.> ner's dlsease") produces pulmonary edema, hemor-
:nagic pnP11monia, ano somi>timPs mi>ningitis. Mort;:ility
:zces approach 100%.
:oidemiology
-. soil rich in calcium and nitra te, with a pH range of 5.0 to
:..O, favors sporulation and bacteria] prollferation at te1n-
:;-ieratures above 15.SºC (60ºF), especially after flooding.
~e geogiaphy a11d seasonalily of oulb1caks rellecl such
:ucumstances. ln cattle, sheep, and possibly horses, out-
=:eaks begin with a few cases contractcd from thc soil.
..!ter excretions and postmortem discharges seed the area,
;econdary cases occur. Floods and industrial effluents
::om rendering works, tanneries, carpet milis, brush facto-
-::es, or wherever else carcasses are salvaged may contami-
--:3te areas. Bone mea!, an animal fee<.l supplement, is a
""lmmon vf>h ir lP in noni>nc1Pmir ilrPilS. \.;:irnivores (m ink)
?.;e usually exposed via infccted n1cat.
Human exposures are contracted in occupations deal-
..::g with animals and animal derived material such as im
X>rted h1des, wool, and bone. Anthrax occurring under in-
.::.istrial conditions is often the lethal airborne version.
11munologic Aspects
::~i>erimmune sera can prevent and alleviate disease.
-_'1tibactenal and antitoxic factors are thou¡;ht to be in-
"'Olved . In most species, immunity is directed against the
-:otecuve anti.gen. Capsular polypeptlde falls to stimulate
:-· otPctivP ;intihnn y
Artificial immunization of livestock has utilizcd 1nostly
~dified live spore vaccines. Currently these are derived
-om avirulent (noncapsulated) mutants. The most widely
-Sed is the Sterne vaccine (a strain of B. anthrads that lacks
·~e pX02 plasmid). A cell-free vaccine consisting of con-
_culrateu l:ulture filtrate has l.Jeen used on humans ex-
- sed to industrial anthrax. lt prod11cP~ tPmporary p rotPC'-
-'ln against cutaneous infection. Protective antigen,
Chapter 30 Bacillus 173
produced by microorganisms lnto which the encoding
8PnP h;:i<; hf>Pl1 placP<I, shows promi<:P, il.<: c1o pJ;:intS into
which the PA encoding gene has been cloned.
Laboratory Diagnosis
Sample Collection
During sample collection, precautions against contamina-
tlon ofthe environment are important. Blood maybe aspi-
ratcd from a superficial vessel. Aqueous humor has the
added advanlage oí ren1oleness f1on1 sou1ces of early posl-
mortem contamination. Por direct examination, bloody
dischargcs fron1 orilices are samplcd.
lf the carcass has been opened, spteen nlaterial may be
collected .
Direct Examination
Blood and organ smears are stained by Gram stain and a
capsule stain such as McFadyean's methylene blue. Chains
of encapsulated, gram-positive, non-spore-torming rods
(see Figs 30.l and 67.1) suggest B. anthracis. Contaminant
Baclllus spp. are usually not encapsulated and lack the
clipped, squared-off appearancc of anthrax bacilli. Fluore-
scent antibody helps in. the diffcrenlialion.
lsolation and ldentification
Bacillus anthracis grows on all common media. Presump-
tive ictentitication can be made by the characteristics out-
li ncd in Table 30.1 and the "stri ng of pearls" test (the
characteristic blebbing that occurs when B. anthracis con-
tacts penicillin). Definitive identification is by specific
bal:lcriu¡Jhagt: (ga11Lllla ¡Jhagc). Fluu11:::.cc11l a11libody a11d
IPctin<> of appropriate specific:ities are helpful. Bacillus
cereus is a commonly cncountcre<.l contan1inate that is
similar to B. anthracis in appearance. These two microor-
ganisms can he differentiated using variou s tests (see
Table 30.1).
Experimental animals (mice, guinea pigs) are injected
subl:Utétut'uusly vvitl1 suspt'ct 1uat1::rial. Dt'ath frou1 éUJ-
thrax occ.11rs aftpr 24 hours. T.f>sions incluciP hemorrh;:igi>s,
gelatinous exudate near the inoculation site, and an en-
gorgcd spleen. The encapsulated agent is demonstrable in
blood and tissuc.
lmmunodiagnosis
Bacillus anthracis antigens can be demonstrated in extracts
of contaminated products by a precipitatíon test using
high -titPrP<I :intise1·urn (Ascol i tP.st) .
Molecular Techníques
Vanous gene sequences have bcen targeted in the design ot
primers that are used to amplify segments of DNA by using
the polymerase chain reaction. Examples include portions
of the gene encoding the 16$ ribosomal RNA, and parts of
pOXl a11d pOXZ.
174 PAJo 11 Bacteria anc.l Fungí

Treatment, Prevention, and Control Prevention ot anthrax exposure through animal prod-
ucts imported from endemic areas requires disinfection of
Badl/us anthracis is susceptible to penicillins, chloram- such material as hair and wool by formaldehyde. Bone
phenicol, streptomycin, tetracycline, fluoroquinolones, m ea! is sterilized by dry heat (lSOºC/3 h) or steam (llSºC/
and erythromycin. Treatment should continue for at least 15 1lli11).
S days. In sorne areas, antiserum is given simu]taneously.
Antiserum is not available ín the Unítec.l States. In acute
anthrax, antimícrobial treatment is often unsuccessful.
Pupuldliuu:. dl 1i:.k. dtt:: VdLLÍlldlt::ll. a11uually.
BACILLUS CEREUS
When an outbreak or a case of anthrax has occurred,
Bacillus cereus can cause opportuniStic infections, most n o-
animal hcalth authorities are notified to supervise control
tably abortions and bovine mastitis. This is often acutel)
mcasures. Carcass disposal involves incineration (pre-
fcrrcd) or dccp burial (>6.5 ft) undcr a laycr of quicklimc gar1grer1ous anti rapi<.lly fatal or <.lestructive to e~1tire 4uar-
tPrs. FrPq11Pnt initiíltors ílrP 11clclPr surgPry ancl tntrama m -
(anhydrous calcium oxide) . Surviving sick animals are iso-
mary medications.
lated and treated. Susceptible stock are vaccinated. 'fhe
Bacillus cereus is also responsible for several forms of
premlses are quarantlnect for 3 weeks subsec¡.uent ~o t~1e
11uman food poisoning manifested by diarrhea or vomit-
last cstablishcd case. Milk from infected anunals 1s d1s-
ing, the former being assocíated with various foods, t ~.,­
carded undcr appropriate precautio11s. Barns a11d fe11ces
Iatter m ostly with rice. Toxins are involved: an emetic
are disinfected with lye (10% sodium hydroxide). Boiling
tuxin (cereulide) aud three ~ecretury t:I1Leru1.o.xi11s (HBL
for 30 minutes will kili sporcs on utcnsils. Surfacc soil is
NHE, '!").
cleared of spores by treatment wíth 3(1/o peracetic acid solu-
tion at the rate of 8 liters (2 gal) per square meter. Sorne
other material can be gas-sterilized with ethylene oxide.

•
 Corynebacterium
DWIGH'f C . HlRSH
\{embers of the gen us CorynP.hactp_ri11m arP plPomorphir,
:Jon- spore-forming, nonn1otile grarn-positive bacilli.
Corynebacteria occur in packets of paralle! ("palisades") or
crisscrossing cells ("Ch in.ese letters") that include coccoid,
:od, club, and filan1entous shapes. This pattern is called
iiphtheroid after the type species C. diphtheriae, which is
-he CélUSe of hUIIlélil <Jipl1tl1eria (Fig 31.1).
()f the more than 10 species i ncluded in the genus, two
:!Ie.regularly associated witl1 diseases of veterinary in1por-
:3nce: the facultatively intracellular bacterium C. pseudo-
:-".berculosi$ (the cause of abscesses in ruminants and
-a1ses), and C. renale (the cause of disease of the ruminant
=ogenital tract). Briefly mentioned in this chapter are
-..4nubuculum suis (élt 011e tiu1e céllled Eubucleriurn suis ;u1u
- rvnehacteriurn suis), the cause of urinary t ract infection
sows, and C. auricanis, associated with otitis externa and
~1natitis in dogs.
-
~:R YNEBACTERIUM PSEUDOTUBERCULOSIS
: =scriptive Features
: ·phology and Staining
~11ebacterium pseuduluberc:ulusis (Fig 31.1) is a typical
-,thProio. "!'he cP.lls range from coccoid to filamentous,
~'.!ffi by >3.0 µm, are non- acid-fast a nd nonencapsu-
-=-j· and often contain granules.
_::nrre and Composition
_ ..:ell Wélll is t yµical vf gra111-posi live bacleria, 1-vitl1 tl1e
- . :non of mP.so-diamino-pimelic acid (DAP), arabino-
.. . ctan, an.d 1nycolic acids (branched chained fatty acids
-'":a chain lengt h for members ot tbe genus c;orynebacte-
of C 22 C3 8) . ·rhe cell vvall contains a high concentra-
of lipids.
- - ?.r Products of Medica! lnterest
·hll. The gran1-positive cell 1-vall contains polysaccha-
and lipids that are of medical interest. The lipotei-
~ ;icíd$ ond pcptidoglycan of the gram positive cell
"'lteract 'l<Vit h 1nacrophage cells resulting in the release
- :inílamrnatory cytokines. The high concentration of
- .:u11tril.Jutes to intraµl1agocylic survival and leuko-
.
.. .
ERNS'l' L. BIBERS'J'EJN
F.xntnxins_ r:nrynehacteriurn pseudntuherculosis producc•s
at lcast two exotoxins: a phospholipase D and a serine
protease.
l. Phospholipase D (PLD) . PLD (a sphingomyelinase
related to t he toxin of the b rown recluse spider) is a
membranP-active toxin with activity on endothe-
lial cells, lyses erythrocytes by activating com-
p lement, produces dermal necrosis, and is .l ethal to
a variety of experimental anirnals. In vitro, PLD
lyses sheep and bovine erytl1rocytes, inl1ibits beta
toxin of S tc1phylococcus aureus (Fig 31.2) and
Clostridium per(ringens alpha toxin (see Chapter 36),
and potentiates 11em olytic activity of "R . equi fac-
tors" (see Chapter 34) (Fig 31.3).
2. Serine Protease. The role played by the serine pro-
tcasc is unk11own, but antibodies to it are protcctive.
!ron Acquisition. Corynebacterium pseudotuberculosis pos-
sess a cluster of four genes, fag.4.- D (for Fe acquisitiongene)
encodiug µroteiu~ ll tal are i11volved wilh iro11 uplake. Tl1e
uptake systern is similar to the one used by enterobactin (a
catecl1ol-type of siderophore found in members of the
family Enterobacteriaceae). Deletion oí this gene results i11 a
decrcase in virule11ce.
Growth Characteristics
Corynebacterium pseudotuberculosis is a facultative anaer-
obe. Tt grows best on rnedia contaiiliug l.Jlovd or serun 1. Al
48 ho11rs on h loocl agar (1S- 17ºC:), colonies are off-white,
duU, faintly hemolytic, and about 1 mrn in dia1neter. They
can be pushed across the agar without disintegrating
(son1e describe this as pushing a "hockey puck"), and dis
perse poorly in liquids .
Biochemical Activities
'fhe agent ls catalase-posltlve. Sorne carbohydrates are fer-
mented.
Resistance
Disinfectants and heat (60"C) kill C. pseudotubercu/osis, but
organisms survive well where moisture and organic matter
abound. Penicillins (inc!uding ceftiofur), erythromycin,
chloramphenicol, lincomycin, tetracycline, enrofloxicin,
and lrin1eLhopri111-sulfonanlide are inhibilory in vilro.
·rhe agent is resistant to aminoglycosides .
175
176 PA10· 11 Bacteria and Fungi

F 1G U RE 3 1 . 1 . Corynebacterium pseudotuberculosis in pus from an equine chest

obscess. Note club sh tJpes, ptJlisading, uchinese letter" patterns, pt.::utl/VlfJltÍ>lll. <310111 >loi11,
1000X.

.....
- .,,,.. •
- - -
, - ,,-
,
~, •
_.,,.-! ,,
e ..... -
- •
' .. ... .,,

... . •

F 1G U RE 3 1 . 3 . Syncrgistic hcmolysis by Corync~:2"'

F 1G u RE 3 1 . 2 . lnhibition of slaµl1yluuJlldl úitld luxi11 (µho}- pseudotuberculosis (center) and Rhodococcus equi (pee ;:;-=
pholipase CJ activity on bovine blood agar by Corynebacterium Hemolysis is maximal where the diffusion zones of the :--. _
pse11dotuberculosis exotoxin (phospholipase D}. organisms overlap.

Variability Ecology
There are two biotypes that differ biochemically, serologi-
cally, and cpldemiologlcally: the "equine biotype," which Reservoir
isoli'!tPs from equine, and most bovine sources reduce ni- The ovine biotypc (scc abovc, "Variability") C. pseudotuh,
trates to nitrites (nitratc-positivc), a11d ll1e "ovü1e bio- culosis is found in lesions, the gastroi ntestinal tract of n Oi-
type," which isolates from ovine, caprine, and sorne mal sheep, and the soil of sheep pens. The reservoir of th .
bovinc sourccs do not (nitratc-ncgativc). equine type is not known.

-_:- íssion
_; become infected through brcaks in thc skin (e.g.,
_. . nds acqui1ed du1il1g sl1caling), goa\s probab\y follow-
~ ::auma related to butting, and horses following breaks
-e skin (e.g., biting flies).
· -::i9enes1s
. "breastbone tever." <;orynebacteriurn pseudotuberculosis
·umisms. Coryncbactcriurn pscudotubcrculosis is a facul-
- --=elv intracellular parasitc whose resistance to phagoly-
m al disposal is relatcd to its surface lipids.
- ~rulence Is attrlbuted to PLD (and perhaps the serine
-Pflse) and ccll wall lipids Roth the cPll w::ill an<I t h e exo-
...::is initiate and aniplify an inflan1n1atory response. PLD
..::::-.ages neutrophils, macrophages, and endothelial cells.
:0Howing in(ection, absccsses fo rm but remain localized
- a exotoxin and/or protease are aL')sent or neutralized.
-.1tholog)I. Ne utrophilic infiltration an d endothelial
- ,<1ge clk1racterí:le early cltanges. Tl1e lesions are ab-
'3.SeS . D istrihution, progrC'<;s, ;in<i ;i ppe;i r;i n c.e <i iffp r wi t h
. d es and routes of inoculation, but lymphatic involve-
- -=-:t is con sistcnt. Thc naturc of thc pus varies largely
;:.~ age of the lesion, which grossly appears creamy to d ry
~ crumt>ly ("cheesy"). C)ld abscesses consist of dead
-~ophages with peripheral neutrophils, giant cells, and
~u!> tissue. LesíOil!> alrnost always coutaíH just C.
-- -iotuhP.Trulnsi~ .
: :;: : se Patterns
- ·-..e. lllcerative lymphangiti'> of horc:P<: (rflrPly c;ittle) :is-
---:ds tl1e lyrnpl1atics, usually of the hind limbs, starting at
-= fetlock. Tts progress toward the inguinal region is
F 1G u R E 3 1 . 4 . Caseous lymp hadenitis in a sheep. Lyn1ph nodes, lungs, spleen. Note
abscesses with concentric ring pattern, especial/y in t he lymph node, top right. (Photograph
courtesy nf flr í n rriP Rrnwn .)
Cf1apter 31 Corynebacceriun1 177
marked by sweUing and abscesses, which rupture to Jeave
ulcers along its course. Hematogenous dissemination is
rare. Areas other than extremities are occasionally af-
fected. Contagious acne (Canadian horse pox) is an un-
conu11011 equine folliculitis due Lo C. pseudotuberculosis
infection.
Pectoral abscess is also called "pigeon fever" and
causes abscesses, usually in the muscles of the chest an d
caudal abdominal regton of horses. ·rhe infective mecha-
nism is not understood, but the seasonal peak (autum n)
a11d geograpl1ic rest1iclion (111ait1ly California) suggesl ru1
arthropod vector. Thc lcsions of cutaneous ven t ral
habroncmiasis anda midventral derma titis du e to horn fly
(HaernatotJius irrita11s) activity are possible portals of entry .
Sign s such as swell ing, pain, and la 1ne ness depend on the
locatlo11 and slze of the abscesses. septicemia occurs rarely,
b ut may rcsult in abortions, renal abscesses, de bilitation ,
and death. Thc superficial lesions resolve slowly afler
drainage.
Sheep and Goats. Caseous lymphadcnitis of sheep and goats
is usually traced to a break in the skin. After introduction,
the agent ellcits a dlffuse tnflammation, followed by the
formation of an absccss that coalesces and undergoes en-
capsulation. Inflan1111atory cclls traverse lhe capsule pe-
ripherally, adding a layer of suppuration and a new cap-
sule. Severa! such cyclcs givc thc lcsions, especially in
sheep, an "onion ring" appearance (rig 31 .4). Old lesions
acquire thick, fibrou s capsules. The general health is unaf-
fected unless disseminat1on occurs to other lymph nodes,
viscera, or the central nervous system, causing progressive
debiliLaLion (thiu ewe sy11d1on1e). MosL for1ns of it1fection
are chronic.
178 PART II Bacteria and Fungi
Cattte. Cattlc occasior1ally develop skin infections with
.lyruµl1 r1uut.: iuvulverueut. Sucl1 epi:;ode:; are ofter1 acute
and can he epidemic. The most con1mon site is the lateral
body wall, suggesting that t raun1a initiatcs the dlsease by
producing breaks in the skjn.
Human Beings. A relatively benign lyn1phac.!enitis may result
foilowing infection in human patients. These infections
generally follow ani rnal contacl (e.g., sheep sheare1s).
Epidemiology
·rhc currcnt vicvv is tl1at C. pscudotubcrculosis is a11 anin1al
parasite and only an accítiental soil inhabitant. ln sheep,
shearing, docking, and dipping are significant factors in
the spread of infection . ln goats, direct contact, ingcstlor1,
and arthropod vectors must be consldered . Thc prev;i-
lence of infection l11crcases with age. Caseous lyn1-
pbadcnitis is onc of the important bacteria! infections of
sn1all run1inants.
·rhe hypotheses concerning equine exposure have been
considered. No age predilection h.as been noted. Ulcerative
lyn1pl1angitis is thought to reflect poor management and
is uncommon nowadays. "Pigeon fever" is limited to the
f<1r wt.::>tt.:ru Uuiteu State~. A1111ual µre v<1le11ce va ríes, :1ee1n-
ing highest after a \·v et "''in ter.
lmmunologic Aspects
'J'ht> roli>s of ;intihncly ;incl ci>ll -mt>cli;itt>cl ri>sponsi>s th;it
occur during the infectious process are undefined.
Antitoxin limits d issemination of abscesses. Caseous lym-
phadcnitis can be progrcssivc, and cquinc absccsscs rccur.
Hacterin-toxoid con1binations prevent cüssemination
(11ot iI1fection) i11 sheep for at least a year and evoke pro-
tective responses in goats and horses. Purlfied serlne pro-
tease il1 adjuvant elicits a protective response in sheep.
Muta11ts co11structed by knocking out specific ge11es that
encode products related to virulence (e.g., PLD; aron1atic
a1nino acid production) rcsult in a n1odificd livc product
that elicits antibody and cell-mediated in1n1une re-
sponses. 'fhese modified live vaccines are effective immu-
nlzing product s.
Laboratory Diagnosis
lntraceHular or extracel 1ular diphtlleroict-shapeci organ-
isn1s may be demonstrable in stained direct smears of n1a-
terial from lesions (see Fig 31.1).
13lood agar plates inoculat ed with abscess material ancl
incubated for 24 to 4811ours (35-37ºC) produce sn1aU, off-
white, faintly l1emolytic colonies tha t can be pushed in-
tact ("hockey puck effect") over thc ugar surfucc.
lnhibitior1 ot staph ylococcal beta toxin (see rig 31.2)
and sy11ergistic hen1olysis with R. equi (see Fig 31.3) con-
ftrm 1cten11flca11on of c. pseudotuberculosts. Suppress1or1 of
tht>SP ri>actions hy antihorly (s11ppliP.cl hy SP.rtJ m from a n
infected animal) forms the basis of serodiagnostic tests
(synergistic hemolysis inhibitio11 test).
Other tests utilize agglutination, complement fixati.
indirecl he1naggluUi.1alion, hen101ysis inhibilion, gel
fusion, ar1d toxin neutralization. Members bclonging
tl1is gcnus can be identified followlng 1111alysis of cell ~
acid analysis (this method has not been as usefttl in s~
ation within the genus). Genus- and species-spec
primers llave been developed to an1plify DNA l)y usin g -
polymi>r:isi> ch::iin rf>::iction.
Treatment and Control
In sl1eep a11d goats, antibiotic treatment is ineffec&
Control is aimed at limiting exposure by segregatioc
culling of affected animaJs and at scrupulous sanitary C"2
uuriug <tctivitie:> like shea1ir1g, dippü1g, a11d surg:~
procedures. Bacterin-toxoid combinations 1nay be he.-
ful in limiting infections. Modificd-livc products sh
promise.
Equine abscesses are handled surgically. Prolon:;--
perlicillin therapy is used to prevent or treat dissemina¡__
disease.
(ORYNEBACTERJUM RENALE GROUP
Diphtheroid bacteria, traditionally called Corynebacterr:
renale but actually constitutlng thrcc spc.cics (:;ce belo-
colonize the lower genital tract ot cattle and someti:::::.
sheep..J\ssociated diseases are bovine pyelonephritjs :o ..
ovine posthitis ("pizzle rot"). At one time, C. renale -
considered to be comprised of three serotypes ('lype ;
anti !Il). Subset1uent DNA <1nalysis revealeti tl1at t Z-
si>rotype c.onstitntt>cl a sP.parate spP.c.ies: r:. rena/e (Typt>
C. cystidis (Type 11), and C. pilosu111 (Type III); together th::
are referred to as the C. renale (}roup.
Descriptive Features
Morphology and Staining
Members of the C. renale Group are typical diphthero...:..
The cells range from coccoid to fila1nentous, are non-ac
fast, nonencaps11l;ited, and nfti>n contaín granules.
Structure and Con1position
'!he cell wall is typical of grarn-posítive bacteria, w itb -
addition of meso-dia1nino-pimelic ac.id (DAP), arabir
galactan, and n1ycolic acids (branchetl ct1ained f<1tty a~
with a chain li>ngth for mi>mhP.rs of the gen us r:oryneb..;.;:;;.
riu111 of C 22- C: 38) .
Cellular Products of Medical lnterest
Adhesin. Members of tl1e C. renal e Group express a fibr>
prutel11 atil1e~l11 (pili, fi1111Jriae) uu Lheil :>u1fc11..e;:,.
\,e/l Wall. The gram-positive cell wall contains pol~
charides, and lipids that are of medica! interest. The lir
ichoic acids and peptidoglycan of the gran1-positi,-e

\Vall interact witl1 macrophage cells resulting in the release
of proinflammatory cytoldnes.
Urease. Meruuer:; uf tht: C. renule C;roup p1oduce urease,
;.;h ic.h is assoc.iated with the virulence o f this group.
~Iowth Characteristics
"'1embers of the C. renale Group are facultative anaerobes
i::apable of gro,"ling on. most common laboratory media as
-LOnl1e111ulytic, opaque, off-·wI1ile colo11ies ll1al develop
i thin 48 hours at 37ºC= on blood agar.
.: ~ochemical Activities
• ~embers of tl1e C. renale Group have impressive urease
;ctivity, which is demonstrable in most strains v.rithin
din ules of conlacl wilh its subst1ate. Glucose is slowly
_ddified, otber carbohydrates variably so. All strains are
:::a:alasc-1Josjtive. Mcmbcrs of thc C. renalc Group produce
:::. protein, renalin, which results in a positive CAM1' test
see St-reptococcu.~ agalactiae, Chapter 28).
~ ;.sista nce
-"lt' agents are not particularly resistan.t to heat, disinfec-
-:=_.-¡ts, or a11tin1icrobial age11ts.
.:rology
"IDbers of tl1e C. renal e Gro11p in h;i hit t he lovver gen ita 1
-;!et of cattle and son1etin1es other run1inants. Occa-
:maily they are implicated in urinary tract disease of
-,eep, horses, dogs, and nonhuman primates. No human
~=ections are known. Distribution is global.
-":. :sm1ss1on
5 anisms pass between animals by direct and indirect
-:::act. Many clinical cases are probably endogenous.
~:.1ogenesis
' h esin-mecliated attachments to urothelium and urea
~drolysis are considered critica! in pathogenesis. Urca
-eakdown with. production of ammonia initiates an in-
--"-':UTlatory process, high alkalinity in urine (pH 9.0), and
~pression of antibacterial defenses, possibly througl:l
~::.:11plement inactivation by ammonia.
Palhulugy. A chruuic iuflau1u1alory process successively
...,.olves the 11Jadder, ureter(s), renal pelvis, and renal
.:-;.~renchym.a in pyelonephritis in cattle. A similar inflam-
-atory response occurs in small ruminants, but is usually
x alized to the distal urethra.
-·~ease Patterns
~.,ttle. Bovine pyelonephritis is an ascending urinary tract
..:ifectiun, lJeginniI1g witl1 cy~titiS, ¡;roceeding LO ureteriliS
Chapter 31 Corynebacterium 179
and pyelonephritis. Rectal palpation reveals thicke11ed
bladder and ureteral walls, distended u.reters, an.d enla.rged
kidneys wilh obscured lobulatio11s. Early cases show pol-
lakiuria, hematuria, and increasing degrees of abdominal
pain. Chronic infections progress to debilitation and
death due to ure1nia.
Small Ruminants. Ovine posthitis ("pizzle rot"), the more
common form of infection in sheep, is a necrotizing in-
flammation of the prepuce and adíacent tissues in wethers
or rams. Disease clevelops in the prese11ce of the urealytic
age11l il1 a11 area constantly irrigated witl1 urine. An1n1onia
is thougl1t to initiate the il1fla1nmatory process. A sirnilar
condition. occurs in goats. Only C. renale and C. pilosurn
have been tound in ovine posthitis.
Epiden1iology
Bovine pyelonephritis is found rnostly in cows near partu-
rition, appearing asan opportunlstic infection by a com-
n1ensal organlstn. Bulls are rarely affected, but are com-
mi>ns<il hosts of ;ill three types and the sole co1n111ensal
source of C. cystitidis (Type III).
"Pizzle rot" occurs typically in animals on rich legume
pasture that is high in protcins, which incrcasc urca cxcrc-
tion, and estrogens, which cause preputial swelling and
u rine retention in the sheath.
lmmunologic Aspects
No useful in11nu11ity develops in the course of the infec-
tion. SeruJn antibody is present and antibody coating
(mostly IgG) of bacteria in urine occurs in bovine pyelone-
phritis (not cystitis).
No in1n1unizil1g age11ts exist.
Laboratory Diagnosis
Gross examination of urine may revea! the presence of red
blood cclls nnd high nlkalinity (pH 9.0). Microscopi.cally,
packets of pleomorphic gram-positive diphtheroid rods
are seen (Fig 31.5) . '['he agent is readi\y cultuted from sedi-
ment. A generous lnoculum of colonial growth planted iu
one spot on a Christensen's ureil ilgar slant vvill procluc.e an
alkali11e shift, it1dicati.ng urea hydrolysis, within minutes
of inoculatio11. A diphtheroid isolate from urine capable of
producing this rcaction and fcrmcnting glucosc probably
belongs to the e.;. renale Crroup.
Treatment and Control
Mcmbcrs of thc C. renalc group are susceptible to pe11i-
cillin, but antimicrobic therapy is successful only ín the
early stage of the infection.
Ovine posthitis is treated by surgical care of leslons,
local antiseptic applications, dietary restriction, an d
LesLosLero11e adn1inislralion.
180 PART TI Bacteria and Fungi

F1G U RE 3 1 . 5. Corynebacterium renale in urinary sediment
of a cow wilh µyelu11eµ/11ili;. Nutr: "diplrtheroid" configurations,
including palisades and " Chinese Ietters. " Gram stain, 3000X.
ACTINOBACULUM (fUBACTERIUM,
(ORYNEBACTERIUM) SU/S
Ar.tinnhar.1J.l1J.m is an anaerobic diphtheroid, whic-':
Sli.ÍS,
was first described as Corynebacteriurn suís. lt is assoclatec
with urinary tract infection of sows (see Fig 74.3). L~
bovinc pycloncphritis, thc discasc is an apparcntly ascenc-
ing iI1fection by an urealytic diphtheroid agent, limited ~
females and often related to breeding operations, pre_;-
nancy, and parturition. Boars are frequent carrlers. Tt~
clist>ast> is rt>cognized prima.ri.ly in Brita.i n a.n d continen~
Europe but also in North America, Australia, and Ho::;
Kong. 'f'reatment is rarely successful.

(ORYNEBACTERIUM AURICANIS
•

í:nrynPhartPri11m a11rirani~ i<; ;i typir;i 1 rliphthProirl i<;oJ;i--

from various disease processes in dogs, mainly otitis e:>.-
ter11a and pyoderma. It has been isolated from the vag::-_
of normal canincs.
 Erysipelothrix
RICI-IARD L.
- rh11siopathiat? is the typP species of the genus
~ipe.lothrix
-~is the species of prünary irnportar1ce. A second spe-
~. E. tonsillarun1, 11as been described for sorne strains pre-
-=ly de~;ignated as serotype~; of E. rhusiopathiaa. Erysipa
ix tonstllarum is biochemically and morphologicany
....;lar to E. rhusiopathiae but is genetically distinct by
.,-DNA hon1ology. E1ysipelothrix tonsillarutn is only oc-
- ü nally involved in clinical disease and is nonpatho-
-c for swi.nc. An additional species, E. inopinata, has re-
~..tty been described.
~'.Sipelothrix rhusiopathiae can be isolated from a wide
-=~ry of environmental settings as well as trorn the ali-
- --~ary tract and lymphoid tissue .of healthy animals.
..,.,. d isease, erysipelas, occurs il1 various anin1al species,
-: swine the inost frequently and severely affected.
cr susceptible animals include turkeys and sheep.
~·e.al presentations include septicemia, a generalized
- form, arthritis, and vegetative endocarditis. In hu-
- --::s. ttle .m ost commonly recognized form of infection is
">eloid, a self-limiting infection of the skin, usually in-
Tilg the hand.
--=rriptive features
-: -:>logy and Staining
; _lothrix rhusiopathiae is a gran1-positive, non1notile 1
~- acid-fast, non spore-forming bacillus, which n1eas
.z to 0.4 µrr1 t)y 0.8 to 2.5 µm in size. On subculture,
- -_-: colonies may develop and produce filamentous
óO pu1or111ore iu leugtli.
I::::::-:-·e and Composition
-e!oth rix rhusiopathiae exhibits a cell waU typical of
:==,-positive organisms. It contains murein of the B I
:;pe. The diamino acid of cell vvaH peptidoglycan is
-=- Tbe DNA tJase con1position is 36- 40 mol'31> G +C. A
~ccharidc capsule has heen desr.riheci :ind rPl:itPcl to
--ce. Little is kl10V1'l1 about thc surface proteins of
---dt:Jthrix. A protective protein, SpaA, shares similari-
- the e terminal regi.o n with choline-binding pro-
_: Srreptococcus pneumoniae.
=.....::--,._, Ptoducts of Medica! lnterest
- - ' - See "Neuraminidase," below.
c'4/e. Erysipelothrix rhusiopathiae produces a polysac-
WALKER
charide capsule, whicl1 protects the microorganism from
phagocytosis.
Ce/l l!Vall. The cell wall of the 1uembers of this genus is
onc typical of gram positivc bacteria. Thc li.potcicho!c r
acids and peptidoglycan of the gram-positive cell ,.vall in- 1
teract wi.th macrophage cells resulting in the release of
¡.iroi11 ílau1111i:ltury cytokines.
Neurarninidase. Nr.ur:im in icl:ise prociuction varies di-
rectly with virulence of E. rhusiopathiae. Cleavage of sialic
acid residues on endotl1elial cells leads to thrombus forma-
tion. Neuraminidase is also responsible for adhcrcncc to
cell surfaces.
Miscellaneous Products. Most strains of E. rhusiopathiae
produce hyaluru1üdase auu coagulase, tJut there lloes not
appear to be a relationsh ip ben-ve.en vi ru lencP :inci thPsf'
enzymes.
Growth Characteristics
Growth is best on inedia supplemcnted with glucose.
Erysipelothrix rhusiopatlliae is a facultative anaerobe prefer-
ri.ng an enviro11ment co11tainiI1g 5% to 10% COz. Optima!
growth occurs at 30ºC to 37ºC and at a pH of 7.2 to 7.6;
however, it is capable of growing overa temperature range
of 5ºC to 42ºC anda pH range of 6.7 to 9.2.
Resistance
Erysipeluthrix rhusiupathiac is resistant to drying and with.-
st:incls s:ilting, pickling, :incl smoking. Tt s11rvives for 11p to
6 months in swine feces and fish slin1e at cool ten1pera-
tures. ft is killed by moist heat (SSºC) in 15 minutes, but
grows in thc prcscncc of potassium tclluritc (0.05%), crys-
tal Violet (0.001 º/o), phenol (ll.2o/o), and sodiu1n azide
(0.1 %). lirysipelothrix rhusiopathiae is susceptible to peni-
cllll n, cephalosporin, clint1amycin, and fluoroquinolones
but is resistant to novobiocin, sulfonarrlides, and amino-
glycosides. Resista11cc Lo eryll1ron1ycin oleando1nycin ox-
1 1
ytetracycline, and dihydrostreptomycin has been ob-
scrvcd. Rcsistancc is apparently not plasmid-mediated.
Variability
Common heat-labile antigens account for cross reactions
betwee11 strains. Heat-stab.le, somatic antigens account for
the existence of at least 23 serotypes. No relationship be-
t1-veen 11osl species a11d :serotype has tJeen recognized.
Serotypes 3, 7, 10, 14, 20, 22, and 2~ exhihit ;i highPr <iPgrPP
181
182 PART II Bacteria and Fungí
of DNA-DNA homology with the type strain of E. tonsil-
laruni than with the type strain of E. rhusiopathiae. Cul-
tures dissociate on passage fro1n convex, circular, smooth
colonics vvith entire edges to rough colonies with undulate
edges. L-forms 11ave been reported.
Ecology
Reservoir
Erysipelothrix rhusiopathiae is often recovered trorn sewage
cffluent, abattoirs, surface slime of fresl1 and saltwater fish,
and soil. It has been recovered from over 50 species of
mamtnals and 30 species o f wild birds, and it can be iso-
lated frorn tl1e tonsils of apparently healtlry ~vviuc, 1J1e
most prominent reservoi r.
Transmission
1·ransmission among anin1als is mostly by ingestion of
contaminated n1aterial (surface \-vater, fish mea!). Wound
infcctions and arthropod bites are other possible routes.
Pathogenesis
Mechantsms. Strains ot E. rhusiopathiae vary in virulence.
Virulent strains produce 11igh \evels of neuran1i11idase,
wllicl1 i!> con!ii<.lere<.I an important viru lence factor in acute
septic.emic. infec.tions. Ni>11r;:iminicl;isp clf';:ives sialic acid
present on cell surfaces, leading to vascular dan1age a11d
hyaline t hrombus formation. Neuraminidase plays a role
in bacteria! attachment and invasion into cells. Antibodies
to neura1nil1idase are protective against experimental i.n-
fections in mice. The presence of capsules has been de-
scribed and appears to play a role in resistance to phagocy-
tosis by polymorphonuclear leukocytcs. Survival inside
professional phagocyLes is in.1µo r la11l for µalhuge1licity. 111
t he presence of normal serum, acapsular mutants do not
survive inside phagocytes, whereas encapsulated orga11-
isms survive and multiply. In the presence of i1nmu1.1e sera,
organisms are readily killed.
Partial immunity of the host and Jow virulence of
strains are felt to account for the localized skin form in
:;wilre.
Localization of R. rhusiopatlziae in joints of S\"line leads
to fibrinous exudation and pannus formation. Subsequent
damage to the articular cartilage and an iinmunologic re-
sponse to persistcnt bacteria! antigcns in synovial tissuc
and chondrocytes are responsible for the chronic articular
changes.
Valvular endocarditis, presumably initiate<.l by bacteria!
emholi and vascular infla rnmation, res11lts in c.hronic.
changes and damage to the heart valves.
Pathology. Pigs dying from acute erysipelas infections
exhibit hen1orrhages of thc gastric serosa, skelctal and car-
diac muscles, and renal cortex. Congestion of lungs, liver,
spleen, skin, and urinary bladder is frequent. Vascular
<.lan1age with inicrot hrombi is observed microscopically. A
mononu clear infiltrate p redominati>s in most r;isi>s .
Raised, pink to purple cutaneous lesions result from vas-
culitis, thrombosis, and ischemia.
In joints, acute synovitis often proceeds to rnore chronic
articular changes. Synovial membranes becomc bypcrplas-
tic and villous anct are il1tiltrated with mononuclear cells.
Spreading of granulation tissue over articular surfaces and
erosion of articular cartilage may occur. Ankylosls of the
joint may be the ultimate outcome. Localization to inter-
vertel>ral <.li:;k~ leaJs LO a deslruclive diskospo11dyl itis.
In valvular endocarditis, the Initral valve is most com-
monly involved with development of Jargc, valvular vcgc-
tations dueto fibrin deposition and connective tissue pro-
liferation. Emboli Jnay produce infarcts in the spleen anc
kidney.
In turkeys, the pathology associated vvith erysipelas in-
fet:tiu11:; l:; generally 1narked by co11gestion and intramus-
r.ular and suhpleural hemorrhages (Fig 32.1). The liver anc
spleen are often swollen and tl1e abdo.m inal fat is pctcchi-
ated. A swollen, cyanotic snood anct ctiffuse red.dening o:
the skin are frequent.
Disease Patterns
Swine. Swine with the septicemic form present with fever.
anorexia, depression, vomition, stiff gait, and reluctanc.:
to walk. Urticaria! Iesions in the skin may be palpai)le bc-
fore becorning visible.1' hey may be pink or, in severe. cases
purpl1sh, espec.ially on tn.e abó.ornen, thi¡shs, ears, and tal..
1n severe cases, the skin becomes necrotic and is slough.e c
If untreated, this form has a high mortality rate.
T..n a less severe form of erysipelas in swine, lesions are
lin1ited to tl1e skin bu t n1ay be accon1pa11ied by a n1ilc!
fe.ver. Skin lesions <l rf' ri>d to p11rpli> rhomhoid;i ls-
"diamond skin disease." Lesions n1ay progress to necrosis
or resolve, leaving a mild scruffiness to the skin. Mortality
is seldom associated with this forrn.
Localizat ion to certain tissues leads to chro11ic forms.
which may occur as sequelae to the acute stages or without
previous illness. A vegetative endocarditis is manifeste<l b \·
signs of r;ircli;ir ins11ffiriP.ncy or suddf'n death. Arthriti.s is
the other chronic forn1 seen in swine. Signs include lin1p-
ing, stiff gait, and enlargement of the affected joints.
Infreque11tly, sows abort dueto Erysipclothrix infection.
Birds. Erysipelas in birds, especially turkeys, is usually a
:;epticernia. Turkeys <.levelop a cyanotic ~kin, l>ecumt:
F J G U RE 3 2 . 1 . Mu/tiple petechial hemorrhages on t he muse/E
surface of a turkey with an Erysipelothrix septicemia.

c1roopy, and may subsequcntly die. A swollen cyanotic
snood, if present, is considered almost path.og11on1onic.
~1ortality rates range from 20/o to 2.'i%. C-:hronic manifesta-
tions include vegetative endocarditis and arthritis. Turkeys
"ith endocarditis appear weak and emaciated or die sud-
denly without prior signs. Other affected avían species in
elude chlckens, chukars, ducks, emus, parrors, peacocks,
an<I phi>;:i<;ant<;.
Sheep. Polyarthr1t1s is the most common presentation ot
E. rhusiopathiaa infection in shcep. Entry is thought to be
rhrough the umblllcus or wounds associated with castra-
~ion, rlocking, or shearing. Affected animals have a stiff
gait and, often, swollen joints. Thcy n1ay 11ave Lro uble geL-
ting up and dov•n. A cutaneous infection following d ip -
ping also occurs in shccp. Pncumonia has bccn dcscribed
in ewes.
Miscellaneous Species. Erysipelothrix rhusiopathiae causes
arthritis and endocarditis in dogs. Erysipelothrix tonsil-
larum can also be a. ca.ni ne pathogcn and has bccn isolatcd
from dogs w1th enclocarditts. Septicemia and urticaria due
to E. rhusiopathiae have been reported in dolphins. Hu man
:i1fecliu11:; uf :.kü1 autl sulJcutls art' callt'tl crysipeloid and are
seen mostly in animal an<I fish-han<lli>r<;_ Septicemia, en-
docarditis, and polyarthritis are rare. l luman "erysipelas"
is a streptococcal infection.
:pidemiology
?igs less ll1a11 3 i11onlhs and ove1 3 year:; uf age art: lt:a:;t
susceptible. Variable passive and active immunity proha-
bly accounts for agc-rclated susceptibility. Predisposing
:actors include environmental stress, d.ietary change, fa-
tigue, and subclinical aflatoxicosis.
In turkeys, the male is most frequently infected, possi-
bly through fight wounds. Tnsemination of hens with co11-
ia!I1inated semen is an 1·m portant source of infcctlon.
lmmunol og ic Aspects
lmmune Mechanisrns in Pathogenesis
Persistence ot antigen in thc joint tiss ue acts as a chronic
stimulus for immune reaction and dcvelopment of arthri
ilS. In addition, an autolmmunc proccss secondary to the
erysipelas infection may be responsiblc for sorne of the
.:hronic joinl changes.
Yechanisms of Resistance and Recovery
'='ell-mediated and humoral responses occur, directed at
-;;euramiltidase, pi olecli vt: :.urface protein, anú other cell
- ·ali components. Serum opsonins apparently play a <leci-
~1\·c role. Phagocytosis is carried out primarily by mononu-
ctear phagocytes.
.:.rtificial lmmuni2ation
_-,ttcnuated vaccines and bactcrins have been used for vac-
2 nation in swine. While effectivc against the acute forms,
r:hnjJter 32 Erysipelothri:x 183
neither type appears to be highly protective against
chro11ic t:ry:;i pt:la:.. Attenuated vaccines are given orall)',
pari>nti>rally, or, in some countries, by aerosol. Whole-cell
bacterins and soluble anligen a1e given subcuta11t:uu:sly ur
intramuscularly. Most commercial vaccines are prepared
from serotype 2. Ccrtoin strains havc becn rcfractory
to vaccine-1nduced 1mmunity. Formalin-inactivated,
aluminum-hydroxide-absorbed bactcrins appeaI to be ef-
fecUve i11 lurkt:y:..
Laborat ory Diagn osis
Specimens
Specimens are collectcd from appropriate sites according
tu :sign:s. Bloutl lultures from severa! affected ani.mals ate
useful in d i agno.~ing <;ppticemia . Necropsy specimens in-
clude liver, spleen, kidney, heart, a11d syn ovial Lissue.
Recovery of the organisms from skin lesions is also possi-
ble. In the more chronic forms, cultures from joints or
heart valves are Iess successful.
Direct Examination
Specimens are examined by Gram stain for the prese11ce of
gram-positive rods. A negativc result does not preclude
infection.
Culture
SamplPs arP plati><I on hloo<I agar and incubated at 37ºC in
10% C0 2. Colonies are often nonhen1olytic and pil1poil1l
after 24 hours' incubation. At 48 hours, a greenish hemol-
ysis may be apparent. E. rhusiopathiae is catalase- and
oxidase-negative and nonmotile. Tnoculation of triple
sugar iron agar slants will show an acid reaction and H2 S
production along the stab line (Fig 32.2). A "pipe cleaner"
type of growth occurs in gelatin stab cultures of rough
coloJ1ies held al ioon1 Le111veralurt: for 3 tu 5 <.lc1y:s.
Erysipelothrix rhusiopathiae does not hydrolyze esculi n o r
urca, reduce nitratcs, or produce índole. Fermentative ac-
tivity is weak. fermentable carbohydra tes include glucose,
lactose, levulose, and dcxtrin. Ji,rysipelothrix tonsillarum
usually ferments saccharosc while E. rhusiopathiae ctoes not.
Selective media containing various aminoglycosides
a11d vanco111yci 11 111ay U\:! usetl to isolate E. rhusiopatfziae
frorn contaminated tissue. Othi>r .~f'li>ctivi> media contain
sodium azide (0.1 %) and crystal violet (0.001 %).
Serology is ot little value in diagnosing erysipelas infec-
tions. Polymerase chain rcaction assays have becn de-
scribed for diagnostic use.
Treatment, Control, an d Prevention
Treatment with penicillin for at lcast S days is effective
againsr the acure forms of erysipclas in swine. 'letracycline
and tylosin are alternatives, although resistance to oxytet
1acycli11t: autl sorne macrolides has been reported.
Antiserum (e<Juini> origin) is sometimes used in conjunc-
184 PART 11 Bacteria and Fungi

tion vvith antibiotic therapy. Treat1nent of ch..ro.n ic forros :...

F 1G U RE 3 2 . 2 . Hydrogen sulfide production by Erysipelothrix
rhusiopat hiae a/ong srab fine in triple sugar iron agar slanr.
Jess successful.
Good sanitation and nutrition are beneficial in p:-.:-
venting outbreaks. Infected carcasses should be dispose_~
of in a proper manner and replaceme11t aniinals isolata'..
for at least 30 days before il1troductio11 into tl1e h en:..
Vaccination is recommended in areas wlth prevlous bis-
tory of erysipelas.
In turkeys, pei.1lclllin. is the drug of choice. Subcuta:i-
eous in jection of penicillin and vaccination with erys:-
pclas bacterin are recommended, if practicable. Penicill!n
in the drinking water for 4 to 5 days has been effective ~
controlling sorne outbreaks. lnjectable erythromycin is -
recomn1ended alternatlve treatme11t..
<] ood n1anagement practices .including pr<"Vf'ntir;
flghling an1011g lo111s, ensuri.ng proper insen1il1ation prac-
tices of turkcy bens, rotating turkey ranges away frorr:
contuminatcd arca~, and using vaccinotion in a rea¡; v<i th::
history of erysipelas are useful preventive and contrc
n1easures.
 Listeria
RICHARD L. W ALKER
Listeriosis is an infectious disease affecting humans and
many specles of anlmals and blrds. Of slx recognlzed
species of l.i.~teria-1 .. innnr.ua, l.. ivannvii, l .. grayi, l .. mnno-
cytogencs, L. sccligeri, and L. welshi111eri-L. 111onocytogenes
and L. ivanovii are important pathogens. 1Wo subspecies of
Listcria ivano~·ii are recognizcd-subspccics ivanovii and
subspccics londoniensis.
Run1inants are the most frequently affected domestic
anlmals. Principal forms of listeriosis tnclude septicemia,
encephalitis, and ahortion. Tn sheep ancl cattle, ahortion is
the usual manifestation of L. ivanovii infections. Listeriosis
occurs \.VOrldwide, espedally in temperate climates.
Descriptive Features
Morphology and Staining
Listeria are gram-positive, non-acid-fast, non- spore-
furruíug, acaµsular ru<.l-shaµe<.l IJacterla, wliich rneasure
O.S to 2 11m hy 0.4 to O.:'i pm.
Structure and Composition
Listeria has a typical gram-positive cell wall. Meso-
diaminopimelic acid is t he major diamino acid. Cell wall
polysaccharldes determine 0-antigen. Perltrichous flagella
an d motility are present at 22ºC. Motility is poor at 37ºC.
Cellular Products of Medical lnterest
..lctA .ActA protein is important in intracellular movement
by actin polymerization and is also thought to play a role
in ccll trophlsm (adhesion) and lnvas1on.
Adhesins See "Internalins," below, and "ActA," above.
Ccll Wall. The cell wall of thc n1en1bers of Ll1is gei1us is
one typical of gram-positive bacteria. The lipoteichoic
acids and pcptidoglycan of thc gram-positivc ccll wall in-
teract w1th macrophage cells resulting in the release ot
p roinflamn1atory cytokines.
Hemolysln. See "Listerlolysin O," below.
Tnt·P.rna lins. Tntf'rnalins are surface proteins. res.ponsible
:nr adhesion and entry in to target ce lis.
Listeriolysin O. LLO (for listerio/ysin O) is a pore-forming,
::holesterol-dcpcndcnt cytolysin that is a major virulence
jeterm1nant for th1s species (mutants defictent in this pro-
·ein have reduced virulence; antibodies to it are protective).
:ne ma)or role for LLO is in the release of L. 1nonocytogenes
-om the phagosome into the cytosol fol lowing phago-
sorne acidification, under which condition s LLO is most
active. Other roles include lysis of fcrritin vacuoles and per-
haps its affect on s.econ dary vesicles fo rmed during L.
111onocytoge11es n1oven1enl fro1n ceil to cell. LLO l:s al:su
thought to ind uce apoptosis in hPp::itocytPs. lv;.inolysin,
another ch olesterol-dependent cytolysin, is the counter-
part in L. ivanovii. See also, streptococcal streptolysin O, in
Chapter 28; clostridial perfringolysin O, in Chaptcr 36; and
arcanobacterial pyolysin, 1n Chaptcr 29.
Phosp11olipnses C. Phosphatidylinositol-specific phos-
pholipase Canda lecilhi11ase a1e iJ11¡.io1 La11l i11 u11::uiatiI1g
membrane lysis.
Misccllancous Products. A bilc salt hydrolase may pro·
mote survival and persistence ot Listeria in the intestinal
lu1ncn. A p rotein, termed p60, may play a role i11 adher
enceto target cells.
Growth Characteristics
Lisceria are facultative anaerobes that grow best under re-
duced oxygen and increas.ed carbon dioxide concentra-
llon. Growlh occurs al 4ºC lo 45ºC, wilh a11 o¡.ili1J1u1u al
30ºC to 37ºC. Simple laboratory media support growth,
preferably at an alkaline or neutral pJI. On sheep blood
agar, most strains of L. monocytogenes produce a narrow
zone of hemolysis. Colonies are usually 1 to 2 mm in di-
ameter and appear blue-green in obhquely transm1tted
light on solid media such as tryptose agar. Colonies of L.
i vunuvii t y pi call y pru<.lucc a largcr a Htl r11ure in tense zone
of hemolysis.
Listcria tolerates 0.04o/v potassium tellurite, 0.025~1
thallium acetate, 3.75% potassium thiocyanate, lOo/o
NaCI, and 40% bile in media. Most strains grow ovcr a pf-T
range of 5.5 to 9.6. It has greater heat tolerance than other
non-spore-forming bacteria; however, short-time high-
terr1p<::rature pasteurization is effectlve at killlng Listeria.
Variability
There are 16 recognized serovars in the genus Listeria based
on somatic (O) and flagellar (H) antigens.. 'I'here is no cor-
n::laliu11 uctweer1 :serovars and speclcs, cxcept that strains
of serovar .'i are 1.. ivannvii. No rPl:itionship between sero-
vars and host specificity has been recognized. Various i1u-
cleic acid-based methods have been used to f11rther clic;-
criminatc bctwccn Listeria strains for epidemiologic
analysis and strain tracking purposes. Whole genome se-
quencing of specles '\.\rithin the genus has recently identi
185
186 PART 11 Bacteria and Fungi
ficd nu1ncrous genes found in virulcnt spccics but absent
in avirulent species.
Smooth an.d rough colonial variants occur. In rough
colonies, filan1ents 20 µm or n1ore 111 length may be ob-
served . T..-forms develop on media containing penicillin
<tnd have been isolated fron1 clinical cases in hun1ans.
Ecology
Reservoir
Listeria have a worldwide distribution and have been iso-
lated from soil, silage, sewage effluent, stream water, and
over 50 species of animals, including rum.inants, s~vine,
horses, dogs, Ci:lt:s, a11u variou:, sµecie:> of IJirds. 111 sorne
an~as. up to 70°Ai of hun1ans are reported to be asympto-
matic fecal carriers. Many isolates from environme11tal
sarnples, previously called L. monocytogenes, would now be
identified as one of the nonpathogenic species based on
current taxonomic criteria.
Transmission
Soil conta1nination an<l ingestior1 of co11tC11ui11ateu feeu
;:irf' thf' prim;:iry modes of transmission of Listeria. Poor
quality silage, with a pH greatcr than 5 .5, is conunonly im-
plicated and accou11ts tor listeriosis often being referred to
as "sil<ige disease." An asy1nptomatic carrier can be a
source for furtl1er co11tami11ation oí the e11vironrnent and,
therefore, an iI1direct source of infection.
Pathogenesis
Mechanisrns . Exposure to Listeria occurs vi.a the oral route.
Most Listeria are destroycd by gastric acids. Use of antacids
and J-12 blockers increases survival rate and are considered
riskfactors for developing listeriosis. Intestinal translocation
appears to be a passive process that can involve bot h intes-
tinal epithelial cells and M-cells overlying Peyer's patches.
Ii1te111alin, a su1face prolein, a11d ils l.nte1acllo11 vvitl1 11ost
cell receptors rnediate entry. After passage th rough the intes-
tinal barrier, Listeria can be observed in phagocytic cells
within the lamina propria. Further clissemination occurs vía
.. -
-·
~
..,._ ...
- .. .. ??•
- ... ...
• ~
•• .. lt¡, el Aj)
p~
• "'
thc bloodstrcam. Various bactcrial ligands have been identi-
fied for adherence and include proteins oí the n1ulti-gene in-
ternalin farnlly, A.ctA and p60. Nonphagocytic cells can in-
ternalize listeriae through a zipper-type rnechani:;ni. After
intern;:ili7.;:ition, listeri;:ie esc;:ipe from the phagosorne, he-
corne associated with actin filaments in the cytoplasm, and
propels itseJf to the cell's plasma membrane via polar assem-
bly of actir1 filamcnts (t\ctA). In this way, it is able to pass to
neighboring cells in plasma membrane protrusions and
tl1us avo.id host defense mechanisms.
An alternative route of entry has tJee11 µroµoseu Ior CNS
infections through o;:im;:iged oral, nasal, or ocular mucosa!
surfaces vía the neural sheath of peripheral nerve endings,
particularly the trigeminal nerve. lt is postulated that cen-
trípeta! migration along cranial i1ervE~s leads to infection
of the central nervous systen1. Organisms have been de-
monstrated in the myelinated ax.ons of the trigeminal
nerve an<l cytoplasm of rnedulli:lry IH::urur 1:,. 'flte lack of
viscf'r;:¡J involveme.nt supports a route other than hemato-
genous although a primary hematogenous route cannot
be discounted.
l'athology. With cc11tral nervous system involvement,
the cerebrospinal fluid may be cloudy and the meningeal
vessels congested. Occasionally, areas of softening in the
medulla are seen. Iiistologically, perlvascular cuffing pre-
rl()min;itprl hy tymph()rytp<; ;in<i hi!';tiocytf's is commonl}'
observed (Fig 33.1). Focal necrosis a11d 1nicroglial and neu-
trophilic infiltrates are seen in parenchymal tissue_
Result ing microabscesses are charactcrizcd by Jiqucfaction
of the neuropil. The rhomboencephalon area of tl1e brain
is most commonly involved.
In the septicemic form, multifocal to diffuse necrosis in
the liver (Fig 33.2) and, less frequently, the spleen m.ay be
110Led.
In t he aborted fetus of ruminants, gross lesions are min -
imal. Autolysis is usually prcscnt as a rcsult of thc dcac.
fetus being retained for a period betore bei11g expelled.
Disc;i~c P;ittcrn5
Clinical outcon>e depends on the nun1ber of organisn1s in-
gested, pathogenic properties of the strain of Listeria, anc
the immune status of the host. Septicemia, cnccphalitis
and abortio11 are the major disease forms.
~
$>
F 1G U RE 3 3. 1 . Section lro1n the brain of a cow with the
encephalitic form of /isteriosis. Perivascular cuffing is present (H&E
~ Listeria monocytogenes was iso/ated

FIGURE 33.2. Section of liver from a 5-week-old foal that
died of Listerii:l rnonocytoye11es sepiite111ia. Asevere, diffuse necro-
tizinq hepatitis was present.
Monogastrics and Neonates. Tlle :;eµtice1uic for1u rnarked by
depression, inappetence, fever, and deatl1 is the most com-
mon in monogastric anin1als and in i1eo11ates.
Chinchillas. Chinchillas are particularly susceptible to liste-
ri;.i J sf'ptic.f'mi;:i.
Horses. Septicemia in neonates is the most common pre-
seutation in horses.
Ruminants. Encephaliti.s. The encephalitic form, sometimes
called circling disease is the most common form in run1i-
nants. In cattlc, it is subacute to chronlc. Signs include de-
pression, anorexia, and tenden cy to Circle in one direction.,
head pressing or turning of the head to one side, unilateral
facial paralysis, and bilateral keratoconjunctivitis. Similar
signs are seen in sl1cep al1d goats, but tl1e co·urse is more
acute anct frequently fatal.
Abortion. Abortion is common in ruminants, but also
occurs in other species. AtJortion is usually late tern1-
;:iftf'r 7 mnnths in cattlf' ;:inci 1?. wf'f'kS in sheep. The fetus
m ay be macerated or delivered weak and moribund.
Retained placenta and metritis may result. Systen1ic signs
are rarc i.n thc cow unlcss thc fctus is rctaincd and triggcrs
a fatal septice1nia. Although abortion is usually sporadic,
abortion rates of up to 10% have been noted. Tt is uncom-
won tu fir1<l the encephalitic forrn and abortions occur-
ri ng in ;.¡ singlf' 011thrf';:ik.
Conjunctivitis. Conjunctivitis in run1inants \'Vi.t l1out as-
sociated abort ions J1as bee11 related to feeding on contam-
inated silage in elevated fecd bu.nkcrs.
Humans. In hun1ans, meningitis is the most comn1on of
ihe three major forms of listeriosis. Still other disease man-
ifestations include infective endocarditis, oculoglandular
d iscase, and dern1atitis.
Chapter 33 Listeria 187
Epidemiology
·r11e widespread distribution of environmental and animal-
associated occurrence of Lísteria makes localizing the
source of a particular outbreak difficu1t. Contaminated
silage is a classic sou1ce oI infecLion. O Lher sources include
particularly organic refuse (e.g ., poultry litter). Stress fac-
tors prcdisposing to clinical disease include nutritional de-
ficiencies, environmental condi tions (including elevated
iron concentrations), underlying disease, a11d pregna11cy.
Cases are usually sporadic and may involve up to So/o of cat-
tle herds or 10% of sheep flocks over a two-month period
oI lin1e. Lisleriosis il1 a11ilr1als usually occur::. i11 Ll1e wi11t.er
and spring.
Most humal1 cases occur in urban environments in the
summer. ·l'here are occasional reports of listerial dermatitis
in veterir1arians and others after handling tissues from lis
terlal abortlons. Otherwise, animals are unlikely direct
sources of h uman infections. Human epidem.i.cs have been
traced t o food sources of anin1al origiu, i11clu<.li11g 111ilk,
Latin-style cheeses, 11ot dogs, and liver paté. Coleslaw
rnadc from cabbage originating on a farm with recent his-
tory of ovine listeriosis was thc source in one outbreak. In
many instances, postprocessing con.tamination is found to
be the source for Lisreria contamin.ation. Frequently there
is also opportun ity for selective growth of L. rnonocytogenes
Lo occur duriug loug periu<.ls of refrigeration.
lmmunologic Aspects
The 111ajorily of ltu111an ca:;es of Usteriosis are associate<.l
V\'Íth immunosuppressed individuals, the eldf~rly, the very
young, and pregnant women. Likewise in anin1als 1
neonates and pregnant animals are predisposed; however.
in sorne cases a predisposing immunosuppressive factor is
not apparent.
As a facultatively intracellular parasite, Listeria is prirna-
rily contained t>y cell-mediated responses. Humoral fac-
to rs may play somf' limitf'ci rnlf' in hnst ciefense.
No immunizing preparations have met vvith ~ignificant
success. Killed preparations have been ineffective, while
live attenuated vaccines afforded SOlne protection in shccp.
Laboratory Diagnosis
Specimens
Laboratory dlag11osls ls based u pon isolation of the organ-
ism. Spinal fl11id , blood, brain tissue, spleen, liver, abo-
masal fluid, and/or n1eco11iun1 are cultured, dependiI1g on
signs, lesions, and tissues available.
Direct Examination
A direct smear of infected tissue may reveal numerous
gram-posi.tive rods in septicen1ias and abortions; however,
fewer nun1bers of organisn1s are Lypically ol>serve<.l iu the
encephalitic form (Fig 33.3). Negative findings are inc.on-
clusivc. Immunol1istochemical staining with .specific anti-
sera is also usetul in diagnosir1g encephalitic listeriosis.
188 PART fl Bacteria and Fungí

F 1G U R E 3 3 . 3. Gn'lm-st;iinPrl im{lrPssinn smP.ilr frnm thP.

brain stem of a goat with /isteriosis. Rare to small numbers of gram-
positive, regular rods are present (arrows).

lsolation
Samples are plated on sheep blood agar a11d incubated at
35ºC in 10°/o C0 2 . Isolation of L. rnonocytogenes from brain
tissuc may be cnhanccd by pour platc mcthods. Aftcr thc
initial isolation atten1pts, remaining tissue is stored at 4~C
for "cold enrichment." Such tissue is subcultured weekly
for up to 12 weel<s. Cold e11ricllrne11t i s 11ot necessary for
isolatio11 fro1n listerial abortions or septicemias.
For samples where contan1il1ation is likely, enricl1111e11t
and the use of selective media (lithiun1 chloride-
phcnylcthanol-moxalactam mcdium, Oxford medium, or
PALCAM Listeria selective 1nedium) are advisable. Various
DNA-based and antigen capture methods for detection of
Ltsterta llave been described, especially tor detectio11 ill
food products.
lmmunodiagnosis
Serology has not been useful for diagnosis due to the
prevalence of positive titers in apparently normal animals
and cross-rcactions with Staphylococcus aureus, Enterococcus
raecalis, and Arcanobacteriurn pyo¡;:enes.
F1G URE 3 3 . 4. Positive CAMP reactions of Listeria monocyto-
genes (LM) with Staphylococcus aureus (SA) and L. ivanovii (LIV)
wilh Rhodocuccus equi (RE). A weak reaclion ís seen between L.
monocytogenes and R. equi. No reaction is detected with L. innocua
(LIN). The variation in the degree of intensity of hemolysis of L.
monocytogenes compared to L. ivanovii is apparent. Listeria innocua
is not hemolytic.

ldentification
fypical colonies consisting of gram-positive, regular rods
are suggestive. Listeria is catalase-positive, n1otile at 25ºC,
and hydrolyzes esculin. Listeria rnonocytogenes is CAMP-
rositivP WhPn Cf()<;<;. <;trPilkPrl With ;¡ hPtil -tOJl'in produCin3
Staplzylococcus aureus on 5% washed sheep blood agar. A
similar phenomenon is observed when L. ivanovii is cross-
streaked with Rhodococcus equi. 1\ weak CJ\MP-like reaction
is sometimes observed between L. monocytogenes and J<.
equi. (Fig 33.4). In semisolid n1otility media incubated at
room temperature, a characteristic umbrella pattern of
motility develops 3 mm to 4 mm below the surface, dueto
LM
lhe n1icroaeropl1ilic nalure o[ Listeria (Fig 33.5). A11 e11d-
over-end tumbling type of motility with intermittent peri-
ods of quicsccncc is sccn in hanging drop prcparations.
Acic1 is produced from glucose anc1 1-rhamnose but not d -
mannitol or d -xylose by L. nionocytogenes. Listeria ivanovii
differs by fermenting d -xylose but not 1-rllamnose.
Fluorescent antibody staining or agglutination with spe-
cific anliserun1is11elpful.
Mouse inoculation causes death vvithin 5 days, with
ncccotjc focí prc:;cnt in thc livcr. Thi:; proccdurc diffcrcnti-
ates L. rnonocytogenes from nonpathogenic species of ,4 . 7.

Listeria; however, it is rarely necessary for definitive identi-

fication.
 (;hapter JJ Listeria 189

Treatment, Control, and Prevention

F 1G U RE 3 3. 5. Umbrcl/;i typc motility of Listcria monocyto-
genes In semi-so/Id motilíry media incubated at room temperature. Listeria 1no11ocytogenes is susceptible ín vítro to penícíllin,
a 111plclll1 n, erythromycln, tetracycllne, and rifamp1n.
C.hlortetracycline and penicillin may he~ <~ffr~ctive in t imely
treatment of cattle with meníngoencephali tís. 1·r eatment
1-i of sheep has been Jess successful. In human patients, the
combination of ampicillin and an aminoglycosidc (gcn-
tamicin) is the primary treatment approach.
Control measures includc rcductíon or elimínatío11 of
feedlng of silage, particularly poor-quallty sllage. Ali forms
of stresc; should he minimi1.c>rt. Affectc>rt animals should h<'
isolated and infected material disposed of properly.
Vaccination has not proven to be híghly successful and
may not be warranted due to the sporadic nature of the
discase.
 Rhodococcus
DWIGHT C.
Me1nbcrs of the genus Rhodococcus are pleomorphic,
11on-spore-forming, nonmotile, facultatively anaerobic
gram-positive bacilli. 1'h ere are 15 members of the genus
Rhodococcus, but only R. equi is ret,'t1larly associated wit!1
dl:st'.a:sc uf a1liu1al:s, particularly cquillt'. fual:s, wl1ere it
causes pneumonia. Rhodococcus equi is a facultat ive int ra-
cellular parasite of macrophages.
RHODOCOCCUS EQUI
Descriptive Features
Morphology and Staining
Rhodococcus equi cells measure about 1 µin by up to 5 ~un.
In sn1ears, ::;Lro11gly gra111-µu:siti ve rud:s a11d cucci (:sorne
being 1nisshapen appearing as "'vatermPlon SPPcis") ari> oh-
served. l'hey are encapsulated and are someti mes weakly
acid-fast.
Structurc and Composition
·rhe cell walls conlaio 1r1eso-dia1ui110-µi1uelic aciu (DAP),
arabinogalactan, and mycolic acids (branched chained
fatty acids \Vith a chain Icngth of C 30- C 54 in I~hodococcus).
111e capsules are polysaccharic.ie anc.i torm the basis of type
specificity within the species.
Cellular Products of Medica! lnterest
Capsule. In rr1ost species of gram-positive bacteria, t he role
of the capsule is to inhibit attachment to, and ingestlon
by, phagocytic host cells. Rhodococcus equi is a facultative
li1traceilular parasite of macropl1ages, which implies that
cith.er the capsule is not expressed in vivo, or it has a differ-
ent role than the one men tloned above.
Ce// Wulls. The gra111-1Ju::;ilive cell vvall c.:ur1tai.r1:s µuly:sac-
charides and lipids that are of medica! interest. The lipote-
icl1oic acids and peptidoglycan of the gram-positive cell
wall interact with .macrophage ce lis resulting in the release
of proinflammatory cytokines.
Virulence-Associared J)roteins (Vaps). Virulent strains of R.
equi possess a large plasmid (85-90 kb) tl1at contains 64
upe11 re<tLli11g fr<true:;. Wilhir1 Litis pla::;111iu is <t Patltuger1i-
city Island (a cluster of genes encoding virulence determi-
nant(::;), an integrase protein, a specific insertion site, and
mobility) that contains genes encoding the seven known
190
HIRSH ERNST L. BIBERSTEIN
Vaps (A- G). Vaps are responsible for the n1icroorganism s
ability to survive within phagolysosomes (resistance ro
acid and reactive oxygen intermediates) of macrophages
Whether Vaps are also responsible for interference vviti:.
pl1aguly:so:surue fu:sior1 i:s likely, l.Jut ur1µruvt:1i. 111 adilitiun
Vaps down regulate expression of gamma interferon b::-
CD4 and CD8 lymphocytes of foals (but not of adul~
horses). With increasing age, there is a gradual increase ir:
the ability of CD4 and CD8 Iymphocytes to respond ane
secrete effective levels of gamma interferon in order to
mou11t an effective immune response. Gamma-interferoi:
is rcsponslble for activatlng macrophages needed to de-
stroy phagocytosed R. equi.
Miscellarzeous Products. Rlzodococcus equi produces dif-
fusible /1 R. equi factors" (a phospholipase C and cholestero~
oxidase) which lysc crythrocytcs in sy11crgy with ph05-
pholipase V of C. pseudotuberculosis (see Fig 31.3). ·rhest:
factors probably play little if any role in the pathogenesís
of discase.
Growth Characteristics
Rhodococcus equi grows aerobically overa wide temperature
rauge (:startiug at lOºC) ou ruost rue<..lia. lvlucoi<..l colonies
("spit-Hke") clevelop in ;ihout 48 hours, may reach l<irge
sizcs (> 70 mm), and may acquire a pink pigmentation.
Biochemical Activities
The agent is catalase, urease, and nitrate-positive but does
not acidify routine fermentation media.
Resistance
The orga11ism withstands up to 2.5% oxalic acid and 5%
sulfuric acid for 60 and 45 Ininutes, respectively, a feature
utilized in attempts at isolation from soil and feces. It is
killed at 60ºC within an hour.
Rhodococcus equi i:s :susceptil.Jle tu rifarnpin, erytl1rurny-
cin, gentamicin, and usually to chloramphenicol, tetracy-
cline, and trimethoprim-sulfonamides, but not to beta-
lactam antibiotics. The combination of rifampin and
erythromycin is synergistic in vitro, forming the basis for
the use of this combination for treating affected animals.
Variability
Twenty-seven capsular types are described. ·rheir preva-
lence varies somewhat geographically.

Ecology
Reservoir
Rhodococcus equi occurs in soll and animal manure and,
perhaps secondarily, the i.ntesti.nf' of mammals ancl hircls.
Disease is see11 n1ost frequently in horses, less fre-
quently in swine, and rarely in cattle, sheep, goats, cats,
humans, crocodilians, koalas, buffalo, and llamas.
Transmission
fufection is acquired by inh.alation, ingestion, or congeni-
tally vla umbtlical or mucous mernbrar1e exposure.
Pathogenesis
M echanis1ns. After infection, R. equi is opsonized by com-
plement components (C3b) generated by the alternate
pathway. Opsonized R. equi associates with macrophages
by way of tl1e Mac-1 complem.ent receptor, and is phago-
cytosed. Rhodococcus equi is a facultative intracellular para-
sile, :surviviug i11 1uacropl1ages Ll1rougl1 suppressio11 of
p hagolysosomal fusion, and if fusion occurs, survival
within the phagolysosome (Vap directed). ln adult horses,
an tibodies to Vaps (from constant subclinical exposure)
which presumably "blocks" these proteins from interact-
ing with their targets (CD4 and CD8 lymphocytes), along
\vith CD4 an.d CD8 lymphocytes t hat secrete effective lev-
els of gamma interferon, result in activation of 1nac10-
phages resulting in the elimination of the microorganism.
fu (oal:;, 11ul. 011ly is Llle leve! of anlibodies Lo Vaps low dur-
ing the time of susceptibility (see below), but also the se-
crction of gamma-intcrfcron is down regulated (by Vaps).
r11e prognosis in foals is related to the amount circulating
anti Vaps antibody (passively acquired from the dam), the
number of R. equi that reach the lung, and the virulence of
the microorganisn1. The numbers that reach the lung are
related tu tl1e cu11ce11tratiu11 of Lhe n1ic!'oorganisn1 il1 ll1e
environment, as vvell as the degree in which the defe.n ses
of the lung are compromised (see "Epidcn1iology," bclow).
Pathology. ·rhe organism is a parasite of macrophages.
The lesions of R. equi infection are abscesses and granulo-
mas. Elements of gran.u lomatous inflammation include
macrophages and giant cells, with neutrophils predomi-
Hali11g i11 Llte caseopurulenl porlions.
Dísease Patterns
ci¡uine. Pneumonia in foals is the most significant mani-
:€station of infection with R. equí. ·rhe agent affects mainly
foals aged 1 to 6 months and causes a suppurative bron-
chopneu1nonia, p roducing large abscesses in Lhe lungs
and hilar lympl1 nodes. Occasionally there is localizatior1
ín joints, skin, and spleen. Ulccrative intestinal lesions
.gaining entrance via M cells overlying lymphoid nodules)
~vith abscesses in mesenteric lyinph nades are common.
The prognosis varies inclirectly with the age of t he foal
at the time of onset and is poorest for t hose affected at less
than 2 n1onlhs of age. The case faLaliLy rale exceeds 50o/o.
Extrapulmonary disease occurs in foals and older
Chapt~r 34 Rhndnr.nr.r.1is 191
horses. Rare uterine infections in mares are possibly re-
lated to perinatal expo:sure of foals.
Swine. Rhodococcus equi is recovered from. both tuberculo-
sis-like lesions in cervical lymph nodes of swine and nor-
mal nodcs. Its pathogen.ic role is disputed.
Humans. Rhodococcus equi pneurnonia is s.een in immuno-
:;uppressed individuals. Affected fual:; are a11 u11cuuw1un
source for humans.
Miscellaneous. Sporadic suppurative diseases have been de-
scribed in diverse mammals. They are com monly localized
in lymph nodes, lungs, and u terus. Ful.minating bactere-
mias are observed in crocodiles and American alligators.
Epidemiology
Rhodococcus equi is part of the equine environment. Tts
concentration varíes with the history of equine use of the
premises. On problem farms, it is highest in the foaling
and rearing areas. Suscepti bility coincides with the fading
of n1ater11ally transrnitted irnrnunity and precedes natural
immunization hy s11hclinical exposures. Gamma inter-
feron production by CD4 and CD8 lymphocytes from
young foals is repressed upon contact with Vaps.
The seasonal peak in summer is attribut ed to the abun
dance of l) susceptible foals and 2) heat and dust, which
impose added burdens on respiratory tract defenses.
Human infections are not uniformly linked to anirr1al
contact.
lmmunologic Aspects
Fu nctional CD4 (T tti) and CD8 T lymph.ocytes (see
Chapter 2) are necessary for protective immunity by "acti-
vating" macrophages by way of gamma interferon.
A11Libody, perl1aps lo lhe virule11ce-associatcd proteins
(Vaps), is needed since maternally derived antibodies as
wcll as passivcly administcrcd antibody appcar to be pro-
tective. 'l'hus, bot h cell-mediated and humoral immunity
are important.
Horses past infancy show signs of immunizing expo-
sures to R. equi, resulting in humoral and cell-mediated
respouses. BoLh appear Lo be involved i11 enabling n1acro-
phages to kili infecting microorganisms. Antibody, de-
tecta b le by enzyme-linked immunoadsorbent assay
(ELISA), appears at 5 months and is passed by colostrum
from mare to foaL Such passively acquired antibody de-
clines by age 6 to 12 weeks, which is roughly the time of
the highest prevalence of this disease.
No 1Iu1uu1liz.i11g µruduct:; are curn1nercially ul.Jtal11al.Jle.
Attempts at immunization with Vap have heen disap-
pointing.
Laboratory Diagnosis
Dernunstration of R. equi in sarr1ples from respiratory tracts
of pneumonic: foa Is constit11tf'S a rliagnosis of R. eq11i pneu-
192 PART ll R;:icteria and fungi

• •
•

•
•

'
f 1G U RE 3 4. 1 . Rhodococcus equi in transtracheal aspira te
trom a pneumonic toa/. Note cocea/ and bac11/ary shapes. Cluster
on /ower left is suggestive of intracel/ular co/onization. Gram
stain, 1000X.

monia. In smears, organisn1s appear as il1tracellular and
extracellular clusters uf gr<1u1-pu:;iti ve coccl ur rou::. (Fig
:~4 .1 ) . 'fhe ictentity of typical gro>vth on hlood agar
(35- 37ºC) i:i confirmcd morphologicully, biochcmicully,
and by synergistic hemolysis (see f ig 31.3), Specific
prin1ers are available enabling amplification of DNA by
n1eans of the polyn1erase chain reaction, Such techniques
have sho\.vn to be useful in d iagnosis, as v11ell as detection
o[ l11e 11ticroorga11is111 iu the eu viro11n1e11L
'to\rith nodular or cavitary lung patterns and hil;.ir lymp h
11ode e11Iarge1nenl respond less frequc11tly to therapy tharr
those with diffuse alveolar or interstitial reactions, The
prcfcrrcd trcotmcnt is crythromycin con<bincd wíth ri-
fampin,
Preventive measures include colostrum. intake, dust
control, and removal of foals from contaminated grounds .
Prophylactic antimicrobic treatment is justifiable in epi-
denlic silualions,

Treatment and Control

'l'he prognosis in R, equi foal pneumonia is always guardecL
Chest radiographs give valuable prognostic clues. Animals
 N on-Spore-Forllling
Obligate Anaerobes
DWIGHT C. HIRSH
Non-sporc-forming obligate anaerobes are a con1po11enL
of approximatcly 33o/o of bacteriologically positive sam -
ples of pyonecrotic material obtaincd from normally stcr-
ile sites of animals. On average, therc will be two species of
obligate anaerobes admixed with facultative spccies in
:;a111p!c:> of :>LH.:h material.
Thf' g r;im-nPgativP, non- -;porP-forming oblígate anaer-
obe Dicllelobacter nodosus, an linpo1lét11l cuulriliutor to
"footrot" of srnall ruminents, will be discussed separately.
Descri ptive Featu res
Morphology and Staining
The non spore-forn1ing oblígate anacrobes com.prisc a
\\'ide vanety of gram-posit1ve ano gram-neganve bacteria
and include rods, cocci, filan1ents, and spiral organisms.
Cellular Anatomy and Composition
Capsules, flagclla, and adhesins (also known as fimbriae or
pili) are cxpressed by some. The cell wall composition is
;:he samc as that ot their iacultative and acrobic counter-
parts.
Cellular Products of Medica! lnterest
Capsule. Capsular polysaccharides, if produccd, are impor-
tant for thosc microorganisms that come in contact with
tbe proctucts and cells ot the host. Capsular substances
protect the outer membrane from the membrane attack
complcx of the complement cascade (In the case of gram-
"Pgative anaerobes), and inl1ibit the microbe from attach-
.:neul llJ, a11c..l i11ge:slio11 uy, pl1ago<.:ytl<.: llost cells. The cap-
.xi]e is thought to endo•v a dcgrcc ofhyclroph ilicity rPlativP
~.., thc mcrnbranc of phugocytic ccll s. ?vfost capsules are
- et;atively charged, as are the membrancs ot phagocytic
-ells. ·rhe capsule of the gra1n-negative anaerobes Bacte
"'fides (ragtlts, plgmented Prevotella, and Porphyrornonas in-
'*' an intense inflammatory response.
Ce// Wa/I. The ccll walls of lhe obligaLe auaerubes are
-:e same as their facultative countcrparts. Gram-negative
-..,aerobcs produce a typical wall composed of lipopolysac-
~r1de (Ll'S) and protein. ·rhe LPS in the outer memhrane
is au i11 1JJurla11 l vlruleucc tlcterrnlnant. Not 011ly Is thc
lipid A component toxic (cndotoxi n), h11t thP IPngth of t he
side chain in the 0 -repeat unit hindcrs the attachn1ent of
the mcmbrane attack complex of the complcment system
to the outer membrane. LPS binds to lipopolysaccharidc-
bindJng prorein (a serum protein), wh1ch u1 turn transfers
it to the blood phase of CD14. l'he CD 14-LPS complex
IJiIH.ls to ToIJ-like receptor protclns (see Chapter 2) on the
surfacP of macrophagP cPll<; triget•ri ng the release of proin-
flummutory cytokine:s. Gram-po:sitiv<: 11nDcrobe:s produce a
cell wall containing !ipoteichoic acids and peptidoglycan,
which interact \Vith n1acrophagc cells, resulting in the re-
lease of prointlammatory cytokines.
Míscellaneous Products. Toxins and metabolic by-products
vvil11 Loxic activity llave uccu <.lcu1onstrated. These include
a cytotoxin produce.el hy Pusohar.tl'riu1n nP.r.rophor11m, an
enterotoxin-like entity secrctcd by Dacteroides fragilis, and
succinic acid, which is inhibitory to polymorphonuclear
neutrophil leukocytes (PMNs). Many produce protcolytic
and other enzymes that may play a role Ln their patho-
genic activities.
Growth Characteristics
Obligate anaerobes do not u se oxygen as a final electron
acccptor; in fact, molecular oxygcn is toxic to this group of
m icrobcs. W11en exposed to molecular oxygen, oblígate
anaerobes forro 11ydrogen peroxide and superoxide an-
ions. Thcsc toxlc molecu!es are formed from the interac-
tion of oxygen with various flavoproteins within the bac-
teria! ce!L U11líke aerolole1a11l bacte1ia, uliligale a11acrouc:.
do not produce superoxide dismutase, nor do they usually
produce catalase enzymes that break down superoxide to
oxygen and hydrogen peroxide, or break down hydrogcn
peroxide to oxygen and water.
Ecology
Reservoir and Transmission
Tl1t: non-spore-forming obligare anacrobes implicated in
pyonP.crotic procps-;p<; are usually part of the normal flora,
but thcy are sometin1es transn1illed by bites 01 ulher
trauma involving contaminated fomites.
193
194 PART 11 Bacteria a11d fungí
Pathogenesis
Disease results from the extension of the normal flora
(both obligate and facultativc anacrobic microorganisms)
into a compromised site, either by contamir1ation ot a
wound '~'ith nearby normal flora or from inoculation into
tissue wíth contamlnated instruments or teeth. The kinds
of microbes found in samples of such material reflect the
sile oI u1ju1y 01 lht:'. 111iL1uuial ¡.>upul¡iliuu uf tht: i11uculiit·
ing agency. Proliferation of anaerobes depends on the es-
tablishment of anaerobic conditions by trauma, vascular
brcal<down, or concurrent intection with (facultative)
aerobes.
Anaerobic bacteria cannot live in healthy tissue because
they cannot survive in the presence of oxygen. In compro-
1ui:;eu ti:-;~uc, i11fla1n1u<1tury cell:; anu co-ínoculated facul-
tative microorgan i s m .~ lower the F.h (a me<is11rP of oxygen
conccntration) sufflcicntly for anacrobcs to grovv.
Anaerobes e licit inflam matory responses clue to compo-
nents of their cel 1wall (lipopolysaccharide, gram negative
species; peptidoglycan, gram-positive ancl gram-negative
species). Some anaerobes produce capsules that, due to
theír chemístry, are potent inuucers of abscess formation.
There ic; c;ome evirlence th<it co-inoc11l<iterl f<ici1l tative mi-
croorganisn1s induce capsule production by anaerobes.
Synergy occurs between facultative aerobic and anaero-
bic microorganisms. Aside from triggering capsule forma-
t1on1 tacultat1ve spec1es scavenge oxygen, curtail phagocy-
tosis of the anaerobic component, and may produce
enzymes (beta-lactamase, for example) that mlght protect
a penicillin-susceptiblc facultative or obligate anaerobic
partner (and vice versa).
The most commonly isolated species of oblígate anaer-
obes are shown in 'lable 35.1. The most common sitcs or
processes that contain obligate anaerobes are shown in
Table 35.2.
Table 35.1. Most Common ly lsolated Oblígate Anaerobes
Gré11'!1-Ne9éltíve Rod~ Gram·Positive Cotci
Bacteroides Peptostreptococcus
Prevo te/la
l'orphyromonas
Fusobacterium
Dichelobac:cer
Ta b 1e 3 S. 2. Relative Frequency of Oblígate Anae robic Bacte ria
wnh Kespect to u1sease t'rocesses
Process Percentage with Anaerobe
Oraining tract
Abscess
Pleural effusion
n ... ~ . . . . J~.. I .. ((. . _: ....
Association of enterotox1n-sccreting stra1ns of Bacte-
roides fragilis with diarrhea in calves, lambs, piglets, and in-
fa11t rauuits ha~ l.Jeen llescribell.
lmmunologic Aspects
Immune responSC'S pl;iy a minor part in resolving thP rr-
onecrotic processes involving oblígate anaerobes.
Laboratory Diagnosis
sample Collection
Anaerobic cu lture is timr-cons111ning <inri expensive <in<I
should be used only when it holds a reasonable pron1ise of
supplying useful information. Material obtaincd from
sites that posscss a normal anaerobic flora (feces, oral cav
ity, vagina) is not usually cultured anaerobically. Routine
anaerobic culture of urine specimens orear, conjunctival,
or nasal :;wabs Is very rarely justifieu. Suppurative and
necrotic proccsscs are the most promising so11rres of clini-
cally significanl anaerobic bacteria.
Sa1nples of fluids for anaerobic culture are collected in
vcssels containing littlc if any molecular oxygcn. Thc casi-
est way ís to collect the san1p1e Clirectly into a syrtnge and
expel ali the air. Materials collected onto swabs o r
bronchial brushes musr be placed in culture immediately
or into an anaerobic environment (anaerobic transport
111t:Lliu111). Rcfrigeratiou is detrilueutal to recuver y o:'
anaerohíc haC'tC'ria; th11s, samplec; sho11ld not hP placPd a-
tcmpcraturcs lcss than 4ºC. Howcvcr, most samplcs that
contain oblígate anaerobes contain tacultative species as
well. Most facultative microorganis1ns grow in samples
11eld at 25ºC but poorly at 15ºC (a temperature harmless to
obligate anacrobcs).
Direct Examination
Gram-Positive. Rods Examination of stained smears prepared directly from the
collected material may give valuable clues regarding the
Clostrídium presence ot anaerobic bacteria. Ma11y o bligate a11acrobes
have typical, unique morphologies: rods are usually nar-
row and thread-llke in appearance; sorne havíng pointeé
ends or bulges. Most of the gram-negative species stair:
poorlywith the saffranine used in the gran1 slail1 (tl1us v.' ih
be pale staining in gram-stained smears). The 1nateria:
may havc a vcry rcpugnant odor if anacrobcs are prcscnt
whereas material without oblígate anaerobes ís usually no·
especially malodorous.
lsolation
Succcssful isolation depends on the care taken by the labo-
40-50 ratory in shielding the bacteria from oxygen. If the sample
30-40 is not processed immediately after collection, it must ~
3~ held in a container free from oxygen, usually a containe;
1-• - - · -'- •-t.. _ ____.. ___ .e---_..... 1 .... ,..,. ~ c ...................~ ..... .- ...:J;- .. . ! J - · -
 <;h.apter J S
been fresh ly 1nade and stored in an anaerobic environment
(a spccial sclcctivc mcdium for isolating B. fragilis from
feces contains polyrnyxin il, trichlosan, novobiocin, and
nalidixic acid). After the plate has been inoculated, it is
p laced into an anaerobic environment and lncubated at
37ºC. 1'he anaerobic envi.ronment is conveniently estab-
lished by tl1e il1teractio11 of a hydrogen.-containiJ1g gas
>vith the oxygen found in air in the presence of a palladium
catalyst in a closed container, su ch as in a11 anaerobic jar or
iI1 a glove box. A major advantage ot a11 anaerobic glove box
1~1ith a built-in incubator is that inoculated plates can be ex-
amt.ned at any t1me without being exposed to oxygen.
Most ob.li3ate anaerobes grow slo•vly, especially during
the early st ages, and plates are not e.xan1ined for the first
48 hours unless they can be examined in an 0 2 -free envi-
ron men t (e.g., in a glove box). Sin ce facultative species will
grow anaerobically, colonies gro\-ving in an anaerobic env:i-
ronment must be tested for aerotolerance.
ldentification
After an isolate has been shown to be an obliga te anaerobe,
t he genus to which it belo1i.gs is determined by shape,
gra1n-stai11ing characteristics, growth in the presence ot
various antibiotics, and met abolic by-produ ct s formed
from an assortmer1t of substrates, as determined by liguid-
gas chromatography. Reactions in prereduced anaerohi-
cally sterilized media containing different substrates help
deter1nine the species. Miniaturized identification systems
are commcrcially available. Gas chromatographic analysis
of cell fatty acids is someti mes used to identify an isolate.
Treatment, Control, and Prevention
Treatment of infectious processes that contain an anaero-
bic component most importantly involve drainage and
the use o.f antimicrobial agents.
Susceptibility data are usually not available for at least
48 to 72 hours after the sample is collected. Prior to this
ri1n e, if the ptesence of oblígate anaerobes is suggested by
lhe cli11ical presenlalion, direcLsn1ear, a11d olher circun1-
stances (odor), one of the following can be used : penicillin
1ampicillin, amoxicillin), chloramphenicoll, tetracycline,
met ronidazole, and clíndamycin. "l"hough most anaerobes
\vill test "susceptible" to t rimethoprim-su.lfonamides in
vi tro, this combination 11as unpredictable activ:ity in Vivo
dueto tl1e presence of thytuidine i.n necrotic material. The
obligale anaerobes are resislant Lo ali of lhe au ü1Higlycu-
side antirnicrobial agents as well as to most of the fluoro-
q uinolones (trovafloxacin is the exception) . Approxi-
mately lüo/o to 20% oí the isolates, usually members of the
B. fragilis group, will be resistant to the penicillins (peni
cillin G, ampicillin, a1noxicil.Hn) and first- and second-
generation cephalosporins due to the production of a ce-
phalosporü1ase, and ofLen lo lelracycline as we!L Re::;i::;tartt
isolates are susceptible to clavulanic acid-amoxicillin, clin -
damycin, mctronidazole, and cl1loramphenicoll. Antiini-
crobial therapy shoulct be aimed at both the tacultative
and the obligate anaerobic microorganisms. Between 70%
and 80% of pyonecrotic processes containing an obligate
Non-Spore-rorming übligate .i\.naerobes 195
Ta b 1e 3 5. 3. Facultative Microorganisms Found in lnfect ious
Processes Conta ining Obligate Ana erobes
Facultative Microorganism
Dogs/cats l'asteurella, en.terícs•
Horse Beta-hemolytic Streptococcus, entericsª
Ruminant Arcanooacrerium pyogenes, enterics•
anacrobc will also contain a facult ative one. 1'he most
comm on are shown in 'l'able 35.3.
D I CHELOBA CTER NODOSUS
Descri ptive Features
Structure
Footrot is a contagious infectious disease affecting the epi-
dern1al portio11s of lhe fooLof sheep and goats. The fully
developed lesions cause a crippling lameness. 'fwo gram-
negative non- spore-forming anaerobes a re primarily
implicated: Dichelobacter nodosus and Fusobacteríum necro-
phorum.
Dichelobacter nodosus, the specific cause, is a nonmotile
rod 1neasuring 2 to 10 pm by 0.5 to 1.0 pm. ln smears from
lesious, iLs eucls are cu111111ur1ly ::;wullt:o (Pig 35.1). Pili
(Type 4, as are those of 1\1oraxella bovís, Neissería gonorrheae,
I'asteurella ni.ultocida, and Pseudon1onas aeruginosa) act as
adhesins and confer t vvitching motility.
Cellular Products of Medical lnterest
Adhesins (Pili, Fimbria). Adhesins (pili or fimbria) are in-
volved with adherence of D. nodosus to interdigital epider-
mis that has been damaged by the proteases secreted by
Fusobucle1 iurn necrophorurn.
Cell Wall. ·rhe cell wall of the n1embers of this genus is
one typical of gram negatives. The lipopolysaccharide
(LPS) in the outer membrane is an ilnportant v:irulence de-
tcrminant. Not only is thc lipid A componcnt toxic (cndo-
toxin), but the lengt h of the side chain in the 0 -repeat
unit hinders t he attachment of the m embrane attack com-
µlex uf tbt: cu111µle::rne::r1t syste::rn to the outer membrane.
LPS binds to lipopolysaccha ricle-h incling protP.in (a Sf>n1m
protein), "vhich in turn tra11sfers it to the blood phase of
CD14. The C~D 1 4-LPS complex binds to 1011-like receptor
proteins (see Cl1apter 2) on the surfacc of macrophagc cclls
triggering the release of proinflammatory cytokines.
Proteases. Severa! proteolytic enzymes (serine and basic
µrutea:;es) are secrete<.! by D. nodosus. The proteases are reg-
ulate.d by the proclucts of two Pathogenicity Islands (a ch.1s-
ter of genes encoding virulence detern1inant(s), a11 inte-
grase protein, a specific insertion site, and mobility)
named Vap (for viru lcncc-associatcd proteins), and Vrl (for
virulence-related locus).
196 PART 11 Bacteria and Fungí

• .... ,..• . f ' •..

•
•
•
••
• • •
• ,. • - .. il
•
.
- .. ' ,,
•
• .. ..
•
•• •
•....

.. 1 •
.. ~
..¡ F1G U RE 3 5. 1 . Exudate from ovine footrot. Mixture of ba<-
, •
,/' (
• • terial species, with Dkheloh;irtP.r norlo'IJs rP.cngnizable as /arge
• rods with swo//en ends: "dumbbells" (<irrows). Gram stain, 1000X

Growth Characteristics Pathogenesis

JJichelobacter nodosus is a strict anaerobe requiring added l'athogenic mechanis1ns include tl1e píii-n1ediated attach -
carbon dioxide and a rich n1edium, preferably containing ment to host cells, proteolytic activity, and synergy with F.
protein. After severa! days, smooth colonles about 1 m1n In necrophorurn, to wl1ichD. nodosus ::;upplie5 growth factors.
clia1neter are produced .
Disease Patterns
Resistance
Tl1e seque11ce of eve11ls is lypically as follows :
Dichelohacter nodosus survives ü1 the ei1vironment 2 to 3
days and is killcd by disinfcctants and n1any antibiotics. 1. The in.te.rdigital epidenui.s is dam<igecl, most com-
n1only by 111aceration dueto persistent soaking.
2. Fusobacterium necrophorum, a constituent of the
Variability fecal flora, infccts the macerated skin a11d produces
Colonia l variation a:tid virulence are related to abu11dance superficial inflammation, hyperkeratosis, paraker-
of piliation. Virulence also varies with proteolytic activi- atosis, and necrosis (ovine interdigital dermatitis
ties of strains. ·ren n1ajor serogroups (A-T, 1vf) are recog- [OID]).
nized, and are based u pon ctiffcrcnccs in thc antigcnic con- 3. Dichelohacter nodos11s from a footrot lt:>sion colonizes
stitution of thc fimbria! adl1esins. Examination of the (with the aid of its pili) and proliferates i11 the lesior:
organization of the genes encoding tbese adhesins reveals initiated by F. necrophorurn, producing in rerdigit~
that atll1esir1s tJelunging to serogroups A, B, C, E, F, G, I, swelling. Invasion of epider1nal structures begins a!
and }.tf are organized differently than those encoding acl- the medial aspect of the claw ai1d, probably witl1 thE
hesins belonging to serogroups D and TI. Strains express- help of bacteria] proteases, advances to the epider-
ing adhes.ins ot the tormer are termed "class l" and the 1nal 1nalrix of Lhe lloof, evenlually :>epa1aLing il froll.
Jater, "class II." the underly.ing dermal tissues ("underrunning").
Secondary invaders help maintain or aggravate f11e
Ecology process. The result is extreme lameness, which becomes
immobilizing when two or more feet are involved. Affecte<i
Reservoir animals may starve.

Tl1e sig11ifica11t reservoir is the infectec.l foot of sheep or Epidemiology

goats. Cattle ancl swine strains are of low vir11lf'nCf'.
Footrot OCCUIS 011 ali co11tine11ts. It is lllOSt SCIÍOUS in re-
Transmission gions with a mild clin1ate and periods of abundant rainfall
(>20 inchcs fSOO mm)). Disscmination of D. nodosus cssco-
Trans1nission is by direct or indirect contact. The brief en- tially ceases at ambient mean temperatures of less than
vironmental survival time of tl1e agent requires p ron1pt SOºF (lOºC), a11d footrot does not occur in arid regions and
colonizaliou of 11ew ho:.ts. improves durlng dry perlods in endemic areas. Ali ages o:
 t.hapter .~.<;
animals beyond nursing stages are susceptible, b ut genetic
differences in susceptibility exist. Fine woo.l breeds are
most severely affected. The agent is eli.minated from con-
tan1lnated pastures wit hin 2 weeks.
lnununologic Aspects
Re:;i:;taJJCI:! i:; r1::lateLl lu ci rculaliug a11 lifi111brial a11 Libody; il
is serogroup-specific. Natural infection produces no im-
munity, but oil-ad juvant vaccines of appropriate speci-
ficity induce temporary protection and improve existing
cases.
Laboratory Diagnosis
Diagnosis is usually clinical. Dircct smcai:s from thc lcsion
revea! stou t rods with terminal swellings (see rig 35.1).
Other mícroorganisms are usually also present , sorne of
w hlch- small gram-negative rods- often coaggregate
aroun d D. nodosus. Immunofluorescence confirms identi-
fication .
c:u lture (on selective media) is not routinely done.
Molecular techniques utilizing t he polymcrasc chain
reaction together with DNA primers specific for D . nodosus
(e.g., the genes encoding the fimbrial adhesins) have been
described for demonstration of the bacterium In samp1es
obtained frorn infected feet as well as identification of iso-
lat1:::; gruvv 11. u11 ar Lificial 111edia.
Treatment and Control
Treatment begins with removal and exposure of d iseased
tissue by hoof trimming, followed by topical application of
disinfectants or antibiotics, su ch as repeated treatme11t
•vitl1 5% to 109/o formalin, 5% copper sulfate, lOo/o to 20ºA>
Non- Spore-Forming C)bli.gate Anaerobes 197
zinc sulfate, o r 5% tetracycline tincture. Use of chloram-
phenicoll (10%) is not permissible in the United St ates.
Formalin , copper sulfate, and zinc sulfate are used in foot
bat h:;. Three 1-hour 20%>z.inc sulfate soaks at weekly inler-
vals have proven effective without foot paring.
Systemic treatment with largc doses of pcnicillin and
streptomyc1n nas neen successful 1n t he absence ot topical
therapy.
Coulrol i:; aclJ.i1::v1::u by a cu1ubi11atiun uf repeatec.1 exa n1-
ination, vaccination, treat 1nen t of active ca.ses, ancl segre-
gation of active cases from the healthy flock. Care must be
taken not t o add infected animals to the flock. Contami-
nat ed lots should not be restocked for 2 wccl<s. Control
programs should be instituted d u ring dry weather.
f OOTROT OF (ATTLE (IN FECTIOUS PODODERMATITIS:
f OULS)
Dichelobacter nodosus combined wit h Fusobacterium
necrophor111n can cause a coronary and interdigital der-
n1aLilis iu caLLle.
What is often called bovine footrot is etiologically ancl
patl1ologícally distinct from the ovine disease. The chíef
agent is F. necrophorum. Other bacteria, particularly pig-
mented anaerobic rods (Prcvotclla) are implicated. Thc
p rocess involves the derm is and subcut1s, and produces a
diffuse, often febrile necrotizíng cellulitis, which may ex-
te111.l tu joiI1ts ur spreatl l1e1natuge11uu:;ly. Sinus tracts de-
velop in the foot region .
Injury, irritation, and maceratlo.n are likely pcedispos-
ing factors. Their presence, rather than transmissibiJity,
probably determines disease prevalence.
Cases are t teated topically and by h oof trimming. Un-
complicated cases respond to systemic sulfona1nides or te-
tracycline.
 Clostridium
DWlGHT C. H IRSH
Members of the genus Clostridiurn are gram-p osit ive,
spore-forming, anaerobic rods. In this chapter the d iseases
produced by members of this gcnus (Jable 36.1) will b e dis-
cussed under two catcgorics: the invasive diseases (includ-
ing t11e e11tcrotoxem i;:i~ ;1 ncl cfiarrhP;:is) p rnd11r Pd by (~. per-
f ringens, C. 11ovyi, C. h11e1110/yticu1111 C. septicu111, C. chauvoei,
C. dífficile, and C. pili(onne; and the noni nvasive diseases
produccd by C. botulinun1, nnd C. tetani. Briefl y discu ssed
will be C. sorrlelli1, C. collnun1, and C. spiro(orrne.
Descriptive Features
Morphology and Staining
MPmhPrs of thP gPn11~ l.lostridiu1n are gram-positive rods
measuring 0.2 to 4 µm by up to 20 µm. Location and shape
of endospores are consistent within a species.
Structure and Composition
Linle of medical relevance Is known of the ultrastructure
;:incl composition of clostricli;:i. A surfacP-a~socia ted struc-
turc characterizcd by ordcrly paracrystalline protein arrays
(S layer) is found in the cell wall of C. difficile. The role
plnycd by thc S !ayer proteins is unknown. Sorne clost ridia
have been sl1own to produce pili or fi mbriae (<.:. difflcile),
and oth ers produce adhesive stru ctures, presumably cell
wall p roteins. c:onsiderable cellular intraspecific antige1lic
rlivPrsity ::inc1 intPrspPcifir rross- rPartivity exist hu t a re of
less int crcst than thc scrologic properties of toxins (see
under respective species). Those that are motile have per-
itrichous flagella. Of pathogenic species, C. perfringens and
C. di!ficile are encapsulated.
Growth Characteristics
Strictness of anacrobic rcquircmcnts varies among clostri-
dial species. In addition to an anaerobic environment,
clostridia prcfer 2% to 10% CO'-in their atmosphere.
Most pathogen1c cloStridia require complex media in-
cluding amino acids, carbohydrates, and vitamins. Blood
or serum is beneflcial. Anear-neutral µH a11u temµeralure
of 37ºC ari> optim;:il.
CrrO\'\'th is usually visible within 1 or 2 days. Colonies
are oftcn irregular in shape and contour. Severa! clostridia
S'i'Var1n ncross moist ngar media \.Yithout forming colonies.
Most clostr1d1a produce hemotysis when gro~vn on blood
agar.
198
ERNST T.. R rRF.RSTF.TN
Tn liquid media, clostridia often grow in air provided a
reducing agent Is present (cooked ineat pieces, t hioglyco-
Iate), though growth occi1rs only in red ur.ed p ortions of
tl1t 111tc.liuu1.
Biochemical Activities
Clostridial cultures typically cmit putrid odo rs d ue to
products ot pcpticte catabolism, which is a comm o~ mode
of energy production.
Most clostridia attack carbohydrates, proteins, lipids, or
nucleic acids. Biochemical reactions and their end prod-
ucts furui~ll a ua~i~ for ~µtcit~ idtnlificaU011.
RGsistancG
1'he vegetative forro is as susceptible to environmenta.
stresses and disinl:ectants as other ba<.teria. Endospor5
impart resistance to drying, heat, irradiation, and disir:-
fectants.
THE INVASIVE CLOSTRIDIA
(LOSTRID/UM PERFRINGENS
Descriptive Features
Clostridiu111 perfringens is a g ra 1n-positivc, sporc-forming
nonmotile, encapsulated obligately an aerobic rod tl1~
produces a varicty of toxins (see below, "Cellular Product
of Medica! Interest"). Four of these toxtns are u.sed rr
"type" members of this species. There are five typPS <li>sig-
11ateu A lhrough E (Table 36.2).
Clostrídiun1 perfringells is associated with wound infec-
tions (gas gangrene), cntcrotoxemias in ruminnnts, and c.·
arrhea in a variety ot species.
Ccllular Products of Medical lnterest
Aclliesin.s. Closll idiu111 perfri11ge11s possesses a numbcr ::.
chromosomal genes whose sequences are similar to thos...
encoding adhcsins in other bncteria. ·rhcse include gen<::"
encoding two tibronectin-binding proteins, and a prote:.::
responsible for binding collagen. Whet her these st-
quences truly encade functional adhestns remains to ~
demonstrated.
 Chapter 36 Clostridiu1n 199

Ta b 1e 3 6. 1 . Selected Members of t he Genus Clo~lridiurn <:111tl Their U~u<:t l Sour<.e o r

Associated Condition

Usual Source or Assodated Comjltion

Clostridium botulinum Botulism

(. chauvoeí B.lackleg in ruminants, p1gs
C colinum Enteritis·andilepatitis in birds
e difficile AntibiotiC/stress-induced diarrhea in horses, dogs, cats
e haemo'lytíaim R;idll;iry hP.mogtohinuri;i in nimin;ints
C. nuvyi Gas gangrene; big head in rams; black disease in rumina11ts
C. perfringens Gas gangrene; enterotoxemla in ruminants. pigs; horses; necrotic enteritis in
chíckens; lamb dysentery; struck in sheep; pulpy kidney disease in ruminants;
diarrhea in dogs, cats, -aná human patients
e piliformc Tyzzer's clisease
e septfcum Malignant edema in ruminants, 1Jigs; braxy in sheep; necrotic enteritis inchfckens
(, sqrdellíí Myositis and hepatitis in ruminants, horses
e 5piroforme Mu~uid enteritis uf rabbits;<1ntibiutic·induced enteritis in rabbits, guinea pigs,
foals; enterocolitis í11 foals
C. tetaní Tetanus

Tab le 36.2. Clostrídium perfríngens Types in Animal Disease
Tyfle Majot foxln Present
Af11J1c1 Beta
A +
B ~ + +
(
o .+ +
+
E +
Type Estralnsoften presem in canle and sheep intestines, but are 1arelyimplitated i11
enterotoxemia.
Capsule. 'fhe role of the capsule in disease produced by
C. per(ringen::; i:s unLl1::fi11ed, bu.l iL p1obably acls as a deter-
r ent to phagocytosis. Encapsulat.ion is an important viru-
lence determinant in '-vounds (e.g., gas gangrene), but
probably not in the intestinal ca11al (e.g., enterotoxemias,
d iarrl1eal diseases).
Toxins. c:/ostridium perfringens produces a variety of pro-
t ein toxins. Most, if not all are regulated by a global regula-
tory sy:stcui ("VirR/VirS," :;ee IJeluw):
l . Alpl1a toxin. Alpha tox.in (Cpa or Ple for C. perfrin-
gens alpha toxin or phospholipase e, respectively) is
¡;roduced by ali C. perf'ringens. Il is a phospholipase
e (a lecithinase) that hydrolyzes phosphatidyl-
choline and sphingomyelin, both of which are con-
stituents ot host cell membranes. Alpha toxin also
displays the "hot-cold" lysis phenomenon (see
staphylococcal beta toxin, Chapter 27).
2. Beta toxin . 'fhe genes encoding beta toxin (Cpb for
C. perf1ingens beta Loxin) are locaLed on a pla~111id.
Beta toxin is a pore-forming toxin, which da1nages
host target cells (intestinal epit helial cells, endo-
thelial cells). In addition, beta toxin affects nervous
tissue by influencing the distribution of caldum
ions across thcir mcmbrancs t hcrcby disrupting
normal nerve conduction. It is susceptible to the
proteolytic activity o f trypsü1.
3 . .Bet d2 toxin. The l>etd 2 toxil1 (Cpl>2 for C. perfriu-
gPns hPt<i 2 toxin) is nPwly dP.sr.rihPd, h11t thP. role.
this toxin plays in associated disease is not well
characterized. The gene encoding this toxin is
sometimes located on a plasmid. Its n1echanism
+ of actton is unkn.own. It is produced by C. per{rin-
gens isolated from t he intestinal contents of pigs
wit.h necrotic enteritis, horses With enterocolitis,
and dogs with diarrhea (beta 2 toxin-producing
strair1s dre isolat.t::c.l h::ss ofLe11 fro111 clinically nor-
n1al animals).
4. Epsilon toxin. Thc gene cncoding cpsilon toxin
(Etx for epsilon toxin) is located on a plasmíd.
Epsilon toxin "targcts" lipld (cholesterol and
sphingolipids) rafts found in eukaryotic cell mem-
branes, though the toxin co11centrates in brain and
kidney. IL is a pern1ease tl1at acts by affecting tl1e
cellular cytoskeleton resulting in an increase in the
permeabílity of epithelial and endothellal cclls (cs-
pecially the microvasculature ot the brain, leading
to leakage of toxin into t hat organ). Etx is secreted
as a protoxin t hat is activated by p roteolytic en-
zymes.
5. lola Loxin. lola Loxin (1Lx for íola toxin) is a binary
toxin composed of a binding portian (Ib) that
binds the toxin to target epithelial cells, andan en-
zymatically active portion (la). After the toxin
binds to specific receptors on the cell surface, la
gains entry into the cytoplasm. ·rhough it is not
clear precisely how entry occurs, it seems that a
µurt:: (<.:orupo:;ec.l uf lli) is forruec.l ir1 tl1e cell rnern-
hrane through wh ir.h Ta travPrsPs. Ta is an ADP-
ribosylating toxin that ribosylates actin within the
200 PAnT 11 Bacteria and Fungi
host cell, resulting in disorganization of the cellu-
lar cytoskeleton and death of the affected cell.
6. Kappa toxin. Kappa toxin (Col for collagenase) is a
collagenase. Col is thougl1t t o aíd spread of clostri-
dial cells t hrough the tissue.
7. lvfu toxin. lvfu toxin (Nag for N-acetylgalactosaml-
niclase) is a hyaluronidase. Nag is thought to aid
spread of clostridial ceJls t11rough the tissue.
8. Theta toxin. See perfringolysin O, belO\"I.
9 . SioJidar.c. Siali da:;c (ncur a1ninidaoc, or }~an) re
moves sialic acid residues from glycoconjugates on
cell walls of eukaryotic cells resulting in disrup-
tiuu:; of tl1~ iutt:!f(.:~llular utatrlx.
10. Hemolysins. A number of hemolysins are produced
by C. perfringens. 1'heir role ü1 disease is unk11ow11.
11. Enterotoxin. 'fhe genes encoding C. perfringens en-
terotoxin (Cpe for C. perfringens enterotoxi11) are
either cl1ro1nosome- (isolates trom 11uman cases ot
food-related gastrointestinal disease), or plas1nid-
based (isolates from ctogs with diarrllea, or frorn
human patients with non- food-related gastroin-
Leslinal disease). Enleroloxin is produced during
sporulation of cpe-contain.ing (~. per{ringens (less
than 10<*.> of Type A strains, the strains in which
Cpe is most commonly found). When the endo-
spore is released, enterotoxin is also released into
tlle surrounding milieu. Ent erotoxin is a bifunc-
tional toxin, first forming a pore in the apical por-
tiu11 uf :>111a.ll ir1te:.tiual eµitl1elial (.:ells It:!:>ultiug ir1
fluid and electrolyte ahnormalities, as vvell as pro-
viding access to tight junction proteins (specifi-
cally claudins and occuldiI1s). lnteractions of Cpe
with tight junction proteins result in. further losses
in control of tluid and electrolytes.
12. Perfringolysin O (also kno\.vn as theta toxin) .
Perfril1golysin O (Pío íor perfrü1golysin O) is a cho-
lesterol-binding cytolysin (see also novyilysin and
chauvcolysin, below; streptococcal streptolysin O,
Chapter 28; listerial listeriolysin O, Cl1apter 33; and
arcanobacterial pyolysil1, Chapter 29). Pfo binds to
cholesterol-containing rafts in the eukaryotic cell
membrane, and forms a pore, which results in the
<.l<::atl1 uf tl1e c~lL 111 au<.litior1, Pfu is respur1sible fur
the lysis of memhranes enclosing l-. perfringens-
inside of phagoly::;osomes, resulting in tl1eir escape
in to the cytoplasm of the phagocytic cell.
Regulation o( Toxin Genes. c;1ostridiurn perfringens gtob-
ally regulates its toxin production by the t>vo-component
r~gulatury syst<::u1, VirR/VirS. VirS i:. a hi:>ti<.liue kiua:;e tl1at
acts as a "sensor" of environn1ental cues resulting in au-
tophosphorylation of one of its histidine residues. This
phosphate .is then serially transferred to an aspartate
residu e, then to another histidine befare being used to
phosphorylate VirR, the "regulator." Phosphorylated VirR
is a trascriptional activator of the ge.nes encoding t11e prod-
U(.:tS ruentior1e<.l above. Wl1at euviruurnt:!utal cue:; are
"sensed" hy r:. perfringens, is unknown, hut the con1posi-
tion of t he intestinal contents appears to play an impor-
tant role in the pathogenesis o f the enterotoxen1ias (see
below, "Epidemiology") .
Ecology
Reservoir
Clostridium perfringens, Type A, occurs in intestinal tracts o:
humans and other animals and in most soils. 'lypcs B, C
D, and E are fou nd mostly in the intestinal tracts of ani-
mals, and their survival in soil is variable.
transmission
Transn1ission is hy ingestion. and wound infection.
Pathogenesis and Disease Patterns
Clostriclium perfringens has the follo>ving disease patterns:
l. Wound In(ections (Gas Gangrene). Clostridium perfrin-
gens Type .A., alone (}r with other bacteria, causes
anaerobic celluliti~ anc.l ga!> ga11gre11e followi11g i11-
0111lation into a normally sterilc site. The me111-
b rane active toxins (alpha and perfringolysin O) ac-
count for the tissue destruction. Spread of the
proccss is aidcd by collagenasc (kappa toxin or Col 1 •
sialidase (Nan), and hyaluronidase (Mu toxin o:
Nag). Encapsulated C. per{ringens resist phagocyto-
SiS. Th.ose tbat are phagocytosed escape the phago-
lysosome by secretion of perfringolysin <). The
p rocess is a necrolizing cellulilis or n1yo11ecrosis
with edema, hemorrhage, emphysema, and a fe-
brile, often fatal, toxemia. 'fhis type of C~. perfríngens
intecti.on in animats is rare, but when it occl1rs it is
associated n1ost often with injection sites deep in
m u scle (mainly horses).
2 . Enterotoxemias. Most animal diseases due to C. per-
fringens are inleslir1al a11d. involve 1"ypes /i., B, C, or D
(rarely E):
a . Clostrídiun·zperfringens Typc A cntcrotoxcmia has
been i1nplicated in outbreaks of gastritis and h e-
n1olytic disease of run1inants (enterotoxemic
jaundice, the "yellows," "yellow lamb disease"};
iI1 hemorrhagic enteritis in cattle, horses, and
i11fa11t alpaca!>; 11ecn.>lice11lerilis i11 pou!Lry; food
poisoning in humans; and antibiotic-associated
diarrhea in humans; and is associatcd with dlar-
rhea in dogs and cats (see belc)\v). ·nssue destruc-
tio11 is probably due to the n1embrane active
toxins (alpha and perfringolysin ()), and the
toxi ns that affect connective tissue (collagenase,
l1yalul'onidase, and sialidase). Diarrl1eal d.isease
in human patients and in dogs and cats is asso-
ciated with Typc A strains that contain the gene
e11coding Cpe (enterotoxin).
b. Clostridhun perfrin_r5ens ·rype B enterotoxentia is
;n1 "Old World" d isease. Closrridiurn per(ringens
Type B causes "lan1b dysentery" in newborn
lan1bs. Occasionally focils, calve::;, and n1ature
s!1eep and goats are affected. Beta toxh1 is consid-
crcd thc principal factor producing hcmorrha¡,"Íc
enteritis affeci:ing the small intestine. Its trypsin
susceptibility explains in part the predilection of

the dtsease for the newborn (colostrum contalns
antitrypsin substances). The signs are depres-
sion, anorexia, abdon1inal pain., and diarrhea.
·rhe course is rapid, with mortality rates ap-
proachit1g lOOo/o. A chronic form occurs in oldcr
anunals. ·rhe characteristic intestinal tesion is
hernorrhagic enteritis. Extraintestinal lesions in-
clude congestion, edema, serosa! effusio11s, and
hemorrhages in various organs. The sign.s and
pall1ology associated with this disease are dueto
the action of the membrane active toxins (alpha,
beta, cpsilon, and pcrfringolysin O), as well as
those products that ctestroy the connective tissue
components. Epsilon toxin, being a permease,
increases intestinal permeablllty, ensurlng lts ab-
sorption into the circ11latinn whf'rf' it ;ifff'cts v;is-
cular endothelium, leading to fluid loss and
edema, as well as damage to kidney function.
Beta a11d epsilon toxins also affect the nervous
system, and tbe severe depression, .lack of re-
sponse to corrective therapy, and high 1nortality
may be clue in part to this activity. Since epsilon
toxin rf'qnirf's ";ictiv;ition" hy p rotf'olytic f'n -
zymes, its role in disease caused byType n strains
is less Ílllportant than that played by beta toxin.
c. Clostridium pcrfringcns Type C ente:rotoxemia in-
voJ.ves neonatal calves, foals, piglets, and lambs
worldwide. Clostridium perfringens Type C causes
hemorrhagic enteritis ln these specles. This rype
is associated witl1 necrotic e11teritides in humans
and birds and an ofle11 rapidly fatal toxen1ia-
bacteremia of older sheep called struck. Beta
toxin is considered the principal factor produc-
ing hemorrhagic enteritis affecting the small in-
testinc. Its trypsin susceptibility explains in part
the predilection of the disease for the newborn
(colostrum contains antitrypsin substances).
The signs are depression, anorexia, abdominal
pain, ;ind cliarrhe;i. 1'he course is rapid, with
mortality rates near 100°16. The signs and
pathology associated with this disease are dueto
t.he action of the membrane active toxins
(alpha, beta, and perfringolysin O), as well as
those products that destroy the connective tis-
sue cornponents.
el. (:tnstridiu.m JJerfringens, ·rype 1), produces an en-
terotoxemia ("overeating disease," "pulpy kid-
ney disease") in older lambs (<l year) and occa-
sionally in goats and calves. ·rhe key epsilon
toxin is secreted as protoxin activated by intes-
tinal proteases (expl.aining predilections for
ulder auiiuclls siuce culustruu1 cu11talns a11tit-
rypsin activity). Epsilon toxin increases intes-
tinal permeabi1ity, ensuring its absorption into
the circulation >vhere it da1nages vascular en-
dothelium, leading to fluid loss and edema.
When toxin levels are high, affected capillary
en.dothellal cells in the brain are damaged, and
Ll1e re::;u)laul etleuia greatly i11creases tl1e in-
tracranial pressure. When the amount of toxin
i.s .lower, bowever (as might be the case in a par-
Chapter 36 Clostridiurrz 201
tially lmmune animal, or \"lhen the an1ount of
toxi.n produced in the intestinal c;in;il is less) it
dan1ages the capillary endothelial cells in the
brain so that toxin levels are increased in that
organ. 1'his rcsults in a focal symmctrical cn-
cephaloma1acia (see Fig /1.1). ln addition to
these changes (which are toxin-dose related),
epsllon roXln rrlggers catecholamlne release, re-
su lting in adenylyl cyclase activation, cAMP-
relatecl hyperglycen1ia and glycosuria, a fre-
1
quent finding in enterotoxemia.
Gross lcsions may be abscnt. Postmortem au-
tolysis is rapid. Subserous and subendocardial
hemorrhages and excess fluid in the body cavi-
tles are sometlmes seen. Cerebral hemorrhage
and rlPgener;:itive lesion.s are common in less
acute cases. I-Iistopathology n1ay reveal enteritis.
Lambs may die without premonitory signs.
Convulsions occur in agonal stages and diarrhca
in protracted cases. Cattle and older sheep show
neural manifestations. In goats, diarrhea is com-
mon . Death rates are high 111 lambs. In calves
;inc:J goats, nonfatal subacute and chronic cases
occur.
e. Clostridium perfringens ·rype E produces a rela-
tively rarc for1n of entcrotoxcnlia in calves,
lambs, and rabbits. The membrane active toxins
(alpha, iota, and perfringolysin O) together>vith
those toxil1s affecting connective tissue sub-
stances combine to produce this disease. Hem-
orrhagic enteritis, and ulcerative abo.n1asitis
(gastritis) are the pathologic lesions.
3. Nonenterotoxe1nic Diarrhea. Nonenterotoxemic diar-
rhea occurs subsequent to the interaction of entero-
toxin (Cpe) with epithelial cells of the small intes
tine following sporulation of the microorganism in
that environment. Though any type of C. perfrin-
gens can harbor the genes encoding Cpe, Type A is
the most common. This disease is one of the most
commonly occurring food -related diseases in hu-
man patients. ·rhe disease also occurs in dogs and
cats. Signs range from watery to h.en1orrhagic diar-
rhea. In addition to altering fluid and electrolyte
flow of the epithelium, Cpe damages epithelial cells
aud tight juuctiuu::; leatliug lo sluughiug witl1 ac-
companying inflarnmatory changes.
Epidemiology
Some normal animals, especially adults, commonly carry
C. perfringens in their intestinal tracts. During outbreaks of
tliarrl1eal tlisease, pathogenic strains survive in soil long
enongh to inff'ct othf'r ;inim;ils.
·rhe determinant of enterotoxen1ic disease is the intes-
tinal environment, which is influenced by diet and age.
Ovcrcating, especially 011 protein and energy-rich food
(milk, Jegume forage, graü1) is aln1ost a prerequisite. In
young animals, the excess feed is often passed, inade-
quately digestecl, into the intestine, '"'here lt provldes a rich
mf'cii11m for proliferation and toxigenesis (up regulation of
the VirR/VirS systen11 see above) by ingested or reside11l

highly heat-resistant spores. ·rhere are thrce types (A, B,
and C), which differ biochemically, epidemiologically,
and pathogenlcally. Sorne would add C. haemolyticum as
Type D (see below)
Clostridiu111 novyi produces gas gangrene, "big head,"
and "black disease" of ruminants.
Cellular Products of Medical lnterest
Toxins. Clostridiu1n novyi produces a number of protein ex-
otoxins that are responsible for the diseases assodated
~vith 1t. Of these, the alpha and beta toXins, and the
cholesterol-binding cytolysin novyilysin have proven
roles in thc tliscascs protluccd by tl1is :.pccics. Toxir1s 11ave
not hccn associated with strains of C. novyi Type C::
l. Alpha toxin. The alpha toxin is produced by C. novyi
Types /\ and B. lt is a glycosyltransferase that gluco-
sylates llho GTPases rendering them ineffectual in
interacting with their substrates (i.e., they become
biologically ü1aclive). In addilion, glycosylalio11
blocks interaction of Rho GDP with guanine ex-
changc factor, and the interaction of Rho c·rr with
G·rPase activating tactor, thereby preventing mem-
brane cycling. As a consequence, severa] signaling
pathways are disrupted, result1ng in a breakdown of
the cytoskeletal components of the affected cell, fol-
lowed by its c.ieath.
?.. Beta toxin . The beta toxi.n is procluced by C. novyi
Typc B. Tt is a pl1ospholipase C (a lecithinase) that
hydrolyzes phosphatidylcholine and sphingomye-
1in (both of which are constituents of host cell
membranes), resulting in death of the cell. This
toxin is also hemolytic.
3. Delta toxin (Novyilysin ). Delta toxin or novy11ysin
is produced by C. novyi Type A, ;inrl is ;i rholesterol-
binding cylolysi11 (see also perfringolysin O,
above, chauveolysin, below; streptococcal strep-
tolysi n O, Chaptcr 28; listcrial listcriolysin O,
Chapter 33; and arcanobacterial pyolysin, Chapter
29). Novyilysin binds to cholesterol-contah1ing
rafts ln the eukaryotic cell membrane. Once
bound, it for1ns a pore res11lting in thP death of
the cell.
-l. Miscellaneous toxins. Clostridium novyi produces a
numbcr of othcr protcin "cxotoxins" with unccr-
tain roles in the pathogenesis of dísease. ·rhese in-
clude a lecithinase (gamma toxin) produced by
Type A strains, a llpase (epsllon toxln) produced
hy ·rypP A str;:iins, hPmolysin (7Pt;i toxin) prod11ced
by Type D strains, and a myosinase (eta toxin) pro-
duced by 1ype B strains.
:cology
~:oServoir and Transmission
• ~ A Is common ln soil. Types A and B occur in the nor-
_,~¡ intP<;tine and liver of 11erbivores. Ali enter their hosts
ingestion or wound infection.
Chapter 36 Closr:ridium 203
Pathogenesis
()f <;PVPr;:il toxins procl11<Prl hy r.. nnvyi, ;:ilph;:i, het;i, ;inrl
novyilysin, which are lethal and necrotizing, are of estab-
lishcd pathogenic significancc. 1'he pathogenic roles of
thc othcr toxins-Iccithinasc, hcmolysin, lipasc, and
1nyosinase-are uncertain:
l. Clostridium novyi Type A is implicated in gas gan-
grene of humans and wound infections in animals.
One of these, "b1ghead" of rams, starts as a fight in-
jury at the top of the head. Toxic endothelial dam-
age (by alpha toxin and novytlysin) produces edema
involving he;:irl, nerk, ;inrl cr;ini;:il thorax _Death oc-
curs in 2 days. The yellow tinge of the eden1a fluid,
which is clear and gelatinous ,.,,ith little hemor-
rhage, is a postmortcm changc.
:l. Closlridiurn novyi ·1ype 8 causes intectious necrotic
hepatitis ("black disease") of sheep and cattle, rarely
horses and swine. Spores orlgtnating in the intes-
ti ne reach the liver ;inrl rem;iin there dormant
within Kupffer cells. When liver ce!Js are injured, as
by fluke migration, resulting anaerobic conditions
allow the spores to gcrminatc. Vcgctativc growth rc-
Sults in toxin producnon (alpha and beta toXins)
and dissemination. Death may be sudden orwithin
2 uay:s of clirlical on:set. Signs lnclude depression,
anorexia, and h ypothermia. Necropsy rPvP;ils
eden1a, serosa! effusion, and one or n1ore areas of
liver necrosis, containing bacteria. Subcutaneous
vcnous congestion secondary to pericardial edema
áarkens tbe un<lersiáe ot t11e s.1<i11, suggesting the
name "black disease."
3. Closlridium novyi Type C, the reported cause of os-
teomyelitis of \>\ iltPr h11ff;ilo in Southe;ist Asi;i, pro-
1
duces no toxins or experimental disease.
Epidemiology
'fhe agent occurs world\.vide. "Bighead" is recognized in
Australia, South Africa, and North America . Distribution
of "blac:k disease" largely coinci.cles with that of the liver
fluke Fasciola lzepatica. Both diseases occur mostly iJ1 adult
sheep, during summer and fall. "Black disease" affects
prcfcrably wcll-nouri.sbcd animals.
lmmunologic Aspects
Circulating antitoxin (antibodies to alpha, beta, novy-
ilysin toxins) and antibody to ccllular components of the
organism presumably are thc basis of inlmunity to C. novyi
infect1ons. Whole culture bacterins and toxoids have pro-
phylactic value.
Laboratory Diágnosis
Liver lesions contain large gram positive to gram-variablc
rods with oval subterminaJ, targe spores, 1dentiliable by
fluorescent anti- C. novyi conjugates.
Culture rcyuires tl1e strictest anaerobic conditions, es-
204 PA1tT JI Dactcria and Fungí
pecially for Type B, v.ihJch is also nutritionally fastldious.
r.1nstridi111n nnvyi m;:iy hP rlt>monstr;.ible in normal livers of
herbivores within hours after death.
'lbxu1, in serosa! effusions, can be demonstrated by ani-
1nal tests (sce undcr C. pcrfringcns, abovc), but antitoxins
far use 1n protcctton tests are notas readily available.
Dctcction in tissuc or identification in culture can be
ac~ompllsh~d by uslng molecular techniqu es. DNA
pruners des1gned to amplify species-specific portions of
tli.e gene cncodi ng the flagellar protein flagellin li.ave bee1i.
used successh1lly to differentiatc C. novyiTypes A, and B, C.
hacmolyticurn, C. chauvoci, and C. scpticum.
Treatment and Control
Therc is no effcctlve treatment. Control is directed at elim-
üi.ating fl ukes and other hepatopathic agents. Prophy-
Iactic vaccination (two 1n¡ectlons a mon th apart) with a
bacterin -toxoid con1bination is generally effective.
(L05TR I D/UM HA EM O LY TI CUM
Cl~stridiu111 /1ae111olyticu111 is a no1i.e1i.capsulated , n1otile,
stnct ana<'rOhE', producing largc, oval, highly heat-
resistant sporcs. Clostridiu1n /1ae111olyticu1n (C. 11ovyi, Type
D) resembles C. nolfyi Type B in practically ali phenotypic
traits. Its toxin, a phospholipase C, is identical to C. novyi
Type B beta tox1n (see above), but 1s produced in much
larger amounts. Serologic and toxigenic variants of C. hac-
mu/ytic:u111 have l>een notetl.
. <J~strid~~1111 /1nmnnlytir11m proc111C'f><; h;ici lI;iry ht>moglo-
b1nur1a or red water" díscase of ruminants.
Ecology
Reservoir and Transmission
c:1ostridi11 rn hae1nolytir11n1 exists in the r uminant digestive
t ract, in the livcr, <ind in soil. Appearance of the disease in
n e\.v, widely separated regions suggests that movement of
cattic piays a part in its disscrnination. ·rran smission is by
1ngest1on.
Pathogenesis
1·11c pathogcnesis of bacillary hemoglobinuria mvolves in-
gestion of spores, colonization of the liver, liver in jury,
spore germinatlon, and toxigene:;i:; (:;ee C:. nuvyi Tvpe B
"hl;ick <li<;P;i<;P," ;ihovf'). ·rhP toxin ic; a phospholip~sE' r.
(beta toxin), which produces a hemolytic crisis and deatl1
within hours or days. Other effects include serosal effu-
sions and \videspread hcmorrhagcs. 'fhc diagnostic lesions
are circumscribed areas of llver necrosis ("1nfarcts"), w h ich
are t11e effccts of beta toxin. Clinically there are fever, pale,
lcteric mucous membranes, anorexia, agalactia, al>uorn i-
nal pain, hPmoglobin11ria ("rt>cl watt>r"), ;in<f hypt>rp nP:i.
Pregnant cows may abort.
Epidemiology
Bacillary hemoglobinuria occurs in Nort h America in the
Rocky Mountain and Pacific Coast st atcs and alono
thc Gulf of Mexico, and in Latin America, parts of l:.ur~
ope, and New Zealand. Although swampy lowlands are
assoclatcd wlth cndcmlc dlscasc and flooding wlth the
spread of infection, little is known of the agent's persist-
e11cc in soil. Shcddcr ani111als n1ay have a role in dissemi-
nation .
Cases are clustcrcd in thc sccond half of thc ycar, typi-
cally among well-nourished animals ayea r o r more of age.
Correlation with flukc infection is less consistent than in
"lJlack cJl:.ea:.c" (:.ce C. riuvyi Type B, a!Jvve) .
lmmunologic Aspects
Im munity is an titoxic. Animals in enden1ic a reas develop
sorne immu n ity. Wholc culture bacterin-toxoid s are effec-
tlve proph ylactlcally.
Laboratory Diagnosi s
Liver lesions are the best source of positive sm ears for
Cram stains and immunofluorescence tests. Cultivation
requires freshly poured blood agar and strict an aerobic
conditions.
Detectiuu iu ti:.:.ue ur iue11lificatio11 iI1 cullure cau bt:
accompli<;hp<f hy 11<;ing molPrnlar tE'chniq ue_<;_ DNA pri-
mers designed to amplify species-specific portions of the
gene encoding the flagellar protein flagellin h ave been
uscd succcssfully to diffcrcntiatc C. novyiTypcs A, and B, C.
flaernolyticurn, C. cflauvoei, and C. sept irum .
Treatment and Control
Early trf>a tmPn t of sirk an ima Is with a hroarl-spP.ctrnnl an-
tibiotic (e.g., tetracycline), antitoxin, and blood t ransfu -
sion produces good results. Ani1n als in endemic areas are
vaccinated minimally every 6 n1onths and preferably 3 to
4 weeks before anticipated exposure.
( LOSTRIDIUM SEPTICUM
Clostridiu111 septicun1 is a short, stou t, and p leom orphic
gram positive, motile, spore-forming obligately anaerobic
rod. In sorne exudates, long filaments are found.
Clostridiu111 septiann is the leading cause of woun d in-
fections (mallgnant edema) uf farrn a1illual:..
Descriptive Features
Cellular Products of Medica! lnterest
Tnxins. r.1nstridiu111 septicum produces a n umber of proteill
exotoxins that are purportedly re::;pon sible for the diseases

associated witJ1 it. However, alpha toxin has been detini-
tively shown to be the only virulence factor produced by
this species:
l. Alpha toxin. ·rhe alpl1a toxin produced by C. sep-
ticurn is a pore-fo.rming, lethal toxin. Following se-
cretiOn, it is bound to glycosylphosphatidylinositol
(C;PI)-anchored proteins on the eukaryotic cell sur-
face (n1air1ly e!1d0Lhelial cells, Lhough nol exclu-
sively). There, the ccll bound protcolytic enzyme,
furin, clcavcs it, rcsulting in fragmcnts th.at insert
into the membrane forming pores Jeading to deat l.1
of the cell.
2. Beta toxin. The beta toxin of C. septicunz has DNase
and leukocytotoxic activity. 'fhe role played by t his
Lv., .iu i 11 Ll IC:: jJdLliVl:)<::l!<:::>i:> v( UÍ:><::a:><:: i;:, u11defiu1::tl.
3. Gamma toxin. 'fhe gamma toxin of C. septicum has
hyaluronidasc activity. "fhc role playcd by t his toxin
in the pathogcnesis of disease is undefined.
4. Delta toxin (septicolysin O). The delta toxin of
C. septicurn, also kno,~·n as septicolysin O, is a
rholesterol-binding cytolysin (see also perfringoly-
sin O ar1d novyilysin, above, chauveolysin, below;
streptococcal streptolysin O, Chapter 28; listerial
listeriolysin O, Chaptcr 33; and arcanobactcrial py-
olysin, Chapter 29). Septicolysin O binds to choles-
terol-containing rafts in the eukaryotic cell mem-
brane. Once bound, tt forms a pore resulting in the
death ofthe cell. The role played by this toxin in the
patl1oge1H:::sis uf disease produced l>y C. septicum is
11ncli>finPcl.
5. Miscellaneous products. Clostridiurn septi.curn pro-
duces a number of other products (chitinases, neu-
ran1inidascs, lipascs, sialidascs, and hcmagglu
tinins) '"litl1 undefined roles in the pathogenesis of
disease.
Ecology
Reservoir and Transmission
occurs il1 soils worldwide and in Lhe
C lostridiun1 septicur11
animal and human intestine. It is acquired by wound in-
fcction an.d ingcstio11.
Pathogenesis
Wo11n<l infe.ction p rocl uc.e.cl hy r:. SP.fJtir.11m is r;:illerl "111;:iJig-
n ant ederna." Ali oi the toxic products outlined above
probably p lay a role, but only the alpha toxin has been
shovvn to be definitively involved. Systeinic cffccts are
thought to t)e the result of endothe.lial damage through-
out the body (leading to severe fluid and electrolyte imbal-
au ces) as well as the edema seen locally. Membrane active
toxins (alpha and delta), dissolution of connective tissue
components (gan1111a toxjn), alo11g wilh deslruclio11 of
polymorphonuclear leukocytes and release of their diges-
tive enzymes (beta toxin) may account for thc tissuc dc-
srruction observed .
The infectious process radiates from the point of inocu-
latio11 witl1in hours to days of exposure. A hemorrhagic,
Ch.apter 36 Clostridiurn 205
edematous, necrotizing process frequently follows fascial
planes as adjacent muscle is darkened.
With muscular involve1ne11t and emphysema, there
may be close resen1blance to "blackleg" (C. chauvoei,
below) a11d "gas ga11gn::ue" (C. pt:r(rin¿;ens, CJoove).
Crepitant swellings change from pa inful an<l hot to
anesthetic and cold. Signs include fever, tachycardia,
anorexia, and depression. The course may be rapid and
fatal within a day.
"Braxy" (Scots) or "bradsot" (Danish) is a fatal C. scp-
ticurn cold-weather disease of sheep. Clostridiurn septicz.a n
produces a lesion i11 Lite al.Jo1u.asal wall cornparable to the
-;11hr11t;:ineo11s one clescribed above. Clinical signs are
mostly toxemia and g<1strointestinal disliess.
Hu1nan wound infections due to C. septicum may de-
velop into cellulitis or "gas gangrene."
Epidemiology
"Malígnant eden1a" may follow such p roc.e.ct11res as r;:istra-
tlon, docking, shearing, tagging, and injections. Postpar-
turient genital infections are sometimes linked to ctystocia
and unskilled obstetrical assistance.
"Braxy" or "bradsot" ls seen mostly in Scotland and
Scandinavia.
lmmunologic Aspects
Imrnunity is probably antitoxin-dcpcndent.
Laboratory Diagnosis
Sporulated rods may be demonstrable in exudates by
Gra1n stain or immunofluorescence.
Clostridium scpticu1n grows on ruminant blood agar
under reasonably good anaerobic conditions, prc)ducing
witl1ü1 48 hours hemolytic colonies up to 5 mm in diame-
ter, wlth rl1izold contours and a freq uent tendency to
sw;:i rn1.
'fhough recovery and identification of this age11t by
culture are not difficult, positive results should be inter-
pretcd with caution. Clostrídíu1n septicurn is an aggressive
postmortem invader. lts presence may be unrelated to
the problem at hand and may obscure that of more
significant pathogens, such as C. haemolyticum a11d C.
chauvoei.
Den1onstratio11 ü1 Lissue or idenlificaLiou of i~olatts is
possible by using DNA primers that have been designed to
amplify thc gene (or portio ns thereof) encoding the alpha
toxin by means ot the polymerase chain reaction. .i\ll
strains of C. septicum contain the genes encoding this
toxin. Detection in tlssue or identification in culture can
also be accomplished by using DNA primers designed to
a111plify species-specific portions of th.e gene encoding the
flagellar prote.i n fl;1ge JI in. whirh h;:ivp hPPn 11<;f'rl <;11rrP<;S-
fully to differentiate C. novyi 'fypes A and D, C. chauvoei, C.
hae1nolyticurn, and C. septicum.. Likewise, C. septicum can be
differentiated from C. chauvoei by the use of prirncrs to am-
plify tl1e 165- 235 DNA spacer regions.
206 PAKr II Bat:tcria aud Fungí
Treatment and Control
Prognosis sl1ould be guarded. Possible therapy includes
penicillin or tctracyclinc givcn systcn1ically, incision,
drainage, and irrigation of Jesions with ant iseptics.
Calves are vac(inated at 3 to 4 months of age, sheep and
goats at weanlng. Annual revaccination i:> auvi:;aule.
Hygienic precautions at ti1nes of likPly Pxposurf> arf> helph11.
(LOSTRIDIUM CHAUVOEI
Clostridiurn chauvoei is a gram-posittve, rnotíle, obligately
anaerobic rod that produces subterminal or subcentral
spores.
Clostridium chauvoei produces an emphysematous
111::crutit.i11g 111yosilis ("blackleg") i11 cattle.
Descriptive Features
Cellular Products of Medica! lnterest
Clostridíurn chau.voei produces a 11umber of protein exotux-
ins that are purportedly responsible for the diseasPs assori-
aled wilh il:
l. Alpha toxin. The alpha toxin ot C. chauvoei is áe-
scJibed asan oxygen stable hemolysin. This toxin is
most !i1<e1y comparable to the pore-form1ng, lethal
alpha toxin of C. septicurn (see above).
2. Beta loxin. 'fhe beta Loxin of C. chauvoei has DNase
activity. 'fhe role played by this toxi11 in the patho-
genesls of disease is undefined.
3. Gan1ma toxin. The gamma toxin of (_;. chauvoei has
hyaluronidase acti vity. 1'he role played by this toxin
in the pathoge11esis of disease is undefined.
4. Delta toxin (chauveolysin). The delt a toxin of C.
chauvoei, al:;u kI1uw11 as chuuvevlysin, is a cholesLerol-
híndi ng cytolysin (see also perfringolysin O, septi-
colysin O, novyilysin above; streptococcal strep-
tolysin O, Chapter 28; Iisterial listeriolysin ü,
(~hapter 33; and arcanobacterial pyolysin, Chapter
29). Ct1auveolysin binds to ch.olesterol-containing
rafts ix1 tl1e eukaryotic cell 1uen1brane. Once bound,
it for1us a µurc resullu 1g in Lhe dealh of the cell. 'fl1e
role played hy this toxin in the pathogenesis of dis-
ease produced by C. chauvoei is undefined.
5. Neuraminidase (sialidase). ·rhe r1euraminidase of e;.
chauvoei removes sialic acid residues from glycocon-
jugates on cell walls of eukaryotic cells resulting iI1
disruptions of the intercellular matrix. ·rhe role
plc1ytu l.Jy Llü~ Lv-"i 11 i11 Lhe p athoge11esis of discusc j:;
undefined.
Ecology
Reservoir and Transmission
Clostridium chauvoei inhabits the intestinf>, livf>r, anct other
U:;sues of susceplible a11d resistant species. Evidence that
soil transmits "blackleg" is circurnstantial (see under
"Epidemiology," below).
RouLes of infeclion aJe not kI1ow11. Ei1doge11ous an.d
soil-acquired infection via ingestion or injury is assumed.
Pathogenesis
·rhe alpha (necrotizing), gamma (l1yaluro1litlase), delta
(chauveolysin) toxins together with the neura1ninidase,
are uelieved to be iesponsible for the i11itial lesions.
Bacterial metabolism, producing gas from fermentation,
mav be contributory.
SeediI1g ot tissues, especial1y skeletal muscle, wit h
spores from the intestine, presumably precedes disease in
cattle. Co11ditions .favoring spore germination, t>acterial
growth, and toxin productio11 cause formation of l?'ª.I lf>-
siuns llli:lfkctl uy eden1a, hen1orrh.age, and n1yohbr1llar
necrosis. The centers of lesions become dry, dark, and e1n-
physematous dueto bacterial fcrmentation, while the pe
riphery is edematous and hemorrhagic. A ranc1d-butter
odor is typical.
Microscopically, one finds degenerative cha11ges i11
muscle fibers disrupted by edema, en1physem.a, an11 hPm -
orrt1age. Leukucytic i1Jfillralion is 111inor.
(]inirally, there is high fever, anorexia, a11d depress.i on.
Lan1eness is com1non. Superficial lesions cause visible
swellings, which crepitate on being handled. Often lesions
are cntirely internal (diaphragm, myocardium, tongue).
Sorne antmals die suddenly, others within 1 or 2 days.
Epidemiology
"Dlackl.eg" occurs worldwide at rates that differ betwecn
and within geographic areas, which suggests a soil reservoir
or climatic or scasonal factors yct to be defined. Young,
well-fed cattle (<3 years) are preferentially attacked.
Exertion, bruising, or acute indigestion are suspected
triggering eve11ts.
111 sheep a11d sorne othf>r spP<if>_~, r:. chauvoei typically
causes wound infections resembling malignant edema o r
gas gangrene. Other clostridia (C. septicurn, C. novyi, and C.
sordcllii) may be present.
lmmunologic Aspects
Circulating antibody to toxins and cellular componc11ts
appare11tly determü1es resistance to(,'.. cflauvoei. Commer-
cial formalinized adjuvant vaccines include up to six oth€.r
clostrid ial components.
Laboratory Diagnosis
Sporulated gra1n-positive rods can be demonstrated in
s1nears of infected tissues and ide11tified with immunoflu-
orescent reagents (see Fig 70.4).
Clostridiun1 chauvoei requires strict anaerobic con<li-
tiu11:; a11u u11::uia rich i11 cysleiI1e a11d water-soluble vita-
mins. '!'he. agent resemhles C. septicum a11d is frequentlyre-
covered with it. Unlike C. septicurn it fermcnts sucrose but

11ot salicil1 ;u1u will 11ot grow ilt 44ºC. 'fhe use uf DNA
primers to a1nplify the 16S- 23S DNA spacer regions (hy
using the polymerase chain reaction) vvill differentiate C.
septicum from C. chauvoei. 'fhis assay can be used to detect
the microorganism in tissue.
Detection in tissue or identificatio11 in culture can also
be accomplished by using DNA primers designed to am-
µlií y (l>y usil1g tl1e ¡;olyruerase cl1ai11 rea<..:tiou) sµecies -
specific portio ns of the gene encoding the flagellar protei n
flagellin, which have been used successfully to differenti-
ate C. novyi ·rypes A, a11d B, C. chauvoei, e;. haernolyticun1,
and C. septicum.
Treatment and Control
Trcatmcnt is oftcn disappointing. Penicillin should be
given intravenously at first, followed by repository torms
intramuscularly.
Cattle are vaccinated at 3 to 6 months of age and ann u-
a lly thereafter. Vaccination should precede exposure by at
least 2 weeks. During an oulbreak, all cal lle are vacci11atell
and given long-acting penicillin.
Prcgnant cwcs are vaccinatcd 3 wccks prior to parturi-
tion, whe11 infection often occurs. Lambs rnay require vac-
cination during t heir first year.
Change of pasture is advisable when cases are first ob-
served.
[LOSTRIDIUM DIFFICll. E
Clostridiurn difflcile is a gram-positive, motile, encapsulated,
sporc-forming anacrobic rod. This spccics produces ad-
hesins (pili or filnbria), and its ceU wall contains paracrys-
talline arrays (S-layer) visible with the electron microscope.
Clostridium difficile is a significant cause of diarrheal dis-
ease in human beings (antibiotic-associated diarrhea
wlücl11nay ¡;rogn:::;s to µseutlo111t::H1l>rauou:; colitis), l>ut its
significance in other anirnals is less clear. The microorgan-
ism has been isolated from syn1ptomatic as well as asymp-
tomatic dogs and cats. Association with a "trigger event"
such as use of antimicrobial agents has bccn suggcstcd but
not proven. Clostridiurn difficile l1as also been isolated from
normal horses; ho"l'.rever, it is more frequently isolated
fl uu1 l1ur:;e:; wiLl1 lliarrliea a11ll a:s:su<..:ialt::<.l protel11-loslI1g
enteropathy, implying a causal relationship. Pathological
fi ndings in J1orses from which C. difficile has been isolated
include hemorrhagic necrotizing e11terocolitis, typhlocol-
itis, and pseudomembrauous colitis. As with dogs and
cats, there is sorne suggestion of association with antimi-
crobial agents, though C. diffíci/e-associated disease has
!Jeeu reporte<.l in previously normal, unmedlcated foals.
Descriptive Features
Cellular Products of Medica! lnterest
.-idhesins. Clostridium ditflcile produces pili or firnbrial ad-
h esins that probably play a role in adhcsion to targct cells
Chapter 36 Clostridiurn 207
in the large intestine. Clustrídíum dífflcíle also produces a
cell wall protein (Cwp66 for r.e.11 wall proti>in of fi6 kDa in
size) v'lith affinity for intestinal epithelial cells.
Capsule. Clostridiu1r1 difficile produces a carbohydrate
capsule that protects it from phagocytic cc1ls.
Toxins. Clostridium difficile produces three toxins that
are responsible for enteritis observed with this microor-
gauisrn: toxin A ("enterotoxin"), toxin B ("cytolysil1"), and
ADP-rihosyltra nsfe.rasi>:
l. T'oxin A. Clost:ridiurn diftlcile Toxin A (ToxA or 1cdA
for toxin C. d.ifficile) is a glycosyltransferase that glu-
cosylatcs Rho G'fPascs, rcndcring thc.m ineffectual
in interacting with their substrates (i.e., t11ey be-
come biologically inactive). In additio.n , glycosyla
tion blocks lnteraction of Rho GDP \Nith guanine
Px<'hangP factor, and tl1e iI1teraction of Rho G1'P
with c;·rrase activating factor, thereby prevenling
membrane cycling. Several signaling pathways are
disruptcd, rcsulting in a brcakdown of the cytoske-
letal components of t he affected cell including dis-
ruption of the tigl1t junctions between intestinal
epithelial cells. These changes result in death of the
r.ell. 1'hi> Pnte.rotoxic p roperty of ·roxA, in addition
to the cytotoxic effect:; ju:;t de5cribed, is due to ils
ability to stimulate influx of PMNs by way of the en-
teric nervous systcn1 (through thc rclcasc of sub-
Stance P and mast cell degranulation). J>rostaglan-
din synthesis by tl1e recruited PMNs (and perhaps
by affected host cells), as well as activation of vari-
ous inositol-signaling pathways within affected
host cells, resul ts in thc secrelion of cl1loride ions
and water (diarrhea).
2. ·roxin B. Clostridiuni difficílc Toxjn B (ToxB or TcdB
for toxin (~. difficile), like ·ioxA, is a glycosyltrans-
ferase that glucosylates Rho GTPases (see above).
However, ToxB has little enterotoxic activity.
3. ADP-ribosyltransferase. Clostridiinn difficile pro-
duces a11 ,L\DP-ril>u:;yl Lransferase (Ctlt for C. diffi-
cile transferase). Cdt is a binary toxin (see l-. per-
fringens iota toxin, above, and C. spirofor1ne,
below), cornposed of a binding portion (C:dtb) that
binds to the target intestinal epithclial cclls and an
enzymatically active portion (C~dta). After the
toxin binds t o specific receptors on the cell sur-
face, Cdta gains entry into the cytoplasm. Though
it is not clear precise.ly how e.ntry or.r11rs, it si>i>ms
that a pore (composed of Cdtb) is formed in the
cell membrane through \.Yhicl1 Cdta traverses.
Cdta is an ADP ribosylating toxin that ribosylatcs
actin withln the host cell resulting i n disorganiza-
tion of the cellular cytoskeleton and death of th<.>
affected cell.
Variability
Clos1rídiun1 difr1cile is quite variable. There are l2 sero-
groups (A- I, K, X, and S), as well as substantial vai:iability
ü1 the gene e11cotli11g tlte flagellar protei11 flagellin (9 dlf-
ferent groups). Random amplífication of polymorbic DNA
(IlAPD) analysis dernonstrates nurnerous types.
 208 P,\JtT JI Bacteria and Fungi
·r11ese di ffe1 e11Lt::> a11:: u~t:ful il1 c.leterrr1ining the epi-
demiology of this microorganism.
Ecology
Reservoir and Transmission
Clostridiun1 difficilc is found in thc intestinal canal of nor-
mal as well as clinically attcctcd animals. ·rhe spores are re-
sistant to most environmental stresses, ''.rhich results in
their widespread distributlon In Iocations where animals
are l1oused. ·rhere does not seem to be an overlap beh<Veen
ll1ose slrains Lhal produce disease il1 hun1au palie11l:;, a11d
those that are associated with animals.
Pathogenesis
Clostridiurn difficile adheres to mucus or epithelial cells of
the large intest ine by p ili o r fin1bria as well as tl1c surfacc
prolein Cwp66. Disease 111v:sl ofle11 follows a "Lriggt:r"
event (e.g ., antihiotics, nonsteroidal drugs, chemothera-
peutic agents) that results in relaxation of the control the
normal flora has on the numbers of C. difficile. Toxins are
produced (ToxA, ToxB, and Cdt), resulting in dcath of thc
epithelial cells, wh1ch follows d1srupt1on of thelf actill cy-
toskeleton and tight junctions. Prostaglandi n along with
the pruc.luLts uf tile ino!>ltul pathway stemming from the
intense inflammatory rf'<;pOn<;f' ('íoxA) n>sults in fluic1 ;inc1
electrolyte sccretion. Uiarrhea, with or without blood,
results.
Epidemiology
C/ostridii11n diflici/e-assoc1ated d1arrhea appears to be
linked to administration of antibiotics, stress, chemother-
a¡.H::ulh.: age11l:>, ur uou~terolc.laJ anti-inflammatory drugs,
although ncwhorn foi!f<; havf' hf'f'n <>hown to develop dis-
ease without a rccognizable "trigger" event. Whether this
organism moves among hospitalized patients, as with ·
human patients in hum:in hospitals, is unknown.
Clostridiun1 clifficile isolated from dogs a11d cats with di-
arrhea do not commonly contain the genes encoding Cdt.
lmmunologic Aspects
Tn1n1unity is probably anliloxic, allhough lhe role of a11U-
bodies to C. difficile is unknown. Orally administered
antitoxin (made in bovines) is protective for human pa-
tients.
Laboratory Diagnosis
1'he genes encoding the toxin(s) can be detected in feces by
assays based on polymerasc chain reaction (PCR).
Immunologically based tests are available for the detection
of ll1e Loxin (ToxA) in fecal specü11ens. Clostricliurr1 clifficile
can be isolated from feces by using a selective medium,
CCFA (cycloserine, cefoxitin, and fructose agar).
Treatment and Control
Diarrhea-associated C. difficile rcsponds rapidly to metron-
idazole. Unfortunately, metronidazole-resistant strains
exist. Thc alternative antibiotic is vancomycin. There are
no vaccin es available. However, the use of orally adminis-
tereu yea!>t, Sacc:llarumyc:es buulardii, has been shown to be
useful in prf'vPnting thf' clisease in hum;in patients. Tn
human hospitals, hand washing by health care personnel
is a very efficicnt mcchanism fo r curtailing spread.
Disinfectants are n ot effective against the spores.
( LOSTRID/UM PILIFORME ("8AC/LLUS Pll/FORMIS")
An acute fatal diarrhcal dlsease of laboratory mice with
focal liver necrosis (Tyzzer's disease) is associated with a
spure-forrning organlsrn, Cluslridiunz pilifurrne, w!lich uc-
curs in hunclles within hepatocytes. lt is unable to grow o n
cell-frce inedia. Clostridiunz pilifor111e ls linked to identica1
diseascs in rabbits, harcs, gerbils, rats, hamsters, muskrats,
dogs, cats, snow leopards, foals, and rhesus monkeys.
Tyzzer's disease has been reported in a human patient in-
fected with the human immunodeficiency virus, but not
In lmmunocompetent persons.
Descriptive Features
Clostridi11rn pilifor1ne is a large, gram -variable, spore-
forn1ing rod lhal is u1uUl1:: by perilrichuu~ ílagella. Git:ru~a
and silver stains are preferable to hematoxylin-eosin and
Gram stains.
Gro~vth has bccn obtained in embryonated hen's eggs
and on cultured mouse hepatocytcs.
vegetattve cells d.le even when deep-frozen or freeze-
dried . Spores survive m o derate heating, freezing, and
tl1awing. Lille1 ren1aius i11feclive for 111011Lli:.. Slrai.11:. are
pathogenically and morphologically uniform.
Ecology
Reservoir and Transmission
·rhc sourcc of thc agcnt is thc infcctcd animal. Thc agcnt
spreads by the ano-oral route and transplacentally. Many
infcctions are believed to be e11doge11ous and stress
triggered.
Pathogenesis
Lesions suggest hepatic invasion from the intestine via
lymphatics and blood vessels. foci ot coaguJation necrosis
are periportal. ·rherc may be dissemlnation to the my-
ocardi um. Parasltlzed cclls include hepatocytes, myocar-
diaJ cells, smooth muscle, and epithelial cells of the intes-
li11e, il1 which a dysentery-like co11dition n1ay develop.
Lymphadenitis, espccially of hcpatic nodes, is seen in foals.
The course is usually under 3 days.

Epiderniology
Outbreaks are often stress-related (crowding, irradiation,
steroid administration). Morbidity is high in laboratory
animal colonies. case fatali ty rates reach 50o/o to 100%, es-
pecially among yo11ng st ork. In many c.olonie.s, suhc.l ini -
cally il1fected individuals are evidently present; these are
often identifiable serologically.
La boratory Diagnosis
T.a h orato ry cliagnosis re.sts on thf. clemonstration of typic.al
b undles of in lracellular bacilli (0.5 µ111 by 8.0 to 10 µ111) 1 es-
pecially in hepatocytes surrounding lesions (see Fig 68.5).
Fluorcsccnt antibody uids diagnosis.
A comp1en1ent f1xa t1on test that ut1l1zes an infected
mouse liver extractas antigen is used to determine the de-
g.r ee u1 1 i11feclio11 i11 111ouse colouies.
Treatment and Control
Treatm.ent of cli ni cal cases is usually unsuccessful. Prophy-
IacticalJy effective antimicrobic drugs include erythrom y-
cin and tetracycline.
Other Species of Veterinary lnterest
C lostridiunz sordellii is associated w ith a fatal n1yositis and
hepatic disease in ruminants and horses, although the pre-
cise pathogcnic proccss is not known. 'I'he agent produces
numerous toxins and is experimenta1ly pat hogenic for
many species o f animals. Mixed clostridial bacterin-
toxoids usually contain C. sordellii.
Clostridium colinum causes quail disease, ulcerative en-
teritis, and necrotizil1g hepatit is of severa! species of fowl
see l~ig 68.6). 'fhe agent is fasti d ious nutritiona lly ancl
forn1s spores sparingly. Jts life cycle is unknown, and a
:oxin has not been identified . 1'he untreated disease is usu-
ally fatal. St rcptom)'Cin 11as been effective in t he field .
Clostridium spirofiJrrne is isoJated frequently from rabbits
'"ith juvenile enteritis ("mucoid enteritis"). It may be one
of several IIliLrourga11isu1s iiuplicaled. Ils exoLoxi n is iden-
:ical to the iota toxin of e;. perfringens 'Jype E and the ADP-
~bosyltransferase of C. dífficile. It acts by ADP-ribosylating
cetlular actin.
7HE NONINVASIVE CLOSTRIDIA
:::Iostridium botulinum (the cause of botulism) and C. tetani
:he cause of tetanus) produce disease strictly through the
3Ctlon of neurotoxins- boruunum ana tetanus toxins, re-
S?t'Ctively. 'fhough botulinum and tetanus toxins have the
same nlecl1anisn1 of actlon, tl1ey produce differenl dis-
=ases with different manifestations because the two toxins
:llfcct diffcrcnt sitcs in thc ncrvous system.
HotuJ.inu1n toxin and tetanus toxin block neurotrans-
mitter release. Both toxi11s are zinc endopeptidases that in
Chaptcr 36 Clostridium 209
terfere wit h the fusion of vesicles containing 11eurotrans-
1nitter with the me1nbrane lining the presynaptic cleft.
Jntcrfcrcncc is du c to hydrolysis of thc protcins involvcd
with "docking," which precedes fusion of neurotransmit-
ter-containing v esicles ~"1'ith the membrane lining the
cleft . Hytlroly:;is of these prote.ins leatls to degeneration of
t he. synapse. an<l hlorkage. o f ne.11rot ra nsmission. Re.ge.ne.ra-
tio11 of the synapse takes several weeks to months .
( LOSTRIDIUM BOTULINUM
Clostrldium botulinum is a gram-positive, spore-forming,
obli.gately anaerobic rod, which produces the disease botu-
lis111, a ueuruvaraly lic i11luxicaliuu ¡;J 1aracte.rizec.l l!y flaL-
cid paralysis. ·rhe intoxication is caused by any of seven
protcin ncurotoxins (A to G) that are identical in action
but díffer in potency, antigenic properties, and d.istribu-
tion. They are produced by a heterogeneous group of
clostridia, called C. botulinum (C. botulinum Group G has
been renamed C. argentinense) on the basis of the toxins,
which in sou1e types (C <u 1Ll D) a11:: lJa1..leJivpl1age-
encoded.
Botulinurn is scen mainly in ruminants, horscs, mink,
and fowl, particularly vvaterfowL Svvine, carnivores, and
fish are rarely affected.
Descriptive Features
Morphology
c·1ostridium /:Jotulinurn is a gran1-positive, spore-tor1ning
obligately anaerobic rod. Ata pH near and above neutral-
ity, it produces subterminal oval spores.
Cellular Products of Medica! lnterest
Toxirz. Clostridiurrz botulinurrz produces severa! proteins
vvith "toxic" activity (botulinum toxin, C2 toxin, and C3
cxocnzymc), but only botulinum toxin ha5 a central r ole
in the production of botulism:
l. Botulinu1n toxin. There are seven types of botu-
linum toxin (BoNT for botuli11um neurotoxin), dif-
terentiated by antige11ic ditterences. Letters A
through G depict the types. Tl1e type of neurotoxin
characterizes the strain of C. botulinum producing it.
·r hus, a strain of C. botulinum producin.g Type A
BoN'f wo uld be desc1ibed as C. botulinurn Type A.
Ali seven types of BoN"l" are zinc endopeptidases
w ith identical activity, i.e., hydrolysis of the dock-
ing proteins required by neurotransmitter-contain-
ing vesicles to fuse "\-vith t he presynaptic mcn1branc.
Though tl1e end result is the same (blockage of the
release of neurotrans1nitter), the various types of
BoNT hyclroly<.e cliffere11t c.luLklug prott'.ins. 'fypt'.s A
and E hydrolyze SNAP (synaptosomal -assoc.iatE~cl
p rotein), 'fypes Il, D, r, G hydrolyze VAMP (vesicle-
associated membrane protein also known as synap-
tobrevin), and Type C, wh.ich hydrolyzes SNAP and
210 PART 11 Bacteria and Fungi

syntaxin. <)nce hydrolyzed, the synapse degener-
ates, taking weeks to months to regenerate.
BoN1· is a "di-chain" molecule consisting of a
light chain (with zinc endopeptidase activity), a
hcavy chain composcd of a translocation domain
(rcspo11sible for forming a pore through whlch the
light chain passes), and a binding domain (respon-
sible for binding to nerve cells). Secreted with BoNT
are scvcral "accessory" proteins thought to aid the
:.ui vi val uf Lhe Loxin in lhe gasLioil1Lestinal tract.
BoNT binds to cholinergic nerve cells, each type
of IloNT binding to a diffcrcnt receptor. After bind-
ing, the toxin is intcrnalized by way of receptor-
mediated endocytosis. Vcsicles containing BoN1' re-
main at the neuromuscular junction. Folio>'ving a
cleaving event, the light cha in (the zinc ~ndopepti­
dase) translocates acruss tl1e ve:.icle 111en1bra11e inlo
thf' cytosol of th t> n c rve cell where it hydrolyzes the
docking proteins.
2. C2 toxjn and C3 exoenzyme. .Both the C2 toxin and
C3 exoenzyme are ADP ribosyltransferases. C2
toxin and C3 exoenzyme ribosylates G-actin and
llho, respectively, resulting in disruptions in the cy-
toskcleton. Neither enzy1ue a¡.i¡.i1.:ar:. Lu µlay a role in
the disease proces.s.
Resistan ce
Although heat resistance of spore~ varíes lJetweeu culturt::
groups (see below, "Variability"), tc.ixin typt>s, ;:inct strains,
111ui:.l heal al 120ºC for 5 n1inutes is generally lethal.
Exceptions exjst. Low pH and high salinity enhance heat
sterilization. Hcating to 80ºC for 20 minutes inactivatcs
thc toxin.
Salt, nitrates, and nítrites suppress germination of
spores ln foods.
Variability
Thcrc are seven types of toxins; they differ in antigenicity,
hcat resistance, and lethality for dlffcrent animal species
(rr()h;i hly rpl;itprl ti) rPrPptnr rlPn<:ity on thP ·"11rf<'lrf' of thf'
motor neuron). Four Lulture gruuµs are al:.o re1.:og11i.i:ed
(T;:ihlP ~6.~).
Ecology
Reservo ir
Reservoirs of C. botuli11ur11 are soil and aquatic sediments.
Vehicles of intoxication are animal and plant material
contaminatcd from thcsc sources. When animals die, C.
botulinum spores, which are common in gut and tissues.
germina te and genera te tox in. This may be ingested by car-
rion eaters or co11taminate the enviruume1 1l. In rolling
vegetation, a similar prorPS'> orn1rs.
Transmission
Apart from toxin ingestion, spore ingestion and wound
contamination may lead to botulism.
Spore ingestion is im portant in human infant botullsm.
Wound-infection botulism is seen rarely in humans and
horses.
Puthogencsis
I11~e:.Led BoNT is absorbed fron1 the glandular stomach
and anterior small iI1testine and distributed via the blood-
stream. It binds to reccptors nnd cntcrs thc nerve cell after
receptor-mediated endocytosis. J-tow HoNT gets fro1n the
bloodstream t o the surface of nerve cells is not understood.
Vesicles containing toxin remain at the myoneural ¡unc-
tion. A fragment of the toxin (light chain) translocates
across tl1e vesicle 111eu1ura11e i11Lu U1e cylosol of Ll1e nerve
rPll ;:incl suhsequently hydrolyzes a "docking" protein
(which protein depends upon which botulinum toxin
The synapse degenerates and Uaccid paralysis resuJts due
to the lack of neurotransmitter (acetylcholi ne). When this
affects muscles of respiration, death dueto respiratory fail-
ure occurs. No primary lcsions are produced.
Clirlical sigus iuclu<.le 111u~cul a1 i11coordll1alion leadinb
to rec:umhency, extrusion of the tongue. and disturbances

Table 36.3. llotulinum toxin types: Uistribution, Origin, and Pathogeniclty

Type Cultural Geographk Occum!nce Affected Species Toxins

Types Present

A 1 Western U.S., Canada, ex·U.S.S.R. vegetables, fruits (meats, fishf Human~ (thilkeu~. 111i11k) A
B 1, 11 Eastern U.S., Canada, Europe, ex·U.5-S.R. Meat, pork produrt~ (v!!l]etables, físh) Humans (horses. cattle) B
Ca 111 New Zealand, Japan, Europe Veyeldlio11, i1wertebrotes, carrion Waterfowl C1(Cvb
CR 111 Australía. S. Africa. Europe. U.S. Spoiled feed. carrion Horses, cattle, mink, doqs (humans) Ci, D (C¡)
o 111 5. Afríca, ex-U.S.S.R., Southwest U.S., f rance Carrion C~ttlc, 1hccp (horses, humans) C2, O
E 11 N. America, N. Europe, Japan, ex·U,S.S.R. Raw fish, marine mammals Humans (f1sh) E
F 1, 11 U.S., N. Europc, ex U.S.S.R. Meat, fish Huma ns F
G' IV Argentina Soil Humans G

• Parentheses iodieale tnfre<¡uency or varlablllty.

b c2is nota neurotoxin but an ADP-ribosylating toxm attectmg Hurd movementacr~ membcanes. ns etfeas are cardiopulmonary.enteik, and ns role In animal botulism is poorly defirred.
<Str.1ins pcoducing this toxin are part of the SJlE(ies Oostridium Mgentinense.

in food prehension, chewing, and swallowing (see Fig
71 .5). No changes iu co11sciousness occur. 1'l1e te111pera-
h1rf> ri>mrii ns normal unless secondary iníections such as
aspiration pneumonia supervene. In nonfatal ca5c5, rccov-
ery is slow and residual signs may persist tor months.
Equinc grass sickness, a commonly fatal dysautonomia, af-
fecting tlle enteric nervous system ls associated With the
ingestion of BoN"l' secrcted by C. botulínum Type <.:: living
in associatioII wi.tl1 l>lalles uf grass (as a biofiln1).
In birds the disease has hP.Pn nan1ed "limherneck" after
the drooping head posture, which often causes waterfowl
to drown.
Epidemiology
Ty¡.¡es A a11cl B a1e found in all soils, il1cluding virgin soils;
C, D, E, and f are linked vvith \.Yet environments- that is,
m uddy soils or aquatic scdimcnt. In nnimals, ·rypcs C and
V predominate. 'J'ype (; living in biofilms on the surface of
blades of grass is the source of BoN'f seen in equine grass
sickness.
Dead cats or rodents in feed can be sotirces of outbrea ks,
as car1 chicke11 111a11ure when used as a cattle feed supple-
ment. Outbreaks on mink ranches are usually due to
r.ainted meat, and thosc in fish hntchcrics to fish food con-
raining C. botulinum ·rype E spores that germinate in bot-
<-0m sludge. Decaying vegetation triggers outbreaks in ,~,a­
terfowl. As Iakes recede in summer leaving muddy shores
or shallov.r pools, rotting plant material be.comes accessi-
ble. Clostridiurn bululinurn a11d i Ls Loxin are ingesLed a11d
after death permeate the carcass, whirh is fP.<i upon hy
blowfly larvae wl1icb. absorb the toxin. Waterfowl ingcst-
!ng tl1ese larvae become intoxicated.
Typc D botulism is classi.cally linked to phosphorus-defi-
d ent ranges, where grazíng animals feed on carcasses and
bones that often contain botulinum toxin. In South African
cattle, tbe condition is called lamziekte ("lame ilisease").
Type B botulism has been seen in catt le rinct m11 les. In
~ Loxo- i11feclious bolulis1n 11
of foals ("shaker foal syn-
d rome") and adult horses, no toxin has been demon-
strated, but thc ugcnt has bccn isolated consistently from
tissue.
Human botulism is usually traced to improperly
p rocessed meat, seafood, or canned vegetables. Infar1t lJot-
u lism involves clostrid.ial growth and toxinogi>nP.sis in the
11
inleslinal lracL, producing the "floppy baby syndro1ne.
Wound-infection botulism results from contaminated ex-
terna! injuries.
lmmunologic Aspects
Resista11ce to botulisn1 depends on circulating antitoxin.
Sorne animals, such as turkey vultures, apparently acquire
immunity through rcpcatcd sublcthal exposure.
Laboratory Diagnosis
Diagnosis of botulisn1 requires demonstration of toxin in
p lasma or tissue before death or from a fresh carcass. Isola-
Chapter 36 Clostridiu111 211
tion of thf> orgrinism, P.spe.c.ially frorn intestinal contents,
or postmortem demonstration of toxin is not definitive.
Demonstration of toxin in teedstuffs, fresh stomach con-
tcnts, or vomitus supports a diagnosis of botulism.
loxin is extracted from suspect material (unless fluids)
overnight with salio.e. The mixh1re is centrifuged and the
clear portlon ftlter-sterilized and trypsinizell (19'ó at 37nc
for 45 mintites). G11inf>a pigs or mirf> arP. injec.ted intraperi-
toneally with the extract, mixtures of extract and antitox-
ins, and extract heated to lOOºC for 10 minutes. Death due
to botulism occurs within 10 hours to 3 wccks (average is 4
days) preceded by muscular weakness, 11mb paralysrs, and
respiratory difficulties..A.ny toxin must be neutralizable by
one of the C. botulinurn antitoxins.
Tsolation of C. hnt11/in11m from s11spi>rt fP.P.ds or tissue hP.-
gins with heating suspect material for 30 rniI1utes at 6SºC
to 80ºC to induce germination. Type E spores require, in
addition, treatment with lysozyme (5 n1g/tnl o f medium).
Culture anaerobically on blood agar plates. Identification
is by biochemical reactions and toxin p roduction. In1mu-
nofluorescence is used· to identify sorne cultural groups. 1
Primer.s di>signPct to arnplify t hf> gi>nes i>nrocting thf> vari-
ous toxins (by t he polymerase chain reactlo11) can be used
to support tl1e demonstration that C. hotulinum has been ~ I
isolatcd.
Treatment and Control
If recent ingestion is suspected, evacuation of the ston1ach
and purging are helpful. Antitoxln treatment following
onset of signs is so1netilues beneficia!, especially for mink
and ducks. Mink aIIu otl1er aui111als al risk should be vaccl-
nated '"'ith toxoid (Types A, B, C, D). L
Removal of affectcd watcrfowl to dry lund savcs many
from exposure and drowning. Placing feed on dry ground
lures birds from contaminated areas.
Guanidine and aminopyridine stimulate acerylcholine
release, and germine intensifies neural impulses. Clinical
reports 011 tl1ei r u~e are few and 1nixed. - '
(LOSTRIDIUM TETAN/
CJostridiuni tetani is a gram-positive, spore-forn1ing, oblig-
ately anaerobic rod, wlúch produces the disease tetanus, a
neuroparaly tic lntoxlcatton characterized by tonic-clorlic
convulsions. The intoxication is c111f> to a proti>in ni>11ro-
t<>xin.
All mammals are susceptible to varying degrees to
tctunus, •vith horscs, ruminnnts, nnd s'.vinc rnorc suscepti-
ble t han carnivores and poultry. In al! animals, the mortal-
ity rate is high .
Descriptive Features
Morphology
Clostridiu111 tetani is a gram-positive, spore-forming, oblig-
ately anaerobic rod. /\ distinguishing morphologic feature
212 PART II Bacteria and fungi
11'
-
-.... -
of C. tetani is the spherical shape and terminal position of
its spores ("drumstick," "racket," Fig 36.1).
Cellular Products of Medical lnterest
Toxi11s. (;/nstridiu.m tP.tani procl n cP~ two toxins (tetanolysin
and tetanus toxin), but only tetanus toxin is of clinical sig-
nificance:
1. 'letanus toXin (tetanospasm1n). l he genes encod.ing
tetanus toxin (1eNT for tetanus neurotoxin) are lo-
calt:ll v11 <t large plasu1ill. TeNT Is released upon lysis
of the clostridial c:elL
1'here is only one type of ·reNT, a zinc e11dopep-
ticlase, which hydrolyzcs the docking proteins
required by neurotrans1nittcr-containing vcsiclcs
to fuse With the presynaptlC membrane. leN·r
hyrlrolyzes the docking protein VAMP (11esicle-
~1ssociated 111cn1bra11e p10Lei11 1 alsv k11vwu a~ ~y11aµ­
tobrevin). Once tl1e docking proteins are hy-
drolyzcd, thc synapse degenerates, taking weeks to
months to regcnerate.
TeNT is a "di-chain" molcculc consisting of a
light chain (with zinc endopeptidase activity), a
heavy chain composed of a translocation domain
(re~µu11:;iulc fur furrning a pore through which thc
light chain passes), ancla hin<ling clom;iin (respon-
sible for binding to nerve cells) .
TeNT binds to cholinergic nerve cells by virtue of
receptors that are diffcrcnt from thosc rccognizcd
by BoNT. These reccptors are composed of lipid con-
taining rafts and glycosylphosphatidylinositol
(GP!)-a11<.:hured proteins. After blnding, the toxln Is
internalized by way of receptor-mediated endocyto-
sis. Vcsiclcs containing TeN! travel by retrograde
axonal transport to the inhibitory interneurons in
F 1G u RE 3 6. 1. Clostridiun1 telani in lissues of a lao1b
recently castrated by the "elastrator" (rubber band) method.
'irrotiil impression. Gram rtain, 1000X.
the ventral horn of the spinal cord. Follo,.ving ~
clcaving cvcnt, thc light chain (thc zinc cndopcpti-
dase) translocates across the vesicle membrane int;..
the cytosol of the n erve cell where it hydrolyzes the
"docking" proteins invo lved with vesicles contain-
ing the neurotran smitters gan1ma arninobutyri...
acid (GABA) aud glyci11t::.
2. 'fetanolysin . Tetanolysin is a c:hoJp_c;terol-hin<lin •
cytolysin (see also clostridial pcrfringolysin O
septicolysin O, novyilysin, chauveolysin, abo,·e·
streptococcal streptolysin O, Chapter 28¡ listcrial
listeriolysin O, Chapter 33; and arcanobacte-
rial pyolysin, Chapter 29). Tetanolysin binds to
choh:sterol-<.:u11tairliug rafts in the eukaryotlc ceU
memhrane. Once houncl, it forms a pon" rf's11lting
in the death of the ccll. Tctanolysix1 has no known
pathogenic signiticance in t l1e production of
tetanus.
Growth Characteristics
Clostridium tetani grows on blood agar under routinP
anaerobic conditions. There may be swarming. Its differ-
ential reactions (carbohydrate fermentation, proteolysis.
índole productio n) vary with the medium used.
Spores resist boiling up to 1.5 hours, but not autoclav-
Lng (121 ºC/10 min). Disinfection by sorne halogen com-
pounds (3% iodine) can be effeclive willii11 severa! l1vur~,
b ut phenol, lysol, and formalin in the usual concentra-
tions are incffective.
Variability
Ten sc>rologic typPs, hasPcl on flagPllar antigPns, arP some-
what related to geographic strain origin. TeNT is antigeni-
cally uniform.

Ecology
Reservoir and Transmission
C/ostridiun1 tetaní is \-videly distributed in soil and is often a
transient in the intestine. Spores are introduced in to \.YOunds.
Pathogenesis
Spore gern1ination requires an anaerobic environment as
found in tissues devitalized by crushing, burning, lacera-
tion, breakdown of blood supply (e.g., umbilical stump or
placen tal re1nnan ts), or bacteria] infection. U11der these cir-
cumstanc:es, C. tetani proliferates and its toxin diffuses via
,·ascular channels or peripheral nerve trunks. The toxln at-
taches to receptors on the nearest cholinergic nerve and is
!ntcn1alized within a vesic:lc, which travels retrograde in-
side the axons to the cell boc.1ies in t h e ventral horns oí the
spinal cord. The light chain of the toxin translocates ac:ross
the vesicle membrane into the cytosol where it hydrolyzcs
~<locking" proteins and suppresses release of afferent in -
:tibitory n1esse11ge.r subslances (glyci11e, ganuua a11ü11obu-
:yric acid) causing the innervated muscles to remain in sus-
~ained clonic or tonic spasn1s. Thc tox.in also travels within
the corc.1 to ot her levels affecting additional muscle groups.
Synapses degenerate following hydrolysis of the "doc:king"
protelns, taking several weeks to regenerate.
The process described ("ascending tetanu s") is typic:al
of anilnals noL h ighly suscepLible Lo Lela11us Loxiu (e.g.,
dogs and cats). Only nerve trunks near the toxigenic site
absorb sufficient toxin to produce overt si.g ns.
"Descending tetanus" is typical of highly susceptible
species (horses, humans) in which effective toxin quan ti-
:!es are disseminat ed via vascu lar channels to nerve end-
·::igs in areas remote froln tbe toxigen.ic site. Toxin enters
:he central i1ervous systein at many levels producing ge11-
eralized tetanus, frequently begin11ing cranially. The se-
~ue11ce .reflects susc.eptibility of various 11euro11s.
Disease Patterns. Early signs, following an incubation
x riod of a few days to severa! weeks, are stiffn ess, m u S<-'U-
ar tren1or, and increased responsiveness to stimuli:
1. Tn horsP<::, r11min;:int~, and swinP , whirh 11s11;illy de-
velop descending tetanus, retraction of the third
eyelid , erectness of ears, grinding of teeth, and stiff-
ness of the tail are observcd. Bloat is common. in ru-
n1inants. feeding becomes impossible ("lockjavv").
Rigidity of extremitíes c.a uses "sa"l-vhorse" attitudes
and, evennially, recumbency. Tetanlc spasms occur
fi rst in rPsponsP to stim111i, h11t latPr become pern1a-
nent. 'rhere is fecal and uri11ary retention, sweatil1g,
and high fever. Consciousness persists. Death, due
to rcspiratory arrest, occurs in lan1bs and piglcts
within the first week, in adult animals in l to 2
weeks. Full recovery requires weeks to months.
2 . In carnivores, the incubation period tends to be
longer, an<I lora 1 (asrPnd ing) tetanus (stiffness,
trcroors) is frequently seen near the original wouJ.1d.
Progressio11 may be slower than in ungulates, but
signs and coursc are comparable.
Yiurtality is at least 50% an<l highest In the you ng.
<;hapter 36 (;fostridium 213
Epidemiology
()ccurrence of tetanus is linked to the introduction of C.
LeLuni spures in to traurnatized tissue (see une.ter "Patho-
genesis," ahove): pPnetr;iting nai 1 \.Vo11nrls of t he foot,
barnyard surgery, t he use of rubber bands for castratin_g
and docklng sheep, ear tagging, injections, shearing
wounds, postpartum uterine infections, perinatal umbili-
cal infections, and s1nall an1n1al fights and leghold traps.
lmmunologic Aspects
Ac:quired resistance to tetanus depends on circulating anti-
toxln. Small amounts have been demonstrated in normal
ru minants. Survivors of tetanus w ith the possihle Px<Pp -
tion of dogs and cats are susceptible to reinfection. 'l'he
amount oí toxin needed to result in tetanus in dogs and
cats is son1etin1es large enough to elicit an antitoxin Je-
sponse.
Passive and active protection is p rovided by administra-
lio11 uf aJ1liluxi11 ur i1nu1urlizatiun vvith toxoid , respec-
tively (see under "Treatment and l.ontrol," hPlow).
Laboratory Diagnosis
A gran1-stained smear from a suspect wound may revea.!
the typical "drumstlck" type of bacteria (see Fig 36.1).
Their absence does not exclude tetanus, and their presence
is 111erely suggestive as the n10.rpl1ology is 11ol un.ique.
Wound exudate is plated on blood agar for anaerobic
culture. Increased agar contcnt (up to 4%) inhi.b its swarm-
ing, anda drop of antitoxin will inhibit hemolysis on that
portion of the plate.
Replicate cooked meat broth cultures are incubate(1 and
sorne are heated at 80ºC for varying periods (up to 20 min-
utes). Ali are iucubaLeu al 37ºC fur 4 uay:> a11u :;ul>culturetl
to blood agar periodically during that ti1ne. Previonsly un-
heated ones are heated before subculture. Suspect isolates
are ide11titied by differential tests and confirmed as tetanus
toxin producers by intran1uscular injection of a 18 hour
broth culture il1to two inice, one of which 11as received
anti.toxin .
Prituer:> uesiguell tu aiuplify tl1e gene encolling tetanus
toxin (hy the poly1nerase rhain rPartion) r;:in hP nsed to
support the demonstration that C. tetani has been isolated.
Treatment and Control
·rherapy aims at 1) neutralization of circulating toxin, 2)
suppression of toxin production, and 3) lifc support and
symptomatic relief to the p atient .
The first objective is pursued by il1jection of adequate
dose:; uf a11Utoxil1: 10,000 tu 300,000 unlts for horses.
So me suggest intrathec:al aom in istration .
Wound care and large doses of parenteral penicillln or
metronidazole are aimed at stopping toxin production.
Supportive treatment includcs use of scdativcs and 1nus-
cle relaxants and exclusio11 of externa!. stimuli. Artificial
214 PART II Bacteria and Fungí
feeding by stomach tube or intravenously may be neces-
sary after t h e hyperesthetic phase. Nursing care is most
importan t.
Prevention
Wounds should be properly cleaned and dressed. During
surgical procedures, especially 011 a 111ass scalc undcr farm
conditions, appropriate h ygienic precautions should be
observed. 1-lorses. un less actively in1munized, are given an-
titoxin after injury or surgery, and depot penicillin.
Active immunization employs formalinized toxo id.
given twice at 1-to-2-1no11tl1 intervals and annually t here-
after.
P;issive immunity passes from immunized mare to
nursing foals and appea.rs to providc protcction for abou.
10 weeks, when toxoid can be given.
 Filalllentous Bacteria:
Actinomyces, Nocardia,
Dermatophilus, and
Streptobacillus
ERNS'f L . BJBERSTEIN
:Members of the genera Acti110111yces, Nocardia, Dennatoplzi-
illS, and Streptobacil/us produce filamentous forms at sorne
time in their growth cycle.
ACTINOMYCES ANO NOCARDIA
~1embers of the genera Actinornyces and Nocardia are gra111-
positive bacteria tl1at b'TO>v in branching fila1nents. Apart
:Iom this pattcrn, thcsc two genera havc littlc in common.
:iowever, their morphologi.c resemblance and similarities
~"l pathogenic patterns (pyogranulomatous inflammation
· ith fistulous traets) ¡ustify their conslderarton under a
~om mon heading. Table 37.l summari7.es their differential
..:haracterlstics.
ACTINOMYCES SPP.
Jescriptive Features
''orphology and Staining
fembers of the genus Actinornyces are gram-positive
.uphtheroid or filamentous rods, about 0.5 µrn in width.
- ·anrh ing fi l;imPnts, often bea<led due to u neven stain-
_,g, are most readily demonstrable in pathologic speci-
::Jens (Fig 37.1). In culture, diphtheroid forms predo-
::iinate (see Chapter 31 for a discussion of this mor-
:-hotype). As of this writing, there are approximat ely
:s species of Actinomyces, 1nost of which are associated
ith tliseases of human patlents. The few that are in-
:>lved with pathologic ronditions in animals are listed
~Tab le 37.2.
:::ucture and Composition
-rinomyces spp. have distinctive cell wall constituents (see
- ..111~ 37.1). Surface fibrils in A. viscosus rnay be adhesins for
DWIGH1' C. HIRST-I
host cells or oll1erbacle1ia ("cuaggrcgaliur1"). Surfcit:e anti-
gens are related to chernotactic and mitogenic activiti<'-~.
Ench nnmcd spccics of Acti110111yces has group antigens
and severa! serologic subtypes.
Growth Characteristics
Anin1al Actinonzyces spp. a1e capnophilic or facultalivr.:
anaerobes. Ali require rich media, p referably containing
scrum or blood. No growth occurs on Sabouraud's agar or
at room temperature. i)evelopmcnt of rnacroscopic colo-
nies may require severa! days of incubation at 37ºC. Colo
nial morphotogy varíes berween and within species. He-
molysis is rare.
Bioche1nical Activities
Aclinomyces spp. are cataJase-negative, with sorne cxccp-
tions (e.g., A . viscosus and A . can is) .
Resistance
Actinomyces spp. are read1ly l\tlled by heat and CUsintec-
tants and require frequent passagc to survive in culture.
Ecology
Reservoir
Mcn1bers of Lhe genus ALlinu1nyte:; ar<:: fuund un oral mu-
cous membranes and tooth surfa<:Ps, thP m11rn11s mf'm-
branes of the urogenital tract, and secondarily (presum-
ably) in the gastrointestinal tract.
Transmission
Most actinornycotic infections are endogenous, tl1al is,
they are caused by introduction of a con1mensal strain
into susceptible tissue of its host. Bites are another means
of transmission (rare).
215
216 PAHT 11 Bacteria and Fungl

Ta b 1e 37. 1. Contrast1ng Characterist1cs of Actinomyces and Disease Patterns

Common Pathogenic Nocardia
Ruminants. ''Lumpy jaw." In "lumpy jaw" of cattle (and oc-
casio11ally oll1er ru111i11ants), A. bovis and (exceptio11ally)
Charactéris¡k Actinomyces ~rtlia
'
A. israelii are introduced from an oral reservoir by a trau-
Source EFJdogenous Exogenous matic cvcnt (c.g., poor quality fccd) into thc alveolar or
Atmospheric requirements Anaer-0bidmicroaerophilic Aerobic paraJveolar region of tl1e jaw initiating a chronic rarefying
Gatalase - or-t -t osteomyelitis. This leads eventually to replacement of nor-
Growth on Sabouraud's agar + mal bone by porous bone, wbich is laid down irregularly
Acid' fostr>css + and honeycombed wit h sinus tracts containing pus. 'rhere
.
Penicillin susceptible Resistant• n1ay be dislodgen1e11l of teell1, inabilily lo chew, and
Cell wall contains meso OAPb + mandibular fractures. 'fhe lesion expands but has little
Lysine + tcndcncy for vascular disscmination. Sim ilar infcctions
Myc-01i( acid + occur in humans and m arsupials.
lymph nooe involvement Ra.re Common
Granules iri exudate {"sulfur Common Rare Horses. "Poll Evil" and "Fistulous Withers." Supra-atlantal
granules") ("poll evil") and supraspinous bursitis ("fistulous withcrs"),
so1netimes contain Actinom.yces, usuaJJy alongside other
"Nocardja nova" are sus,:eptible to penicillin.
il bacteria (e.g., Brucel/a abortus).
bDiaminopimelic <icid. Cervicul Lyrnphudenilis. A.11 u11sµecia ted Actinornyces is
sometimes isolated from cervical abscess, n1imicking Strep-
tococcus equi subspecies equí infections.

Dogs and Cats. Actinomyces are couu11u11 i:>ulatt:'.) frou1 suµ-

Pathogenesis
Pathology. Mernhers of the genus Actinomyces evoke pyo-
gra11ulon1atous reactions by unkno,-vn mechanisms.
Bacteria] colonies form in tissue. triggering suppurative re-
sponses in the immediate vicinity. Peripheral to this, gran-
ulation, 1nononucJea.r infiltration, and fi brosis furnish the
granulomatous element s. Sinus tracts carry exudate to the
outside; the exudate often contains yellowish "sulfur gran-
11lt>s," which arP colonial masst>s s11rro11nrlP<i by a micro-
scopic fringe of "clubs" consisting of mineral and possibly
antigen-antibody complexes. They are also called rosettes
(Fig 37.2, and see Fig 69.2). J\lternatively, Actinomyccs spp.
may be found, usually in mixect culture, in abscesses,
empyemas, or suppurative serositis.
p11rativP SPrositiclt>s of small carnivores. Tn dogs, actino-
mycosis may be associated (e.g., by licking) wlth foreign
boclies, particularly migrating grass awns, especially of
the genus Hordeu1n ("foxtails''), whicl1 so1nctilncs lodge
near vertebrae, causing actinomycotic discospondyli-
tis. Cutaneous actinomycosis in dogs is a rare nodulo
ulcerative Jymphangitis.
Swine. Mastitis. Mastitis in SO\.VS is sometimes associated
with Actinon1yces.
Pneumonia. Actinornyces is sometimes recovered, along
wlth other bacteria, fro1n lung lesions in swine.
Abortion. Actinomyces is sometimes recovered fron1
abor ted fetuses.

• •

.
<t.
.. • •
F 1G U RE 3 7 . 1 . Actinomycc5 spp. in aspira te from retrobul-
bar abscess ín a horse. Gram stain, 1000X.
 Chapter 37 Filamentous Bacteria: Actinom)'ces, Nocardia, Dermatophilus, and Streptobacillus 217

Ta b 1e 3 7. 2. Members of the Genus Actinomyces Affecting lmmunologic Factors

An imals and Their Usual Source or Associated Condition

Tnfected humans show cell-mediated and humoral im-

Sl ecies . ____..,..._
....-,¡............... Usual source orAssociated ondjtiep mune respon ses .
Circulating antibody, produced during infection, con-
Actinomyces bovís Ruminant jaw ("lumpy jaw")
fers no protection. Speciflc res1stance, if any, is probably
A. canis Pyonecrotic processes of dogs; vagina of dogs
cell-mediated. Phagocytes kill Actinornyces. No artificial
A. catulí Pyonecrotfc processes of dogs
irnrnurlizatior1 is available.
A. colcocanis Normal vagina of dogs
A. hordeovulnerís f'lant awn-assofiated pyonecrotic conditions in
dogs and cats
A. hyovaginalis Abortion in swlrie; pneumonia in swine
Laboratory Diagnosis
A. ísrae/ii Ruminant jaw ("lumpy jaw"l- rare; PY.Onecrotic
processes in dogs-'rare
Sample Collection
A. marimammalium Disseminated disease in marine mammals ;\spirates from unope11ed lesions or tissues, p referably in-
:i\. naeslundii Abortion in sw.ine-rare cluding granules, are optimal.
A. suimasritidis Mastitis in swine
A. vacómaxillae Ruminant jaw ("lumpy jaw")-rare
A. viscosus Pyone<rotic p1oces~es of dogs a11d c,its Direct Examination
Suspected ex udates are exa1nined for "su]fur granules"-
yellowish particles, varying in firn1ness, up lo severa! n1il-
limeters in diameter. Granules are washed in saline and
placed o n a slidc in a drop of salinc. A. cover slip is gently
Epidemiology pressed dow11. rhe p reparation is examined unc1er sub-
dued light (see Fig 37.2) . Rosettes are suggestive of act ino-
Actlno.mycosis is no11communicable, except via bites mycosis, especially if bacterial-sized filaments extend into
(which is a rare forin of transmission). Contamination of the clubbed fringe.
bursae ur lJuuy cavilies 111ay l>t: 11<::111dtuge11uus ur result C ru:;l1eu grd11ules, exullate, or tissue irnpressions are
from r~rforations of the ;¡]jmentary trac.t, Or l)ocJy Vvi!ll. stained with Gran1 ancl ac.icl -fast sta ins (Kinyonn's).
Actinomycosis associated w ith plant awns ("foxtails") oc- n ranch.ing, gram-positive, beaded, non- acid-fast fila-
curs in outdoor dogs in semiarid areas. m ents suggest Actinornyces spp. (see f ig 37.1).
Actino1nyccs-associntcd discascs occur in goats, shccp,
wild ruminants, monkeys, rabbits, squirrels, harnsters, lsolation and ldentification
marsupials, and birds. Except for occasional reported isola-
tlons of A . ísraelii in dogs and cattle, there is little to sug- Most strains obtain.e d from antn1als do not require anaer-
gest interspecific transmission of Actinomyces spp. obic incubri tion but benefit from incr.eased car.bon diox-

F 1G U RE 3 7 . 2 . "Sulfur granule" in bovine pleural fluid showing an amorphous center

and clubbed fringe. Unstained we t mount, 400X.
.,,)i.~ ,.. - -•
• '
·~

•
...• 'f.Í1
~~
• .iw
218 PART 11 Bacteria and Fungí
ide. c;ranules offcr the hest c.hancf' of isolation from the
usually mixed flora. Colonies develop in 48 hours or more
atter incubation at 3S- 37ºC. Organlsms of suggestive
morpl1ology and staining characteristics are tcstcd for
catalase activity. Isolates grown on blood agar som.etunes
give false-positive catalase results.
Precí:;e id.eutification inclucle~ cell wail analysis (see
Tahle :~7.1 ) ano hiochemical tests. M;iny ;inimal isolates
cannot be assigned to existing species. DNA prin1ers based
upo11 the sequence of genes encoding the 165 ribosomal
RNJ\ have bee11 desig11cd for spcciation of mcmbcrs of this
genus by using the polymerase cl1ain reaction.
Treatment and Control
In bovine actinomycosis, iodine compounds given orally
(1 to S gn1) duily or intruvcnou:;ly (75 rog/l<g) wcckly urc
used. 1reatment must be interrupted when signs of toxic-
ity appear (hypersalivation, anorexia, vomiting) but can
be resun1ed sorne weeks later. Accessible soft tissue lesions
can be drained or excised.
Pe11icillin a11d an1inoglycoside co1nbinalio11s are oflen
used on bovine actinomycosis. 1' he contribution of
aminoglycoside is uncertain. Isoniazid per os has been ad-
vocated. Bacteriologic cure of lumpy jaw will not restore
normal bone structure, but the process can be arrested by
syste1nic medication aided by drainage, lavage (iodine),
and debridement of lesions.
Iu docs é111d cal:s, :>U.le t i y ( u1ciiuage, li:.1Vd1$t, e.11.ci:.iu11, 1e-
moval of foreign bodies) is required, supplemented by
long-tcrm antimicrobic therapy. A penicillin derivative
(e.g., a1npicillin) is the drug of choice. Alternatives are
eryth.romycir1, rifarnpin, cephaloridine, and minocycline.
Am inoglycosides a11d fluoroquinolones are ineffecttve.
Remnants of plant awns togetl1er with the propensity of
F 1G U R E 3 7 . 3 . Noca rdia in pleural effusion of a dog. Without an acid-fast stain,
Actinomyces could not be ruled out Gram stain, 1000X.
•
'
. -·
•
Actinornyces spp. to form cell wall- deficient variants (L-
forn1s) n1ake lrealn1e11l of lhis condilion difficu!L
Where plant awns (foxtails) are prevalent, conscien-
tious grooming is a critical preventive step.
NOCARDIA
Nocardiae are aerobic, saprophytic, gram-positive, par-
tially acid-fast fi lamcntous bacteria prcsc11t in most cnvi-
ronments. Diseases att ributed to mem bers of this genus
occur in rnammals, fish, mollusks, and birds (rarely).
Ge11eralized suppurative and pyogranulon1atous processes
occur in immunosuppressed and massively exposed indi-
viduals. Currenlly, lhere are 30 species ü1cluded will1Ln
the genus. Nocardia asteroides, the species most often asso-
ciotcd in thc litcroturc witl1 disco:;c:; of unimol:;, h a:; bccn
shown to be composed of severa! species (N. asteroides, N.
a.bscessus, N. 0macigeorgícci, N. fa.rcinica, N. nova, and N.
rransvalensts) . For t his reason, in the discussion that fol-
lows, a species designation will not be given to the nocar-
dial agenls associaled wilh lhe various diseases ascribed lo
memhers of this genus, unless isolates were identified by
techniques (most1y DNA-based analyses) currently ac-
cepted for identification of this group of bacteria.
Descriptive Features
Morphology and Staining
Gram-st ained Nocardia spp. are indistinguishable from
Actinomyccs spp. (Fig 37.3). Nocardiac alt crnutc bctwccn
the coccobacillary (resting phase) and the actively grow-
ing fi lam entous forms.
 Chapter 37 Filamentous Bacteria: Actinomyces, Nocardía, Dernu:i.tophilus, and Streptobacillus
Structure and Composition
TI1e cell wall .is typ.ical of gram-positive bacteria, with the
additio11 of meso-diamino-pimelic acid (DAP), arabino-
galactan, an.d 1nycolic acids (branched chained fatty acids
w iLl1 a Lhai 11lt:11gLI1 of C 46-c 60 for iYocardia). ·rhe cell wall
contains a high concentration of lipids. N'o exotoxins are
known, but a superoxide disrnutase acts as a virulence fac-
tor. Soluble antigens- largely protein- are type-specitic.
Growth Characteristics
l'alhoge1licnocardiae are obliga le aerobes growi11g on sil11-
ple media (e.g., Sabouraud's) overa wide temperature range
(10 to 50ºC). Colonics, which appcar aftcr severa! days, are
opaque anct variously pigmented. ·rhe colony surface,
'"'ªxy to powdery to velvety depending on the abundance
of aerlal growth, becornes wrlnkled wltl1 age. Colonial di-
<imeters may reac:h several centin1eters. Tn b roth, a surface
p eUicle or sedin1er1t is produced, but little turbidity.
Biochemical activities include catalase production,
acidification of various carbohydratcs, and usually urca
hydrolysis.
Resistan ce
Nocardiae thrive iI1 the e11viro11n1ent. Ali are susceptible to
chlorine disin(ecta11ts (100 ppm/5 min) and benzalko-
ni.um chloride (100 ppm/10 min).
Variability
Colonial dissociation is seen in single strains. Scrotypes are
uased ou soluule antigeu:;.
Ecology
Reservoir
Pathogenic nocardiae are saprophytes fou11d in n1any cli-
mates in soils and water, eitltcr as indigcnous flora or con-
taminants.
Transmission
1·hree main routes of infection are inhalation, tratima, and
ingestion. Dust, soil, and plant material serve as vehicles.
\Jocardiac producing bovi11e mastitis are introduced an.d
disse.m tnated by equipment and personnel.
J>athogenesis
Mcchailisms. Pathogcnic nocardiac survivc within phago-
cytic vacuoles by preventing phagolysosome formation.
This is attributed to a surface Jipid. Other cell walI lipids
may trigger granulomatous reactions. Variations between
strai ns an<l growth phasP.s in c.P.11 PnvPlopP ronstitnents <ire
paralieled by changes in virulence and infectivity.
Superoxide dismutase and lysosomal er1zyme inhibi-
tion protcct against phagocytic killing.
[Jathology. Nocardiosis is a predominantly suppurative
219
process with variable granulomatous features. Lympl1
nodcs are consistcntly involved. Hcn1atogenous disse1ni-
nation may result in osteomyelitis and widespread abscess
formation. Central nervous involvement is rare in anim.als
(thougt1 fairly common in human patients). Tn dogs, the
common fonn is thoracic empyema \<Vith granulomatous
se1osilis. Exudales a1e sauguinopu1ulenl a11d scn11eli111es
con tain small (<1 mm cliameter), soft granules consisting
of bacteria, neutrophils, and debris ("sulfur granule-like").
·rhey usually Jack the microstr ucture ot sultur granules
sometimes see11 with Actinomyces infections (see above,
"Actinomyces").
Disease Patterns
lnfections ca11 be regional or disseminated. Local wound
intections may extend to contiguous a.reas or regional
lymph nodes.
Ruminants. Mastitis. Bovine nocardial mastitis is often initi-
a Led will1 "ho111en1ade" udde1 iuf usio11s ( co11La111i11aled
with Nocardia). Onset is sudden with fever, anorexia, and
abnormal milk secretion. '!'he affected gland is swollen,
hot, and paintuL l)ischarging listulous tracts may develop.
Lymphadenopathy is coinmon, and there is occasional
dissemination. The affected gland usuaUy beco1nes 11on-
f1.1nctionaL Fatalities (SºA:. to lOºA:.) may occur during the
acule slage or upo11 r uplure of lhe udder.
Bovine "Farcy." Bovine farcy is a chronic suppurative in-
fection usually starting at the lower limbs. lt involves the
lymphatics of the extremities or head region and the asso-
ciated lymph nodes. Ulcers and discharging sinuses form
along the path of lnfection. Affected a11imals remaln in
general good h ealth unlcss dissemination t o interna) or-
ga11s uccurs. Tl1e tlisea:;e is re:;trictetl to the tropi<..::; autl al-
trihuted to N. farcinica or Mycnhacteriu1n farcinogenes (see
Chapter 38) .
Miscellaneous. Pneumonia, abortion, an d lymphadcni-
tis are somctimes (rarely) encountered associated with
Nocardia.
Horses. in horses, rare local or ger1eral infectior1s with
Nnr.ardia are seen, the lattP.r seron<lary to profonnd c.onsti-
tutional disturbances (equine c:ushing's, combined im-
munodeficient foals) .
"Norcardioform placcntitis.,, "Norcardioform placcntitis"
is a relatively uncomrnon condition of mares. ·rhe agent, a
gram-positive branching, filamentous bacteria, is associ-
ated With placent ltls and abort l.on. 1'hough slmilar to
members of the genus Noca rdia in morphology, the cause
of this condition is the bacteriun1 Crossiella equi, a 1111-
croorganism unrelated to Nocardia.
Dogs and Cats. Dogs and cats develop debilitating, febrile
illness. Pneumonia and suppurative pleuritis with em-
pyema ls the common finding. Disseminati.on occurs to
Ji.ver, ki.dn eys, bones, joints, and, rarely, the central nerv-
ous syste1n. Case falalily rale exceeds 50o/o. Nocurclíu nuvu i::;
the nocardial agent most con1monly encountered.
Myccto111a. Nocardial inycetoma ("actinomycotic myce-
toma" see Chapter 47) has been reported in dogs and cats.
220 PART JI Bacteria and Fungi
Swine. Pneumonia, abortion, a11d ly1nphadenitis are
sometimcs (rarcly) cncountcrcd associatcd with 1'-Jocardia.
Miscellaneous Species. Nocardiosis observed in birds (rare),
whales, and dolphins are mostly respiratory with signs of
dissemination.
Epiderniology
t>athogenic nocardiae occur worldwide, suggesting con-
stant exposure. In huma11s, dlsease is assocíated largely
with immunodeficiencies. This association ts established
in horses. In dogs disease may occur as a sequela toan in1-
1n unosuppressive vir us in fecliou, e.g., ca11i11e llisle111per.
Canine nocardiosis is 1nost comn1on in pups.
Ilovine nocardial n1astitis is most often traceable to
unsatisfactory hygienic practices. Typically infection is in-
troduced during the "dry period" with intramammary
mastitis therapy. Ac"Ute mastitis is triggered when onset of
lactation flushes the organisn1 fron1 li1nited foci tl1rougl1
tl1e lactiferuu::; duct ::;y::;te11i. .1\lter11atively 1 HUt:ardiu::;i::; a¡.>-
pears spontaneously in one anin1al anct spreads in the
course of milking operatíons.
lmmunologic Aspects
AntitJolly ancl cell -mediated immune responses, inclucling
hypt>rst>nsitivity, con1monly develop du rin g n ocardial in-
fections. Yet severe nocardiosis in several species is asso-
ciated with immunosuppression, particularly of cell-
mcdiated responses.
A.n tibody apparently confers little p rotection. ~pecific
resistance is largely cell-mediated.
No ptacticai immuniz.ation methot\ is ptesently available.
Laboratory Diagnosis
Branching, gram-positive, acid-fast beaded fila1nents,
along 'INit h shorter, c::occobacillary forn1s, are fou11d in
s1ue<ir:s iu<tllt'. fruJI1 r1ucarúia-ir1ft:cted ::;a111ples, irnpre::;-
sions, and sections. t-;ranules are sometimes ohservect.
Specimens for culture should not be chilled or frozen.
NocardiaJ colonies on blood agar, incubated at 37º(~ . will
be dull opaque, waxy to velvety, and hard to dislodge by
loop or needle. Stains confirm nocardial morphology.
Acid-fasti1ess may be lost 011 culture. Catalase tests are pos-
i ti vt'., a11d ll1t: age11t fail:s tu grow ::;erially unller a11.<it:ruuic
con.d itions.
Serologic (immunodiffusion, complement fixation,
enzyme-linkcd immunosorbent assays) and cutaneous hy-
persensitivity tests employing extracellular antigen to de
tect nocardial infection in cattle and dogs are of uncertain
sensitivity and are not generally available.
Id1::11tifica tiou uf lsulates i11vol ve::; ueteruli11atiu11 of cell
wall constituents, as well as determination of restriction
length polymorphism (RfLP) of DNA encoding the heat
shock protein, GroEL. RfLP data con1bined with susce1)ti-
bility to a battery of antimicrobial agents (piperacillin,
ampícillin, erythromycin, imipenem, ciprofloxacin, cefo-
taxime, and tobratnycin) aid in the identification of iso-
latcs. Dctcrminution of thc scqucncc of t hc DNA cncoding
tl1e 165 ribosomal lZNA has also been used.
Treatment and Control
Antimicrobic therapy of nocardial tnastitis 1nay produce
témporury clinical rclicf and ccssation of shcdding, but no
permanent cures. l~ontrol involves removal of infected an-
ilnals, thorough disinfection of premises and equipn1ent,
and scrupulous stabling a11d rnllking 11yglene.
Tn other forms of nocardi.osi.s, tri.methoprim-s11lfon-
an1ide therapy has produced in1pressive resul ts, especially
when combiued with surgery.
Altcrnativc drugs includc minocyclinc and doxycy-
cJine. Nocardiae are fairly resistant to the fluoro-
quinolones. Nocardia nova. responds to the penicillins and
macrolides (erythromycln, claritl1romycln), and the tetra-
cyclines (doxycycli.n e, inl11ocycline).
Abscesses, e1upyen1as, and serosa! effusions are treated
by drainage and lavage. Granulomatous proliferations re-
q u1rc cxc1s1on.
DERMATOPHILUS CONGOLENSIS
Dermatophilosis or strept othricosis (cattle and other
species), rain scald or rain rot (horses), grease heal (horses),
lumpy wool (sheep), and strawberry footrot (sheep) are in-
Ieclions duelo Derrnulophílus congolensis, a gia1n-posilive,
filamentous bacterium. In temperate climates, dermato-
philosis is often a cosmetic problem controllable by man -
agement practices. In sheep and cattle, especially in tropi-
cal areas, it can severely affect productivity.
Descriptive Features
Morphology and Composition
The reprolluctive unit of D. cungulensis is the motile coc-
coid "zoosporP.," ahout 2 pm in d iamP.tP.r. llpon gP.rminat-
ing, zoospores sprout a ger1u tube, about 1 µm thicl,;,
which elongates and thickens, d ividing both tran sverselr
and longitudina.lly, forming a strand severa! cell layers
thick. Enclosed iI1 a gelatinous sheath, constituent cells
become coccoid as they differentiate into 1ntdtiflagellated
í,',UUspure::;, wlúcl1 are liuerated a::; tlie stra11tl tli:;i11legrate:.,
completing the life cycle.
Derniatophilus has a cell wall containing n1esodiamino-
piinelic acid, but lacking glycine and arabinogalactan.
Growth Characteristics
Derrnuluphilw; cun<'<ult:n:;i~ grows 011 b lood and infusioi
media, hut not on Sabouraud agar. It is aerobic and capno-
philic. Hemolytic colonies, which develop in 48 hours.
vary trom 1nucoid to viscous and waxy, whitish-gray ta
yellov<, and smooth to wrinkled. Glucose a.n d sorne other
carbohydrates are attacked nonfer1nentatively. Catalase.
 Chapter 37 Filamentous Bacteria: Actinom,vces, Nocardia, Derm.atophilus, and Streptobacillus
urease, and proteases are produced. The agent survives
vvell in soil and on fomites.
l'here is colonial and antigenic variability, but ali
strains appear to share antigens.
Ecology
Reservo ir
Dermatnphilus congnlensis <loes not mult iply saprophyti-
cally. Its reservoir is infected an.imals. Cattle, sheep, goats,
and horses are con1mon hosts. It has been d~agnosed in
swine, dogs and cats, turkeys, primates (including hu-
n1ans), and ~vild man1mals, including marine man1mals.
The distribution of Ll. congolensis is worldwide, but its
greaLest e<.:u110111ic s.ig.rüHcauce is i11 trupic<1l Africa.
Transmission
Sprcad is by direct and indirect contact. Transmission oc-
curs t!uough tlying and r1onflying, biting and nonbit ing
arthropods. Tnjury by thorny range plants and shearing
cuts may creare portals of infection or inoculare tl1e agent.
Pathogenesis
The disease is an exudalive epidern1ilis. ILs pri1nary aclivily
is confined to the living epidermis. As access to this is li1n-
ited by hair, wool, sebaceous secretions, and the stratum
corneum, infection depends on their disruption by soaking
ortrauma. Dcpositcd zoosporcs, rcsponding to a C02 grad.i-
ent, "home" into deeper ceu layers. Upon germination,
germ tubes and filaments arborize \<Vithin the epidermis
and colonize hair follicles. A laver, of inflammatorv , cells
(largely neutrophils) forros under thf' infected epidermis,
'vhicl1 keralinizes (see Fig 69.1). Beneatl1 tl1e i1eutropl1ils
new epidermis forms, whicl1 in turn is invaded. The even-
tual result is a scab consisting of Jayers of neutrophilic exu-
f 1G U RE 3 7. 4. Dennatophilus congolensis in s111ed1 flu111
lesion ot "rain sca/d" in a horse. Gram stain, IOOOX.
221
date and infected keratini7.ing epiclermis. Thf' sr;ibs ;ire eas-
ily lifted by the hair, wl1ich protrudes from both surfaces.
The primary lesions are painless and nonpruritic.
Wetting favors their expansion. Biting arthropods can ln-
fect areas protected fron1 soaklng rai11 (aXilla, nank, ventral
trunk). Soaking provides the preparatory step for lesions
on tl1e back (equine "rain scald"), feet , and legs (ovine
"strawherry fontrot," eq11inf' "grf'a~e heel"). The extent
varíes from a few scabby areas of roughened hair or "lun1py
wool" to widespread loss of epidermis, causing secondary
parasitisms or infections and eventual loss of the a11imal.
·rhis progressive forn1 affects Afncan cattle. Neglected
cases of "lumpy ~<Vool" may lead to complete solidification
of the fleece.
Dermatophilosis in nonepioerm;:il tiss11e-tongue, the
urinary bladder, ly1nph nodes, and subcutis- is reported
in cats (rare).
Epidemiology
TJ1e prevaleu<.:e uf llerrnatophilosis depends on 1) infected
animals; 2) dissemination, for ex;:imple, by artl1ropods or
tho rny plants; and 3) susceptible hosts' epidern1i5 ren-
dered accessible by traurna or '""etting.
lmmunologic Aspects
Antihody is vvidespread among r;ittle in endemic areas. Its
protective role, relative to that of cell-mediated resistance,
is at present unscttled.
Attcmpts at artificial immunization have been incon-
clusive.
laboratory Diagnosis
Gram or Giemsa stains of ground-up scahs usually show
typical phases of the life cycle described (fig 37.4). In suba-
i:i
222 PART II Bacteria and Fungt

cute and chronic cases, bacteria! elements may be rare and
lack, for example, multicellular branching filaments.
Zoospores resemble large cocci. They and other Dermaco-
philus fragments in skin debris may be identified by fluo-
1esc1:ul <11.tlibvdy.
Dermatophilus congolensis is cultured on blood agar
undcr S'J.6 to 10')6 carbon dioxidc at 35-37ºC. Colonics ap-
pear within 4ts hours and when gram-stained are seen to
consist of branching filaments and zoospo.res. A wet
mount reveals motile zoospores.
Treatment and Control
Acute cases are often self-limited. Mild cases respond to
grooming and rcmovnl to sheltcr. Severe cases can be
t reated parenterally with penicillin G, tetracycline, chlo -
ramphenicol, or spiramycin.
Control should be allned at mlnlmlzing s.kin t rau ma
and exposure to rain and a rth ropods.
5TREPTOBACILLUS MON/l/FORMJS
Ecology
'fhe reservoir is the pharynx of rodents, particularly rats,
and possibly sn1all carnivores and swinc. Tnfection is by
bite or other contact with contaminated material, includ-
ing ingestion, as in milk-borne "Haverhill fever" among
humans.
Lesions are inflammatory and often purulent or
necrotic. Rats and m1ce develop bronchopneumonia and
guinea pigs cervical lymphadenitis. In turkeys and mice,
sepUce111ic itúeclioos lead to polyartl1ritis or synovitis and
often death.
ln humans, prostrating fevcr, accompnnied by a rash on
the extremities, precedes localization in joints, lymph
nodes, \ungs, or heart val ves. Mortality rates may approxi-
mate 10º1<:> in untreated cases.
lmmunologic Aspects
Little 1s known about immunity. Antibodies are produced
during infection.

The taxonomic position of Streptobacillus moniliformis, an

Laboratory Diagnosis
agent of rat-bite fever, is uncertain. In rodents, it causes Diagnosis requires demonstration of the agent. It can be
respiratory infections, and in rodents, turkeys, and hu-
visualized directly by immunofluorescent reagents and
u1<Uis, systt::uiic i11f1::ctiu11s tliat cu1111nu11ly lucalize iii
cultivated in thioglycolate broth and blood agar or other
joints.
rich media in a moist chamber. Identification is by mor-
phologic and biochemical traits. The agent produces a
un!que array of cell wall fatty adds that can be d etermined
Descriptive Features chromatographically. Production of L-form colonies is fa-
vored by special media (e.g., Rogosa's).
Streptobacillus monili(ormis is a nonrnotile, gram-negative,
¡.ilcu11iur¡.ilúc ruu that uftc1i gruws iii lung, lJeac.led filarnents
hearing knohhy irregularities. Tts cellular proteins varywith
gcographic a11d pathologic sources. ft requires blood or
Treatment and Control
serum-enriched media and incubatio11for 48 hours or more
The obvlous preventlve measure ls avotdance of direct and
at 35 37ºC to produce v isible gray, mucoid colonies.
ind.ir.ect c:ontact with likely carriers. Effective an timic:ro-
Dissociation into cell wall-deficient L-forms produces
bics include penicillin G and tetracycline.
xnycoplasma-like colonies that are recognizahle among the
cur1vc11tiu1Hil culuuit:!s. lt is a fC1cultC1tiV4:! a1iat:!rulJe, oxidase
and catalase-negative, and ferrnents glucose and sorne other
carbohydrates. Its resistancc is not remarkable.
 Mycobacterium
DWIGHT C. HIRSH
Members of the genus Mycobacteriurn are aerobic, acid-fast
rutls. 1'here are a µµrux ir 11alc::ly 100 1111::1111Jer:; oJ Lhi::; gen us,
and although most are saprophytic organisms that live iI1
the environment, sorne are strict parasites inhabiting the
mucous membranes ot thcir host. Diseascs produced by
various species of mycobacteria include tuberculosis, lep
rosy (in human paticnts), and granulomatous diseases in
prcviously norn1al or compromised mammals, birds, rep-
J le:., a11d fisl 1.
Descriptive Features
~orphology and Staining
:\fycobactcria are predominantly rod-shaped, about 0.5
µm wide. and variable in length. Spores, tlagella, and cap-
sules are absent.
Mycobactena, though cytochemically gram-positive,
often resist stain i ng with the Gram stain. ·rheir most noted
:.-ralnl11g µruµerty Is l l1t:l1 al'.it.l fa:.L11e:.:.-i.e., u11\.t :.lai.ueu,
·ht>y rt>sist <lisc:olorat ion with 3o/o hydrochloric acid in
eth anol. Mycobactcria can be stained with fluorescent
dyes (e.g.. auramine-rhodamine).
Structure and Composition
:\1ycobacteriaJ cells abounct ln lipids, especially in t 11eir
'"alis. Lipids accol1nt fnr acirl f::i~tnPss and sorne patho-
genic and in1111unologic properties.
Thc surface rnycosides (mostly glycolipids and peptido-
~!ycoli pids) determine colonial characteristics, serologic
speciticities, and bactcríophage susceptJbilities.
Subsurface laycrs of long-chain branched mycolic acids
and their esters make up the bulk uf cell w¡¡ll liµids. Acid
fastness somchow rlt>pt>n<I' 11pon tht>sP c:ell wall con-
stituents. Mycolic acids are Jinked to the innermost pepti-
~ '1glycan !ayer by way of arabinogalactans (see aiso
-._._11cbactcrh11n, Chapter 31 ¡ Nocardia, Chaptcr 37).
The presence of carotcno1d p1~ents in sorne mycobac-
-c:~ a ("chromogens" vs. "nonchromogens") and their
...!ght dependence ('~photochromogens" vs. "scotochro-
-;ogt>ns") i' a hasi' for r!a,~ifying nont11ht>rc11lo11s m y-
.;.obactcria.
ERNST L. BIBERSTEIN
Cellular Products of Medica! lnterest
Alkyl H ydroperoxídase Reductase. Ahp (for alkyl hydroperox-
idase rcctuctasc) is rcsponsiblc lor resistance to superoxides
and reactive nitrogen intermediates found within 1nacro-
phage phagolysosomes.
D i 1nycof)1f Trehalose (Cord Factor). Dim ycolyl trehalose,
aniong 0 Lhe1 effec L::., iuuuobilizes ueuLropl!ils, i!CLS as a11
adjuvant, evokes granulomatous responses, and causes mi-
tochondrial disruption lcadi.ng to disturbances i.n cellular
rcspiration. lts re!ation to thc "corct" pattern ot mycobac-
terial gro,vth is not proven.
!ron Acquisirlo11. See next secrton, "Mycobacrtns and
Exochelins."
Myc,ubac,tin:. u11tl E.xuc,/1c:li11:.. M ycobacti11s a11d exoche-
lins are cell \Vall aminPS involvt><I in iron acq11isition. F.xo-
chelins are proteins that remove ferric iron from ferritin.
Mycobactin is a complex lipid residing in the cell mem-
brane of thc microorganism and is rcsponsiblc fo ( transfer
of iron from iron-exochelin con1plexes to the bacterium.
S11lfolipids (or S11l(ntides) and fJhosphatid)'l Inositol Mnnno-
sidc (PIM). Sulfo!lpiLls anti phus¡.>l1atid yl iJ1usitul u1a11 11u-
side (a pho,pholipicl) ::ii<I in rreventing thc respiratory
burst and phagolysosomal fu sion, and intcrfere \-vith func-
tion of reactive oxygcn inter mediates following ingestion
by macrophagcs.
Sur(ace Mycosiclcs. Surface mycosides (mostly glycolipids
and peptidog!ycolipicls) are considered helpful in ensuring
l.Jacterial :.urvival willtill 1nacrophages.
Waxes. Waxcs of various kinds are contained within the
cell 'l'\•all, have adjuvant activity, and actívate macrophagcs
Jeading to granuloma formation .
lmmunity to Members of the Genus
Mycobacterium-General
Mycobacteria display a variety of surface proteins, carbo-
hydrates, and lipids on thcir surfaces. These substances
(mannose residues; lipoarabinomannan) or substances
that are bound to them (e.g., opsonins such as comple-
ment protelns) react wlth receptors on the surface of
macrophagec: (Pe , mannose receptors, complen1ent re-
ceptors, To!l-!ike receptors).
223
224 PART 11 Bacteria and Fu11gi
A numhP.r of fvfnt<; occ11r following binding of my-
cobacteria to the surface of nlacrophages; tl1e t"l-vo n1osl
important are 1) direct activation, and 2) phagocytosis
leading to ac.,'tivation. Dircct activation is thc rcsult of com-
ponents of the bacteria (n1ai11ly lipoarabir1omar1nan)
binding to macrophage cell surface receptors (e.g., the
CD14/Toll-like receptor complex). Following phagocyto-
si.s, Toll-like r.eceptors in the wall of the phagosome bind to
cell wall-associated lipoprotei11s, rcsullli1g h1 Lhe acliva-
tion of the macrophage.
Activatcd macrophages secrete a varicty of proinflan1-
matory cytokines, the most important being interleukin
12 (IL-12). IL-12 induces the production of interferon y
(lNf-gamma), granulocyte monocyte colony stimulating
factor (GM-CSf), and migration inhibition factor (MIF),
which aLL1acl and aclivale ruacrophages. Aclivaled 111acro-
phages acquire the capacity to kill mycobacteria. INF-
gamma np regulates macrophage expression of reactive
oxygen (peroxidases, superoxides) ar1d nitrogen (nitric
oxide) intermediates that are responsible for killing il1-
gested mycobacteria. In addition to the production of
IL-12, activatéd macropl1ages produce IL-8 (a chemokil1e
respu11sil.Jle íur altractiu11 u( PMNs), 111unucyte clteIJHJ-
attractant protei n (MC.P) re_<;ponsihle for attraction of
monocytcs/n1acropl1agcs, and mo11ocytc inhibitory pro-
tein (MIP), which "holds" n1onocytes/macropl1ages, at-
tractecl to the infectious focus (formation of a granuloma).
In addition to killing by reactive oxygen and nitrogen
intermediates, ingested n1ycobacteria are also a target for
actívate<.! 'l'T-1 1 cells, gamma-delta 1· cells, and CD8 T cells,
which kili hoth thf m;icroph;igf ;incl the n1ycobacteri.a
within (granulysin is thought to be the inaterial responsi-
ble). These T cells probably "recognize" stress proteins ex-
pressed by macrophages containing mycobactcria.
This cell-me<.1iated response is characteristic of the in-
fection, and is central to resolution of the infectious
µrucess.
THE AGENTS OF ANIMAL TUBERCULOSIS
Tuberculosis is a chronic graJ1ulomatous disease produced
by M. tuberculosis (the agent ot t he disease in prilnates), M.
bovis (in other mammals), and lvf. aviurn subspecies aviurn
(birds, hereafter referred to as M. avium).
Descriptive Features
úrowth Characteristics
Tubercle bacilli are strict aerobes that grow best on com-
plcx orga11ic medié! such as LowensteiI1-Jensen's, which
contains, among other ingredients, whole eggs and potato
flour. A dye, n1alacl1itc green, inl1ibits contan1inants. Olcic
acid-albumin medía, such as Middlebrook's 7Hl0 agar, are
al so used for isolation, often in (:on1bi nati.on ""ith
Lowenstein-Jensen's. Simple syntl1etic rne<.iid containir1g
ammoni1J1n salts, ;isparagine, citrate., glycerol, minerals
and vitamins are unsuitable for isolation but support
grovvth from large inocula.
The presence of glycerol favors growth of M. tuberculosis
(eugonic) and M. uviurn, l.Jul uul M. buvis (<1ysgu1lic).
Generation times of tubercle bacilli range fro1n 12
hours upward, and it may take weeks before colonies are
visible. A wetting agent, t'-veen 80, expedites grovvth in liq-
uid media, and transparent oleic acid-albumin agar media
permits early discernment of colonies. Mycobacteriurn
avium grows more rapidly than mammalian types.
The 1na1n111alia11 species grow al 33nc lo 39nc, wltile
avían and related mycobacteria (see below) grow at 25ºC to
45ºC, with the optimum being 11ear tl1e top of that range.
<.:olonial growth of mammali.an tubercle bacilli is dry
and crumbly. Avían forms grow in do1ne-sb.aped colonies.
Resistance
Tubercle bacilli survive exposure to 1N Na OH or HCl for 15
to 30 minutes, a circumstance utilized i11 decontaminating
diagnostic specilncns. Mycobacteria are resístant to many
antimicroblal dr ugs, bacteriostatic dyes, and disinfectants_
Pheuolic disi11fectanls are lile n1osl effeclive.
Tuhercle hacilli resist drying and survive for long peri-
ods in soil. They are ki.lled by sunlight, ultraviolet irradia-
tion, a11d pasteurization.
Variability
Genetically, the .n1a111n1alian tubercle bacilli are variants of
one species, M . tuberculosis: human, bovine, and murine.
The vaccine strain BCG (bacillc de Calmctte et Guérin) is a
1noditied M. bovis.
The avian tubercle bacilli constitute one species within
the M. intracellulare group of saprophytic mycobacteria_ Of
the approximately 20 numbered serotypes in the M.
aviut11-intracellulure con1plex, 1 lh1ough 3 are aviau Luber-
cle bacilli.
Ecology
Reservoir
'["hP. SOl JTCf Of tllhfrC]f h;ici Jli i<; t11hfrC11 ]011<; Í no iVic111;:¡ ]$ .
Ilumal1S perpetuate M. tuberculosis, cattle M. bovis, and
chickens M. aviurn. The latter two can infect wild 1na1n-
mals and other species of birds, respectively, which occa-
sionally become sources of infection for domestic animals.
Transmission
Tubercle bacilli are transn1itted via the respiratory anda.Ji
mentary routes through contaminated atrborne droplet
nuclei, feces, urine, genital discharges, milk from infected
rn<urunary gla11u.s, ur cu11tar11i11ate<1 feeu a11<1 water.
PP.rc11t;inP.011s, tr;insplacfnta l, ;inct transova ria n (hi rds) in-
fections are unusual. lntrauteriI1e infection oí calves oc-
curred when bovine t uberculosis was comn1on.
Pathogenesis
Mechanisrns and Pathology. The lnfcctious process begins
witl1 deposition of tubercle bacilli in the lung or on pha-

;yngeal or int estinal mucous membranes. In p reviou sly
u11exposcu a11 i ruals, local inulliplicalio11 occurs wilhiI1.
Resistance to phagocytic killing (cell wall chemistry and
shunting to endosomal compartme11ts rather than those
m at fuse witb lysosomes) allows continued intracellular
and extracellular multiplication. An infla1n1natory re-
sponse (stemming from activation of macrophages, as well
as sternming from the irritating nature of the mycobacter-
!al <.:ell wall) i11volvi11g Jargely n1onocyles (wilh son1e
PMNs) develops around the infectious focus. Infected host
cells and bacteria reach draining lymph nodcs, whcrc pro-
liferation and intla1n1natory responses continue.
After tl1e first week, cell-mediated in11nu ne reactions
b egin to modify the host response from essentially a for-
eign body reaction (resulting from the irritating nature of
i he inycobaclerial cell wall) Lo a reaclio11 characlerislic of
!nfectious graoulomas. Activated macrophages acquire
thc capacity to kili mycobactcria. Thcir cfficicncy in doing
so depends on the adequacy of the immune response and
t he virulence of bacteria.
Epit helioid cells appear among the macrophages. They
h <ive oblong vesicul<ir nuclei <ind pale, poorly deline<ited
cytoplasn1; they often becon1e the predon1inant cells. A
~hird, less co1nmon cell type is the Langhans giant cell,
~v-hich is probably a ccllular fusion procluct. It has a largc
amount of pale cytoplasrn and, peripherally, many vesicu-
_ar nuclei (Fig 38.1). Both cells are probably macrophage
derivatives. ·rhey do not seem to be effective phagocytes.
Th.ei r bacteria] content presu mably carne from their
u nu.:ropJ ta!'jt: precur~ors.
At the. cc~nte.r of the. Je.sion, caseation necrosis develops.
_A. fu11ction of the hypersensitive state, it may proceed to
calcification or liquefaction.
J\t the periphery of the lesion are unaltered macro-
phages mixed with lymphocytes. ribrocytes appear, and a
fibrous layer eventually invests the lesi.on, which is called a
rubercle (Fig 38.2).
1'ubercles may enlarge, coalesce, 8nd evenh1;i ny or.c11py
sizable po.rtions of organs. Such tubei:cles consist mostly of
caseous material.
F1G U RE 3 8. 1 . Li:inghans giant ce// with tubcrclc bacilli
(arrow) among epithetíoíd ce/Is in a bovine tuberculous tymph
node. Zieh/-Nee/sen stain, 7OOOX.
Chaj1t1~r 3R Myr.nhr.u:t1~ri11.m 225
The process clescribed is typical of human and rumi-
11a11l tuberc ulosis caused by n1<n11n1¿1lia11 tubercle bacilli. Tt
is chro11ic; the lesion is called productivc or proliferativc.
Occasionally an acutc cxudativc proccss takcs place,
marked by predominantly neutrophilic responses and
fluid effusioo. Tt is thought to be favored by such factors as
a large inJecting dose, focally deUvered; high virulence of
the infecting strain; constitutional predisposition of the
l1osl; a loose lissue arcl1ileclu1e as in Lhe l ung, serous
membranes, or meninges; and a higb. degree of tubercu-
lou s hypcrscnsitivity. Onc such acutc proccss is tuberculous
pneurnonia, which may cause extensive necrosis (Jargely
due to an overly exuberant imn1une/inflammatory r.e-
sponse) and be rapidly fatal ("galloping consumption"),
resolve almost completely, or subside into the chronic
pallern.
Ccll-mediated responses int1ue11ce the course of the dis-
casc in scvcral ways. Primary i.nfcction is disseminatcd via
lymphatics to lymph nodes and beyond these tbrough the
bloodstream, seeding many reticuloendotheli.al tissues.
Cell-mediated immunity and macrophage activation
elirnin<ite these foci, except '.vhere they have developed
furtl1esl-tha l is, Ll1e poinl of prin1a1y exposure and Lile
adjacent lymph node. Here primary lesions (Ghon or
Ghon-Rankc complcxcs) pcrsist. Som.ctimcs, cspccially
w ith alimentary tract exposure, they are "incomplete"-
that is, or1ly tl1e lymph node lesion is discernible.
Once cell-mediated reactivity is established, sub-
sequent reinfection follows a different course: antigen-
specific 'f' ly n1phocyLes and acliva led n1acrophages
prornptly converge on the site, contain tl1e infection, and
prevent lymphatic spread.
A.ntigen-speciíic ·r lympl1ocyte responses, ho,>Vever, also
n1ediatc cytotoxic reactions and cause extensive tissue de-
struction, which is characreristic of progressive tuberculo-
sis. While the lymphatic disse1nination is lünited by the
irrnnune response, tiss ue Llaruage facilitates !Jacterial
sprea<i hy c.ontiguous exteosion, or erosion of hronchi,
blood vessels, or víscera, introducing infection to new
areas. Wherever microorganisms lodge, this reaction will
226 PART ll Bacteria and fungi
F 1G U R E 3 8. 2. Sector of tubercle showing a centrul ure<J of culcifícution (1 ),
surrounded Oy casearlon necrosis (2). Adjolnlng Is a zone of eplrhe/lold (3) and gldnl lt!//)
(arrow). The outermost zone (4) contains other mononuclear ce/Is, particular/y lymphocytes,
among which tracts of fibrous tissue are discernible. Hematoxylin-eosin stain. 1OOX.
•.
·~
.
i~~ ..
~~--
be rcpcated with c11n111l;itivf> rnns1,q11Pnrf>s. He1natogen-
ous dissen1ination may produce 111iliary tuberculosis: multi-
focal tubercle formation throughout an organ.
"Reinfection tuberculosis" is most often endogenous;
that is, it results from reactivation of previously dormant
foci.
Disease Patterns
Clinical tuberculosis is typically a debilitating illness
charactcrizcd by progrcssivc emuciation, erratic appetite,
irregular low-grade fever, and occasionally by localizing
signs such as enlarged lymph nodes, cough, and diarrhea.
Many uf these signs are tl1e cu11:.1:ljue11c..:e o f released
cytoki nP~.
Cattle. Cattle are usually infected with i\ 1. bovis, and the in-
fcction is centercd on the rcspiratory tract and adjacent
tymph nodes a11d serous cav1t1es. The disease is commonly
progressive via air spaces and passages. Hematogenou s dis-
scmlnatlon involving liver and kiuucy uccur:.. The ute1us
may ~PrvP ;is port;il for fpt;il infection, a pattern v irtually
unknown in other domcstic animal:>. Surviving calves
com 1no nly develop liver and spleco lesions. Udder intec-
tion is rarc ( <2CJ.6 of cases) but has obvious public health
implications.
Infection with 1\1.. aviun1 is generally subclinical. Abor-
tions rcsultlng apparently frorn localimliuu uf 1\1. uviurn il1
thc uterine w<ill orr11r, in ~om<' instances repeatedly.
Mycobactcriu111 tr.wen:11losis causes minor, nonprogres-
sivc Iesions in cattle.
.~
:·. ..:~
.:,.• .
.".
... ~. 1~. ~ , • •
...:_ ..;
. . _.
. .. . ..;. • •
Sheep and Goats. ShPf>P ancl goats a re susceptible to 1\1. bovis
and perhaps slightly less so to M. aviun1, but they are resist-
ant to progressive infection by i\1. tuberculosis. Visease pat-
terns resemble those described in cattle.
Horses. Horses are rarely infected, but relatively more
often with M . avium thau wilh Jvl. fJuvi~. fnfeclion enters
usu;illy hy thf> alimf>ntary tract, \vith primary complexes
related to pharynx and intestine. Secondary lesions may
be in lung, liver, splecn, and serous membranes. Lesions in
ccrvicul verteb.rae may he due to a secondary, nonspecific
hypertrophic periostitis.
Cross lesions are tumor-like. They lack caseation anci
gro:.s calcifici:ltiou i:111u cu11li:1i11 few lyn1pl1ocytes. There is
fihrohlast proliferation but usually no firn1 encapsulation.
Swine. 5wine can be infected by tubercle bacilli, usually
via the alimentary route, but only M. bovis causes progres-
stve disease with classical lcstons. Mycubacteriurn lubi:n.ulu-
sis infections do not adv;inrf> ril~t rf>gional lymph nodes.
1'vlycobacteriur11 aviu111 infection, thc predominant form in
1nany countries, may disseminate to viscera, bone, and
meninges. The lesions lack the organization of tubercles,
b ut contain granulomatous elements. c:aseation, calciflca-
tion, or liquefaction is ncgligible. Bacteria may b e abun-
dant. Epidemiologlcal studles as weU as gc11ctic a11alysis of
M . avium isolatcs from ~\IVinP have shown that "bird A1.
aviu111" is different from "swinc M. aviu1n" and "human 't.1.
aviu1n." For this reason, it has been proposed that swine
hun1an strains be designatcd as M. aviurn subspecies ho-
1niniss11is.

Dogs and Cats. Dogs and cats are readily infected witl1 M.
buvis but rarely witl1 JvL uviurr1. Dugs are al su suscevLible Lo
M. tuherculnsis. Intestinal and ahdominal localization of
infection is more con1mon in cats than in dogs, reflecting
a likely alimentary route of exposure.
Hypcrtrophic pulmonary ostcoarthropathy (Maric's
disease, acropachia), a nonspecific periostitis most notice-
ably affecting the long bones, sometimes affects tubercu-
lous tlugs (autl hurses). Tlte eliulugy seeu1s relaLetl Lo pul-
monary i11capacitation rather than to specific agent s.
Ulcerative skin lesions are more common in cats than
in other hosts, as is eye involvement, with tuberculous
choroiditis leading to blindness.
Lesions, especially in dogs, often more closely resemble
a foreih1l1 -body reacti.on than tubercles, since they may
lack Lypical epiLhelioid and gianL cells, and nei Lhe1 case-
ate, calcify, nor liquefy. The course is usually progressive.
Primates. Primates are susceptible to the t'"'º mammalian
tubercle bacilli but resistant to M. avium. unless severely
compromised, as by human immu nodeficiency virus
(HIV) infection or p reexisting bronchopulmonary disease.
Sucl1 ü1dividuals also becon1e infected witl1 nonlubercu-
lous mycobacteria.
In humans, infcction is usually contractcd by inhala-
tion of droplet nuclei originating from "open" human
cases (t hat is, respiratory shedders). When tuberculosis
was widespread, most cases did not develop beyon<.1 the
primary complex in the respiratory tract. A minority be-
<.:arne progre:ssi ve <.:lini<.:al t ul.>e rculosis, reseu 1!Jli11g tl 1e
hovi ne. ciise.ase descrihed a hove.
Infection with M. hovis typically occurred follo,-ving in-
gestion of unpasteurized .m ilk .from a tuberculous cow.
Prirnary invasion, through pharynx or intcstinc, rcsulted
in lymphadenitis ot adjacent nodes. Hematogenous d is-
se1nination to vertebrae could result in a hunchback, a
condition now rare thanks to pasteurlzatioo of m!lk and
eradication of bovine tuberculosis in in dustrial countries.
Mycobacteriutn tuberculosis and M . bovis cause progressive
disease in captive non human p rimates, which are resistant
to M. aviurn, although intestinal infcctions rcscmbling
Johne's disease of ruminants (see belo,.\T) are associated
with members of the M. avium-intracellulare complex.
Imrnunosuppressed hurnans, especially tl1ose with ac-
q uired immunodeficiency synd rome (AIDS) , appear sus-
ceptible to a variety of n,ontuberculous n1ycobacteria,
for example, J\1. gen.avense, mernbers of tl1e M. avium-
intraccllularc complex (scc abovc rcgarding M . aviurn from
swine and humans, and the designation M. avi urn ssp. ho-
m inissuis), M. si1niae, }.{. marinum, and M . haernophilum.
Birds. Rirds are naturally susceptible primarily to M.
aviuni. Most poultry mfections occur via t he alimentary
canal and dissen1inate to liver and spleen. Bone rnarrow,
lung, and pcritoncum are oftcn affected. Although thc
agent 11as been isolated from eggs, transovarian infection
' genavense affects canaries
o f chicks is rare . }.lf}1cohacterium
ancl parroLs.
·rubercle formation stops short of calcification.
Although M. aviznn affects many species of birds,
psittacines are resistant hut are susceptible to M. tuberculo-
Chapter 38 Jvlycobacterium 227
sis. Ca11ari.es also are more suscepti.b le to mammalian t han
to aviar1 bacilli.
Epidemiology
With eradication of cattle tu berculosis in industrial coun
tries, the traditional reservoir in domestic mammals has
disappeared . Garne farn1s, animal park s, and zoos remain
foci of M . bovis in technically adva11ced cou11tries. Spo·
radie cases of canine tuberculosis often prove to be M.
tuberculosis infcctions traccablc to human contacts-
"reverse zoo11oses"- that are also fotind in 11onl1uman pri-
mates in laboratory colonies and zoos.
ln commercial pou ltry establishments, rapid popula-
tion turnover ( <1 year) eliminating transgenerational
tTansn1ission has eradicated M. aviu1n. It ren1ai11s a prob-
lem. in barnyard flocks, however, particularly since t he
agcnt can survivc in soil for severa! ycars.
Tuberculosis is typically a disease of captivity anct cto-
mestication. When wild a11d captive infected populations
are compared, cllnical improvement and lack of spread in
free-living animals contrasted with deterioration and high
con1n1unicability in confined gi:oups. Tuberculosis 111 wild
populations seems to be a relative rarity. Nevertheless, M .
bovis, probably origir1ating from cattle, is endcmic in badg-
ers of southern Englan d and in brush-tailed opossums of
Nevv Zealand, both of '"'hich are considered sources of in-
fection for livestock.
Tmmature inclivicluals often clevelop inore severe leslons
than older ones. Breed susceptibilities differ: zebu cattle are
more resistant than European breeds, and fox terriers and
lrish settcrs are 111orc often infectcd than dachshunds and
Uoberman l'inscher dogs. !'he higher p revalence in <.tairy
than beef cattle may reflect closer confinement, longer life
spans, and greater productivity stress among dairy cows.
Exemption from pregnancy and lactation may explain t !1e
lovve1 disease p revalence in bulls Lhan crn'\Ts, allhough in
dogs the reverse sex ratio is observecl.
lmmunologic Aspects
ln1111une Mechanisn1s of Disease
'l'he key role of cell-mediatec! immune responses in the
pathogenesis of tuberculosis has been discussed (see above,
"Irnrnunity to Men1bers of the Genus Mycobacteritun-
c:re.ni>r;:il). In thPir ;:ibsPníP t hP cliSP.::lSP m;:iy progress ;is a clis-
seminating inflammatory d isease (as it does in athyn1ic
mice) vvithout the development of typical lesions.
Recovery and Resistance
Acquired resistance depends on cell-mediated responses.
Undcr natural conditions this develops along '"'1th hyper-
sensitivity, both ot ,.vh.ic.h are demo11strated in the Koch
phenomenon: an already tuberculous guinea pig suffers a
rapid, destructive, but limitecl reaction at the site of reex-
pos11ri> to t11berc·le b;1cilli, \Nhile a virgin animal develops
persistent, p rogressive, disse111inating, and evenlually fat al
disease when injected at the same anatomical site.
228 PART II Bacteria and Fungí
lromune-mediated hypersensitive reactivity and protec-
tivc responses are separable: immunity can persist in ex-
perimentally desensitized an1mals and can be absent in
sensitized subjects.
Anlibudy lo tulJercle bacílll does not protect agalnst
natural infection.
Artificial lmmunization
Vaccination of huma11s 'l>vith BCG produces te111poraiy i111-
munity and hypersensitivity. The benefits of vaccination
havc been greatest wherc exposurc was most intense and
negligiblc where prevalence was low. vac<.ination in hu-
mans is focused on infants and tuberculin negative indi-
viduals antlcipating exposure.
BCG has been used in calves. 'fhis practice is inappro-
pda lc in counlries alle111¡;Lü1g tu eratlicate t uberculosis
becausc it interferes with the intcrrrctation of t hf> t11bPr-
c u li n test.
Myco/Jacterrurn rn1croti, the vole bacillus, stimulates im-
munity to bovine a11d l1un1an tuberculosis. Tts virulence is
too variable to pcrmit lts use as a vaccine.
Among subcellular experimental immunogens, a ribo-
sou1al pr1:¡;araliur1 is of ir1terest because lt produces protec-
tion wit)Jout tuherculin hypPr<:Pn<:itivity
laboratory Diagnosis
Sample Collection
Samplcs in clude tracheobronchial and gastric lavages;
lymph node, thoracic, abdominal. and other aspirates;
and urine, feces, and biopsy specimens. At necropsy, mate-
rial Is obtal11ed from Jesions.
Direct Examination
Flt1ids are concentrated by centrifugation in tightl y
cappcd conlainers. Sai11vlc:. ü1tentletl for microscopy only
are digcstcd and disinfecteci with hyporhlor.ite (bleach,
Clorox). S1ncars of sediment or l'issue are stained with a11
acid-fast stain, or with auramine-rhodamine where fluo -
rcscence microscopy is available. Histologic scctions are
:.ta1111.·u w1t11 11emaroxyun-cos1n ano aciO-fast stams.
PositivP rPs11lts should be culturally confirmed .
Culture and ldentification
Oi~1::.liu11a11tl :.clectivt> tlt><.:ontamlnatlon are advisable es-
pccially with specimens likPly to cont:iin a mixtuie of mi-
croorganisms.
ldentitica tion is based upon growth characteristics (fast
vers us slow), pigmentation (in thc prescncc or abscncc of
light), and biochemical rcact1ons. 1hese maneuvers are
bcst left to a reference laboratory.
DNA. µrobes, ~pecific for thc maln gro ups, are comn1er-
cially avai lahle. Amplific;:ition of myco bacteria-specific
fragments of DNA by use of the polymerase chai n reaclio11
is an important \.Yay to demonstrate or identify members
of this genus.
Animal inoculation s are somctunes used.
lmmunodiagnosis
Tuberculin Test. Ccll-mcdiatcd hypersensitivity, acquire<l
through infection, can be ctemonstrated systemically b~
fever, ophthalmjcally by con junctivitis, or dermally by
lol:al swelling, when tuberculln or its purifi ed protein de-
rivativP (PPD) is give11 by the subcutaneous, conjunctival.
o r intradermal route, respcc lively. 'fube1culi11;:, are IJacter-
ial peptides liberated into culture media during growth. To
sorne of them, orto their parent protcins, dclaycd-typc hy-
persensitivity develops during 1nfect1on.
In cattle, tuberculin, the equivalent of a 0 .2 to 0.3
11¡g/dose of 1Juvi11e PPD, i::. il1je<.:ted inttadermally 1n the
ca11cial, v11lv;:ir, or anal skin or, in sorne sitt.tations, the n eck
region. In positive cases, a swelling (>5 n1n1) develuj.1::>
within 72 hours. Wbile tuberculin cannot induce the hy-
perscnsitivc statc, it may dcscn sitizc animals for weeks or
months.
A positive test implies past or present infection, requlr
111~ Lllt: reacli11g a1111ual tu IJe slaugt1tere<.I and necropstc<1.
Where tuberculosis is rarc, no lcsions a re often fonnrl in re-
actors (NVL, forno visible /csion reactors). Such apparentl y
false-positive reactions are explained by hypersensitivity
to nontubcrculous, related agents such as other mycobac-
teria, or nocardiae. Simultaneous use of avian tuberculin,
which detects hypersensitivity to severa! nontuberculous
mycobacteria, often helps to decide, by comparative s1ze
assessment of the two reactions, whether sensitivity is due
prin1a1ily Lo 1ua1111ualiau or a t1eterologous tuberculln.
Other explanations for NV 1. ;:irP p;:irly states of infection, re-
mote location of lesions, or microscopic sizes of lesions.
False-nega tives occur in an imals too recently infected
and in advanced cases in which ancrgy dcvclops due to
antigen excess or 1mmunosuppression. Nonspecilic tac-
tors, such as malnutrition, stress, and impending or recent
parluriliu11, are alternative causes of anergy.
Rules goveming thf> 11sP nf t11berculin tests in eractica-
tion programs vary from country to counlry.
Tuberculins of appropriate specificity are used on swine
and poultry. In s'-vinc thc cars are injcctcd, in poultry the
wattles (Fig 38.3) . TI1e reliability of tuberculin tests on
horses, sheep, goats, dogs, and cats is not established.
SPrnlngy. .'\Prologic tests hllve not been useful in diagnosis
of mammalian tuberculosis. As a Iirsl slep in poull1 y Lu1J1:r-
culosis eradication, a whole-blood agglutination test is
availablc. It is scn sitivc but lacks specificity.
Treatment and Control
first- line drugs for tuberculosis thPrapy arP strPptomycin,
isoniazid (INrI), et hambutol, and rifampin. Second-line
drugs are pyrazinamidc, para-aminosalicylic acid, kana-
mycin, cycloserine, capreomycin, and cthionamidc. Bc-
cause resistance often develops undcr a single-drug regi-
men, a combination is commonly used; a favored onc in
hu111d11 111etlici11e IJ1;:i11g !NH-tthambutol-rifampin. Tteat-
men t is 9 months with rifamrin incl11ciPcl, 18 to 24
months without it.
Because of the public health hazards inherent in the

F1G U RE 3 8. 3. Positive tuberculin reaction in the left watt/e
of a ch1cken expenmen tafly 1nfected three weeks earl1er w1th
Mycnh;irtpri11m avi11m
retention of tuberculous animals, antituberculous chemo-
rherapy of animals is discouragcd. Prophylactic trcatrncnt
·,\;th INH may be considered for pets recently exposed to
tuberculosis. Sorne experimental successes with fNH for
prophylaxls and treatment of calves have !)een reported. Tn
countries with eradication programs, treatment is gener-
ally di$couragcd or illegcil.
Bovinc tuberculosis is controllcd by idcntification and
elimination of infected animal!;. This app ro¡1ch h as rcsultcd
:n near-eraclication of t he infection in rriany countries.
Contint1cd surveillance is required to prevent resu rgence.
111 µou ltry, tuberculosis in backyard flocks is perpetu-
ated hy rC'tC'ntion of hi ros ano pC'r<;iStC'nC'P of <;Oi f C'Ont;im i-
:lation.
Eradication of bovine, hun1an, and avian tuberculosis
·,·¡u diminish infection hazards for othcr spccics.
TH E AGENT oF JOHNE' S DISEASE
.'11YCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS
!vcobacteríi1111 aviurn contains three subspecies, avium,
.-.lfatuberculosis, and silvaticurn. A fourth suhspecies, ho-
"'li11iss11is has bee::11 :-uggeste;:u tu c11co1npass ~trains that af-
cct swine and humans. Tn gcnC'r:il tf'rm<;, <;nbspecies
'\.1ratuberculosis is differentiatcd from the other nvo by
-eing mycobactin-dependcnt and possessing the DNA in-
Chapter 38 Mycobacteríum 229
sertion sequence IS900 (though neither trait is absolute).
My1..ul;u1..·teriu1n uvium su!Jspe<.:ies paratuberculosis, h ereafter
referrecl to as Mycnbacteriu1n parafuherr.ulnsis, is the causa-
tive agcnt of a cl1ronlc, i.J:reve1slble wasLing disease uf ru-
1n inants called Johne's disease.
Descripti ve Features
Growth Characteristics
Llke the rest of the genus Mycol>actenum, M . paratubercu-
losis is an obligate aerobe. Tn thc past, the growth de-
pendency of M . paratuberculosis for mycobactin (an
iron-hinciing lipi<l, Sf'f' ;ihovr., "Cellular Produ ct s of
Medica! Tntcrcst") h as been used to differenliate i l fron1
other mycobacteria. Tl1is trait is shared, howevcr, with
othcr strains o f M. aviunz. Thc 1ncdium of choice is
Hcrrold's egg-yolk m ed ium . Most stralns are stimulated
by pyruvate.
Gruwtl1 is sluw. Development of visible colonies re-
quires 8 to 12 '"f>Pks of inc11h:ition at 37ºC.
Mycobactcriun1 paratuberculosis survives n1any n1011ll1s
in soil or in organic matter '"'hen protected frorn dircct
sunlight and drying.
Ecology
Reservoir
1'he reservoir for M . paratuberculosis is the intesti nal tract of
i11feclt::d a11i111al::.1 i.Jullt l11e;: ¡;li11ically affe<.:tetl and, rnore
importantly. those infected hut asymptomatic.. Tn an af-
fcctcd hcrd, infcctcd, asymptomatic fecal shcdders may be
:lU times more numerous than thosc showing clinical
signs. An infected animal may also shcd M . paratubcrculosis
into colostrum and m1lk, and may pass the organism to
her fetus in utero. M)lcobact<?riurn parat11hcrc11losis has bccn
lsolated f10111 sen1en, seu üuife::rou:. LulJule;::;, a11 u tlt<:: vro:;-
tatc glands of infected bulls. -
Transmission
Thc infcction is usually acquired through the ingestion or
contact '~ith fccally contaminatcd matcrials (food,
fom1tes). In utero iofection and ingestion of contaminated
colostrum or milk are also possible routes.
Pathogenesis
The pat hogenic m ech anisms appear to involve cell-
n1ediaLed in11 11u1u:: µl1euuu1<::ua. ·r11e granulomatous lc-
sions associated with cli n ical o isri'lsP ri rf' rf'l:!tf'd to cf'l l-
mcdiatcd immunity. 'fhe organism is found w ithin macro-
phages in the submucosa of the ileocecal arca and tl1e adja-
cent lymph nodcs (ileocecal) following ingcstlon. Extrain-
testlnal colonization suggests the ex1stence of a blood
phase, but this probably occurs after establishment of a pri-
n1a1 y fucu:. of llúectiun in the lntest1ne. Whether this oc-
curs soon after infertion or J;itPr ¡., not known. Initially the
230 PART 11 Bacteria and Fungi

animal shows no signs of disease. The inr.ubation petiod be- bloocl monocyt<-'s h;1vP bf>f>n c!Pmonstr<itf>cl ;incl may hP. the
fare overt clinical discase is 12 n1onths or longer. n1anner in which sites outside of tl1e intestinal tract be-
After ingestion, M. paratuberculosis enters M cells over come infected.
lymphoid nodulcs of t hc ileocecal arca. Shortly thcrcaftcr,
macropl1ages \.Vithin thc nodule become infected, v.,ith M. Disease Patterns
paratuberculosis seen within phagosomes and phagolyso-
somes. Ingested n1icroorganisn1s limit production of su- Cattle. Anl1nals sl1owi11g clinical sigr1s present witl 1
peroxides and d iscourage fusion of lysosomes with phago- r.hronic vveight loss, c!i;irr hf>i-l, hut normal appetite and
son1es. Those will1in phagolysoson1es are relatively ten1perature. At this stage of the discase, the submucosa of
resistant to destruction (probably related to the chen1istry the intestinal tract, extendit1g both cranially as well as cau-
of thcir ccll wall), as wcll as thc production of pcroxidascs dally from tl1e ileocecal area is infiltrated with macro
such as Aph (see above, "Cellular l'roducts of Medical phagcs, epithelioid cells, and sorne giant cells that contain
Interest''). lntracellular 1\1. paratuberculosis acquire iron for large numbers of l\lf. paratuberculosis. 'fhe draining lyrn.ph
growth \.vith the excretion of exochelins, •vhicb remove nod.e::; are aln1ost- dl~vays er1large<.1 ar1<.l fille<.l witli 111acro-
iron from macrophage ferritin. Iron is transported c:om - ph<iges cont;iining mycohac:tP.ria . 1'he classical g ross lesio n
plexed w ill1 exochelin across ll1e n1ycobacterial cell •vall to in cattle is a permane11t transversc corrugation of t he in-
the cytoplasmic mernbrane where mycobactin transports testinal mucosa caused by granulo1natous inflammation
the. iron into tl1c cclL Thcrc is cvidcncc that iron availabil- within thc lamina propria and submucosa (Fig 38 .4). ·rhe
ity dictates the d istribution of 1\1/. paratuberculosis, with extent of the 1ofiltration is not always related to the sever-
most iron available within macrophages of nodules ill the i ty of the el inical disease. 'fhe sympton1atic disease usuall)-
ileocecal area. progresses to a fatal outcorue.
Reli:;ase of cytokines by activ ated macropl1ages initiates
lite i11fla1n111alo1 y and in11nu11e responses. The rcspo.n scs Sheep and Goats. Diarrhea is nota co111mon c linical sign of
are initiated hy t he recruitment of specific TH 1 lympho- tl1e clisease in these species. Herd unthriftiness, however,
cytes and subsequent release of cytokines leadiI1g to the at- should prompt cxumination for M. paratubcrculosís. I ntes-
traction of rnacrophages to the site ot interaction oí 10.. tinal lesions in sheep and goats are less obv1ous than in
paratuberculosis and macrophage. Nor111ally1 gan1n1a inter- cattle. Weight loss has been a consistent finding with af-
feron (one of the released cytoklnes) would activare fected goats.
macrophages to deal effectively witl1 ingested mycobacte-
rid. A<.:ti v¡1tio11 is LOUlIJfOUlÍS<.!U, ltowever, l.iy galJHl li:I uel ta T
Epidemiology
lymphocytes that are cytotoxic for the specifíe T fil lympho-
cytcs. 'fhi:; cytotoxicity i:> rcgulatcd by C DS T lyn1phocytc:>. 'fhc young animal is thc 1nost :iusccptiblc to infcction. Oldcr
ln addition, gamma interferon released by· l' lymphocytes is animals can be infected, but only v•ith very large inocula.
a poor activator of lYf.. paratuberculosis-infected macro- Many ruminant species are subject to infection. Clin-
pl1ages. Tbe interaction between these cell types results in ical disease occurs mostlv, u11der conditions of don1estica-
t h e formation of a slO\>Vly progressing granulon1atous reac-
lion as evidenced by Lile accurnulalion oí 111acroµbages io
the subn1ucosat region. Sorne n1acrophages die subsequent f 1G U RE 3 8 . 4 . Bovíne íntestine affected by Johne 's disease
to the m ultiplication of mycobacteria within them, result- show1ng permanent corrugat1on. (Photograph courtesy of Dr. Murray
ing in the release ot microorganisms and phagocytosis by Fow/P.r.)
other 1nacrophages. The granulon1atous response and cel
lular i1úiltrate lead ultimately to sloughing of tl1e n1ucosal
epit helium and release of infected macrophages and n1y-
cobacleria in lo lhe 1Lu11en. Son1e have "slaged" Lhe progres-
sion of this disease in ternlinology that is used in describ-
ing lcprosy (scc bclow, "Fclinc Lcprosy"): the carly,
Jo calized granulomatous lesion being tern1ed the "tubercu-
lo id " stage, and the later-occurring diffuse infiltrative
process being termed tlle "lepro1natous" stage.
The d isease in sorne affected animals progresses to mal-
absorplion, prolein-losing e11leropalhy, a11d overL cli11icaJ
disease. However, only 3o/o to Sº;ó perce11t of the anirnals in
an infected herd progress to clin.ical discasc. Othcr cco-
nomic losses are traced to breeding problems (e.g., longer
calving intervals), to reduccd 1nilk production due to an
in creased incidence of inasritis (not caused by l\1. paratu-
berculosís), and to general ill thrift.
ExlrainlesLi11al localizalion (e.g., n1an1 n1acy g!and,
fetus, sex organs of males) suggests a blood phase. There is
a direct correlation betwcc11 tl1c dcgrcc of disscmination
and the numbers ot 1\1.. paratubercutosis in feces. lnfected

tion or captivity, especially in association ~vith stress fac-
tors such as parturition, crowding, shipment, or faulty di-
Ptary regimes.
Genetics n1ay play a part, as c.ertain c.allle breeds
(Guernsey, Jersey, Shorthor11) seem to be preferentially
affcctcd.
High prevalence ot inapparent shedders, chro11icity of
infection, and persistence in the environn1ent favor dís-
seminatio11 of the agent. The overall prevalence in U.S.
bovines is approximately 1%to3% (tl1e herd prevalence in
W esle1n Eu1ope <tlld Nu1LI1 A1111::1 h .. a l1a:. 1Ji::t::11 e:.Lü1111 Led lu
b e as high as 50o/o). The i1uportance of transmission
through milk and placenta is unccrtain.
Bacteria resembling M. paratuberculosis have been iso-
!ated fro1n humans with Crohn's disease (regional ileitis),
a tlisease remarkably similar to Johne's disease.
tmmunologic Aspects
The lesions and host responses are ma11ifestations of cellu-
lar immunity. 'fhe cell-mediated immune response proba-
bly atcuunts fur the luw percentage of anlmals that
progress to overt disp;ise. OthPr problPms, such as de-
creased milk production and increased calving intervals,
m ay be indirectly due to the generalized immunosuppres-
sion observed in infected anirnals.
Artificial in1munization is practiced in sorne areas of tl1e
-\·orld, and this practice seems to reduce the losses that
occur dueto this disease, but it does not e1iminate the prob-
Pm. In the TJnited States, vaccination is not practiced be-
::ause of its real or perceived ü1terfe1c11ce wilh diagnosUc
:ests (see below), since vaccil1ated anünals will test positive
:Or this discase and occasionally for tuberculosis as well.
F1G U RE 3 8. 5. lmpression of mesenteric lymph node from a qoat with Johne's disease,
showing masses of minute intra- and extrace//ular acid-fast bacteria. Ziehl-Neelsen stain, about
2000X.
•
.,
-·
Chapter 38 A1ycobacterium 231
·rhough cell mediated imn1une responses are generatcd
followi11g infection, antibody responses are also observed
mainly during the "leprornatous" stage ofthe disease.
Laboratory Diagnosis
Sample Collection
From cattle, samples from the ileocecal area (intestine or
ly mph nodcs) ore bc:;t, but mucosa! scrnpings from thc rcc-
tum in the live anirnals are easier to obtain. Biopsies ot
ileocecal lymph nodes are performed on valuable cattle. In
sheep and goats, exarninatio.n of the ileocecal Iy1nph
noc!Ps is thP most rP\<V'1rding. Sam p les of intestinal content
or scrapings are least useful in these spec.ies.
Direct Examination
Impression smears of lymph nodcs, or srncars of rectal or
intestinal scrapings, are stained by the Ziehl-Neeisen pro-
cedure. lvf.y cobacteriun1 paratuberculosis will stain acid-fast.
Tltc::y art: ~lturt, slender rocls, occurrlng in bunches (Fig
38.S). Other acid-fast st;iining stn1ctures in sélmples (sapro-
phy tic mycobacteria or bacterial endospores) will be soli-
tary and quite large. Sect ions stained with hematoxylin-
eosi11 reveal the typical granulomatous lesions. If staincd
by acid-fast procedure, masses of io tra- and extracellular
organisms can be demonstrated.
lsolation
Growth of the organism in vitro is ;i clPpPncl;ihlP \·vay to
make the diagnosis. Fecal samples or lymph node biopsies
..
•
o#ft.,- •
•
232 PART ll Bacteria and Fu11gi
are first decontaminated with hexadecylpyridiniunl chlo-
ride. The deconlan1inaled san1ple is placed i11 egg-yolk
medium containing mycobactin (mycobactin J produced
by M. paratuberculosís is prefcrred). Because son1e strains
are Lnhibited by sodiun1. pyruvate, egg-yolk n1edium vvith
and without this substance should be inoculated, as
sl1ould the same mediuJn with and without m ,vcobactin.
Tl1c tiJne required to develop visible colonies is about 8
wtek::.. Currt:1 1L Lt:Ll111i4ue::. alluvv fur Llie LleLeclio11 of one
organism per gram of material.
A radion1etric p rocedure (BAC'fEC:-RCM) utilizing
14C-palmitic acid followed by polymerase chain reacti.on
amplification of the DNA insertion sequencc 15900 (a M.
paratuberculosis-specific sequence of DNA) l1as been. de-
scribed and allows detection in f18:es in 2 to 4 ,,veek$.
Molecular Techniques
A number ot l)NA tests have been developed for the
demo11stratión of the presence of M. paratuberculosis. Ali
utilize a specific sequence for the developn1ent of probes
or for the design of primers needed to an1plify DNA by
u:sillg Llie polyu1erase chain reaclion. Specific sequcJ1ccs
that have heen exploited are those found in the gene en-
codil1g the 165 riboson1al RNA; the insertion sequcnce
IS900 (proper p rimer selection is crucial siI1ce there are
scgn1cnts within this gcnctic clcmcnt whicl1 are sh.ared
with 151626, an insertio11 sequence found in subspecies
aviurn; in addition, 15900 has 949/o identity with a DNA
sequence from Mycobacteriu1n cookii, a saprophytic tny-
cobacteria); hspX, a gc11e c11codil1g a 11eat shock pro-
teül unique to M. paratu/Jerculosis; and F57, a fragn1ent of
DNA that does not hybridize with DNA f rom any ot her
bacteria.
·1ests h ave been designed to determine the presence of
J\1. paratuberculosis DNA it1 fcccs, tissuc, and b lood 1nono-
cytes. Methods using feces, tissue, or n1ill< suffer from a
lack of se11sitivity, wl1ich is due to inhibitors found in
these substances. Steps taken to ren1ove inhibitors found
in feces, tissues, or milk greatly increases sensitivity. One
m cans of doing this is to "capture" lvf. paratubcrculosis with
paramagnetic beads coated vvitl1 antibody specific for tl1e
orga11is1n. Subsequen t passage overa n1agnet removes the
n1icroorganisn1s, whicl1 are then analyzed t>y molecular
(or othcr) mea ns.
lmmunodiagnosis
Immunodiagnostic proced ures are run in parallel '"'ith cul-
tures of thc organlsm and examination of direct smears.
Available tests rely either nn t he presenre of antihody
(forn1cd during tl1e lepron1atous stage of the disease) or on
delayed-type 11yperscnsitivity to extracts of t he o rganism
(elicitcd throughout thc discasc proccss).
Delayed-Type Hypersensitivity Tests
'l'here are tl1ree ge11eral types: ;:in intr;:i<1ermal test, an IV
jol111il1 (jolu1il1s are bacteria] peptides liberated into cul-
ture n1edia during growth .,.,1. paratuberculosis), and a lym-
phocyte proliferation assay. The intradermal test requires
lhe i1 1Lraden11al i11jeclio11 of jol111in, followed by exan1i-
nation of t he injection site for swelli11g 48 to 72 hours
later. This test 1nay result in as h.igl1 as 75% falsc-positivc
reactors.
The IV johnin test is more specific. 10 sorne johnins, or
to their parent p roteins, delayed-type hypersensitlvity de-
velops d uring t he infectious process. Reacting anin1als de-
velop a feve1 of l .5ºF or l1igl1er Iollowing IV il1jection of
johnin. ·rhis test detects about 80º!& of the cases showing
clinical signs, but not many asymptomatic shcdclcrs.
·rhc t h ird type ot test 1neasures t he cell-rnediated
in1mu ne status of t he animal to M. paratuberculosis. Jm-
munity can be measlired by an In vitro ly1nphocyte-
stim11J;:itinn tP~t PPripher;il hlnnrl Jymphocytes ari> ro l-
lectecl and exposed to johnin (or to its purified protein
derivative). lf tl1e anünal has developed cell-mediated im-
munity to thc antigens of M . paratuberculosis, these lym-
phocytes '"'ill respond by p roliferating and releasing cy-
tokines. Proliferation can be measurcd by the addition of a
radio-labeled nucleic acid precursor, or cytokines can !Je
ni.easured imm11nological ly (e.g., commercial kits are
available to n1easure interferon-ga¡nma by an ELISA spe-
cific for t his cyto kine).
Serologic Tests
Tt is important to note t hat serologic rests are designed to
detect anti body. Anti bodies are formed during the lepro-
matous stage of the <lisease, i.e., uuriug tl1e lCJter :stages oI
the d ise;:ise aftf'r shf'<1<1ing h;:is rom m enred. ·rhe comple-
ment fixatio11 test will result in about 75% false-positive
reactors. 'fhe other serological tests, thc agar gel i1n1nun-
odiffusion (AClD) test and thc cnzyme-linkcd i1nmuno-
sorbent assay (ELISA), are more specific. ELISA detects
sh edders before the .A.GID test, '"'hich becomes positive
only after large numbers of lv.f. paraluberculusis are in the
feces.
Both the ELISA and A(i!D tests have been shown to be
useful in d iagnosing this disease in sheep and goats.
Treatment, Control, and Prevention
At present there a re no economically useful antimicrobial
agents that are effectivc agail1st M. parat1Jberculosis in vivo.
'fhe 11ewer n1acrolides, such as clarithromycin, and sorne
experimental í1uoroquil1olones are effective in vitro, but
are too expensive to u se clinically. Hov.rever, clofazamine,
1.vith or without either ethan1butol or rifabuti n are used
vvl1e11 cirLun1:;ta11L<:::; warra11L (resulls have bee11 disap-
pointing with cure ra rely, if ever, occurring). M,vcobacte-
riutn paratuberculosis is resistant to isoniazid in vitro, and
probably so in vivo.
Elin1inating infect!;!d animals and preventing possible
spread within the herd J)est attack the disease. Husbanctry
procedures that n1ust be implemented in elude segregating
tl1e neuuate fru111 tl1e lla111 a11d oll1e1 adull a11in1a.ls, ensur-
i ng that parturition takes place in noncontan1ináted areas,
and taking p recautions against feeding neonatcs potcn-
tially infectious, unpasteluized colostrum or 1ni lk. ln1mu-

::iologic and cultural tests are applied to promptly iden-
dfy those animals t hat are infected and shedding. Cul-
- ure is used to confirm the results of the serological assays;
~' is also used ü1 vaccü1aled 11erds wl1ere serological lesls
ire not reliab le. Molecular techniq ues t hat utilize n or1-
~al samples (sucl1 as peripl1cral blood) show great prom-
~e in speeding u p t he process ot identitying aftected
anima Is.
Pasteurlzation ls an important step in control on
dairies, not only in assuring that calves receive n1ilk or
colostrun1 that is free of M . paratuberculosis, but also fron1
a public health perspective since the relationship b et,-veen
),J. paratubcrculosis and Crohn's Discasc is unclcar. High-
temperature, short-t ime pasteurization has been shown
::iot to be 100º/o effective i11 killing 1'1. paratubercu/osis in
naturally infected milk, but only if the numbers of m i-
croorgan isms in t ht> milk arP. largt>. This probably has .Uttle
?ublic health conseq uence i11 areas where large quantities
of milk are mixed together from several dairies, thereby
dtluting any infected milk. I-Iowever, this may be vcry
:rrtportant for producers that p rocess milk from a single
-
rarm.
Preveutiu11 uf tl1e <.liseélse is <.lifficult . Since t here are few
:-e-gulations governing tht> salt> ancl shipmt>n t of possibly
:.Ofected livestock, a producer 11as 110 way o f ascerLai1Ji11g
whether a purchased an imal is unin.fect.ed except by test-
ing . Until i.tniform tc:;t :; an<l rcporting proccdurcs are cs-
:ablished, the assurance that an animal is free of M. tuber-
culosis is not possible.
Parenteral vaccines, Uve and killed, are used in sorne
countries but are not universally available.
MI SC ELLAN EOUS MYCOB ACTERIAL
INFECTIONS
FELIN E LEPROSY
:llere is no true leprosy in domestic animals. Since a d is-
ea.se of cats is called feline lepras)', key features of the proto-
.y pe (hu111a11) co11diLiOll are suu11 11ari;¿;1::d:
l . Mycohacterium Jeprae, the acid-fast causative bac-
tertum of human leprosy, has not been propagated
in vitro. Lin1ited infections can. be produced in ro-
dt>nts. Tht> nint>-banclt>cl armadillo is susceptible to
natural and experin1ental it1fecti o11.
2. Leprosy attects superficial tissues, mainly skin an.d
upper respiratory tract. Only in advanced cases are
internal organs coloniZed.
3. Particular t argets of infection are peripheral nerves.
4. 'fhe clisease IJlcture nu1ges lJetween two extremes:
a . In lepr0!1'1atOu$ leprosy, hacterial prolift>r<ition is
profuse. Poorly circumscribed nondestructive
Jesions ctevelop, dominated by a monocytic re-
sponse but little other lnflammatory reactivity.
Cell-rnediated immunity is suppressed, l)ut cir-
culating immunoglobulins are h igh.
b. In luberculuiu levrusy, lJacteria are scarce.
Lesions are granulomatous, ancl ct>ll-m ecli;ited
Chapter 38 Mycobacteriurn 233
infla1n1natory responses are well developed.
They cause i1eural damage, leading to anesthe-
sia, paralysis, dystrophy, disfigurem ent, and
1nuLilalio11.
S. The course extf~nds over years ;incl clt>r<iclt>s. Af-
fected in.dividuals die of complications traceable
directly or indirectly to imn1une disorders or neural
destruction.
feline leprosy is a chronic noduloulcerative, nontuber-
culous rnycobacterial infection of the skin.
Its acid-fast agent is believed by m any to be
Mycobacteriu"1 lepraemuriurn, a cause of leprosy-like dis-
ease in rodents. It is cultivable on a special medium,
which also ~upvurts gruwth uf the feline isolate. Knowi1
M. lepraeniurium has failed to in ft>c.t c.ats. Howt>vt>r, st>-
qucnce con1parisons of DNA obtained fro1n tissues of cats
wit h feline Jeprosy show significant identity with M. lep-
raemuriu.rn .
Sources, n1ode of spread, and pathogenic m echanísms
are unknown. Transmission by rodent bite or arthropod is
suspecled.
Judged from experimental infectlons, lt>sions of ft>line
lcprosy dcvclop i112 to 6 mo nths. Nodules occur in cutis or
su bcutis in many sites, and are freely movable and pain-
less. Ulceratio11 and lymph nodc involvcmcnt are frc-
quent . The general health is unaffected. MicroscopicaJJy,
the lesions are granulomas consisting largely of monocytic
With. variable I1eUllüphiJiC, ly lIIIJ]IOCytiC, IJlaSIIlCI, an<.J
gianr c.t>ll aclmixt11rt>s. Cast>ation necrosis and irregular
neural involvement occur. Acid-fast bacteria abou11d
within histiocytes.
Limited obscrvat ions suggcst thc cxist cncc of lcproma-
tous and tubercu101d forms.
Examination of stained (Ziehl-Neelsen) biopsies of af-
fecle<.l siles revedls large 11urnlJer!> uf aci<.l-fast organisms.
Routine c.ulturt> for rapiclly growing mycohactt>ria or for
mycobacteria ca using tuberculosis are negative. Use of
DNA technology (polymerase chain reaction amplifica-
tion of specific sequences¡ DNA probcs) aid in making a dc-
finitive diagnosis.
·rreat ment includes surgical excision of affected sites (if
pussilJle). Ant i!Jiotics such as clofazimine J1ave been trled
with limitt>cl s11cct>ss. Postsurgical relapses are com1non.
(ANINE L EPROID GRANULOMA SYNDROM E
Car1ine Leproid Granulom a Syndrome is caused by an
unculturcd, saprophytic spccics of Mycobacteriun1 (based
on sequence comparisons of IJNA obtained 1rom infected
tissue with other mem bers of this genus as regards the
gene encoding the 165 ribosomal RNA). Diag11osis is
b;ised upon demonstration of nun1erous acid-fast or-
ganisn1s in stail1ed (Ziehl-Neelsen) b iOIJSie'.i ur su1eélrs
of affected tissue or sites. The condition affects the suh-
cutis and skin of thc pi nnae, fa ce, and body extrem ities.
It is freq uently self-limiting (though su rgical excision
or antib iotic t herapy will basten resolution). A c.on1-
bination of rifampicil1 and clarit h romycin has bee11 ef-
fective.
234 PART II Bacteria and Fungí
ULCERATIVE DERMATITIS OF (ATS ANO 00GS DUE TO
RAPIDLY GROWING MYCOBACTERIA
Tl1e n1ost common prese11ting complail1ts are cbronic
(111011tlis lo years), 11onlie<tli11g ski11 le:>ious. Hislopal ho-
logic evaluation of hiopsies of affected tissue reveals a pyo-
grun ulo1nut ou:s inflummution. Micr oorguni:ims muy :ituin
poorly ("ghosts," or "speckled" structures, thought to rep -
rese11t nonstaining or poorly staining mycobacteria, re-
spectively) when stained with Gram's or a Romanovsky-
type stain (e.g., Wright 's, (";-iemsa). When samples are
stained with acid-fast stains (Ziel1l-Neelser1), orgarlisu1s ar<::
not oftPn Sf>Pn.
Culture on standard blood agar medium will result in
t he isolation of rapidly growing (48- 72 hours incubation)
colonics. Isolatcs are idcntificd by biochemical traits, and
by UNA tests. The most commonly isolated in vvestern
Nortl1 An1erica is M)'cobacteriurrz fortuitu.n1. (sporadically
isolated are M. chelonae-abscessus, Jvf. flavescens , M. phlei, M.
stnegrnatis, M. xenopi, lvf. u/cerans, M. therrnoresistible) .
Percutaneous wound infection is the likely portal of
cn.try. Trcattncnt (surgical, antibiotics) has bccn disap-
pointing. Mycobacteriurn tórtuitum is susceptible in vitro to
amikacin, cefoxitan, ciprofloxacin, clarithromycin,
kanamycin, an<.1 minocycline, but resistant to doxycycHne,
Pryt hromycin, gPntamic.in, tohra myc.in, t rimethopri1n/
sulfonamides, and vancomycin.
BOVINE MYCOBACTERIAL ULCERATIVE LYMPHANGITIS
Nodulo-ulcerative skin lesions occur in cattle, particularlr
on the lower extremities and ventral trunk. ·r11ey resemble
tubercles and typically cont ain noncultivable acid-fast
bacteria. They are of interest n1ainly because of their asso-
ciation with false-positive tuberculin reactivity.
 Chlamydiaceae
DWlGH'f C.
Mcmbers ol the ramily Chlamydiaceae ("chlamydiae") are
oblígate intracellular gram negative bacteria incapable of
obtalnlng energy by rnetabolic activit ics. Their life cycle al-
terna tes between noninfectious proliferativc stages and in-
fectious nonproliferaLive sLages. Tl11:: far11ily cuuti:lins two
genera, Chlamydia and Chlarnydophila. (: hlamycliaP a rP as-
sociatcd with a variety of diseases affecting an assortment
of animal species ('l"able 39.1).
Descriptive Features
Morphology and Staining
C hla n1ycliae are coccobacilli 200 l>y up Ll> 1500 111n ir1 size.
Though cytochemically gram-negative, they stain best
vvith Gimcncz, Macchiavcllo's, Castancda, and Gicmsa
stains.
Life Cycle
Elen1e11 Lary liodit:S, 200 to 400 TI TI 1 i 11 :.izc, en lcr suscepti-
ble cells hy receptor-rnediated endocytosis. The enclosome
~traffics" to pathways not involved with endosomc-
lysosomc tusion. Elementary bodies change into nonin-
fectious, mctabolically active reticulate bodies, measuring
600 to 1500 nm, that generate, by binary físsion, a new
crop of elementary hodies that are relcascd u pon ccll lysis
•by lysosoutal hosl e t rL:yr ne:::;). T11 vi lru, ll tc cyclc requires 30
to 40 hours.
F.lcmcntary bodies appear red by Macchiavello's and
(jimenez, blue by Castaneda, and reddish-purple by
Giemsa stain, while reticulatc bodics stain blue, green, red-
dish, anCI blue, respectively. Giemsa s tain is best for the
Pmonstration of both stages.
: tructure and Cornposition
""'"ñe cell envelope ot clcmentary and reticulate hodies rc-
"'mblcs a gram-negative cell wall but lacks peptidoglycan .
trilam Inar outcr membrane consists of protein and
:x-ipolysaccharidcs. Proteins confer species and type-
'"':'CCificity, and n1ay acl as ad11esi11s. He::i>a• i1 r-like:: ucrivate:;
;::oduced by chlamydiae are also proposed to act ac; ad-
- esins by associating with heparin-binding reccptors on
- ost cells. ·rhc lipopolysaccharides, which havc endotoxic
HIRSH ERNST L. BTRF.RSTF.TN
properties (scc Chapters 7 and 8, are specific to the family
Chlamydiaccac.
Thc gcnome size, 6.6 by 108 L>a, is among the smallest in
prokaryotcs. 'J"here is much RNA in the reticulate but little
in the clcmentary boelies. In the reticulate body, electron-
rlense material is fair:ly uniformly dispersed throughout
the cell inlcrior, wl1ile i11 Lht: Ll1::v1::lopi11g clc1nentary bocly
it becomes increasingly concentrated into a compact nu-
clcoid, which, in thc maturc clcme ntary body, occupies
most ol the cell (fig 39 .1 ).
Cellular Products of Medical lnterest
Adhesins. r Tcparin-like derivates actas ad hesins by bindil1g
to heparin receptors on cells of the host.
Ce// Wall. Though the cell walls of the members of this
genus lack peptidoglycan, rhcy do contain lipopolysac-
charide (LPS). As in other gram-ncgative bacteria, LPS is an
i111pot Lanl vi1 ule11ce de::te::rruiuaul. LPS 1.Ji11tls to lipupoly-
saccharide-hinding protein (a sc rum proti>in), which in
turn transfers it to the blood phase of CD14. The CD14-LPS
complex binds to Toll-like receptor proteins (see Chapter
2) on the surface of macrophage ce lis Lriggcring thc re le ase
of proinflammatory cytokines.
Míscellaneous Product~. A soluble hemagglutinin and
ce::ll-lJou11d l!eat-li:tlJile tuxic effect, tlemonstrable ln mlcc
and on rnacrophagcs ano neutrti lizti hlP hy typP-specific
antibody, is associated with elementary hut not reticulate
bodies.
Growth Characteristics
Chlamydiae do not grow on cell-frcc 111edia. 'fhey grow
well in thc yolk sac of embryonated chicken eggs and in
tissue culture (c.g., L-cclls, McCoy cclls), where they form
intracytoplasmic colonies.
Reticulatc bodies transport adenosinc triphosphate
(A' l'P) lnro cclls and adenosine dlphospl1ate (ADP) out of
ci>lls. Tn thP presence of A'I"P, peptides are synthesized. ·rhe
agents are "energy parasites." Ele111enlary bouic:; show lit-
tic biochcmica1 activity.
Resistance
There is liltle iesislauce lo curru11011 tlisinfectants, heat,
and sunlight. Elementary hodip_c; survivP in water for sev-
235
236 PART 11 Bacteria and Fungi

Ta b le 39 . 1 . Members of the Genera Chlamydia and Chlamydophila and Their Usual 5ource or Associated Condition

Usual 5ource or Associated Condition Obsoiete Taxonónw

Chtamyaia trachomatis l'rachoma, sexuafly transmitted disease, and lympilogranuloma venereum in

human patients
C. muriélarum Respirator¡ disease in mi<e and hamsters C. tracho¡¡;¡atis of mice
e suis Conjunctivitis, enteritis, and pneumonia in swine e trachomatis of swine
Chlamydophila abortos Abortion in rumiria11ts C. psittací abortíon 9roup
Cph. cavíae ¡:oojunctivil:is in guinea pigs c. psirraci inclusion conjunctiviJis agent
Cph. fe/is Conjunctivitis ano upper· respiratory·tract disease in cats e psittaci feline pneumonitis agent
Cph. µelot um Al.Jurliun, Lu11ju11divili~, enleritb, p11eumunia, dru.l ,i1C!11ilb j11 1urni11dnts (. petO/Ulfl
Cph. pneumoniae Respiratory disease in human patients, koala bears, and horses C. pneumoníae
Cph_psittaci Ornithosis/psittacosis in .birds, and human patients C. psittací avian group

F 1G U R E 3 9. 1 . Ch lamydophila p sittaci inclusion containing re ticulate bodies (RB), condensing

(011ns (CF), and e/ernenla1y bodies (EB). The nuc/eoid of the ele1nenta1y body /las a polar eccentric
locatíon but occupies most of the ce//. Transmission e/ectron micrograph, 15,000X. (Reproduced by
permission of Storz, J., Chlamydia/es: Properties, cycle of deve/opment and effect on eukaryotic ho5t
ce/Is. Curr Tapies Microbiol lmmunol 1977;76:167.)

eral days at ambient temperatures and apparently persist
in dried a11imal cxcrctions for long periods.
Variability
Son1e of the chlamydiae show considerable variability as
n1easured by ll1e disease and 11osl (biova1iely 01 biovar),
and by antigenicity of surface str uctures (serovariety or
serovar). Bclo'"' is a description of tl1osc chlamydlae that
exhibit variability (serovars, biovars):
l . Chla1nydia trachomatis. There are tvvo biovarieties of
C. trachon1atis: thc trachon1atis biovar and thc ly1n-
phogranuloma venereum biovar. ·rhe trachomatis
biovar contains 14 serovari.eti.e s (A through K, Ba, Da,
and Ta). Serovars A, B, and C are associated with tra-
cho1na, and serovars D through K are associated with
sexually lra11s111illed diseasc. ·n1c lyn1phogranulon1a
venereum bi.ovar contains four serovars (L1, L2, L2a,
and L3), which are associatcd wit h thc scxu ally
transrnitted disease lyrnphogranuloma ve11ereum.

2. Chlamydophila pneumoniae. There are three biovars
of Cph. pneurnontae- biovar 1'WAR (named from the
straiI1 designations of the original isolates TW- 183
and Ar-39), biovar Koala, and biovar Equine- lhal
are associated, respectively, with respiratory tract
disease in hlunan patients; conjunctivae, urogenital
tracts, and respiratory tracts ot Koala bears: and res-
piratory tracts of horses.
3. Chlarnydophtla pstttact. ·rhere are eight serov ars of
Cph. psittac:i, design.ated A throug!1 H . Serovar A af-
fects psittacine birds and h.u n1an patients in co11tact
with them; serovar B is associated with pigeons,
turkcys, and an unusual cause of bovinc abortion;
serovar e affects a variety of avian species and h.u-
man. patients in contact with then1 (mainly slaugh-
terhouse contact); serovar D affects turkeys, para-
keets, and human patients in cúntact with th.em
(n1ai11Jy slaugh.terhouse co11lacl), :>erov a r E affecLs
mainly human patie11ts; serovar F affects parakeets;
serovar Gis associated with bares and muskrats; and
serovar H affects cattle.
Ecology
~e servoir
The respiratory, intestinal, and genital tracts of n1a111111als
and birds constitute the reservoir.
- ransmission
E..xposure is through inhalation or ingestio11 of infectious
m a terial. Egg transmission occurs in sorne birds.
::athogenesis
.Jechanisrns. Elementary bodies attach to and are endocy-
.o:s<::d oy (:!pitll(:!Jia] c(:!ll:;, W}l(:!r(:! tl1ey UlUltiply, 11aving
somehow averted phagolysosomal fusion hy trafficking to
e.."ldosomes that do not readily fuse to lysosomes. Synthe-
sis of host DNA, RNA, and protein ceases and cells eventu-
ally disintegrate through host enzyme action. Microbial
:nxicity (LPS) and tissue damage elicit inflammatory re-
sponses.
Pu.lhulugy. Pall!ulogic clia11ge:s vary wiLIJ lucalizatiu11 of
~ection. In pulmonary chlamydiosis, an exudative hron-
chiolitis and bronchopneumonia develop mostly in the
3I11erior lobes as reddish-gray areas of consolidation with
~urulent exudate in the bronchioles. Microscopically, the
~'f! allest bronchioles contatn predon1inantly neutrophilic
exudates. In the acini, alveolar macrophages predomina te.
~den1a 1nay be 1narked . E¡;iLhelial dau1ag<:: iI1 th(:! l.Jron-
::~ioles is variable. Mononuclear cells gradually replace
2cutrophils, and ly1npl1oreticular cuffing is common in
-i.:minants.
Ocular infections take the form of a catarrhal conjunc
:1\"ltls.
Chlamydial arthritis involves ali soft tissucs near af-
:ected jolnls. Suppuralion, edeH1a, a111.l 11eu1orrhage are
rucceeded by granulomatous and fihrotic reactions.
Chapter 39 C'h/amydiaceae 237
Chlamydial abortions ofru1ninants are associated with
a placentitis with cotyledona.ry necrosis and edematous or
leathery intercotyledonary thickening. Lesions in the
n1ale geniLal l1acL of r uu1iuauls (epidi<.ly1ui:;, tt:::;ticle, U(:!fer-
ent duct) are granulomatous.
(:hlan1ydial enteritis is associated with a granuloma-
tous inflammation of the lamina propria and submucosa.
Chlamydiosis ofbirds is n1arked by fibrinous serositis of
body cavities, air sacs, and organ surfaces. Lung, spleen,
and liver are enlarged and congested. Microscopically,
ll1ere is fibrinonecrolizing infla11u11atiu11 ~vitl1 u1u11u11u-
clear and heterophil leukocytes.
Disease Patterns
Ruminants. Abortion. Chlamydial abortion (almost al\-vays
caused by Cph. abortus) occu(S a1nong previously unex-
¡;u:.eu 1;;we:, (e11zuulic a!Ju1 Li<J1 1 of ew<:::;) autl goat:;. AIJor-
tion occurs sporadically in cattle and rarely in other
spccics. Ewcs abort typically late in p regnancy without
premonitory signs or atter effects, except for a vulvar dis-
charge and occasionally a rctaincd placenta. Bovine abor-
tions follow sin1ilar patterns.
Polyarthrítís. Chlamydial polya(thritis (Cph. pecorum)
affe1..l:> 1uaü1ly lau1lJ:, aull calve:,. lll ":;Liff la1u!J tll:sease"
morbidity n1ay be 80o/o, but the mortality rate is 1°Ai. Tn
calves, therc may be systernic complications and high
mortality.
Nervous Systern. Sporadic bovine encephalomyelitis
(SBE) is a febrile disease predo1ninantly of young cattle
that is caused by Cph. pecorurn. Clinical signs include loco-
rnotor, postural, and behavioral dlst urbances. ·rhere may
he milrl c.011gh. n;is;il cfi.;ch:irgf', ;incl di;irrhPa ThP cl i~<'ilSP
lasts fron1 days to weeks, and morbidity and case fatality
rates may reach 5ü<Yo. Sorne outbreaks 1nay continue for
months, with new cases appearing irregularly.
Avían. Avían chlamydiosis (ornithosis, psittacosis) is
cau:;etl vy Cph. psittaci, antl is most significant economi-
cally in tnrkeys. Onset is insiclio11s ;incl m:iy occnr >vef'kS
after exposure. .Early signs are mappetence, weight loss,
and the voiding of gree11ish-yellow, gelatinous droppings.
Egg production is reduced. Severity varíes with strair1 v iru-
lence (toxicity); 1norbidity 1nay range from .)o/o to 80o/o,
mortality from 1ºA:> to 30%. Geese and ducks are sometimes
affecte<.l, chickens exceptionally so. Subcllnical forms pre-
rlomi n:ite in p igf'ons and psittacine birds.
Miscellaneous. Pneumonia. Chlamydial pneumonias, pro-
duced by a varicty of chlamydiac (sec Table 39. l) are seen
particularly in pigs, cats, sheep, goats, and cattle, and are
often subclinical or part of mixed infections (enzootic
sheep pneumonía, shlpping fever). l'he cUnical impor-
tanc.f' of 11nromplir.ated infections (e.g., "feline pneu-
monitis") is uncertain.
Conjunctivitis. Chlamydial conju11ctivitis, although re-
ported in cattlc, dogs, pigs, guinea pigs, and koalas, is most
noted in cats and lambs (see 'Jable 39.1). lt may pass
through acute and prolonged chronic phases. In lambs,
where it accompanies poiyarthritis, secondary complica-
tions, keratitis, and corneal ulcerati.ons are seen.
238 PART II Bacteria and Fungí
Epidemiology
The natural reservoirs for the various species of chlamy-
diae appear to be the animal species in which the natural
disease occurs. Asympton1atic carriage is common.
Zoon otic act1uisitior1 of several species of chlarnyc.liae
occ11rs. Most importantly is í.ph. psittar.i fron1 clinically af-
fected or asy rnptomatic birds (slaughterhouse workers, pet
owners). Unusual transmission for hurnan patients in-
cludcs Cph. fclis from cats and Cph. abortus from affcctcd
sheep.
lmmunologic Factors
Recovery and Resistance
Ewes that have aborted rarely abort again, a11d calves re-
covered f1on1 SBE resist reit1fectio11. In other cbla111ydial
d iseases, which are often chronic and marked by remis-
sions and relapses, thcre is little evidence of heightened re-
sistance.
Ccll-mcdiated an d humoral immune responses are
de1nonstrable by skin tests, lymphocyte stimulation tests,
and serological reactions. Protective responses appear to
be cell-mediated.
Artificial lmmunization
Vaccination of anin1als agait1st chlamydial iI1fcctio11s
often produces or1ly short-llved part1al protect1on, except
against e11zootic abortio11 of ewes (see under "'l'reatme11t
<u1<.l Coutrol," bt:lvw).
Laboratory Diagnosis
Diagnosis requires laboratory confirmation. Chlamydial
inclusions are often demo11strable by appropriate stains
(see "Life Cycle," above; Fig 39.2), including immunofluo-
rPscPncf within inff'r.ted epithf'lium and mac:roph.ages. An
enzyn1e-li o ked i.mm u11oadsorbent assay (F.T.TSA) ter.h -
nique detects chla111ydial antigen in vaginal discharges.
lsolation
Chlamydiae are isolatcd on tissue culture line cells (1-Iel.a,
McCoy for Chlamydia spp.; HEp-2, 1-Il., BGM, McCoy for
Chlamydophila spp.) or in fPrtilP chickPn Pggs. l)econtami-
nation of suspect feces, placentas, urine, semen, and con-
junctival fluids can be accomplisl1ed by treatme11t with
combinations of gcntarnicin (SO µg/n1l), vancomycin (75
µg/ml), and n ystatin ('.)00 units/n1J). With tissue culture,
centrifugation of the inoculum onto the monolayer on a
cover slip is beneficial, and addition of cyclohexiruiue (2
pg/n1l) provides an addeci hnost to ch larnydiae. Growt:h oc-
cur~ ""ithin 2 to J days of incubation.
Eggs are inoculated into the yoll< sac and candted daily
thereafter. Yolk sacs of e1nbryos dying 3 or more days after
inoculation are examined for chlamydial inclusions.
By either method serial subpassages are reguired to rule
out chlan1yd.!osis.
Presumptive isolates can be cnnfirmPd serologically by
in1n1u11ofluorcsccncc, con1plen1ent fixation tests, or by
molecular means (see below).
Serologic Tests
Complement .tixation and enzyme-linked immunosor-
bent assay (ELISA) tests are not generally available to vet-
erinarians, although genus-sped.flc a11r 1gens are markete<l.
The presenc:e of antibody confirms a diagnosi.s only if its
titcr increased fourfold during the course of illness.
Molec.ular methods
Molecular tcchniqucs havc bccn dcvelopcd that utilize
DNA primers designed to amplify genes (or portions
thereof) er1coding the 16S and 23S riboso1nal RNA, an.d
outer membrane protetn 2 (omp2) by the polymerase chain
reactlon. These .methods allow the mfans fnr ciPtt>ctinn of
F 1G U RE 3 9. 2 . Chfamydial inc/usion (arrow) in a conjunctiva/
epithelial cell from a kitten. Wright sta1n, about 1uuux.

chlamyciiaP in tis<;nf'<: nr f'X11rlates as well as identification
o f isola tes.
Treatment and Control
'i'reatment
Tetracycline is the drug of choice in the treatrnent ot
chlamydiaJ infections. In birds, flock treatment involves
_111.:orµorating the drug in feed. Moderate doses (200
g/ton) are adec¡uatP for <li<;Pa.<;P rontrol in turkeys. Higher
doses (2800 g/ton) are needed for tissue clearaJ1ce. To li111iL
o utbreaks of abortion in ewes, intramuscular injections
20 mg/kg) of long-acting forms are given at 2-week inter-
vals. Ietracycline is given orally to lambs (150 to 200
mg/kg) to prevcnt arthritis. For pet birds, tetracycline con
taiuir1g millet (0.5 mg/g) ls avalla ble.
Cha11ter 39 Chlamydiaceae 239
Prevention
Vaccination against chlamydial infections has had vari-
able success. rormalinized vaccines are beneficia! in pre-
venting enzootic ovin e abortion (Cph. abortus). Feline
chl<unydial vacclnes concaln!ng modified live or killed or-
ganisms attPnuiltf' rlinic-;:il illncss without curing or pre-
venting infection (Cp/1. {e/is). A vaccine for avían chla111y-
diosis is not availablc (Cph . psittaci).
Prompt disposal of infected material and scgrcgation of
affectcd animals is helpful in control of ovine abortion.
Young turkey poults should be placed in clean quarters,
wi ll 111u access tu lnfected dropplngs and unexposed topo-
tentia lly contaminated air currents.
Regulations gove1uiug i111µorl uf exutic t>irds are de-
signed to minimizc in troduction of psittacosis in to ho11sP-
hold and residcnt bírd populations.
 Mollicutes
R ICHARD L.
The mollicutes are members ot thc order Mycoplas1natales
and class Mollicutcs (soft skin). Thcy are the sma.llest of the
lrcc-Jiving prokaryotes and are devo1d of cell walls. Only
1ncn1bers belonging to the genera i\lf}'coplasma and Urea-
plas1na are important In veterlnary me<.licine. Achule-
plas1na are son1etirnes cnco11ntPrP<1, h11t 11s11<1l ly <is c.ontam-
i nan Ls. Mollicutes is the correct term to use when
collectively rcferring to members in this order; however,
thc triviol numc "mycopla:;ma(s)" i5 also used for this pur
pose. l<cclasslf1cat1on of haemotrophic rickettsial species
of the genera Haen1oharto11elln and Eperytlzrozoon to the
genus Mycoplas1na has been proposed based on 165 riboso-
mal RNA (rRNA) gene sequence analysis. These have col-
lL·cti vel y 1Jee11 gi ven Lhe Ll ivial na111c "l1aen1oplasn1as."
The "nonhaemotrophic" mollicutes ("mycoplasmas")
infcct a wide range of animal spccics. lnfcctions rangc
frorn subclinical to severely debilitating and sometimes
f::it::il diseases. Clinical 1nanifesta tioi1s in elude respiratory
and urogenital tract infect1ons, arthrltls, mastitis, anc.I sep-
ticemia. Most pathogenic speciPs Pxhihit <i high <lPgrPP of
host spccificity. Infections in hun1ans usually present as
respiratory or urogenital tract disease.
"fhe "haemotrophic" mollicutcs ("hacmoplasmas")
also intcct a widc range ot animal species (espedally the
very young, or stressed). Hemolytic anemia is the most
con1 monly cncountered manifestatlon follo,·v ing infec-
tion wilh these bacteria.
Descriptive Features
Morphology and Staining
Thc ccll morphology of thc mollicutes is extremely pleo
morphic. Cell shapes include spherrcal, ring-shaped, pear-
shaped, spiral-shaped, and filamentous forms. Cells
sometlmes appear as chalns of beatls, tl1e result uf asy11-
chroni1ed gPnomir rPplir<ition <ind c.cll division. The di-
a111eter of the spherical forrn rangcs from 0 .3 µm to 0.8 µm.
Mollicutes stain poorly by the Gram method. Giemsa,
Custnncda, Dicnes a11d n ew methylene blue stains are
preferred.
Structure and Composition
Thc n1ollicutes are not only devoid of cell walls but lack
the genetic capacity to produce one. They are bound by a
si ngle trilaminar membrane composed of proteins, glyco-
proteins, lipoprotems, phospholipids, and sterols. Choles-
240
wJ\LKER
terol in the membrane provides for osmotic stability. A
polar bleb 11as been demonstrated in sorne species and has
a role in adherence to host cell surfaces. Capsules have also
been desc ribed for sorne species.
Tl1e ruollicules have a sn1all genome (600-1,400 kb)
compared to other bacteria. The base compositio11 is poor
in guanine and cytosinc with thc molo/o G 1 C of DNJ\
ranging trom 24 to 40o/o relating them 1nost closely to the
Clostridium-Streptococcus-Lactobacillus group. Sequence
analysls of 165 rRNA indicates that 111ullicule=> a1e 1nost
closely related to the genus C/n~tridi11n1. The entire genome
for sume species 11a:. l>ce11 :.ey ut:11Ct:d. "l11e n1oll icutes ap-
pP<ir to h;ivp evolved hy genome reduction. Transposons,
plasmids, and bacteriophages have been dcmonstratcd in
sorne species.
THE NONHAEMOTROPHIC MOLLICUTES
·rhe "nonhaemotrophic" moll1cutes include members of
the genus Ureaplasn1a and those of the genus M}'coplas111a
that are not associated wlth red blood cells. Unlike the
"haemotrophic" mollicutes (see below), they can be
gruw11 i11 vitro 011 Hfeless inedia. Clinical manifestations
include respiratory and urogenital tract infections, arthri-
tis, mastitis, and septicemia. Most pathogcnic spccics cx-
hibit a high degree of host spcciticity. lntections in llu-
mans usually present as resp·i ratory or urogenital tract
discasc (Table 40.1 ).
Cellular Products of Medical lnterest
Peroxide/Superoxide. Peroxides and superoxides are pro-
duced and may be important In disruption of host cell
intcgrity.
Un:u.)c. U1ease, p1oduced by 1nembers of the genus
Ureaplasma, maybe involved in injury to hosttissue as a re-
sult of Lhc production of ammonia by urea l1ydrolysis.
Miscellaneous l'roducts. f.xper rmental inoculation of a
200kDa protein from thc supernatant of lvf.. neurolyticurn
cultures causes neurologlc slgns In 1nice. Va!;cular <.la111ag1:
in the brain is evident but th<' 1nt>ch<1nism of action je;
u11clea1. Poorly defi11cd products, mostly lipoproteins,
from sorne mollicutcs induce interleukin-1, interleukin-6,
and tumor necrosis factor production from ::ictivated ma
crophages. ·rhis may account for the endotox.ic-like ac-
tivity observed with sorne infections. 13ovine ureapla~­
mas produce IgA protcase, whtch cleaves IgA1 a11<.l1uay aiu
 Cftupler 40 Mo/líe,ule::; 24 1

Ta b 1e 4 o. 1 . Animal Species, Agents, and Diseases Associated with Nonhaemotrophic

Mycoplasma and Ureaplasma

itnimal

.... ..
~
Cats
Ageot
~--~-=-~--~--~-~~"""'~--~--~-----
Mycop/asma fe/is Coniunctivitis
Conimoo Clinical Manifestations
-------------------~

M. gatae Arthritis
Gattle M. a/kalescens Arthriti>. ma.stitis
M. bovigenitalium lnfertility, mastiti;, seminal vesiculitis
M. bovi; A.bsle;~~~. arlhrilis, mcc.titb, oüti;, [')neumonía
M. bovoculi Keratoconjunctivitis
M. calífornicum Arthritis, mastitis
M. canadense Arthritis, mastitis
M. dispar Alveolitis, bronthiolítis
M. mycoides subsp. mycoides Arthritis, pleuropneumonia lCBPPJ•
(srri~ll r.olony v~ri~nt)
U. dive1sum l11fe1 Ulity, fJJ1eurnor1io, vulvov.agini.Lis
Chickens M. galliseptícum Respiratory disease
M. synoviae Airsacculitis, sternal bursitis, synovitis
Dogs M. canis IJroge.nital tract disease
M. cynos Pneumonia
M. spumans Arthritis
Go;¡f~ M. ;ig;i/;irti;¡p Ag-alactia. artfiritis, conjunctivitis
M. caprícolum subsp. cap1 kolum A1 Lf ui li>, 1 11o~liti>, fJfleumurtio, ;eplicemio
M. capricolum suosp. capripneumoniae Pleuropneumonia (CCPP)b
M. conjunctivae Keratoconjunaivitis
M. mycoides subsp. mycoicfes Abscesses, arthritis, mastitis, septicemia
(large colony variant)
M.· myEoides suosp. capri Pneumonia, arthritis
M. putrefaciens Arthritís, mastitis
Horses M. fe/is Pleuritis
Mice M neurolytíc-0m ConjunEtivitis. l)eurological disease
M. pu/monis Respiratory disease
Rats M, arthrítidis Arthrítis
.M. pu/monis Respiratory disease, genitat tract disease
Sheep M. agalactiae Agalactia
M. conjunctivae Keratoconjunctivitis
M. ovfpneumonfae Pneumonia
Swine M. hyopneumoniae Enzootic pneumonia
M. hyorhinis Arthdtis, pnet1monia, polyserosítís
M. byosynoviae Arthritis
Turkcys M. gallisc,oticum Sinusitis, respiratory' di.sease
M. iowae Emhryo mortality, leg deform1ties
M. meleagridis Airsacculitis, decreased egg hatchabilít}\ perosís
M. synoviae Sternal bursitis, synovitis

ªCuntdyiou~ Uuvlc1e µleuruµtteu111011ici.

blontagious caprine pleuropneumon1a.

in avoiding the host imn1une response on mucosa! sur-
faces. Other proteases, hemolysins, and nucleases are also
proctuced.
Growth Characteristics
Tl1e r1onhaernotropllic rnollicutes grow slowly and gen-
era lly rPquirP ~to 7 clays' inr11hiltion hPforP colonif's arP
apparent. Growth is best at 37ºC in an atmosphere of in-
creased C:0 2 . Beca use of tota1 or partiil 1 ina hi lity to syn-
thesize fatty acids, cxogenous sterols are required by
Ureaplas11za and Mycoplasma. They are not, however, re-
quired by Acholeplasma. Gen.era of veterin.ary im.p ortance
are facultative anaerobes. Optima! pH for growth ranges
fro1n 6.0 for Ureaplasma up to 7.5 for other mollicutes.
Molllcute colonies are smaH and difficult to visualize
with thP 11n;:iirlPrl PYP r.olony ~i7P\: v;:iry fron1 O.OJ mm to
1 .0 mm. When observed with a dissecting n1icroscope,
242 PART II Dactcria and Fungí
F1G U RE 4 O. 1 . "Fried-egg" colony m orphology that is typica/
of many Mycoplasma species.
n1any species exhibit a "fried egg" n1orphology (Fig 40.1).
Thls un1bonate appearance is the result of the central
portion of the colon y embedding in to the agar with a pe-
ripl1eral Z(J11e of surface growlh. So1ne species produce
film spots, \-Vhich are composed of cholesterol and phos-
ph<>lipids and vvhich appear as a wrinkled film on the
media surface.
Resistan ce
Tl1e lack of a cell 1'\1all renders H1ollicutes resislanl Lo Lhe ac-
tion of anti n1icrohial agents that affec:t th e c:ell \-val! or its
synthe:sis. They are sen:sitive to compounds that intcrfcrc
with protein and n ucleic acid syntt1esis. Acholeplasrna
species are resistant to l .So/o digitonin, whereas the grov.,th
of otl1er mollicutes ls lnhlbited by thls concentratlon. In
genf'ral, m ollicutes survive outside tJ1e host for substantial
peri<>ds in n10ist, cool environn1ents. Tl1ey are very suscep-
tible to heat and most detergents (tween) and di sin fectants
(quaternary amm<>nium, iodine, and phenol-based com-
pounds).
Variability
VariaLions in nuLrilional and al!nosphe1ic require111e11Ls
account fo r so1ne of the diversity among genera and
species within genera. Differences in colony morphology
and size can be used t o distinguish sorne of the molli-
cutes. Ureaplasma species produce substantially smaller
colonies tl1an othcr n1olllcutcs and often lack the fried-
egg colony morphology. The colony size of M . 111ycoides
subspecies rr1ycoides isolales fron1 goals is consisle11lly
larger than those isolated from cattle. 'fhis size difference
is used to distinguish betvveen the two variants. While
sorne antigens are shared among the mollicutes, anti-
genic differences are sufficiently specific to allow for
species identification. Animal host specificity is stro11gly
exhibited by the pathogenic n101licutes and may be ex-
plained by specific 11osl receplors 11ecessary íor allacl1-
ment or the failure of the host to recognize host-adapted
species as nonself.
Ecology
Reservoir
Thf' major rf'Sf'rvoir for t hf' nonh;lf'motrophic mollicntes
is the host they infect. Asyn1ptomat ically infected anin1als
carry organisn1s on m ucosa! surfaces including nasal, con -
ju nctival, oral, intestinal, und genital n1ucosa. 'fhc car
canal of goats has also been shown to be a reservoir for
sorne of the patl1ogcnic caprinc mycoplas.m as.
Trans mission
Transmission occu rs predon1inately by spread fro1n ani-
mal to animal through direct contact and is mcdiated
through aerosolization o f respiratory secretions or
through venereal transmission . Mechanical transmission
is also lmportant, especially with regara to bovlne and
caprine mycoplas1na mastitis. In poultry, vertical trans-
111 ission Ll1rough halching eggs is an in1portant n1eans of
sprf'ad fnr man y of the pathogenic avian species. Contami-
nated milk can be a source of infection for calves and goat
k ids. Little is known about the role of ectoparasites i n
transmission; hovvcvcr, pathogcnic caprine species have
been isotated trom ear m ites of goats.
rathogenesis
Mechanisms. Attachment to !1ost cells is the fi rst ste¡.> iu es-
tablishing infection anc! is mf'ciiatf'd th ro11gh the anionic-
surf¿1ce !ayer 011 111ost n1ycop!asn1as. In sorne species, spe-
cial attachment structures have been demonstrated,
which appear to be encoded by a commo11 ancestral ad-
hesin gene. Host receptors tor attachment are glycoconju-
gates and allow for colonization of mucosa! surfaces. The
ciliostatic capability of sorne lvlycoplasrna species further
promotf's Psta blishmen t of infpctinn . ·rhf'rf' is Pvidence
that son1e Mycopla:;n1a species can penetrate and ex.ist in-
side non-phagocytic cell~ . Mycoplasn1as have also been
demonstrated to fuse \<Vith eukaryotic cell mernbranes.
Latent infections are commor1. ractors that allow for
persistence include n1echanisms to avoid the immune sys-
tem such as antigenic variability of cell membra11e
lipoproteins or biological 1nilnicry TJnderlyi.ng factors
sucl1 as agc, crowding, concurren.t infections, and trans-
portation stresses lead to ovcrt disease. Breaks in integrity
of thc cpithclial barricr probably account for thc initial
step in breaching l1ost defenses. Lipoproteins in the cell
membrane, acting as modulins, play a major role in cy-
tol<ine induction.
Acute, septicemic forms of d iseasf' rf's11lt in a c-oag11lo11a-
thy and widespread vascular thrombosis, which resembles
a gram-negative septicemia and is, at least in p art, n1edi-
atcd t hrough induction o f cytokincs.
·1·he pathogenesis of chroníc infections 1s dircctly re-
lated to persistence of the organisms in tl1e face of an in-
tense inflammatory response. PeroXldation causes host tis-
sue dam.age. Activation of con1plf'mf'nt anci cytotoxic T
Iyn1phocytes further contributes to host injury.
Virulence varíes among species and strains within
spccics and accounts for sorne of thc variations in disease

manifestation. ln sorne species, virulence is correlated
lvith presence of ano uter surface Jayer. A galacta11 polymer
in M. mycoides ssp. mycoides has been shown to modulare
~he immune response an.d promote di.ssemination. Viru-
lence is rapidly lost by in vitro passage. Mycoplasn1a bovis
induces apoptosis in lymphocytes. Mycoplasma dispar can
inhibit activation of macrophages.
ln addition to specitic ettects related to mycoplasma,
generalized effects on the imrou11e system may increase
susceptibility to secondary infections with other bacterlal
pathogens.
Pathology. The lesions associated with mycoplasma in-
fections vary from acutc to chronic and are dependent on
the agcnt involvcd and thc sitc affcctcd. In acutc infcc-
tions, there is an i11flammatory reaction with an infiltra-
:ion of neutrophils and fibrin accumulation. Geoeralized
!nft:ctiou:s lt:a<l to <t filJrinopuruler1t exudare on serosal
surfaces and synovial membranes. In persistent loc:ali7.ed
i.nfections, tissue destruction can be substantial. Ab -
scesses may develop at presst1re sites in calves and are
characterized by an eosinophilic coagulative necrosis
·.,ith peripheral fibrosis. In cases of mycoplasma mastit1s,
f}OCkets o.f purulent exudate may develop in affected
n1a1111nary Lissut:. Ev1::11 Lually tl11:: aift:ctt:<l gla11d l>ecornes
5.brosed. In the acute stage of mycoplasma in fections of
m e joint, the joint becomes distended with fluid contain-
'.ng fibrin . As infections become chronic, there is villus
::iypertrophy of the synovia anda proliferative and erosive
arthritis develops.
A marked pleura] effusion develops in respiratory tract
!nfections dueto Nf. mycoides ssp. mycoides in cattle and M.
mpricol11m ssp. capripne11rnoniae in goats. The subpleural
tissue and interlobular septa become thickened and fluid
5.lled. Affected areas of lung become hepatized with result-
:.ng scqucstration of nccrotic tissuc.
An intiltration of lymphocytes and plasma cells is often
observed in mycoplasma infections, particularly arou11d
··essels and in the submucosa. Peribronchial and peribron.-
dliolar lymphoplasmacytic cu.ff.i ng is a characteristic find-
-'1g i11 respiralory lracl infeclion. ·r11e profound lyrnpl10-
~lasmacytic proliferation ohserved in many infe.ctions is
éue to nonspecific mitoge11ic effects as well as to a specific
2 ntimycoplasmal immune response.
J isease Patterns
Júectio11s can 111anifesl in a variely of ways. Con11norr
;nanifestations include septicemias, disseminated infec-
2ons involving multiple si tes, or localized infections. Com-
11100 manifestations caused by t11e ditterent pathogenic
!peCies in major animal species are Usted in Table 40.1.
!.vian. Mycoplasmosis in poultry has important economic
:onseq uen ces. lvíycoplusrrtu galliseplicurn caust:s a el trouic
:espiratory disease in chickens, turkeys, and a number of
:nhcr domcstic avían spccics. Clinical signs include cough-
~"1~1 nasal discharge, and tracheal rales. 'lürkeys can de-
-:-etop sinusitis with production of a thick, mucoid exudate
~at results in severe swelling of the paranasal sinuses.
Jccasionally, clinical signs related to brain and joint in-
-ctlven1e11l are recognized. Decrease iu egg vroduction aJso
Chapter 40 Mollicutes 243
occurs. Mycoplasrna synoviae also intects a wide range of
avían species. Synovitis resulting in lameness, swelling of
jolnts and tendon sheaths, and retarded growth are com-
mon presentations. Sternal bursitis is also frequently ob-
served. Airsacculilis, wl1ich is usually subclir1ical 1 is au-
other rnanifestation. Myr.nplasma m1?leagridis and M . iowae
infections are rnostly limited to turkeys. Mycoplasrna. rnclca-
gricliS causes respiratory disease, predominately an airsac-
culitis, which is often clinically mild or i.napparent. Skel-
etal deforrnlties, including bowing or twisting of the
tarsometatarsal bone and cervical vertebrae, are occasion-
ally detected. Decreased egg halchabilily is a serious conse-
quence of M. meleagridis infections. Airsacculitis, leg defor-
mitics, and stunting in poults 11ave been demonstrated
experimentally with M. iowae. Decreased egg hatchability
has also been noted with h.f. iowae infect.ions. Natural out
breal<s of conjunctivitis in finches causect by M. gallisep-
fí(:11n1 hilvf> r;111sed substantial reductions in the finch pop-
ulation on the eastern coast of the Uniled SLales.
Bovine. Mycoplasma mycoides ssp. 111ycoides (small colony
v ariant) is considered the n1ost virulent of the bovine my-
coplasmas. It causes a respiratory disease, contagious
bovine pleuropneumonia (CBPP), in cattle that ranges
from a pPrsistent, subclinical infection to an acute, some-
times fatal disease. Cli11ical sig:ns il1clude respiraLory dis-
tress, coughing, nasal discharge, and reluctance to move.
In severc cases, thc animal will stan.d with its i1eck ex-
tended and mouth open to facilitate breathing. Subclini-
cally affected anin1als serve as a source fOJ maintaining
and spreading infection in the herd. Most infections are
limited to the respiratory tract, although arthritis occurs
in calves.
Mycoplasma mastitis is caused by a number of species.
Mycoplas111a bovis is the most common cause and results in
tl1e most severe disease. Mycoplasma californicum and M.
canadense are also frequently involved. Mycoplasma alka-
lescens and M. bovigenit.alium have also been implicated as
etiologic agents on occasion. 1ypically, there is a drop in
111ilk produclior1. Tltt: r11ilk be::cuH1t:::. tllick a11<.l iutt:rnúxed
~vith a watery sec'retion and n1ay progress to a purulent ex-
uda te (Fig 40.2). 1'he udder is often swollen, although not
painful. Sometimes ali four quarters are involved. It is a de-
structivc 1nastitis and oftcn rcfractory to trcatmcnt. Most
infections are limited to the mammary gland; however,
a.rtl1ritis subseguent to bacteremia occurs. In sorne cases
t.lisSt:I11inate<.l iufection results in periarticular involve-
1nent and fasc:i itis. Spread from c:ow to cow is directly re-
lated to inadequate 1nax1agement and sanitation practices.
Mycoplasma respiratory tract infections in calves often
present as pneun1onia in association with othcr bovinc
respiratory pathogens. Mycoplasma bovis is the predomj-
nant species recovered. Mrcoplasma dispar causes a mild
respiratory disease characterized by bronchiolitis and alve-
oli tis and is us11ally precipit;:itecl by environmental stresses
or a prirnary viral infection. Both J\.f. bovis a11d lvf. dispar
can be recovered as commensals from the upper respira-
tory tract.
Urogen1tal tract i11fections are caused by M. bovigenital-
ium and Ureaplasma diversum. Seminal vesiculitis in bulls
and granular vulV!tls, endometritis, anct abortion il1 cows
244 PART II J3acteria and Fun"i
b
F l G U RE 4 O. 2. Milk from a cow with severe mycop/asma mas-
titis. The mílk is thíckened with a watery component that contains
srna// flakes of 1nateríal. Mycoplas111a bovis was recove11=ú.
are associated with botll o f these orga11isms. Both are
found as normal commensals in the lower uroge11ital tract .
Arlh1ilis in calves occLus sporadically. While a nun1ber
of different species can cause arthritis, i'vf. bovis is most fre-
quently recovered. Other less commo11 presentations in-
clude otitis media and decubital abscesses (fig 40.3). Otitis
media usually occurs in co11junction witl1 respiratory dis-
ease. Dccubital absccsses have bee11 associated witl1 con-
fil1ed 11ousil1g conditio11s. M)lcoplasrna bovis, agaiI1, is tl1e
U:)Ud] dC)t;l ! l..
(¡¡nine. A numbcr of Mycoplasrna spccics havc bccn iso-
lated fron1 dogs; 11owever, little is known ahout ttle role
they p lay in d iscase. Experimental and clinical evider1ce
suggests M. canis can cause urogenital traer disease in-
cluding prostatitis, cystitis, endometritis, orchitis, and
epididy1nilis. 'file role of lvfyi;oplusruu i1 1 reproduclive <.li:s-
orders of the bitch is uncertain. Mycoplasrna spp. have
been associated with pneumonia, often as secondary in-
FIGUR E 40.3 . Abscess ínvolvíng sternal area of calf. The
abscess was related to pressure trauma. Myco plasma bovis was
recovered. (Courtesy Dr. H. Kinde.)
vaders. Mycoplasma spumans 11as l)een reported to cause
arthritis.
Caprine. Mycoplasma infections in goats are economicall:-
important and can rcsult in discasc of cpizootic propor-
tion. Mycoplasrna rnycoides ssp. rnycoides (largc colony var:-
ant) infectio11s present as a mastitis, pneu1nonia, bursitis
or arthrltls in adult a1l11nals. Sorne does develop a genera1-
izecl toxi<· disease th at can be fat;11. A rapid ly frit;il sep-
ticen1ia is co1nn1on in kids. Those that su rvive develop a
chronic, destructive arthritis and/or bursitis (fig 40.4 .
Mycoplasma mycoidcs ssp. capri causes a plcuropncumonia
similar to that of goat strains of M. rnycoides ssp. rnycoides.
It l1as been proposed that M. rnycoídes ssp. m)'COídes (largf:
coJony variant) be combined wit h Mycoptasma mycoides
ssp. capri as a single subspecies capri b;isi><l on thi> high
DNA hon1olc>gy they share. Septicemia, arthritis, and m as-
titis occur with M. capricolurn ssp. capricolurn infections.
Mycoplasma capricolurn ssp . capripncitrnoniac (formerly M/
coplasma sp. F-38) causes conta¡.,>ious caprine p leuropneu -
monia (CCPP), '"'hich is s.i.milar to CBPP in cattle. Both ~L
agalactiae and M. putrefaciens cause mastitis. ·r11e rnastifu
d ue to M . putrefacíens is purulent in n;i11 1ri>, whili> infPc-
lions will1 M. agalactiae rcsult in a decrease or total cessa-
tion in 1nilk production. Both species can cause arthritis.
Mycoplas11·1a conjunctivae causes a kcratoconjunctivitis th ar
p resents with lacrimatton, conjunctival tlyperemia, anc
keratitis. Paru1us is sometim.e s evident.
Equine. Mycoplasrna felis is the 011ly species that has been
~ulidly (:l~~ulici leu vvill1 lli~ed~e ü1 ll1t:: 1 1u1~1::: . IL i~ 1e1...uve1t::tl
from tl1e upper respiratory tract as a commensal b ut can
ca use a p leuritis, usually related to soJnc cxcrt.ional activ-
ity. 'ltle p leuritis is selt-limiting and trequently resolves
spontaneously.
Feline. A variety of comme11sal n1ycoplasmas have been re-
<.:overe<.l IroII1 ruucusc1l :surface:s uf cat:s. Relc1tively few art
associated with d iscase. -,.,1ycoplasma gatae has heen reco~·-
F1G U RE 4 O. 4 . Bursit is in a Z-mont h-old goat kid. The bursa is
distended with an amher-cnlnred fluid anda white fihrinous mate-
rial. Mycopl asma mycoides subsp. mycoid es (larg e co/ony varíant)
was recovered. (Courtesy Dr. M. Anderson.)

ered trom cats with arthritis. Mycoplasn1a fe/is has been as-
sociated with a serous to mucoid conjunctivitis. Typically
the con¡unctiva is edematous; however, the cornea is not
M-ivolved. A mycoplasma-like organism has been associ-
aled wilh subcuLaneous absces:ses, bul neilhe1 Lile disease
nor the organism has been well characterized. Mycoplasma
5pp. have bce11 isolated as part of thc polymicrobial com-
?Onent trom pyothorax exudates.
•.1urine. Mycoplasma pulmonis causes a low-grade respira-
-nry disease in rats. Infections involve the nasal cavity,
::niddle ear, larynx, trachea, and lungs. Thc most commo11
clinical sign is a low-pitched wheezing or snuffling result-
.ng from thc purulcnt nasal cxudatc. In micc, clinical signs
are often inapparent, although a chattering sound and
continued rubbing of the cycs and nosc may suggest infec
tton ln the colony. Mortality is low and when lt occurs is
-platcd to pneumonia. G·enital tract infections with M. pul-
"lOnis are also recog11ized ü1 rats. Mycoplasrna arthritiúis
causes a polyarthritis in rats and mice, altl1ough many in-
·ections are subclinicaL Experimental infections in micc
-esult in 101nt swelhng and, in sorne cases, posterior paral-
:·sis. Natural infections with M. n eurolyticum generally do
.iul L"au:.c tliscasc, altl1uugh L"unjunLLiVitis has been re-
;x>rted. Experimental inoc:ulation with M. nP.urnlyf1r.un1 or
cell-free filtrates causes a neu.rologic syndrome referred to
as rolling discase.
Jvine. Compared to otber ruminant spccics, mycoplasma
~1fections in sheep are not as frequent or devast ating.
.f1 coplasrna ovipneumoniae is associatcd with pneumonia
1
.md usually in conjunction with other cornmon bacteria!
• "1Lllvb1.:11:> vf 1111.: vvl111:: lt::>püalv1y 11a1...l. Oulurt:ak:> uf kt:r-
_toco njunctivitis have been attributed to M. conjunctivae.
...galactic mastitis caused by M. agalactiac is similar to that
bserved in goats. Sheep can also be intectcd with many ot
:..'1e oth er species that affect goats.
;orcine. A number of important clinical entities are associ-
.,.¡e::u willJ 111yL"uJJlasrna i11ft:L"liu11s in swint:. T11ft:L"Lit>IJS with
J. hyopneumoniae presentas a chronic respiratory disease,
;eferred to as cnzootic pncu1nonía (Pig 40.5). 'I'here is high
"""'lOrbidity b ut low mortality. ·r11e principal clínica! sign is
_ chronic nonproductivc cough. J\ífcctcd pigs appcar un-
~hnfty and have retarded growth. Mycoplasrna hyorhinis
.auses a S)rstemic infection in pigs between 3 and 10 weeks
- f age. Inilial signs include feve1, iuavJJeLeuce, aud lislle::~-
-ess. Swelling of the joints and lameness frequently fol-
. "-. Thcre is a characteristic polyserositis that involves
..., eural, peritoneal, and pericardial serosa. Synovial mem-
.::-r:i.nes are also affected. Chronic infcctions rcsult in de
...-:eased weight gain. Mycoplasma hyosynoviae causes arthri-
·is in growing pigs 12 to 24 weeks of age. Lameness and
~ocialed difficully wiLh ru obilily are ll 1e JJri11L"ipal L"liní-
._al signs.
~:iidemiology
'le prlmary source of most of the pathogenic mollicutes
the host that they infect. Introductio n of an infected an-
---ial in loan u11i11fecled populaLi011 a1.:L"u u11ls fur disseuli-
Chapter 40 Mollicutes 245
Enzootic pneumonia in a pig caused by
F 1G U R E 4 O. 5 .
Mycoplasma hyopneumoniae. (Courtesy Dr. M. Anderson.)
•
i1atlon of infection. Asymptomatic carriers, usually colo-
nized on mucosa! surfaces, serve as thc source for main-
taining organisrns i11 lhe puvulaliu11. Yuu11g a1ú1uals are
very susceptible to infection and generally develop n1orP
scvcrc discasc than adult animals. As a rule animal path-
ogens are not considered to havc zoonotic potential.
lmmunologic Aspects
lmmune Mechanisms in Pathogenesis
'fhe immune response ofthe host is intimatcly involved in
tlle partiugenests uf t11sease. consequences of borh an ac-
tivP humoral ano CP.l111lar imm11nP Tf'~pon~f', '1S \<VPll ;is im-
ffiUilOSuppressive effects of the pathogen itself, are in-
volved in the pathogenic process.
Sorne mollicutcs havc bccn shown to posscss nonspc-
cific mitogenic properties and are able to induce a poly-
clonal B lymphocyte stilnulation to a variety of antigens,
lncludlng host antlgens. Mycoplas1na arrhriditis possesses a
sm;ill peptide that acts as a supcrantigen and stimulates a
broad population ofT lyn1phocytcs. Thc rcsulling produc-
tion of various cytokines and intlammatory reaction are
detrimental to the host. Shared host and mycoplas1na
antigens, such as the galactans found in thc lungs of cattle
and in 111. tnycoídes ssp. n1ycoidas, can result in autol.cnmunc
dlscasc. Mycoplasmas actívate the complement cascadc by
the classical pathway, which contributes to the inflamma-
lury rt::.¡.iu11:;e::. Wllil~ u~n~fiL"ial in controlling infectlons,
the inflammatory rp_<;ponsP can rP~11lt in <lamage to by-
stondcr host ccll:;. T'crsistcncc of antigen in selected $Ítc.:11
such as joints, allows tor turther damage due to develop-
ment of an imn1une cornplcx mediated inflammatory re-
sponse. Induction of interlcukin l (IL-1), IL-6 and tun1or
1
necrosis factor (TNF) from activated macrophages by
n1any 111ycoplasn1as leads lo acli valiur 1 uf L"ytutuxiL" ·r ly1n-
phocytes and results in an endotoxin-like effect.
Si1pprcssion or lack of the host immunc cesponse is im-
portant in allowing tor persistence and avoidance of recog-
nition by the host. A number of Mycoplasn1a specics havc
been shown to decrease phagocytic actiVIty of neutrophils
246 PART IT Bacteria a11d Fu11gi
and macrophages. Proposect mechanisms lnclude decreas-
ing the respiratory burst (M. Liovis) or decreasing phagocy-
Losis as a resull of capsule produclion (lvJ. dispar) . Anligens
sl1ared between Mycoplasma species and the host tissues
lnay rcsult in a biological i11imicry, v•hcreby the 11ost rec-
ognizes the mycoplasma as selt, leading to persistent ilúec-
tions. Antigenic variabil.lty is a11other mechanis1n em-
ployed to evade J1ost defenses. Tl1e pMGA ge11e family,
\~rhich accounts for 16% of the entire genome of 1 \ 1. gal-
liseplh.urn, geueraLes auligeujc va1 ia11 ts o.f a ruajor sui-face
protein . Incorporation of host antigens by mycoplasmas, a
condition referred to as capping, further aids sorne molli-
cutes in escaping detection by the immune system.
Mechanisms of Resistance and Recovery
Age, environmental conditions, ge11etlc predlsposit1011,
cro,Mding, and concurrent ü1fections are ali .i nvolved in
contri!JutiI1g tu re:;i:;tauce Lo ü1 (ecliou or lack Lhereof.
Minimizing predisposing stresses will minimize disease.
Sorne of the lnnate host mechanisms for protection, such
as tl1e n1ucociliary escalator system in the respiratory tract,
are lmportant in preventing colonization.
'fhe chronlcity of 1nollicute infections suggests that the
immune response is not very effective at controlling infec-
tion once established. Mycoplasmas stirnulate a 11urnoral
rt>sp<Hl!\P ;ind .~pf'cific antibodif's can hf' df'monstratf'd.
Antíbodies have been shown to enhance clearancc. Cellu-
lar immunity is also recognized, althougl1 less is knovv11
about it. In the facc of an inte11sc i11flammatory response,
ho,~rever, m ycoplasmas generally appear able to avoid
elimil1ation.
Artificial lmmunization
Vaccination is e1nployed to control so1ne mycoplasma dis-
cases. A11 attenuated vaccine is used to protect cattle i11
a.reas "''here CBPP is e11zootic. Protectio11 lasts for approxi-
mately 18 mo11ths. Attenuated and killed, adjuvanted vac-
ci ncs have been used with variable success to control sorne
capríne infections, specifícally those caused by M . agalac-
liae, M . mycuides ssp. capri, and 1\!J.. caprilulun1 s:;p. caprip-
neumnniae. J nactivated vaccines afford sorne protection for
swine against i11fection '-vith Ñf. hyopneu111oniae and M . hy-
orhinis. In poultry. live and inactivated vaccines are em -
ployed to control egg production losses and respiratory
disease associated with M. gallisepticu1n infections.
Laboratory Diagnosis
Sample Collection
The appropriate sample for isolation attempt is determffied
by the clinical presentation and includes exudates, swabs
from affcctcd si tes, affccted tissues, and milk. 'fl1e ear ca11als
of goats can be sampled to detect inapparent carriers.
Because of tl1e niollicutes' fast idious nature, samples
should be submitted to the laboratory a:; suur1 a:; pussiule
after collection . D11ring transportation, samplf's shonlcl he
kept cool and n1oist. Various cornmercially available media
(Stuart's and Amies' without cl1arcoaJ) are suitable for
transportit1g swabs. If a prolonged transport time (greater
Lhan 24 ho urs) is expected, ::;CJ111ples sliulLld be sliipved
frozen and preferably on dry ice or in liquid nitrogen.
Direct Examination
The variability in microscopic n1orphology and poor
staíning with t h e Gram method make direct examination
fo1 1.uost 11101Jicutes u1uewarding. Direcl fluorescei1l anLi-
body tests and DNA fluorochrome staining have bee11 de-
scribed, particularly for diag11osing conjunctiviti.s and
mastitis, but t11ey are not vvictely used.
lsolation
No one n1edia forn1ulat ion is suitable for growth of all of
the mollicutes. ·rhe media selected sl1ould be based on the
specific species or group of spccics of intcrcst. In general, a
tairly complex inedia is require<.1. Serum is the usual source
of sterols a11d is required by most species. Differe11t species,
I1owever, grow better with different sources of seru1n. The
exception is tl1e Acholeplasma, \.vhích have the ability to
:;yr1thesiz.e tl1eir uw11 fatty acids and Lherefore do nol re-
quire exogenous sterols. Yeast extract is also included as a
source of growth factors . Growth of sorne species is en -
hanced or requires incorporation of specific substances
such as vaginal mucus (M. agalactiae) and nícotinamide
ade11ine dinucleotide (M. synoviae). Sorne goat mycoplas-
mas groiv on sl1eep blood agar as sn1all colonies and an
alpha-type ("green111g") hemolysis (Fig 40.6). Penici!Un.
thallium acetate, and amphotericin. B are commonly
added to n1ed ia to inhibit contan1inating bacteria and
.f un gi. Specific immu.ne sera directed against commensal
mycoplasmas can be incorporatcd in media to allow for sc-
lecttve isolation for pathogenic species. For optimal reco,--
F1G U R E 4 O. 6 . Growth of Mycopl asma mycoi des subsp.
mycoides (large colony variant) from a goat on a sheep blood
agarlMacConkey agar biplate. The sheep b/ood agar is in the lower
ha/f of the p/ate. Tinv mvcoplasma colonies are evident. No growth
is present on the MacConkey agar (upper half). This is one of the
few mycoplasmas tha t grows sufficiently on sheep blood agar to
produce co/onies that are small but are still visible to the naked eye.

ery, samples are lnoculateú 111to lJotl1 a llqul<.l a11d solld
media and inc.uhated at 37ºC in 5o/o to 10% C02 for at least
7 days. Sorne species require longer incubation times.
Semen and joint fluids may contain inhibitory factors and
should be diluted prior to cttlture to enhance recovery.
Blind passages from broth to broth for up to three passages
may enhance the recovery of poultry pathogens. Urea-
plasrna species are susceptible to pH cl1C1uges as CJ result of
hydrolysis of urea included in the media and must he suh-
cultured frequently to n1aintain their viability when isola-
tion attempts are made.
ldentification
11 1.l<.: ro-
Pla le 111elli<t i:s exa111i11ell willt Ll1e a ill uf a lli::;::;ecll11g
scope. Colonies with the typical l1mbonate morphology
are stai.ncd dircctly witb the f)icn,es stai11 to differentiat e
them from other bacteria (Fig 40.7). ·rhe mollicutes stain
blue beca use of their inability to reduce methylene blue in
The stain. Other bacteria red uce methylene blue by using it
as a hydrogen acceptor iI1 maltose oxidation and therefore
appear colodess with tl1e Die11es staü1. The excepl.ions a1e
L-form bacteria, which exhibit a similar colo11y morphol-
ogy and staining rcaction as tl1c mollicutcs. L-forms must
be different iated fron1 mollicutes by demonstrating rever-
si.on of the L-form bacteria back to a walled form .
Digitonin sensitivity is used to distinguish i\1ycoplasma
and [lreaplasma from Arholeplasma. A large zone of inhibi-
tion around paper disk:; salurated will1 l.5% digilonin will
be present with Mycoplasma and Ureaplasma but only a
small or no zone is observed with Acholeplas1na. Com-
monly used f)iochem ical tests to further characterize iso-
Iates include detection of phosphatase activity, fermenta
n on of glucose, and hydrolysis of arginine or urea.
Definitive identificatio11 is based on reactivitywith spe-
t.i fic antisera. A nurnber uf rnetl1ot1:; are ernployed lJCJseu
on eithP.r the ;ibility of spi>rifir antisi>rri to inhihit growth
or n1etaboli.sn1 or t he den1onstration of reactivity with a
specific antisera using either a fluorescence or chrornogen-
bascd dctcction systcm. Growth inhibition tests cmploy
f 1G u RE 4 o. 7 . Díenes-stained mycop/asma co/onies wíth
dense/y staining centers and /ighter staining peripheries (75X).
Cf1u11Ler 40 Mollic,ules 247
F1G U RE 4 O. 8. lmmunoperoxidase staining method used f or
typing mycoplasma isolates directly on agar media. Two different
types of Mycoplasn1a are present in the sa1nple. One type (M.
bovirhinisJ stains when anti-Mycoplasma bovirhinis antisera is used
(darkly stained colonies); the other Mycoplasma isolate does not
react wit h that antisera.
•
antisera impregnatcd disks or antiscra p laced in wclls in
media and demonstrating a zone of inhibition. Metabolic
inhibition tests use growth inhibltio11 in liquid media and
a color change based on pH asan .indicator system. Other
ti>st prorerlures com m.only used to demonstrate specific rc-
activity are direct or indirect in1n1u11oiluoresce11ce on
colony impressions, colony epitluorescence, and immu-
nopcroxidasc staining of colonics on agur platcs (Fig 40.8).
Nonserologic methods for species identification using
polymerase chain reaction (PCR) have recently been de-
scribed. Amplification of spedfic DNA sequences by PCR
and restriction endonuclease analysis of PCR products
11ave bee11 used for idenlificalion and characlerizal.io11 of
isolates. Randomly amplified polymorphic DNA analysis
l1as been used for strain differentiation. llestriction en-
donuclease analysis, protein electrophoresis, and ribotyp-
ing have also been used to further characterize strains.
DNA sequencing of selected genes, especially the gene en-
coding the 16S rRN.A., is becoming more important as a
Lool fo1 Laxono1nic place111e11L of Myc,uplu:srna specie:;.
Antibiotics susceptibility testing of clinical isolates is
not routinely performed. No standardized method or ap-
propriate control organisms have been described.
Nonculture Detection Methods
J1n111 uuoperuxi<.lCJ:;e a11<.l irr1rr1uuufluurescent staining of
histopathologic sec.tions has heen 11sed suc.c.i>ssfully for
.identification of sorne species in tissues, including M. bovis
in cattle t issues, M. hyopneu.moniae in pig lungs, and sorne
of the poultry mycoplasmas.
A number of PCR methods have been described fo r
identification of pathogenic species directly from clinical
1naLerial. Poly111erCJse cl1aiI1 reaction tests for direct detec-
tion of sorne~ of the pathogi>nir poultry mycoplasm as are
commercially available.
248 PAnT II Bacteria and Fungi
lmmunodiagnosis
.A nurnber of imrnunodíagnostic tests have been t1eve1oped
for many of the impottant mycoplasma diseascs. Ma11y
have not been standardized and are not in '-v1de use . Prob-
lems with sP.nsitivity, PspPci;illy with ;isymptomatic carri -
ers, are common. Lack of specificity as a result of cross-
reacting antibodies is also a problem.
Enzyrnc-linkcd immunosorbcnt assays (ELISAs), platc
agglutination, and llemagglutination inllibition tests are
routinely used to detect flock infections vvith A1. gallisep-
ticum, l.ví. rneleagridis, and M. synoviae in poultry and are a11
importéu1t part of overall eradication progran1s used by
con1n1ercial poultty operations. Con1n1ercially available
tests are available for poultry.
Treatment, Control, and Prevention
Success of treatment varíes depending on the species in-
volved, Lhe affecled sile, and lin1e course of tl1e d.isease.
Alt hough the mollicutes are susceptible to a 11umber of an-
tibiotics in vitro, treatment failures are common. Con1-
monly used antibiotics include tctracyclines, tylosin,
erythromycin, lincomycin, spectinomycin, a11d tilmi-
cosin. Resistance to sorne of these a11timicrobials, particu-
larly of the i11acrolide class, has been noted. Animals that
uo (t'.;)¡JUlld LO Lrt'.alUlt'.llL oflt'.JI l.Jt'.C:Ulllt'. carrit'.C:>.
Control measures depend on the disease status of the
country, specific disease, and animal species infected.
Diseases such as CBPP and CCPP, which affect large popu-
lations of animals, are controlled by test and slaughter of
affected herds in countries attempting to maintain a clis-
ease free status. Vaccination, culling of infected animals,
a11d 1Hauage111t'.u l cha11gt'.:s tu µrt:vt:11t ui;):Sl.'.11li11atiu11 are
employed in countries where the disease is enzootic. Tn
general, because of thc poor succcss in trcating infcctcd
anítnals, culling of clinically ill animals is often employed
as a control measure in infected populations where test
and slaugllter is not feasible. Industry-driven efforts, par-
t icularly in the poultry h1dustry, have outlined measures
Lo elilniuaLe or prevelll i11fecLio11. ,<\Lle111¡;t:, tu erauicatc iu-
fections, particularly in breeding flocks, indude serologic
Table 40.2. Animal Species, Agents, ;incl Dise;i<;es Asso(iatecl with Haemotrophk
Mycoplo5mo
Animal Species Agent
Cat !v!ycop/asma haemofe/is
M. haemomínutum
Cattle M, wen.yonii
Oog 1.4. haemocanís
Llama M. haemolamae
Mice M. haemurís
Opossurn M. haemodídelhidis
Sheep M. haemovis
Swíne M. haemosuís
tcsting and eli1nination of positive flocks and anlibiolic
treatment of hatching eggs to produce mycoplasma-free
chicks. Trcat1nent of eggs involves immersing warmed
eggs in a chillect antibiotic solution, which promotcs an-
tibiotic penetration in to the egg. Routine culturing of bulk
tanks is used to monitor for mammary infections in co'i\·
and goat herds. Animals identified as shedcling organisms
111 ll1e n1ilk are usually culled.
Preventing infection should be based on following
stJ·ict biosccurity practiccs to prccludc introduction of ln-
fected animals into a mycoplasma-free herd. Ne•v animals
should be quarantined and tested before being mixed
'\Nitl1 the herd. 'laking animals to shows and fairs at1d re-
turning them to the herd n1ay also serve as a so urce fo.r tn-
troducit1g infeclio11. 'fl1is is a11 especially con1111011 sce-
11ario for outbreaks i11 goat herds. Good hygiene and
managcmcnt practi.ccs are important in p rcvcnting
spread among animals \.Yhere infections are eI1zootic.
Because n1ilk can be a source of infection, especially in
goats, it sllould be pasteurized to prevent infecting young
animals in thc hcrd .
THE HAEMOTROPHIC MOLLICUTES
T·he haemotrophic mollicutes include microorganisms
pteviously iucluded in Lhe genera Haernobartonella and
Eperythrozoon (Table 40.2). All are parasites of red blood
cells, and produce hen1olytic anemia in the young, or
stressed individuals (Fig 40.9). Subclinically infected ani-
1nals are the source for bae1noplas111as. Several members of
thiS group are transmitted by ectoparasites. Unlike t lle
nonhaemotrophic n10Ilicutes, they have not been culti-
v<1ted u11 lifeless rnellia.
Infections witl1 this group of microorganisms are usu -
ally asymptomatic. Howcvcr, following a strcssful cvcnt
(e.g., concurrent infections with FI V or J'eLV in éats in-
fected with M, hacnzofclis or M. hacn1orninut11rn) sometin1es
leads to the develop1uent of clinically apparent hemolyt ic
anemia.
Taft:ctiurrs witl1 tlre l1<1eu1o¡;la:;u1a:s uiay caust: ictt'.ru:;,
splenomegaly, and hone-marrovv hyperplasia.
Cotnmon Cfinical Ma1Jif&$tatiims
Anemia (Feline lntectious Anemia)
Anemia
Anemia
Anemia
Anemia
Anemia
Anemia
/\nemia
 Chaptcr 40 Mollicutcs 249

;:: 1GU RE 4 O. 9. Mycoplasma haemofelis on feline erythro-

:ytes. Note cocea/ and ring forms. Wright's stain, 1OOOX.

Diagnosis is based upon clinical signs, dernonstration fication using DNA prin1ers and the polymerase chaln
Jf the agent in stained (a Romanovsky-type stain, e.g. 1 reaction.
-.,-Tight's or Giemsa) srnears of peripheral blood, or de- ·r reatment includes correction of the tlemolytic ane-
:~on of genus and species-specific DNA. following ampli- 1nia1 and one of the tetracyclines.
 Rickettsiae: Rickettsia,
Coxiella, and Orientia
]ANET E. FOLEY
DWIGHT C. HJRSH
Members of the order Rickcttsialcs are minute obligate in
tracelJular gran1-negative bacteria. ·rhey are basically para-
sites of arthropods. Two families, Rickettsiaceae and
Anaplasmataceae, within this order are of veterinary inter-
est. The family Rickettsiaceae, which includes parasites of
Liie vascula1 e11d0Lheliun1 (Rickettsia, Co.xiella., a11d
()rientia), commonly referred to as "rickettsiae,'' and para-
::;ite::; of phagocytic cells (Ehrlichia and Neorickettsia), com-
monly referred to as "ehrlichiae." ·rhe family Anaplasma-
taceae, cornposed of the genus Anaplasrna, parasitizes
erythrocytes, pl1agocytes, and platelets.
·rhc el1rlichiae (Ehrlichia and Neorickettsia) are discussed
i11 Cl1a¡.itcr 4 2, a11d 1ue1uuer~ of tl1e fau1ily A11apli:!~rna ­
tac:eae are disc:11ssect in C'.hapter 4:i. 'rhis chapter dPals with
thc rickctt::;iac: Rickettsia, Coxíella, aod Orientia, tl1ougl1
only those associated with anin1al disease are discussed ü1
d.etail (Rickettsitz ricketl'sii, the cause of Rocky 1v:Countain
spotted fever il1 dogs and sheep, a11d c·oxiella burnerti, the
c;111s1~ of Q ff'ver in run1inants.
Descriptive Features
Rickf'ttsiaf' mf'as11re 11p to 0 ..5 pm by 1.0 pm. Although
structurally gran1-negative, better visualiz:ation is achieved
with Gimenez, Maccl1iavello, or Giemsa stai11s. 'l'he forn1er
two stail1 r.ickettsiae red, tl1e latter purple. Electrol1 micro-
scopically a11d chen1ically1 rickcttsiae resen1ble other
gram-negative bacteria. E11dotoxin is present (see Chapters
7 and 8). Cells are nonmotile. The life cycle of Coxiella bur-
netii includes an endospore-like phase. Rickettsiae entf'r
endothelial cells through endocytosis actively initiated by
1netabolizing rickettsial cells. ·rhcy escape fron1 the pl1ago-
so1ne and n1ultiply in the cytoplasm and, in sorne cases,
the nucleus.
Rickettsiae are propagated in yolk sacs of chick em-
bryos, cell cultures, and laboratory animals, notably
guinea pigs or micP. ThP optima! tPmpPrah1rP fnr growth is
33ºC to 35º<:, at which the generation time is about 9
hours. ln cell culture, good growth mayreqliire ir1cubation
for severa! weeks, depending on the rickettsial species
i11volved.
Glutan1ate is a key nutrient for rickettsiae, which utilize
250
ERNST L. BIBERSTEIN
it with the aid of transaminases, del1ydrogenases, and en-
zymes of the citric acid cycle. There is llttle glycolytic actív-
ity. Rickettsial adenosine diphosphate (ADP) is readily ex-
cl1anged for host ade11osine triph<>sphate (A:rP) acro~s cell.
membranes. 'fhis "leakiness" of ric:kettsiaf' ca11sf's loss of
crilic.al n1etabolites, in.Ecctivity, and viability when agents
are removed to extracellular sites, but is probably an adap-
tatio11 to intracellular existencc rathcr than its cause,
which is unknow11. Within hosts, Coxiella burnetii is an ob-
lígate intracellular bacteriu.tn, but it survives extracellu-
larly in the environment for months, formalin, ultraviolet
radiation, classical pasteurization at 63ºC for 30 minutes,
a11<l ucca~ioually even "flasl1" ¡.iastt:urizatio11 (71.ó"C for 10
to 20 sec:oncts).
Many mernbers of the rickettsiae are associated ~vi tl1 in-
vertebrate vectors, and sorne can be passed transovarially
in tick!; and mi tes. The pathogenic and ecologic relatio11
ships of important rickettsiae are shown in Table 41 .J .
·r11ere are three of veterinary interest: R. rickettsii belonging
to the spotted fever group, R. fe/is il1 the lyphus group, and
C. burnetii. Rickettsia felis is inc.luded bec:ause preliminar)
research suggests tl1at it is an important small-animal de-
rived zoonosis, although it is not pathogenic for cats.
RICKETTSIA RICKETTSll
Ecology
Reservoir and Transmission
'fhe agent of Rock]' Mountain spotted fcver (RMSF).
Rickettsia rickettsii infects dogs (and people) in endemic
areas. It is carried naturally by some 20 species of ixodid
ticks, including Dermacentor andersoni (the wood tick) a11d
D. varia.bilis (the American dog tick) in western North
America anc.1 D. variubili.s in tl1e easter11 ¡.iorlio11:> of Norllt
Amf'rica. !t is passe<l transovarially in ticks. Serologic sur-
veys suggest that most canine RMSF infections go unno-
ticed. (.)tl1er tick-borne diseases (ehrlichiosis, babesiosis.
borreliosis) should be considered along with RMSF in tick-
infested dogs. RMSF is limitcd t o the New W<>rlc!. Cas5
occur most commonly (and are increasing in prevalence
 Chapter 41 Rlckettsiae: Rickettsia, Coxtella, and Orientia 251

Table 41.1. some Dlseases due to Rlckertslae: Agents, Reservoirs, veaors, and Horu

Disease Causative Agent Vertebrate Reservoir Vectors Transovarian Passage Oinically Affected Hosts

Epidemic typhus
Classlcal Rickertsla prowazekli Humans (recovered) Uce Huma ns
Sylvatic R. prowazekii Eastem flying squirrel~ FIP~~ Huma ns
Endemic (murine) typhus R. typhi Rats, opossums, cats fleas Hu11 1d11~

Endemic typhus-like R. fe/is (ELB aqent) Opossums, cats Fleas Humaris

Scrub typhus Orientia tsutsugamushi Mice, rats Mitcs + Huma ns
Rocky Mountaín sponed fever, R. ricketrsii Various feral mammals TICks + Humaris (dogs, sheep)"
Q fevl'r Coxie/la bumetii Mammals. birds, fish? Ticks, mites, insectsl> 7 Humans (ruminants)

• Occasionallv affected.
b Airborne transmission commonly ocrurs in absence of vectors..

in castcrn North Amcrica. Scasonal incidcnce parallels
adult tick activity: lJ. andersoni is active in spring and early
summcr, whilc D. variabilis is active from midspring to late
sw11111cr. Tlu.: rcscrvoir for RMSF is smaJI mammals, which
constitute a primary sylvatic: c:yclP with immature ticks.
?athogenesis
Rickettsia ríckertsii replicates in the epithehum of the tick,
and is transfcrred to salivary glands and ovarían tissue.
..\ftcr tl1e tk:k bites a suitable host, R. rickettsii Is injected
anci targPts vasr11lar i>ndothelium, where they are endocy-
tosed, escape the phagoson1e, and n1ultiply in cell cylo-
;ilasrn and n ucleus. Rickettsial phospholipase and pro-
teascs damagc cndothclial ccll mcmbrancs, lcading to
:iecrosis, vasculitis, hemorrhage, edema, perfusion inade-
quacies, thrombosis, and dyspnea. Clinically, the infec-
=ion 1s rarely fatal, wtth affecrea aogs presenting wttn hJgh
:~er (40ºC), anorexia, vomiting, diarrhea, petecchiated or
ecchy111vliL 111uLou:. 111e1111Jra11es, a11u le11ucr11ess ovcr
.:-mph nodes, joints, and muscles. Purpuric and central
~ervous disturbances may occur later. These, and heart
::.'ld kidney involvement, account for most fatalities. Un-
;ortunately, scvere nec.r osis of extremitics may occur whcn
·ne dog appears to be in convalescence.
:nmunologic Aspects
•:rimune complexes are suspected in thc pathogenesis of
-~e vascular man ifestations of RMSF. Humoral and cell
-edlatcd responses occur. The Janer especlally are signifi-
nt far rcmoval of the agents by activated macrophages.
o vaccine is available for RMSF.
_aboratory Diagnosis
me rickettslal antigens, including those of R. rickettsii,
-.,,.,_rpart \vith somatic antigens of certain Proteus (OX)
-ains, a phenon1enon (Weil-Felix reaclio11) ulilizeu iu tl1c
::.:3gnosis of rickettsiaJ infections. This approach generally
'-Cks spccics spccificity. Indircct fluorescent antibody (FA)
and enzymc-linked imn1unosorbcnt assay (ELISA) are
most commonly performed to detecl TgG specific for rick-
ettsial antigens. Negative or Jow titers may occur carly in
disease. Rlckensiae can be demonstrated in cells by direct
immunofluorescence. Polymerase chain reaction-based
tests a1c available Lo evaluate for R. rickellsii DNA in tlcks
and dogs.
Treatment and Control
Rickerrsía rickettsii is susceptible to chlocamphenicol, tetra-
cyclines, and fluoroquinolones. Aífccted dogs must receive
aggre:>:>ivc supportive therapy and posslbly steroids. Pre-
vention of RM.SF in ciogs rPquirP<; tirk <:ontrol. Transmis-
sio11 depends on prolonged feedi11g IJy liLk:. auu 111ay lJe re·
duccd by prompt tick removal.
(OX IELLA BURNETll
Ecology
Reservoir and Transmission
'fhe agent of Q fever, Coxiella burnetii, is distributed world
wlde. lt survives in the environment, unlike other rick-
ettsiae, and can be disseminatcd by the airbome route.
Survival 1nay be 1elaLed Lo Llie ":.111all-cdl-variant," an
endosporc-like growth phase. Natural hosts an<l vec:tors in .
elude sorne 125 mammalian spccics and many spedes of
arthropods, including ticks, mites, tleas, lice, and tlies,
sorne of which support a sylvatic cycle of C. burnetii.
Recent re:search suggests that the organlsm can occur in
amoPhaP anrl crayfish, which may be an important mech-
anism of environn1el1tal persisle11ce, and a source (vr iu-
fection of humans and other animals.
Most hun1an cases of Q fcvcr can be traced to exposure
to infected sheep, cattle, and goats, either directly or via
unpasteurized milk products. Outbreaks have occurred
aftcr wtnd currcnts carried infecttous "spores" from in-
ft>rtPci ciairies. Cattle and small r uminants can shed the
bacteria in feccs, spern1, ai1d u:vrvuuLtive discharges.
252 PA.lrr 11 ilnctcria and fungi
Parturient cats a11d dogs 11avc becn in1plicated as sources of
hun1an infection.
Coxicl/(I burnctii has bccn wcaponizcd, and is consid-
ered an important agent of possible biological terrorism.
Pathogenesis
Coxiella bur11etii is acquired through inhalation, ingestion,
or arthropod bite and gains access to vascular endothe-
lium and rcspiiatory and renal cpithclia. Coxiella bun1etii
multiplies within the phagosome, thanl<s to an enzvme
system adapted to low pH (5.0). After hematogenous dis-
sem l natlon, 50ºA> of humans are asymptomatic or have
mild, self-limiting infection. Less t h an 10% develop severe
<.liscasc ~vi t lt vasculi lis, µ11cu11 1u11 ilis, sµleno111egal y, fever,
and lymphocytosis. In animals, the pattern may be simi-
lar, although mildly affccted animals may have latent in-
fection . persisting particularly in the lactating mammary
glar1d and the pregnant uterus. Latent ínfection ís reactí-
vated during parturition: sporadic abortion may result
from placentitis or thc delivery may be normal and pro-
duce viaule yuu11g. Iu t:!itl1er case.:, rickcttsiae are auun-
rl;:intly <;hPrl . 'l'hP f)íltho logic lrsion in animals w ith mild or
self-limited disease is a granuloma; people and animals
with c11ronic active infcction fail to contain the b acteria in
a granuloma and thcrc are abundan t bacteria •-vith severely
vacuolared macrophagcs. Mortality in human cases is due
to pneu1nonia, hepatic infection, or endocarditis.
In culture, C. burnetii transitions from a virulent phase I
(resists m;:icroph;:igp killing ;:inri m11ltipliPS slo\.vly) typP to
an avirulent phase ll. Phase 11 bacteria have altered expres-
sion of cell wall lipopolysaccharide, do not occur in na-
ture, and are killcd by macrophnges.
lmmunologic Aspects
A phase 11 vaccine is available in sorne countries but has re-
portcd poor cfficacy. A whole cell killed vaccine is given to
rescarcl1ers. Thcrc is considerable interest in thc dcvclop-
ment of a phase T Q rever vacc1ne, whicl1 has proven effec-
tíve experimentally in rum inants and hu1nans.
Laboratory Diagnosis
G1menez or other sta1ns on suspect tissues \Vill not differ-
entiate between C. burnetii and Chlarnydoph ila psittm..-.
Coxiella burnetii can be isulated fulluwing ir1jectiun in:
guinea pigs and mice, embryonated eggs, or cell culturt
but cells dissociate to phase 11. In additíon, the agent :_
considered dangerous to laboratory personnel. l'aradox.-
cally, cven though animnls are infected only with phase.
bacteria, titers of ant1bod1cs to phase I antigen in acute;-
infected people are low, while antibodies to phase II anti-
gens ~hvw a IélJ,.JÍd fvu1fo ld increase. lgM a11libodie
against phase I do occur. If the infection becomes chronic
levels of antibodies to phase 1 becomc ir1creased. This pat-
tern may occur in animals, but long-term observations o.
acutcly and chronically infectcd ani1nals of various species
are scarce. lndlrcct 1m1nunofluorescence, complen1ent fu..-
ation, or El.ISA tests are used to detect antibodies. In ceL
culture, eggs, and experirnental a1liII1al li:.sui::s, direcl iu1·
munofhH)rt>stPnt st;iinin g will idcntify an isolate. Poly-
merase chain reaction-bascd assays employing specific
DNA primees are also used to docun1ent infections witr
thís agcnt.
Treatment and Control
·rhe major challenge for succcssful therapy is that the
phagosome where C. burnetii persists is highly acidic, and
most antimicrobial agents are not effective at such lo"-
pHs. AlkaJinizing cells by using chloroquine has been
shown to improve clinical efficacy of tetracycline. Drugs
with modcratc efficacy for C. burnetii infectlon include
tctracyclinc, chloramphenicol, clarithromycin, enrofloxa-
cin, ru1<.l Lri111i::tltuµri111-sulfa. Lu11g-Len11 lelrdcycli11e feeu-
ing has been used in attempts to control mammary excre-
tion of C. bur11etii, which has met with mixed results.
Pregnant sheep and goats n1ay be treated with tetracycline
to reduce the probability of abortion.
 Ehrlichiae: Elzrlichia and
Neorickettsia
]ANET E. FOLEY
DWIGHT C . l-lIRSJ-J
Descriptive Features
\1.ernbers of the order Rickettsiales are 1ninute obligate in-
ttacellular gra1n-negative bacteria. They are basicaHy para-
:.i te::; of arthropods. 'CWo families (Rickettsiaceae and
Anaplasmatar.eae) w ithin this order are of veterinary inter-
est. The family Ricket-tsiaceae, which iI1cludes vara::>ite::; of
the vascular endothelium (Rickettsia, Coxiella, and Orien-
tia), commonly referrcd to as "rickcttsiae," and parasites of
phagocytic cells (Ehrlichia and 1Veorickettsia), commonly
referred to as "ehrlichiae." 'J'he family Anaplasmataceae,
composed of the genus Anaplasma, parasitizes erythro-
cytes, phagocytes, and p latelets.
T11e rickellsiae a1e discu~::;ed in Cl1apter 41, and 1nem-
bers of the family Anaplasmataceae are disc.nssr<l in
Chaptcr 43. Discussed in this chapter are the ehrlichiae:
Ehrlichia and J'Jeorickettsia.
Ehrlichiae are white blood cell parasitcs that multiply
•vithin membrane-lined intracytoplasmic vesicles. Colo-
nies (morulae) less than 4 µm in diamcter that consist of
~elerne11tary uodie::;" smaller than l µm in diameter are
demonstrahlf~ hy examination of Giemsa-stained blood
smears (Pig 42. l) or immunofluorescence. A cell wall is
present.
The ehrlichiae are comprised of E. canis, E. chaffccnsís, E.
ei.vingii, E. (formerly Cowdria) ruminantium, Neorickettsia
helminthoeca, N. (formerly Ehrlichia) risticií, a11d N. (for-
rnerly Ehrlichiu) sennelsu. Ehrlichia equi has been reclassi-
fied as Anaplasma phagncytnphilum (see Chapter 43).
J:hrlichia canis, 1!,. chaffeensis, N. risticii, N. scnnetsu, and E.
n1tninantiu1n have been propagated in cell culture, which
simulates host conditions in vitro. Thc tick-transmittcd
species (E. canis, E. cha{feensiS, E. eivingii, and t:. ru1ninan-
-i11m) are passed transstadially, but not transovarially,
\vithin ll1e vecLors. Bluuu 1nay rernain inJectious for 10
days at room ten1perature, 14 days at rrfrigerator tempera-
:urc, and 1.5 years if frozen .
ERNST L. BIBERSTEIN
t:/
-.. r
EHRLICH/A CANIS, f. CHAFFEENSIS, ANO f. EWINGI/ r
Ecology
Reservoir and Transmission
Thc brown dog tick, Rhipicepltalus s1~.ngui11eus, is lhe vector uf
E. canis, t11e agent of canine monocytic ehrlichiosis.
Although dogs and other can id hosts of E. canis muy rernain
bacteremic for years, their blood is infectious for ticks gener-
ally far 2 weeks or less during the ac..ute disease. Puppies and
Gern1an shepherd dogs are most severely affected. 'fhe infec-
tion is concentrated in tropiral and subtropical latitudes, but
occurs on ali continents except Australia. Tl1e vecL01 of E.
ewingii, the agent of one fonn of canine and human granu-
locytic ehrlichiosis (but distinct from granulocytic "ehrli-
ChiOsis" caused by Anaplasma phagocytophilum) is Arnb/yom-
ma americanum, the lone star tick. E. eivingii appears most
cuuuno11ly in central North America although there are re-
ports from c.oasta 1a reas of North America as \"7ell. Although
a few rare cases of hun1an n101i.ocytic el1rlichiosis 111ay !Je uue
to E. canis infection, n1ost are dueto infection with R. chaf-
(eensis, which is closcly rclatcd gcnctically and serologically
to E. canis. Its vector is LJermacentor variabi/is, the American
dog tick, and its ma\nmaliar1 reservoir m.ay be deer.
Pathogenesis
Each of these ehrlichiae is inoculated into its host hy tir k
bite. J\cute onsct of discasc follovvs an incubation period of
l to 3 vveeks, during which the agent proliferates by binary
fission vvit hin monocytes (or granuJocytes in the case of E.
ewingii). In severe acute disease, lasting up to severa! weeks,
there may be vasculitis and thrombocytopenia. lntravas-
cular coagulation n1ay occur, aud leukoper1ia anc.! anemla
are common. CUnically, there may he fevrr, n1alaise, de-
pression, inappctcnce, v•eight loss, pale n1ucous n1en1-
branes, lymphadenopathy, and joint pain (the latt er par-
ticularly in E. ewingii infection in dogs and people).
E. ewingii and E. chaffcensis occasionally progress to
chronir or severe disease in humans, with polyarthritis,
CNS disturba11ce, and vulluuuary infiltrates. Canlne
rnonocytic ehrlichiosis appears uniquP. in that affected
253
254 PARl 11 Béi<.:terié1 a11u Fu11gi
dogs may entera late11t :stagc a11u a fi11al ch1orlic pl1asc. In
thl' ch rnnir ph;ise, whirh may occur months to years after
quicscent infection, reduction of ali blood ccll typcs may
accompany 11emorrhagcs (commonly epistaxis), edema,
serosa! cffusions, dyspnea, interstitial pneumonia, ane-
mia, secondary Lnfect1ons, and enlarge1nent of spleen,
liver, and lyn1ph nodes. Hyperglobulinc1nla, glomerulon-
ephrltls, and witlespread plasrna <.:ell il1flllréili011 suggesL
immunopathogen ic mf'rh;:inisms.
lmmunologic Aspects
Progression to Iatent and chron1c can1ne ehrhchiosis oc-
curs more frequently in dogs '"'ith genetic predispositions
and possibly impaired cell-mediatetl lmmunity. Lateut iu-
fection appears to reside in thP spli>i>n. Although hyper-
gan1n1aglobulil1en1ia usually is polyclonal, several cases of
monoclonal ga1nmopathy have been reported, suggcsting
almost malignant transformation of B lymphocyte clones
antl/or suppression of n1ost oll1cr clones.
Cellular and hurnoral imn1une-mediated responses to
infectcd mo11onuclear cells anti ]Jlatelet:s are sus¡;e<.:teu Lu
contribute to bloncl cf'll c1f'str11rtion and hone marrow de-
pression, polyarthritis, and uveitis; mcrlingiti:s may be due
to irnmunc complex deposition. Resistancc to reinfection
is antibody and ccll-mcdiatcd.
Laboratory Diagnosis
Gic rnsa-stained sxnears of buffy co;it <irP ex;imined for in-
lract:llular n1orulae. 'fl1c:sc are scarce and found mainly
during the acute stage.
The agents of hun1an and caninc monocytic ehrlichio
sis may be cultured in canine cell lines.
Serodiagnosis by indirect immunoOuorescence may de-
tcct antibodies to these ehrllchlae, anu pulyrnera:se 1.:liai11
rcaction testing (utilizing specifi<' ONA primers) is recom-
1uc11ueu Lu delect active infcction in blood.
F 1G u RE 4 z. 1 . Neorickettsla helm inthoeto lo/ony (11101 u!d,
arrow) in canine lymph node impression. Giemsa stain, TOOOX.
Treatment and Control
·retracycllne ls eJfective in early 111u11ucytic el11lichiosis bu.
less so irr adv;i nrPn rasP.~. 1m idocarh dipropionate has alsc
1Jcc11 re1.:u111111ended. Lale-stage monocytic ehrlicl1iosis has
a poor prognosis and is 1nanaged with doxycycline anc
stcroids.
For prevention, tick controJ is imperative. People w1th
rcccnt tick b ites receive doxycyclil1e prophylactically for
10 days.
fH RLICHIA RUMINANTIUM ANO f. OVIS
Ecology
Reservoir and Transmission
"African heartwater disease," caused by E. ruminantium, at-
fccts mostly ruminants. The disease is passed only by par-
cnteral 1ntroduction of blood. Tlck vectors are Amblyornrna
spp. Strains of E. ruminanti11111, though serologically 11ni-
form1 vary in viruleu<.:e. TIJe i11fecllon is endemic in tropi-
cal Afric-;i ;:incl p;irts of thc Caribbean.
Elirlichia avis parasitizes bovine or ovlnc mononuclcar
cells, and E. ondiri also invades granulocytes. ·rhe latter
causes bovine petechial fcvcr (Ondiri disease) in the high-
lands of East Africa, a disease marl<cd by fever, lowereu
milk yield, mucosa! hemorrhages, and v;:iri<ihlP P<irly mor-
t<ility. Artl1ru¡;ull vecL01s are assun1ed.
Pathogenesis
Ehr/ichia n11ninantium multipliP_<; in cells lining the sinu-
soids of lymph nodes, disscminates to the bloodstrcrun, and
colonizes endothelial cells, particularly cerebrocortical cap-
illarics. 1'hcir prcscnce induces little cellular inflammatory
 Chupter 42
response, bue does rcsult In wtdcsprcad vasculitis with effu-
-;ion an<I C'pitht>lial an<I Pn<lotht>lial hemorrhage. Pericardial
effusion, v.•hich gave the disease its nan1e, is inconsisle11l.
Affcctcd animals also may develop encephalitis. Splecn,
lymph nodcs, and usually livcr are grossly cnlargcd.
r:Unical signs vary. In the peracute form, a tever ot sev-
era! hours' duration precedes collapse and death under
cunvulslons. In the acure forrn, rever occurs follo\-ved
\Vithin hours hy c1ish1rhancpo; including hyperexcitability,
muscle tremors, ataxia, deficits in conscious propriocep-
tion, head pressing, coma, and seizurcs. Dcath within 2 to
10 days is the rule in sheep and likely in cattlc. A subclinical
form is aJso recognizecL Mortality in sheep is trom 6- 80%.
Little is known about the pathogcncsis of Ondiri
disease.
lmmunologic Aspects
Ncwborn anin1als a11d so111c cattlc lJreetls are relatively re-
sistan! to Ji. rurnínantium.
rn surviving cattle, the agent disappears within 2
months alter recovery, but cell-rnediatcd immunity per-
sists for up to 5 years.
Laboratory Diagnosis
Hcartwater rnay he diagnosed by dcmonstration of thc
agent in a Giemsa-stained smear of cerebral cortex. ln-
fected cerebral extracts ,..,.¡n agglulinate with specific anti-
1.Jutly. Polymerase chalo reactlon-bascd testing utilizing
<;pPcific 1)NA primPrs c::;in doc:ument the presence of E. r11-
tnina11tit1111 DNA in t he sample.
Treatment and Control
Tetracycllne treatrnent is effective if the disease is treated
early. Tick control anrl vaccin;ition 11re important preven-
tivc :stcp.s. Young calves and lani.bs (..:4 vveeks) or gotlls ( <:6
\veeks) a re vaccinated with virulent rickettsiae. Older ani-
mals may be infected and then treated w ith antimicrobial
drugs, thus developing immunity. Heartwater is exotic in
'-'orth America although cornmon in thc Caribbean .
.\t11blyo111rr1a licks have beeu i11 lroúuccú iutu North
.\merica a numbcr of times on imported wildlife, inclu<l-
.ng ungulatcs and reptiles. In 1999, leopard tortoises being
~ported through rlorida were inlcstcd with A. sparsun1
:hat were test-positive for E. runrinantiutn.
NEORICKETTSIA RIST/CI/ ANO NEOR/CKETTS/A SENNETSU
Ecology
~ese rvoir and Transmission
.'otomac horsc fcver (PIIV), an acute equinc dia11heal sy11-
drome first noted in North American horses in Mont-
~omery County, Maryland, is a monocytic ehrlichiosis
Ellrlicfliae: Eflrlicfzia and Neorickettsia 255
causcd by N. ristidi. Neorickettsia ristidi is acquired orally
probahly in association with fluke metacercariae in water.
'fhe i11Ler111eúialc 11ust uf tl1c fluke is a snail, alrhough thc
full range of possihle sna il spc-ciPs as~ociatt>rl with N. risticii
has not heen describcd. Thc gcographic clistribution of N.
rísticií includes .m ost of North America. Dogs and cats can
be experimentally infectcd w ith N. rislicii. Scnnctsu fcvcr
(N. sennetsu) is a disease in Malaysla and Ja pan that targets
human monocytes. Little is known of its ecology.
Pathogenesis
Thc PHV agent infects equine monocytes, intestinal ep-
ithclium, and colonic mast ce lis. Clinically, PHV resemblcs
ehrhch1oses with fever, listlessness, anorexia, variable leu-
kopenia, diarrhea, and, as a late complication, laminitis.
U tcline infeclio11 11a:> lJcc11 ulJservetl. The fatallty rate of
cases with diarrhea is 20o/o to 10'M>. l Jlct>r11tivP g;istroenteri-
tis is thc most notable lesion at necropsy.
lmmunologic Aspects
I1nn1unologic faclors art: uot fully understood. Antibodles
to N. risticii cross-react with N. $ennet~u, hnt not with Ana-
plas111a phagocytophilum (formerly E. equi). Recovered
horses are immune.
Laboratory Diagnosis
Microbiologic diagnosis involvP.s clt>mon.~trati on of the
agcnt in monocytes in Wrigh t-staincd blood smears.
!)crologic diagnosis is by an indirect fluorescent antibody
test or by cnzyme linked immunosorbcnt assay (ELISA).
Neorickerrsia risridi DNA can be detected by the polymerase
chain rcaction.
Treatment and Control
'letracycline, if given early, has bccn crcditcd with rcduc-
ing n1ortality to under iorKl. Supportive care also is irnpor-
tant. J\n effective bacteri11 is commercially available.
NEORICKETTSIA HELMINTHOECA ANO ELOKIMIN
FLUKE FEVER
Descriptive Features
ThP agt>nt of salmon "poisoning," Neorickettsia heln1in-
thoeca, infects lyn1pl1orellcula1 lissut:s of canids. Neurickell-
sia helnlinthoeca multiplies in the cytoplasn1 of macro-
phages, frequcntly forming multiple morulae and filling
the en tire cell. Individual neoricketlsiac may be dispersed
through the cytoplasm. ·rhey are coccobacilli measuring
u:;ually less than 0.5 µm in any dlmension and are demon-
strahlt> hy Gie1nsa stain. Neorickettsía helnúnthoeca has been
propagated in ccll cultu1e.
256 PART 11 13acteria and Fungi
Ecology
Reservoir and Transmission
'fhe resC'rvoir of f\I. hPlrninthnPra is th<' fluke, NannJ>hyetus
sa/111i11cola, "vhosc lifc cyclc includcs passage through a fish
and a snail. ·rhc gcographical distribution ot salmon poi-
soning is limited by the range of the snail intermediate
hosts ot N. saltnincola (Oxyrre111a silícula) and includes
coastal areas of North America (northernmost California,
Oregon, Washington, anti Britlsh Columbia). Infections in
nonPndPmic arp;i<; ilTP ch1P to planting of inff'ci"f'c1 fish.
Dogs (a5 well as bears, raccoons, and other animals) are the
definitive hosts of the fluke, harboring the adults in their
intcstinc, and are infcctcd by N. hclmh1thocca by cating
tluke-i ntected tish . t<luke eggs are shed in canine feces, em-
bryonat<.!, and hatch m iracid ia that invade tl1e s11ail.
Cercaría euH::rge fru1n tllt! sua.il au u iuvade a fi.~11 1 1Jecu111-
ing sPssilP rnE'tacc>rc::iria C'ncysted in the kidney. Upon
being ingcstcd by thc dcñnitive host, they become adults
within 6 days. Uninfcctcd flukes produce little disturbance
i11 dogs.
El0Kom1n lluKc tcvcr, a mtl<.1 form ot sa1mon ct1sease, at-
tacks canids, fcrrcts, raccoons, and bears.
Pathogenesls
From the ingested fish kidney, metacercarial flukes emerge
in the dog's intestine, develop into adults, attach, a11d rc-
lease into the tissues 1V. l1ehni11t/1oeca, \vhich colonizes
Iy1nphoreticular tissues throughout the body. Enlarge
ment of lymphoid organs rcsults from proliferation of re-
ticuloendothelial components. A nonsuppurative menin-
gueuceplialili:. i:. Lu111111u11.
Withln 5 tlays to several weeks of exposure, tlogs de-
vf'lop fPvPr, ;inorc>xi;i, c1Pprf' ~sion , wf'ight loss, swollen
lymph nodes, and often hemorrhagic enteritis. Vomiting
and persistent, eventually hemorrhagic, diarrhea are typi-
cal. Death occurs in up to 90% of untreated cases within 2
weel<s.
lmmunologic Aspects
lJogs recovered from salmon disease are immune. The
agcnt docs not induce immunity to the closely related, epi-
dem10Iog1cally 1dent1ca1 Elokimin fluke fever agent.
Laboratory Diagnosis
Presence oí eggs ot 1V. saln1incola in the teces of a dog shov.--
ing pcrtiner1t clinical signs constitut es strong evidence of
salmon discase, although many affected dogs do not shed
fluke eggs in feces. Demonstration of rickettsiae in Wright-
stained lymph node aspirares is tlefinitivc (see Fig 42.1).
Treatment and Control
Pen1c1111n G, tetracycline, chloramphenícol, and sulfon-
amides given parenterally are usually effective. Supportive
rreatment ts essentlal.
The best p reven ti ve measure is the exclusion of infected
:.al111u11 fru111 llu.: ca11i11e tliel. Flukes aud N . helrninthoecu
are killed hy cooking and hy freezing for 24 hours.
 Anaplasmataceae
}ANE'l' E. FOI~EY
embers of the ordcr Rickettsiales are minute obligate in-
-3cellular gram-negativc bnctcria. Thcy are basically para-
·•es of arthropods. Two families with in this order are of
i::terina ry interest. Thc family Anaplasmataceae, com-
J~t:ll of the genus Anaplas1na, parasitizes erythrocytes,
-hagocytcs- and platf'lf'ts . 1'he family Rickettsiacene, which
.;:eludes parasite::; of vascular endotheliun1 (ge11e1a Ri1.--
.ettsia, <..:oxiella, and Orientia). commonly refcrred to as
~ickettsiae," and parasites of phagocytic cclls (genera
- '1rlichia and iVeorickettsia), commonly referred to as
Phrlichiac."
1'he chrlichiae ar e lli=>~u~~ctl ir1 Chapter 42 and the rick-
.::ttsiae are discussed in C:haptr.r 41. l)isc11ssed in this chap -
:er are mcmbcrs of the genus Anaplasnza.
1·he genus Anaplasrna includes the species Anaplasma
'71arginale, /\. centra/e, A. ovis, A. caudatu1n, A. (formerly
Ehrlic/1ia) bovis, A. (formerly lif1rlichia) platys, and A. (for-
-nerly Ehrlichin) phnsocrtophilum . .4nnplasn1ntnceae para
51tiz;e erylh1ocyles, µlalclct~, and granulocytcs In severa!
1..lasses of vertehrates anrl may rause anemias. The genera
Aegyplianella, Tlaen1obar/011ella, and Eperytl11 oz.oon !Jav<::
been reevaluatcd phygenetically and are classed currently
\Vith the family Mycoplasmntnccac (Chaptcr 40).
Of the erythrocyte-infecting species, only A. rnarginale
is a significant pathogcn. Anaplasrna avis rarcly causes
anaplasmosis of sheep and Is infective for goats and deer.
4naplasrna caudaturn, which has a tail-Hke appendage, has
been sccn in bovine infeclions alu11g=>idc A. rnurginale. Its
significancc is uncertain. Anaplasrna centra/e, a l~o fo11n rl
in cnttlc, is of interest asan immunizing agcnt against A .
margina te.
1'hc agents of human granulocytic ehrlichiosis (previ-
ously unnamed), eqtúne ehrliChiOsis (prcviously Ehrlichia
equi), ;ind ehrlichiosis of n1minants (previously E. phagocy-
topl1ila) have been con1bined inLo a :-i 11glc ~vecies, A .
phagocytophilurn .
ANAPLASMA MARG/NALE
Descriptive Features
In Giemsa-staincd blood smcars, A . rnarginale appears as
purple structurcs (l pm in diameter) near the periphery of
erythrocytes (Fig 43.1). 1'hcse marginal (inclusion) hodies
art: 111c111urane-lined vacuolcs containing up to 10 initial
bodies (400 nm i>arh) Pnrloscd in a two-layered inem-
F.RNST T~ . BIB f.l~S'I'EIN
hranf'. Tnitial bodies contain DNA and RNA but lack cell
walls. The orga1lisn1 has IJt:t:JJ serially propagated in a tick
cell culture. It requires an oxygPnatf'<l atmosphcre, is cata-
lasc-positivc, and utilizes exogenous an1ino acids.
Ecology
Reservoir and Transmission
lnfected ruminants are the reservoi r of A. n1arginale.
Although m.any species can b e infcctcd, few regularly de-
vclop disease. So1ne, such as deer, 1nay be nat urally in-
fc~cted and serve as a source of bovine anaplasmosis. Thc
infection occurs 011 ali ~011tinen ts. Transmission is by par-
enteral introduction of infected blood, in nature probably
most often by ticks and blood-sucki11g ílyi11g insects, but
also bv contaminated instruments, transplaccntal, and
conjunctival exposurc.
Pathogenesis
Tnitial bodies enter crythrocytes by cndocytosi:; aftcr ad-
herlng to the cell surfacc by v.1ay of ma¡or surface p rotein-
1 (M-;p-1) and multiply by binary fission within thc endo-
some. Ncw ir1ilial bodie=> ar<:: releasetl from the crythrocyte
surface, possibly to contiguou.~ cf'lls, \Vitho11t d isr.crnible
ccll damage. Erythrocytc destruction occurs following an
immune response to parasitizcd erythrocytes, resulting in
indiscriminate erythrocyte ren1oval by thc macrophage
sysrem. Anemia, icterus, b1le stasis, splenomegaly, and hc-
patomegaly follow. 'fhere is little intravascular hemolysis
and no hen1oglobi11uria.
Tllncsses, following incubation pf'riod-; of up to 5 weeks,
rangc from subclinical to peracutely fatal. Signs include
anemia, tever, anorexia, depression, weakness, constipa-
tion, and abortion. Severity oftcn varics dircctly with age
and duration from hours to weeks. In mature cattle (>3
ycars), m o rtality may reach 50%. Disease is generally seen
in ca l lle 1 year uf agc or older. Long-tcrm carriage occurs in
ticks.
lmmunologic Factors
Humoral and cell-mediatcd responses dcvclop afler i11ít:~­
tion with A. rnargina/e. Anaplasma ntarginale cxpresses at
least five Msps, all of which display antigcnic variation due
257
258 PART JI Bacteria an<.l Fungi
in largc port to the polymorphic nature of the encoding
msp genes. The antigenic varlatlon may explai11 tht:
chronic naturc of anaplasmosis Antihnrly ri>spnn.~e is di-
rectcd also to host antiger1s, au<.l µlay:s a lole i.n patl1ogen-
esis Tmm11nity following recovery may not be permanent.
Natural ir1fection in calves is subclinical and confcrs rc-
sistance to subseque11t cxposure. It may produce im medi-
atc or dclaycd cli.nical c.lisease in the individual and may
spread to other stock, requiring vector control. Resistance
of calves outlasts maternal antibody and can be termi-
natect by splenectom y.
lnfcction with A. r.mtralt', ri>sulting in mild disease, in-
duces resistance to A . 1nargi11ale. Two commercial vacci.ncs
are available for A . tnargínale. An inactivated A . mar~nale
vaccinc produces immunity of severa! months' duration
subject to rc1nforcen1ent through natural cxposure. The
erythrocyte constituents of thc inactivatec.l vaccine ac-
cou n t fo r occasional imrnu11u!Jer11ulylic problen1s il1
11conat;.il c;.ilvi>s n11rsing in1muni7.ed an imals. A modificd
livc va<.:cine also is produccd and induces lifelong im mu-
nity. 1lowever, this vaccine is contraindicatcd in pregnant
cows and cattlc older than 21 months.
Laboratory Diagnosis
Anaplasn1a rnarginale is dcmonstrable by routine blood
statns, acridtne orange, or immunofluorescence. The latter
is most specific and sensitive. Acridinc orange produces
nonspcclflc stalnlng of nuclelc act<.ls, whereas ruutine
blood stains may not dctect inff'rtion hi>yoncl tht> first ft>w
weeks. Molecular tcch11i.ques su<.:h as the polymerase chain
reaction utilizü1g spccific DNA primers has been devel-
oped a11d are vcry sensitivc.
Scrologic methods include complement fixation, cap-
illary agglutination, radioimmunoassay, enzyme-)jnked
immunosorbent assay (ELJSA), and a carc.l agglutinatiun
t est for field use All are useh1l in df'ti>rting s11hrliniral
cases.
~ 1<.;u K E 4 3 . 1 . A naplasma marginale in bovine e1yt/11otytt:!>.
Wright's stain, 1OOOX.
Treat ment <lnd Control
Tctracycline is effective against A. marginale. Clinical Casé.
are treated by parenteral therapy, whereas the carrier sra:
is treated by feed medicatio n for up to 2 months. VacciP~
tlon and vector control are helpful.
A NAPLAS MA PHAG OCYTOPHILUM AN O A. PLATYS
Descriptive Features
In Giemsa-stained blood smcars, Anaplasma phagnq-
µltilurr1 avpears as n1en1brane-bound morulae consisting
1 to 10 bacteria within ncutrophils and, Jess common:
monocytes of rumi.nants, horscs, dogs, and humans. 11.n.::
plasma platys occurs on the surface of canJne platelets ar-_
causes a cyclic throm bocytopenia.
Anaplasma phagocycophilum has been seri<tlly .(JfUP"·
gated in a tick cell culture ilnc1 hnm;in neutrophilic leuk
111ia cells. ll is aerobic, catalase-posJtive, and utilizes exo •
nous amino acids.
Ecology
Reservoir and Transmission
Tht: lt:se1 voil l1osls of A. phagocytopliilurn include rodents
and possibly larger wildlifc species such as coyotes and
deer. Ecology varíes significantly in diffcrcnt gcographical
regions, with the primary cycle in eastern North An1erica
involving the white-footed mouse (Peromrscus Ieukopus)
and the deer tick (Ixodes scaputaris). In Europe, the maln
vector is l. ricinus, and reservoirs include a variety of ro-
uer 1t:s. 111 Wt:Sler11 Norlh A.111e.rica, ll1e vector to humans
and veterinary patients is thc western black-legged tick
(/. pacificus), although a rodcnt-spccific tick, l. spini
 C1zapter 43 Anaplas1nataceae 259

;.1/pis, may be an important vector in the sylvatic cycle.

:>onkeys, sheep, goats, dogs, cats, and monkeys can be ex- F 1G U RE 4 3. 2 . Anaplasma phagocytophilum (formerly
~rimentally lnfected. [hrlichia equiJ (arrows) in smear of buffy coat from horse. Wright's
The reservoirs and vectors of A . platys are not known. sta1n, 1UUUX.

~athogenesis

!,11aplasrna phaxocytopflilurn occurs in granulocytes and,

ess commonly, monocytes primarily of ruminants in
•
l:.uropc (whcrc lt Is called "tick-borne fcve r," TBF) and
"orses, dogs, and humans in North America . Ali hosts de-
-elop vasculitis associated with thromboses, thrombocy-
:npenia, edema, and hemorrhage, especially in the distal
..mbs. Vascular changes are pronounced in testes and
varíes of ruminants. Leukopenia successively affects lym-
;)hoeytes and neutrophils. Lesions includc splenomegaly 1
.i;1d hernorrhages along tl 1e intestinal tract. Histologically,
::. striking l;:ic:k of ly1nphoid el<~ments is ohsPrVPcl.
Clinically, there is fever accompanicd by depression, ac-
.::elerated breathing, inappetence, hemorrhage, edema,
a.'1emia, icterus, and ataxia (especially in horses), and, in
co•vs, a clrop 1n milk yield. Ewes and cows may abort. 'fhe
.:!isease is mildest in young animals. A heightened suscep-
_!Jili ty to :.ccoudary iufec1:ior1s u1ay IJt: <.Jut' to parasirtza-
:::on of neutrophils, leukopenia, anda lymphopeni;:i t1fft>ct-
.:1g 13 Jymphocytes. Tick pyem.ia (due to Stap11ylococcus Laboratory Diagnosis
~lfection) is commonly linked with TBF. Fatalities are due
orare secondary complications, such as respiratory infec- Morulae in neutrophils can he demonstrated in Wright-
.:ons or Iamini tís. stained blood and buffv coat film s most readily 48 hours
Dogs infected with A . plat}'S develop cyclic thrombo- after onset (Fig 43.2). Índirect immunofluorescent anti-
;:ytopcnla vJa lmmune-mediatcd dcstructlon,. possibly body (11:-'A) and ELISA tests have been developed using in-
ccompanied by mild fever, anci secondary 1mmune- fectcd equine neutrophils and human Ieukemia cells for
:nediatcd discasc such as uveitis. thc If.A and recombinant antigens for the ELISA. 1"he poly-
1111.:ra!>e cl1ai11 reactiou (utilizlug :spt:<.:ificall y <.Jesignt'<.l DNA
prímcrs) is uscd to detect the microorgt1nisms heforf' ;:inti-
mmunologic Aspects bodies are produced.
lndividual A . plat}'s organisms can be visualized on
:,.s for A. n1argi11ale, there is antigcnic strain variability in- Wright-staincd platclets of infcctcd dogs. Prcvious cxpo-
·-olving mainly the major surface protcins, although the sure can be documented by IFA .
..:levaJ1cc in A. phu;sulyLuphilulft ir 1ft:clio11 is u11 k11uwn.
-:Oere is sorne serological cross-reactivity with Ehrlichia
spp. although animals with A . pl1agocytophilu1n in fec- Treatment
tion usually have higher specitic, compared with cross-
reacting, titcrs. Recovered animals are refractory to reinfec Tetracycline is effective. ·rhe first dose can he given intra-
:ion, although rccurring illnesses have been reported in vcnously followcd by daily doses intravcnously or orally
successive years. Recovery occurs after a peak in produc- for l wcck. Reduction of exposure to ticks is desirable .
..:on iu i11le1 fero11 gan11na. No vacciues exisl.
Splenectomy can reactivate or exacerbate A. platys-
associated thrombocytopenia.
 44 Bartonellaceae
BRUNO B. CIIOMEL
Members of the Family Bartonellaceae are small gram-
ncgative rods. Until recently, the genus Rartonel/a con-
slstcd of only one species, ll. bulilli(urrni~ . The gen us now
contains :.ill thf' "PPCiPs thatwerc once included in the gen-
c r<1 Bartonella, Rochalirnaea, <1nd Graha1nella. I'he ge11us
Barto11ella, has been placed into the tamily Bartonel/aceae
(which hos olso been removed from thc order Ríckattsia/e.~) .
Members of the genus Barto11ella belong to the alpha-2
subgroup of the alpha-proteobactcria . Most of thesP h;:irtf'-
ria are erytluucyte-adhe1e11L bacilli.
·rhc present family con'\i'\t'\ of morr than ZO species or
subspecies, of which ni ne are human pathogens:
1. As:>u1..:iatcu w ill1 tli:>ea:>t:: i11 l1 uma11 patients.
/lartnnella hacilliforrnis is the etiologic agent of
Oroya fever, an acu te bacteremic infcction charac-
terized by sepsis and hemolysis, and ot verruga pe-
ruana, mainly a cutaneous nodular vascular erup-
tion reprcse n ting chronic infection.
Bartonel/a quintana, the agent of trench fever, has
also been founc.l to be une uf tl1c agc11t~ uf 1.Jacillary
::ingiom;:itosis (BA), ;:i vascular proliferative lesion
observed i n immunocompromised individuals,
mainly with acquired immunodeticiency syndrome
(A!DS). Bacillary angiomatosis can also be causcd by
!3. /1e11selae.
Bartonella henselae causes cat scratch disease
(CSD) in immunocompetent indivil.lual~.
Rartnnr.lla elizahethaP is associated wit h endo-
carditis in ünmunoco rnpctent patients .
Bartonella vinsonii subsp. /Jerkl1oftii and H. washo-
e11sis are associat ed with endocarditis or 1nyocarditis.
Bartonella grahatnii has been associated \~1 ith neu-
roretinitis.
Bartonella vinsonii sulJ:>p. iirupe11:>i:> was isolated
from tl1e blood of ;:i r::inrhPr \Vith fever and rnild
11eurological symptoms.
Bartonella clarridgeiae is suspected to also be a
minor agent of cat scratch discasc.
2. Associated with a11imal patients. sorne of the
Btirtonl•llo species th<it <1 r1>p:ith0gPnir tn h11m;.ins (R.
VÍllSOrtii subsp. berkhoffli, B. clarrhlgeiae, n. henselac,
B. eliLabethae and B. washoe11sis) have recently bcen
associated with various clinical entities, including
endocarditis, in domestic dogs.
Severa! other Bartonella species- such as B. vin-
sonii subsp. vi11sonii, B. aoshiae, B. taylorii, B. peror11y-
sci, B. birtlesii, B. tribocor11n1, B. alsatica, B . talpae, R .
koehlerae, JJ. buvi:;, B. ~tliuc11/Juchensis, and B.
260
R ICI<IE w . KASTEN
capreoli- have only IJeeu isulatt:tl fron1 Lhe blood of
vario11s ;:inim;:il spPriC's, including various wild ro-
dents, squirrels, rabbi ts, fclids, canids, bovids, and
cervids. At present, these species are not .known to
induce any spccific disease in the infected an imal.
Descriptive Features
Morphology and Staining
Bartonellaceae art! fa:>titliuu~,
ae1obic, sl1ort, pleomorphic
er;:im-npg;:itivP rorrohacillary or bacillary rods (0.6 µm br
1.0 µm) that takc from S to 15 days and up to 45 days on
primary culture to form visible colonies on enrichec..
blood-containing medi•t, as they are highly hem in-
depender1t. In infected tissues, warthin-Starry silver im-
pregnation staiI1 reveals s1nall bacilli, which te11d to ap-
pear as c lumps of tightly co1npacted organisrns. Siulilarl}
sma11 organisms can be identified in rPc1 hlooci rells h;
May-Grü11wald Gien1sa coloration. Bartonellae have a
close evolutionary resemblance with members of the ger.-
era Bruce/la, Agrobactcriun1, and Rhizobium.
Cellular Composition
Bartonella bacillifonnis and B . clarridg<~iae :.irf' thf' only m f'r--
!)1::1:s uf Lhe ge11us that are 1notile by n1eans of unipolar f_
gella. Bartonella quintana and B. henselae have a twitch.ÍJ"' .
motility as:;ociatcd to fimbrioc o r pili. Because of their slo-
grO\>Vth, standard bioch emical methods for identificatio-
are notas useful for identification. The bartonellae are O."Ll-
dase and catalase-negativc. M1::11:>u1e111e11ls of prefor111 _
enzymes :.ind st::inci;:irci tf'sting have revealed differences be
tween species. Most species are biochemically inert excf"'."
for the production of peptidases. The MicroScan Ra;-
Anacrobc Panel (Baxter Diagnostics, Deerfield, IL) has tx._
reported to provid e spec1es identification . Whole cell fa:-
acld (Cf.A) analysis for the gcnus has proven useful for idt-
tlflcatiun b1::1..:aust: Burlunellii ha ve a unlque and character
tic w holP c:Pll fatty acid compositlon. The Bartonella h a:
gas-liquid chromatography fatty acid profiles consistí.:-_
mainly ot C 18 ,09 , C 18, 19, and C11'·0· Molecular genetic me~
ods such as restriction fragrnent length polymorph! -
(!U:Ll') of genes encoding citrare synthase, 165 riboso:-
RNA (rRNA) or 16S-23S rRNA spacer regían, and mor""
cently analysis 1.Jast:tl 011 polyn1erase cl1ain reaction r
of r;inclom, repetitive extragenic palindrornic seque.::
have been used to distinguish strains and spccic,
 Chapter 44 Bartoncllaceae 261

Epidemiology of Bartonella Species or Subspecies Presently Described

Bartonella sp. Reservoir Vector or Potential Vector Current Geographic Distribution

B. biici/liformis Huma ns Phcblotomincs (Lutzomyi~ verrucan;m) Andes (Pcru, Ecuador, Colombi¡i,
(sand tlies) Bolivia, Chile, Guatemala)
B. quintana HtJmans Human body lice (Pediculus humanis corporis) Worldwide
B. flensetae Cats (fefü catus) Fleas (C1enocepflalldes feHs) Ticks? worldwlde
B. clarridgeiae Cats (fe/is catus) Fleas (Ctenocepha/ides fe/is) Cosmopolite
B. koehlerae Cats (Fe/is catus) Fleas (Ctenocephalides fe/is) California, France
8. vinsonii subsp vinsonii Meadow voles (Microtus pennsylvanicus) Ear mites (Trombicula microt1)? Cana da
B. vinsonii subsp arupensis White-footed mice (Peromyscus leucopus) Fleas? ticks? USA (Midwest)
8. Vinsonli subsp berkhoffii Coyotes (canis Jarrans), dogs (Canis familiaris) Ticks? Cosmopolite
R talpae Moles (Tafpa europaea) ? United Kingdom
B. µe1u11Jy)ti Fielú lllÍle (Pl!tOllJ~lW )¡i¡i.) ? United States
B. birtlesii Wood mice {Apodemos spp.) )
France. United Kingdom
B. gr¡¡homii Bank voles (Clethrionomys glareolus) ? United Kingdom
B. taylorií Wood mice (Apodemus spp.) ? United Kingdom
8. doshfae Meadow vales (Microtus agrestis) 7 United Kingdom
B. ellzabethae Rats (Rattus norveglcus) fleas Worldwide
B. tribocorum Rats (RattuI norvegiru1) ? Cosmopolite
B. ~lsatica Rabbits (Oryctolagus cuniculus) fl"dS? Tilk~? Frartle
B. washoensis California ground squirrel (Spermophilus beechey1) Fleas? Ticks? Western USA
B. bovis Oomcstic c~ttlc (Sos tuurus) Biting flies? ticks? Cosmopolite
B. capreo/1 Roe deer (capreolus capreo/us) Bitinq tlies? Ticks? Europe
B. schoenbuchensis Roe deer {capreolus capreolus) Biting flies? Ticks? Europe

ijarfnnt'J/a. R;iT.P nr '>Pí}11Pn<'P ::in::ily<;i<; nf f)NA Pncnciing 16S Pat hO!JP.nesis

:R,'IA, c it ratc synthase genes after PCR an1plification both
directly from spec.:imens or pure cultures have been Jargely
•..ISed for detecting and characterizing Bartonella. More re
Lently, 1dcnt1flcation has also heen performed 'l·\lith the am-
?lification of the DNA encoding the 165-235 rRNA inter-
:;enic spaccr region (ITS) or protein-encotling genes. ·rhe
¿;enes most widely used are those enco<ling thP citrate syn-
thase (gltA), lhc hcal shock protein (groEL), the riboflavine
ribC), a cell division protein (ft~Z). anda 17kDa antigen.
Growth Characteristics
Tradltlonally, members of the genus Bartonclla are culti-
•-,¡ted in St'misolid nutrient ;ig:ir cont;:iining frf'Sh r::ibbit
~lood (or sheep or horse blood) at 3SºC (except for B. bacilli-
·onnis, which gTO\-VS best at 28ºC) in So/o C0 2 . On primary
isolation, sorne Bartoncll.a, such as B. ltcnsclac, B. clarri.dgciac,
B. vinson11, or l3. el1zabethae havc colonics with a white
:ough, dry, raiscd appcarancc and pit the mcdium. They are'
~ard to break up or transfer. Other bartonellae such as B.
quintann h::ivP colonif'S th::it ::irP 11sn::illy sm::i llf'r, gr:iy,
uanslucent, and somewhat gummy or slightly mucoid.
Ecology
Reservoir, Transmission, and Geographic Distribution.
~fost members of the genus Bartonella species are vector-
bo rne organisms. The reservoirs, vectors, and gcographic
d istribution are shown in Table 44.1.
A4echanisms. Bczrtonella quintana and B. henselae are clini
cally associated with proliferativc ncovascular Iesions. ·rhe
pathogcncsis of bacillary angiomatosis involves injury
a11u ¡.¡ruliferaliuu uf the va~cular e11uull1eliu1u l.Jutl1 ~vilh
R. hen.<;Plae and R. quintana. ThcsC' organisms induce en-
dothclial ccll proliferation and migration in vitro, a11d a
protcin fraction was idcntificd as thc angiogcnic factor.
Bartonclla infcction (in vitro) stimulatcd c11dothelial cell
pro! i Feration and induced obv1ous morr>hological changes
dueto modifications of the cytoskeleton. Bartonella hense-
lac has been shown to intluce infec.:ted cells to protluce vas-
cular Pn<lothPlial growth f;:ictnr, wh ich in tum stimulatP<I
the proliferation of endot!1elial cells and the growth of B.
l7enselae.
Bartonclla hcnsclac sccms to sharc with B. bacillifomiis a
common mcchanism for mediating pathogeneSis. A bacte-
riophage-like particle similar to thc bacteriophage ob-
served In B. bacilliformis has bccn found In culture super-
n::it::int from R. hr•115p/np This particle has at least thrce
associated proteins and contains 14 kbp Iil1ear DNA scg-
ments that are heterogeneous in sequen ce. Jt has been
spcculatcd that an anccstor of B. hcnsc/ac a11d B. bacilli-
forrnis acquirect the ability to m ediate angioproliteration as
a mea ns of enhancing it s dissemination or its acquisition
of nutrlcnts within the host. It Is possible that a common
tr::in~<l11cting phage may be thc mcchanism of genetic ex-
change by which the two organis1ns acquited tl1is patho-
genic trait.
Ali Bartoncl/a spccics multiply and persist in red blood
cells. J:Jartonella bacilliforrnis posscss polar tlagella that have
262 PART 11 Bacteria and Fungi
been shown to mediate erythrocyte acthesion. For non-
flagcllatcd Bartonella, bundlc-forming pili as well as sur-
face proteins may play a role In erythrocyte adhesion.
Until recently, mechanisms of persitence of Bartonella bac-
Le1enJia in 1ua1nn1als were 11ot well underslood . Recenl 1e-
ports have revealed the intraerythrocytic localization of
thcsc bacteria, which is a unique strategy for bacterial per-
sistencc. Nonhen1olytic intracellular colonization ot ery-
throcytcs would preserve the organisms for efficient vector
transm1ss1on, protect Bartonel/a from the host immune re-
sponse and contribute to decreased antimicrobial efficacy.
Per:.l:.te11cc uf i11fecliu11 ~va~ rece11lly ueu1u11;)l1dlt:u ii1 d tdl
moclrl 11~ing B. tribocon1n1. After a 5-day "hidden/silent"
phase follo~ving experimental infection, the organism
multiplied until there were an average of eight Bartonella
per red blood ccll. Thcrcaftcr, thc orgonisms ren1ained
within the cell tor the life of the erythrocyte. Nonbemo-
lytic intracellular colonization of erythrocytes is likely a
bacteria! pcrsistence strategy that preserves t he Bartonellu
spccics for potentiaJ transmission by arthropocls, hrcausc
thc rc:>crvuir liusl would serve as a source of infection for
hloocl-fe>rding arthropods, which could then subsequently
infect a new host.
Disease Patterns and Epidemiology
Human Patients. Cat Scratch Diseast'. Cat scratch diseasc
(CSD) i!) cau~ed !Jy Burlune/111 lien~elue. In <:so, 1 lo 3 wecks
Pl~psr brtwrrn the scratch or bite of a cat and the appear-
ance of clinical signs. In 50% of the cases, a small skin le-
sion, often resembling an inscct bite. appears at the i11ocu-
lation sitc, usually the hand or forearm, and evolves from
a papule to a ves1cle and partially healcd ulcers. These le-
sions resolve within a few days to a fcw weeks. Lympha-
tlenltls tlevelups appruxüuatel y 3 week=> dÍler exposu1e and
is grne>rally unilateral. It commonly appears in the epi-
trochlear, cixiUary, or cervical lymph nodes. 5,-velling of thc
lymph node is usually painful and persists tor severa!
wccks to severa! months. In 25% of the cases, suppuration
occurs. The large rnajority of the cases show signs of sys-
temic infection: fever, chills, rnalaise, anorexia, head-
aches. In ger1eral, tl1e djsca:>i;: i:> l.ie11ig11 a11d l1eals sponla-
nro11sly without sequelae. Atypical manifestations of CSD
occur in 5% to 10% of the cases. "fhc 1nost common of
thcsc is Parinaud's oculoglandular syndrome (periauricu-
lar lymphadenopathy and palpcbral conjunctivitis), but
also meningitis, encephalitis, ostcolytic lesions, and
thrombocytopenic purpura may occur. Encephalopathy is
ene of the mo:;t seriuu:; cu111plicalitH1:. o( CSD, wl1icl1 usu-
.:illy ocn1rs 2 to 6 weeks after the onset of lymphadenopa-
thy. l lo'<.vcvcr, it usually resolves with complete rccovcry
and few orno sequelae.
Thcre were an estimated 22,000 hun1an cases of CSD in
thc Unltetl Sta tes in 1992, sorne 2000 of whom were hospl-
talizcd. The estimated annual hcalth cost of CSD was n1ore
than $12 million. From 55o/o tu 80% uf CSD palienls are
undPr thP ;igr of 20 yrars. ·rhere is a seasonal pattern, with
n1ost ca~es seen in autumn and winter.
Ne'v clínica! presentations associated '\'ith H. henselae
infcction have been reported in immunocompetent per-
sons, 1nclud1ng neuroretinitis or bacteremia as a cause of
chronic tati}..'lle syndrome, anda case of aggressive H. /1f!' -
lae endocarditis in a cat owner. Bartonella henselae was a.
recently deterrnined as a fre4ue11t cau:.c uf vrulu11ged f~ •
and fever of unknow11 orig.in in children. Rh P1 1 m~tic mar
fcsla lions of Bartonella infection have been recently ...
scribed in cbjldren, including a case of myositis and a ca.
of arth.ritis and skin n odulcs. Arthritis has olso bccn d.
scribed in a very limited number of cases. Other rheuma· _
manifcstations related to Bartonella infection i_n humar
lnclude erythema nodosum, leukocytoclastic vas<.ul -
fever of unknO\'Vn origin with myalgi;i, ancl arthralgia.
E111Joi::u11iitis. Several Dartr.uH:lla spp. have also been =e-
ognized as causative agents of blood culture-negatiYe e
docarditis or myocarditis in humans, including B. hensel
B. quintana, H. elizabethae, 13. vinsoníi subsp. berkhoffti, an-_
B. washoensis. Bartonella spp. account for approximarc
3% of ali h uman cases of endocarditis, a percentage sim .~ -
to endocarditis cases cau sed by Cnxir~lla humelii, the age
uf Q f1;:v1;:1 (see Ch aplei 41).
Bacillary Angiomatosis. For bacillary a11giomatosis in h--
munocomprornised pcrsons, thc signs and symptoms 1-_
vcry different from U>V. t5acillary angiomatosis, a.
called epithe/ioid angiomatosis, is a vascular proliferatr
d1sease of the skin charactcrized by multiple, blood-ftllec.
cystic tumors. It is usually characterized by violaceous •
colorle:ss papular ar1d uullula1 :.ki11 lesions Lhat clínica:·
m~y <;1Jggrst K;iposi's sarcoma, but histologically resemb
epithelioid hemangiornas. When visceral parcn chymal c·-
gans are involved, the condition is reterred to as bacill;r
peliosis hepatis, splcnic pcliosis, or systernic bacillary a·
giornatosis. Fever, weight loss, malaise, an d enlargem ent
affected organs may develop in people with disserninatE--
baclllary angiomatosis. Endocarditis has alsu 1.Jt'.en r~.
ported in patients with bacill~ry ;ingiom;itosis.
Cats. No major clinical signs of CSD have been reported ·-
cats undcr natural conditions, but infection is very corr
mon, especially in young kittens. It is estimated t hat abo'_
1Oo/o of pet cats and up to 30-50% of stray cats are Bartone/1~
bacteremic at a g¡ven time. In western North An1en ...
(<.:alifornia), a 40% prev;ilencr nf h:irtPrPmic cats \vas folll'
in l he San Francisco-Sacramento a rea. Mino r clinical sigr.
including fever, en larged lymph nodes, uveitis and 1~~
ncurological sym ptoms have been reported in experime:-
tally infccted cats. However, severa! cases of u veitis and _
case of Bartonel/a henselae endocarditis were recently dia~­
nosed in pct cats. Atlditionally, repruducli ve disorders (la-.
of pregnancy or prrgnancy only after repeated breedinl!
slillbirths) have been obse.rved in experimentally infectcc
queens. J3acteremia usually lasts a tew weeks to a to-
months. Th c orgai1isms have bccn reported to be intraer· -
throcytic, and pili may be a pathogenic dcterm1nant i
this Bartonel/a species. Cats can yield more than 1 milli1 -
colony-fornung units (CPU) l'er 1nillililer of blood. Dire..
tr;in~m i s~ion from cat to cat, as well as vertical transm issi -
fron1 bacteremic fernale cats to kittens, was unsucccssfu! -
various experiments. Transmission trom cat to cat \.vas St:~
ccssfully achieved by depositing infected fleas collecte
from bacterernic cats onto nonlnfected kittens. Presence
B. henselae DNA was found in infected íleas. EpidemiolC"_-
cal studies dearly deu1ull~lrC1Lt: Lhal a11libody prevaler:~

nd bactcremia prevalence is thP highest in stray cat popu-
tions living ln warrn cuid hun-iid areas whcre flea infesta-
on is usually highPr.
s. Rnrtonella vinsonii subsp. berkhoffil has b~c11 id en ti-
ed as a n important cause of canine endoc.arditis, espe-
.ally in large brecd dogs. ln a t wu-year prospectivc study
f endocard itis cases, almost onP-third of the 18 cases were
:aused by Barconella :.pecics. Bartonella clarridgeiae and B.
.ashoensis have recently heen associated wit h dog endo-
arditis cases. ·r1ie clinical spectrum of this infection in
ogs h:i" ;:ilso been expanding, as it has been associa led
··itb cardiac arrhythmias, e ndocarditis <ind myocarditis,
.::ranulomatous lymphadenltis, a11d granulon1atous rhini
;,s. In sorne dogs, intermittent lameness, bone pain, or
;ever of unknown urigin can precede thc diagnosis of en-
docarditis far <>Pveral months. Bartonella clarridgeiae DNA
' ·as lk:lc::cled il-i a d og with lymphocytic hepatitis. Rarto-
•1plfa henselae DNA was initially detected In a uog "1-vith pe-
iosis hcpatis, and more recently in a rlog wtth hepatopa-
•hy and in three dogs with various clinical entities. These
:hree B. /1ense/ae- DNA-positive dogs presentcd nonspecific
..:lin ical abr1uri11alities, such as scvere weight loss, pro-
rracted IPthargy, and anorexia. A fourth dog v.ras diaguused
.is being infccted with B. elizabet/Jae by PCR amplification
and sequencing, increasing the n u1111Jer of Bartonella
specics idcntified in infectcd dogs. Serological studies in
:-.:ort h An1erica and F.urupt! indicate t hat Bartonclla infec-
. ion in domestic dag~ is quite rare (less than .)lYo), whereas
high seroprcva.lence has been rcported fron1 dogs living in
tropical cauntries (up to 6So/o of dogs tested from Sudan).
In Nurlh An1erica, espccially in the Southeast, high '\Pro-
"!lrPvalence has been reported in dogs also seruposi tive for
various tick borne pathogens (111ainly Erlic/1i(I, Babesia,
.\11aplas1na). A high seruprevalcnce (35%) has been re-
ported in coyotes from California, and in one spcc1flc Cali-
!ornia c.:uuuly, 28% of the coyotes tested were bacteremic.
Rodents. Experimental infection of prcgnant lahoratory
mice with B. birtlesii showed pathogenic.: t:!fects o n the re-
productive function of these mice. Bartf'remia was signifi-
cantly high er in virg1n femalcs lhan in males. In m icc in
fected befare pregnancy, fPtal loss and resorption \.vas
h 1gher in infecteu 111ice than controls, and the weight of vi-
able fetuses w:is significantJy lower tor infected than for u1t-
;11fc::.cted micc . ·rransplacental transmission w;is also
nPmonstrated, since /6º;ó of tl1e fetal resur¡.¡Lions werc cu l-
ture-positivc for B. birtlesii. The histopathological analysis
of tl1e placentas of infected mi ce sl1uwed vascular lesions in
thc maternal placenta, which ro11ld explain the reproduc-
ti,·e d1sorders observ~u. The isolation and characterization
of the complete virB homologue (virBZ-11) and a down-
strean1 lucaled virD4 gene in B. tribocoru1n has been recently
described. An essential role for rrus v1rB/VlrD-t 1'4SS iu es-
tablishing in tracryt hrocyt ic infection Wil.<: demonstrated.
lmmunologic Aspects
Infcction by Dartonc/la organisms stimulates both thP rel-
lular and the humoral responses. Bartonellu /Jenselac and B.
Chapter 44 Bartonellaceae 263
quintanu i11duce proliferation and migration of cndothe-
lial <"Plls. These effects are due to a trypsin-sensitlvc:: factor
lhat appears to be associated with thc bactc~ria l r rll \\rall or
membrane or intracellular molecul1:::.. Barto11ella lzensclac
infccts and activates endothcli<il r ells. Bartonella hensclae
outer membra ne prutci11s (OMPs) are sufficient to induce
NF1d~ activatian and adhesion m o lecule expression, fol-
lowect by c1ú1anced ro lling and adhesion of leukocytes.
Tn infected individuals, spcc1fic antibodles c.:a11 be de-
lected a few days to a few weeks after infection. Most of the
c.linica1 cases of CS.IJ or BA are associatetl wilh elevated
titers against B. Jie11selae or B. quintn11a. fmmunity is usu-
ally long laSting In ca:>c::~ uf CSD. Tlumnn co:ic:; of endo
carditis are fre<J11Pntly associated with very high lndirecr
fluurescen l anti body (IfA) titcrs (> 1 :800).
ln a rnurine model, spleen cells from infectcu C 57BL/G
n1ice prolifcratcd specifically upan stimulation "vith heat-
killed Bartonella antigen, and CD4 T lyn1phocytes mainly
mcdiate proliferative response<>. 'fhese respo nses increased
dunngthe course uf i11fection and peakcd at 8 weeks post-
jnfection. (}anima interferon, b ut not intcrleukin-4, was
pruuuced in vitro by spleen cells from infected animal<:
upon stimulation with Hartonella antigens. A:. described
also in hurnans, cats, and dogs, Bartonella-~pccific lgG an-
tibodies were detectable in the ~eru111 of the infected n1icc
by the second week, and thc antibody concentration
peaked ar 12 we~ks posl-infection. IgG 21> was the pron1i-
n ent isotype amon g the Bartonella-specific serum IgG
anlil>odic:;. Thcreforc, B. hcnse/ae induces cell-mediatPrl
immune responses ,,rith a -rH 1 phenotype In iuu11unocon1-
pctent CS7BL/6 mice.
In cats, H. henselae antibOdies tlct1::cted by !FA or cn-
zymc-Hnked immunoso rbent a-;~;iy (ELISA) appcar 2 to :~
weel<s afrer exper.IJ11e111al inoculatio n and usualJy persist
for several months. Most infccted cats are bactcremic for
severa! ~veeks despite high antibody titers. Chronic bac-
tPrPmia, despite a hu1noral imn1une respor1sc, Is co ru-
mon ly obscrved among cats. 'fhere is no d irect corrplation
between. antibody titcr and the magiliLudc of bacteremia;
however, cats '"'ith IFA serologic titers of 512 or more are
n1ore likely to be bact~1eu1ic than cats with loy..•er titers.
Dogs with enrlocarditis often show high antibody
titcrs. In ~perin1entally infcctcd dogs, B. l'insonii subsp.
brrkhnffii establishes chronic infecnon, V>'hlch may result
in in-imune supprcssion, characterized by d cfects in mono-
cytic phagocytos1s, an impalrecl suu:.c::l of CDS T lympho-
cytes, and impaired antigcn prcsentation within the
lymph node.
Laboratory Diagnosis
For years, the diagnosis of CSD v.ras based on clilii ca l crite-
ria, history o f cxposurc to a cat, failurP. t() i~nl<itP other bac-
teria, and/or histologlc exa111i11alion of biopsies of lymph
nodcs . .4. skin test using ll ntigen prepared from pasteuri.zcct
exudate fru1u lyn1ph nodes of pat1cnts with CSD was also
used in rli;:ignosing CSD, but this test ~vas n ot standardi.zed
a11d elicited concerns about the safety o f such a product.
Serologic tests, such as IFA or f.LISA, and t~cl111iques to
isolate the organism fro1n human , dog, anc1 c.at specimens
264 PART II Bacteria and Fungí
have been developed since tlle mld 1990s. Because Barto-
ne71a <ire intraerythrocytic bacteria, cell lysis using a lysis-
centrifugation technique greatly facilita tes bacterlal lsola-
tion from the b\ood. However. blood isolation is seldom
obtaincd. from human cases of C'.SD and fro1n don1cstic
dogs. C>n t he contrary, isoJation is more common ly suc-
cessful from human BA cases, for which serology is often
negative. Isulation from the l>loocl of natural reservoirs is
also quitP common with hactPre.mia prPvalPnce. ranging
from 10 to 20 percent (such as for wild felids or coyotes) to
up to 95o/o (beef cattle, deer).
For blood culture fron1 cats, 1.5 n1l of blood is drawn
into lysls-centrilugation tubes (lsostat Microbial Systen1,
Wampole Laboratorles). More recen tly, cat blood collec-
tion in EDTA rubes l<ept frozen at - 70ºC for a fevv days or
weeks has been a preferrcd alternative because of easier
11auul.i111:) a.11u lvvv1::.1 1..v:>L. Fln dog:; lJl caLLle a la1ge1 volu1ne
of blood can be collected (3- 5 mi). The t11bes are cen-
trifugcd ancl thc pcllct sprcad onto infusion agar platcs
containing 5% fresh rabbit blood, which are maintaLned at
35ºC in a high l1u1nidity cl1ambcr wltl1 5% C0 2 for 3 or 4
weeks. c:olonies usually vvill develop in a fe'"' days from cat
blood, although sorne strair1s 1nay require a few weeks.
O Lhe1 1ueau:> uf i:,vlcilivu vf J3u1 luru:llu l1ave lJeeu lJy
using Bactec blood-culture systern or BacTIAlert blood-
culture system. ldcntificat ion of isolatcs as Bartonella can
be perforrned by using enzyme-based identification sys-
te1ns, but is usually confirn1ed by DNJ\ an1plification using
PCR-RFLP ana1ysis. Severa! restriction ene1onuc1eases, such
as TaqI ancl I-JhaI for citrate synthase gene, are used to di-
ge:;L llie siugle pruúucl a 1nplified l>y svecific J!ri1uers. PCR
has ;i lso hPPn nsPcl to iclPntify Rartonflla spp. in tissues, in
absence of culture. Diagnosis of Bartonella endocarditis re-
lies 11eavily on this rnethod in hLtmans and dogs in con-
junction with high antibody titers.
Evidence of infection can be detected in humans oran-
hnals by dctection of antibodies by IFA or ELISA. An IFA
titer uf at least 1:64 is cunsidered positive. Bartunella hense-
lae antihodies can he de.te.ctPd de.spitP conc11rrPnt hac-
tercmia il1 cats and sometimes in humans.
Treatment
In hun1ans, a11tin1icrobial treatn1e11t is ge11erally ü1dicated
for patients with hacillary angiomatosis, hacillary peliosis,
or relapsing bactere1nia. I3acillary angion1atosis patients
respond dcan1atically to macrolide antibiotics. Treat111e11t
with erythromycin, rifampicin, or doxycycline for at least
2 to 3 months in immunocompromised people is recom-
mended, but relapses can occur. ln such cases, patients
s!Jouhl receive Ji(eloug lreal!Heul vvi l!J uue uf Llte::.e aulil>i-
otics. For CSD, antimicrohial treatment is not generally in-
dicated, becau5e mo::;t typical ca::;e::; clo not respond to an-
timicrobial administration. Intravenous administration ~­
gentamicin and doxycycliue and oral adroinistration :
erylhro111yci11 have been used successfully i11 lhe lrt:i:.-
ment of disseminated CSD and therapy of patients ,,-r,.;.
ncuroretinitis. In cases of Bartonella endocarditis, pati~
receiving an aminoglycoside '"'ere 1nore likely to fully ~=­
cover, and those treated witl1 aminoglycosides for at leas;:
14 days were more likely to surv:ive than those wlth shorter
thPrapy ch1r;ition.
In cats, antibiotic treatment (doxycycline, 25 mg to sr
mg twice daily; lincomycin, 100 mg twice a day for 3 weeks
may suppress bactercmla. Various antibiotics (doxycycline.
erythromycin, enroJ:loxacin) have been sho;vn to reducr
the leve] of bacterernia in experimentally infected cats bu:
do 11ot eliminate infection, and t he Jevel ofbacteremia ma...-
•
surpass the initial leve! a few weeks after t he cessation o~
L1ealrnenl. In dogs, n1acrolide:; (erylhro111ycin, a;;itluo111y-
cin) most probably represent tl1e oral antibiotic class of
choice for treating Bartonclla infections, but to date no op
timal protocol has been established.
Prevention
/\. large reservoir for B. henselae and possibly for B. clar-
ridgeiae cxists a1nong thc 68.9 million pct cats rcsiding ir:
one-third of homes in North America. Consequently, neg-
ative publicity about the perceived hazards of cat owner
snip is likely, especially for immunocompromtsed people.
Sero.n egative cats are likely not to be bacteremic, but youné
ki.tt1::1ts, <::spe<.:lally iluµou11<.l1::<.l kitt<::us a11d ílea-i 11 feslt:d kit-
tens.. are more likely to be bacteremic. Therefore, peopb?
who want to acquire a pet cat, especially if they are in1-
munocompromised, should seek a cat raised in a cattery-
if possible, an adult cat coming from a flea controlled en
vironrnent. Unfortunately, there is no correlation between
seropositivity and bactere1nia. Bacteremia can also be tran -
sient w.itb relapses. Declawing cats has also been suggestee
bnt h;is a limited value, because íleas can transmit iniec-
tion from cat to cat. Flea control, therefore, appears to be
011e of the major control measures to prevent cat ir1fectio.E
and its sprcad from cat to cat. The most effective means o:
preventing B. hensetae infection are common sense, ny-
giene, flea control, and, possibly, modification ofbehavio:
of tl1e cat ow11ers tl1en1selves. Wasl1 J:ia11ds after handling
pets and clean any cuts, hites, or scratches promptly with
soap and •vater.
For dog bartonellosis, where tick infestation could bea
m ajor risk factor for acquiring infection, tick and flea con
trol measures should be used duri11g the tick and Flea sea-
son. Systematic inspection of the dog for the presence of
Lick:, afler a vvalk iu infesled areas is 11ighly reco1un1e11d.
 Yeasts Cryptococcus,
Malassezia, and Candida
DWIGI-IT C.
Vhelhe1 a fuugus is caLeguri;1,eu a~ r11 ulu ur yea~t i~ 1.Ja~e<.I
".lpon. the microscopic app earance in tissue or on routi ne
::ulturc media (thc asexual stage). Microscopically, if hy-
::>hal structures are observed, the tu ngus is termed a mold;
I single-celled, budding structures are observed, the fur1
sus is termed a yeast. C)n routine culture media, molds will
!lave a "fuzzy" or wooly appearance, and a yeast will be
oocle1 \a-like ü1 il:. L.ulu1Iial 111u1pl1ulugy auLI cuu::;i~ Ler 11.:y.
.Sorne pathoge11ic fungí >-vill produce either hyphal-like
structurcs or ycast-U.kc structurcs, depending upon the
conditions in which they are growing. Such h1ngi are
ca.lled dimorphic (ungí (Chapters 47, 48).
In this chapter, three yeast fungí wiU be discussed, Cryp-
-:ncoccus, Malassezia, and Candida.
(RYPTOCOCCUS NEOFORMANS
::-ryptococcus neoformans is associatcd with ulccrativc lc-
s~ons affecting t he mucous me1nbranes of the upper respi-
ratory tract (including nasal sinuses), the central 11ervous
system (menin.ges), and eye (chorioretinitis) of cats (do-
~estic animal most commonly affected) and dogs, and is
a.u unco11u11011 cause of 1nasLi lis in caLLle. However, Llü:,
~-east has the potential to affect all animals, including hu-
:nans. ln all spccics, thcce is the tendency foc the central
.::ervous system to become involved.
Jescriptive Feature.s
' orphology
::ryptococcus neo(onnans is a yeast. 1'he spherical cells (3.5
--lll Lo 7.0 µ111 dia111eter) produce single (usual) buds at-
:ached by slender stalks and surrounderl hy polys;:irrh:i-
:!de capsules (Pig 45.1).
Strains of C. neof(Jrrnans have bcen cxperimentally con-
--erted to a mycelial, sexually reproducing phase, Filobasi-
.;;iella neoformans, a basidiomycete.
:.C.llular Products of Medica! lnterest
::.1psule. The polysaccharide capsule (composed prin1arily
-,i a glucu ronoxylomannan) prevents effective opsoniza-
HIRSH ERNST L. BIBERS'fEIN
tion, stimulates suppressor lymphocytes, is toxic to
mac.rophag~s, :in<l rlPrreases inflammatory responses.
Melanin and Mannitol. Melanin and n1annitol are free
radical scavengers (reduce the toxicity of hydroxy radi-
cals, superoxides, and sü1glct oxygcn radicals found
within the phagolysosome). Melanin is producecl from
phenols by phenoloxidase vía the lacease pathway.
Pr1usphul1pase. Phosphol1pase Ls imporrant for survival
,.v ithin mac.rophagPs, ;:inrl is needed for the systemic
spread of thc yeast fron1 the respiratory tract to t he ce11Lral
nervous system. How tl1is enzyme functio11s in this regard
is not known.
Sialic Acids. Sialic acids found within the cell \vall direct
complement proteins toward the degradative pathway,
rather than generatlng effectlve opsonizing fragments and
;in;iphylotoxins.
Growth Characteristics
Cryptococcus ncofónnans grows on co1nmon laboratory
media at ambient or body temperatures. Other members
of this genus do not grow consistcntly at 37ºC. Encapsula-
tion is optimal on chocolate agar plates (see Chapter 14)
incubated under 5º/o carbon dioxjde at 37ºC. Colo11ial
growth rnay be apparent \.Yithin 2 days or requlre several
weeks. C:olonif>_~ :ire gr;:iyish \Nhitf> to white ;'tnd mucoid
and ca11 reach dian1elers of severa! cenli111elers.
Biochemical Reactions
Cryptococcus spp. hydrolyze urea. Their carbol1ydrate as-
shnilation pnttcrns are utilizcd in idcntificution procc-
dures. Cryptococcus neo{orn1ans (but few other c:ryptococcus
spp.) utilizes creatinine and produces melanin-pigmented
colonies 011 rueuia coutaining diphenolic and polyph.e no-
lic con1pounds. These suhstanc.es :irf> 11sP<l in me<lia for se-
lective recovery of C. neofonnans.
Resistan ce
Cycloheximide concentratio11s found in sorne fungal
isolation media inhibit e:. neoformans. Replication ceases
above 40ºC. Higltly itlkaliue e11vironrnents kill the
agent.
265
266 PAKr 11 Bacteria and fungi
.r ')
Variability
four antígenic types, A, B, e, and D based on the antigenic
makeup of the capsular polysaccharides bave been de-
scribe<.l. Phenotypic, genetic, and epidemiological differ-
P.nct>s hP.tvvf'f'n thf' ;:intigt>n ic typt>s hilvf' rP.~11ltPd in thP PS-
tablishment of three varieties of C. 11eofor1nans: var. grubii
(serotype A), var. gattii (serotypes .B and C), and var. neofor-
rnans (scrotypc D). Varictics grubii and ncoformans prcdom-
inatc in tl1e tempera te zone except for an area in Southern
California, where variety gattii is pron1inent.
There are two 111atlng types (sexual stages): MAfa and
MAl'cx.
Ecology
Reservoir
Cryptococcus neofnrmans (v;:ir. gruhii ;:ind neofórrnans) lives
in surfacc dust and dirt. In soil it does not con1pete well
vvith resident microbiota. Acantharnoeba, an an1oeba,
phagocytoses and destroys 1;01ne strains of cryptococci.
lnterestingly, tl1ere are ott1er strains that are capable of sur-
viving withit1 amoeba by using the same intraceJlular sur-
viv<il strategies that are used for their survival withln
macrophagP.s. ·rhus, somP str;:iins arP rlPStroyed, while oth-
crs a.re cndosymbionts using amoebae as an environn1en-
taJ niche. Jn dried pigeon droppings (ricl1 in creatinine,
which inhibits other inicroorganis1ns), the fungus reaches
higl1 conce11tratlons and survives for more than a year at
much reduccd capsular and cell size. Cryptococcus neofor-
rnuns Vdf. ;;uLLii lives Illé!inly in association with <.lecaying
wood of thc red rivP.r gum gro11p of i>11c;:ilypt11s trf'PS.
Though cucalyptus trees are the main habitat for var. gat-
tii, this variety has also been isolated from other tree types,
bat guano, and a wasp nest. Cr}'ptococcus ncoforn·1ans var.
neoformans and var. gru.bii are occasionally isolated from
dccayi.ng wood in hollows of a variety of different species
uf Lrt:t:s.
F 1G U RE 4 5. 1 . Cryptococcus neoformans in nasal granulom~
of a mouflon. India ink wet mount showing encapsulated budding
spherical yeast ce/Is. 400X. (Photograph courtesy of Dr. Roy
Henrickson.)
Transmission
TI1e route of iI1fectio11 is usually res·pi.ratory, rarely percuta-
neous. Cryptococcosis is no.ncontagious.
Pathogenesis
In an environment where moisture and nutrients are plen -
tiful, C. neofi:Jrnzans makcs littlc if any capsular material. l::
arid conditions, t he capsule collapses and protects thE
yeast from dehydration. In either case, the size (approxS
mately 3 µm) is small enough to make lt to the lung alve-
oli. At physiologic concentrations of bicarbonate, CO:-
a11d free iro11, a capsule is produced. The cryplococcal cap-
sule is a very efficient activator of tl1e alternate comple-
n1cnt pathwuy rcsulting in thc dcposition of C3b on i3
surface. ·rhus opsonized, the yeast will adl1ere to the su:-
face of phagocyt ic cells, but is poorly phagocytosed eve::
in the presence of anticapsular antibody. Capsular poly--
saccharide increases participation of suppressor T lympho-
cytes and dccreases a11Lige11-processü1g, leadi11g Lo a poor
antibocty response. Capsular polysaccharide also dinliI:-
ishcs t11c chcmoattractivc cffccts of thc anap!1ylotoxins
C3a and C5a generated by activation of the alternate com-
plement pathway. h1 the event phagocytosis occurs, the
rcspiratory burst is dlminished (capsule), a11d tl1e proctuc-
tion of .m elanin and mannitol by tl1e yeast scave.nge free
radicals and Teduce the hostile environn1ent within the
phagolysosome by inactivating superoxides, hydroxyl,
and singlet oxygen radicals. In addition, phospholipase is
proctucel1, further diminishing the ability ot phagocytic
cells in eliminating the fungus. Thus, inflammatory re-
sponses are minimal and cryptococci grow into Iarge
space-oc<'11pyine "myxo1natous" masses, consistine of
capsular slin1e, yeast cells, a11d few inflan1n1atory cells.
Eventually these n1asses acquire histiocytes, epithelioid
cclls, and somc giant cclls.
Develo_p ment of pulmonary lesions is erratic. lnfections
often localize ü1 the central nervous system (perhaps due
to lower compleme11t conce11tratlons in the CNS a11d to
 Chapter 45
high conccntrations of catechols, a suhstrate for phe-
noloxidasc, thc cnzyme the yeast uses to produce
melanin) tollowing dissemination fron1 the lungs and are
m anifested by neuroloi,>ic signs (see Fig 71.3). E.ye involve-
m ent, leading to chorioretinitis and blindness, is relatively
c ommon.
Disease Patterns
Cats and Dogs. Cats and dogs are most otten clinically af-
fected. Sig11s include ulcerative lesions of the mucous
membranes in nose, mouth, pharynx, and sinuses or myx-
om;:itous nasal 1nasses. Central nervous system involve-
ment is con1111on. Tl1csc lcsions inay arise fnn11 lot:al infet:-
tions. Most skin lesions are probably hematogenous.
l:attle. Cattle acquire cryptococcosis during administra-
C:on of contaminated material of lntramarnmary n1edica
.:on. Tltt::n:! is gross swe!Jing, hardening of the gland, and
gradual changes in the secrt>tions. Destruction of the lact-
ierous epithelium is extensive. Severa! glands n1ay be irre-
--ersibly damaged. 1'he disease rarely advances beyond re-
_;ion al Jymph nodes.
.:~idemio logy
::~yptococcus can proh;i hly ;:ifft>ct any mam mal. Its occur-
-ence is sporadic and worldwide. Birds, particularly pi-
:eons, often carry the agent in their intestinal cont ents
:_-,d contributc to its rcscrvoir (see above). They are rarel.y
iliected clinically, a11d then mostly on mucosal surfaces.
Human cryptococcosis is often assodated with im-
-unosuppressi.on (organ transplants, Hodgkin's disease,
::-:egnancy, acquired immunodeficicncy syndrome) or in-
=e!isive exµosure . Atteruµts tu relate anirr1al infections to
.Zlílar circun1stances have heen spec111;1tive.
Sovine cryptococcal rnastitis usually starts as an iatro-
_.:::ic.ally induced inoculation infection.
Cr yptococcosis in l<oala bears is associatcd with contact
-~ eucalyptus leaves conta:ininated wi tl1 var. gattíí .
--munologic Aspects
-.:nunosuppression is a predisposing factor. 1'he capsular
·-ysaccharides ¡>roduce immune paralysis, complcrncnt
_p!etion, and antibody masking.
Humoral and cell-mediated pheno1nena (TH l subset re-
.lng iu 111at:ropl1agc at:tivatiun) evidently contribute to
;ense against cryptococcal infection. M;:icroph;:iges par-
- "'pate in disposal of the agent.. 1'here is sorne evidence
_.__: r Lymphocytes (CD4 and CD8) as well as natural killer
- s kili or inhibit e;. neoformans directly.
Resu lts of ex perirnent al immunization have been
..ivocal. No vaccines are available .
.::::ryplococcosis does 110L appear tu !Je a d.isease in cats
=cted with feline irnmunodeficiency vi rus (FIV) ;:is it is
!!um an patients infected with human immunodefi-
::FLlCY virus (HlV) . FIV-positive cats respond to appropri-
- antifungal therapy; human 1-IIV-positivc paticnts re-
, :id p oorly if at ali.
Yeasts- Cr,vptococcus, Ma/assezia, and r:andida 267
Laboratory Diagnosis
Direct Examination
A small an1ount of sedin1e11l fro111 exu<.lates, trat:l1eo-
bronchial washes, and cerebrospinal fluids is mixed with
a11 cqual amount of India ink on a slide anda covcr slip is
added. MicroscopicaJJy, the encapsulated organisms ap-
pear as brigh.t circular Jacunae in a dark field, containing
the yeast cells in their centers (see Fig 45. l ). Preparations
stained with Rom;:inovsky-type stains (Wright's, Giemsa)
are also used to den1011strate ll1e capsule (Ll1e !Jo<.ly uf tl1e
yeast will stain darker bluc than t he capsule). Fungal
stains- c.g., pcriodic acid Schiff (PAS) and (.Jomori methe-
na1nine silver (CrMS)- deli.neate the cell wall but not the
capsule, '"'hich is stainable by mucicarmine. With Gra1n
:;ta.iu, cryptococcal cells often appear gran1-positive.
Tn sections proct>ssed by tl1e usual histologic methods,
the capsules are unstained 11alos separaling Ll1e yeast cells
fron1 tissue constituents or from each other.
Culture
Blou<.l agar an<.l Sabouraud's agar cultures (without cyclo-
heximide) arf' inc11h;:itecl, respectively, at 37ºC and room
temperature (see Chapter 46). Suggcslive colo11ies are ex-
amined by India ink wet mount . If found to consist of
encapsulated yeasts, (~. ncofon-nans is confirmed by
demonstration of urease activity, absence of lactose, meli-
biose and 11itra te assimilation, and lt.~thallty for mice upon
intracerebral or intraperitoneal injection.
St>lective media incorporating antibacterial a11d anti-
fungal drugs, creatinine and dlpl1e11yl, are used for 1::11 vi-
ronmental sampling.
Sorne normal dogs and cats harbor small numbers of e;.
neoforrnans in their nasaJ cavities. ·r herefore, care should
be taken when interpreting (Ulture rcsults from samples
obtained from this site. Examination of direct smears (the
numbers of yeast in smears from clini.cally nor1nal animals
are too Jow l.o see) and/or analysis of seru111 fo r t:apsular
antigen are helpful adjuncts to culture. Capsular antigen is
not detectable in scrum of 11ormal dogs, regardless of
wl1ether they harllor (; . neoformans in their nasal passages.
lmmunodiagnosis
Antlgen demonstration in serum and cerebrospinal fluid is
attempted in diagnosis and assessment of patient progress.
Latex par Ucle suspensious cuaLe<.l ~vi tl1 antit:apsular anti-
body are marketed as slide agglutination test· kits.
Antibody is irrcgularly demonstrable bccause of the
"sponging" action ot circulating capsular antigens. lts
presence (d.e monstrated by ü1direct fluoréscent antibody
tests or by Jatex particles coated with capsular polysaccha-
ri.de) is a favorable sign of decreasing antigen levels .
Treatment and Control
1'hc trcatmcnt of choice for dogs and cats is fluconazole.
Alternative therapy is 5-fluorocytosine, but its efficacy
268 l'ART ll Bacteria and funt,.'1
should be tested periodically as strains may be resistant or
become resistant.
'fherapy should be continued until clinical signs are re-
solved and antigen disappears from seru1n and cere
brospinal fluid.
Conta1ni11ated surfaces (pigeon lofts, attics) ca11 be dis-
ir1fected witl1 lirue solutiou (1 llJ l1yuraled lirne/3 gal
water) prior to phys.ical cleanup. Dirt ren1oved is placed in
conttiiI1cr.s ond covcrcd with J1ydrotcd lime powdcr, which
can also be used on exposed floors and beams. Masks are
worn during the operation.
MALASSEZIA PACHYDERMATIS
Me1r1bers o[ Lhe genus Malassezia are parasitic yeasts.
There are seven spccies: M. pachydermatis, i\!f. restricta, M.
globosa, M. obtusa, M . furfur, M. sympodialis, nr1d i\1. sloof
fiae. All, except M . pachydermatís, require lipids for growth
(though lvf. pachydermatis is lipophilic). 011ly M. pachyder-
rnatis is commonly associated with animal disease, most
often otitis externa, and dermatitis in dogs. However,
eaclr uf tl11:: lipid-ueveudeul species 11as bee11 isolated
from skin and externa! ear canals of norn1al and clinically
affected dogs, cats, ruminar1ts, and horses. Thus, thc
reason why M . pachydern1atis is more commonJy tound
may be due to the relative ease i11 whicl1 this species is
demonstrated.
Descriptive Features
Morphology and Composition
Malosse7.ia par:hydt,rrnatis is <in oval h11d<ling yf':ist (2 pm hy
5 µm). In direct smears (and from colonies obtained fron1
culture), there will be a single b ud attached by a bread base
(0.9 µm - 1.1 µm) (Fig 45.2). Filaments are not usually ob-
served, regardless of culture conditions. 1"he cell wall is
composed ofglycoproteins (75-80o/o), lipids (15- 20%), and
chitin (1 2o/o).
Growth Characteristics
Though not requiring lipids for growth, M. pachyde-rmatis
i:> lipophilic, c:tud g1owlh is in1proved vvl1cr1 lipi.d::; are
added to the medium. Most strains of M. pachydernzatis
grow on blood agar plates, though th.e colonies will be
very small (<1 mm in diameter, and sometimes 011ly a
"greenish" tint will be seen on the surface of tl1e plate)
after several days of incubation (optimum temperature Is
37ºC, although it will grow at temperature ranging from
25ºC Lo 41 ºC). T!Je yeasl will grow il1 eilhcr an aerobic, or
microaerophilic atmosphere (it does not grow well anaer-
obically).
Blochemical Reactions
Afalassezia pachyderrnatis assimilates the carbon of glucose
a11d D-ma11nitol, but ctoes not ferment carbohydrates.
Urea hydrolysis is strain-dependent.
Resistance
Malassezia pachydermatis is resistant to cycloheximide. lt is
sensitlve to cold.
Variability
There are a number of biotypes of M . pach)'dermatis as
reflected in variability in D-mannitol anti sorbitol assi111i-
l:ition, hy<lrolysis nf nrf'a, anct Cf'll wall fatty acid con-
centration.
Seven genetic types (a through g) have been described
F 1G U RE 4 5. 2. Exudate of canine otitis extern;¡ rnnt;¡ining
Malassez:ia pachydermatis. Note characteristic "shoe print" pattern
of budding yeasts (arrows). Gram stain, 1000X.
 Chapter 45
LLc;ing thf' c;equenc.e of l)NA encoding the large rihosomal
ubunit as thc basis for comparison. Others have delin-
eated four genetic types (A through D) after using the ran-
jom amplification of polymorhic DNA method, togeth cr
· 1th sequence co1nparisons of the gene encodu1g chit1n
~'--nthase.
,
•
écology
='eservoir
• !alassezia pacf1ydennatis hves on the skln and external ear
canal of hcalthy animals, including dogs, cats, ferrets,
¡.>igs, and rhlnoceros (from v;hich it gets lts nan1e). The sur-
:ace of M. pachydermatis has mannosP-C'Ontaining glyco.
proteins that are responsibk: for binding to mannose re-
-:eptors on the surface of corneocytes. thus allowing
adherencc in this niche. It is rarely isolated from human
skin, or thc cnvironment.
ransm1ss1on
falassezia pachyden1Jatis is an opportunistic fungus, con-
:ributing to disease processes already in progress (e.g .. al-
.oergic dermatitis). Thc source of the yeast is endogenous
..e., a mcmbcr of the patient's normal flora). latrogenic
.:tisease has been reported for the transmission of the yeast
.~Ul ll él uog witlJ. Otitis ext efild to d hurnan patient Via the
'1anrls of a carPgivPr (the rlog's ownPr) who handled a
~pid-rich intravenous solution (for total parenteral nutri-
ion) subsequently administered to the patient.
~athogenes is
.Jalassezia pacl1ydern1nris plays a secondary, but sigruficant
¡ole in otitis externa and dermatitis in a variety of animals,
.... ul 111u:>l l:o111111011ly i11 dogs, aud lo a lesse1 exle11L, cals
see Fig 69.4). The exact role played by M. pachydennatis re-
'"Ilains unclear, and what causes the yeast to change from a
'larmlcss commensal t o one that con tributes to disease, is
unknown. If its presence is ignored when formulating a
rreatment regimen, l1owever, resolutlon of the disease
process is p roblematic.
: pidemiology
'.falassezia pacl1ydennatis is a parasi te of skin (including the
c.xtcrnal car canal) of nonhuman animals. Malassezia
~Jc/1ydern1atis-assocíated dermatitis is more commonly re-
portcd in Australian silky terriers, basset hounds, cocker
<ipanicls, dachsh unds, poodles, and west Highland white
+f'rriPrs. Thc yeast has '""orldwidc distribution.
lmmunologic Aspects
• Jalassczia pachydennatis is ai:1 opportunistic yeast, con-
=::-ibuting to preexisting compromises of the skin and ex-
:.:rnal ear. lt is unknown how M. pachydcm1atis contributcs
~o d1seasc.
Yeasts-Cryptococcus, Malassezia, and Candida 269
Laboratory Diagnosis
Direct Examination
'fhe nu1nbers of yeasts on norn1al skin or in tl1c 11orn1al ex-
terna! ear canal are usually too IO\.Y to visualize il1 samples
takcn from such sitcs. Thus, dctcrmin ing whcthcr M .
pac/1ycten11atis is a co11tributing factor to otitis externa orto
a dermatological condition is relativcly easy, because the
IIU111lJers of yeasts will be high enough to be seen In sa1n-
ple<; taken from affectP<l arf'ac;. HowP\·Pr, sorne atopic dogs
havc been sho,>Vn to have increased nun1bers of M. pacliy-
dcnnatis in normal as well as affected areas .
Samplcs takcn with cotton-tippcd swabs are the casicst
to obta1n from cases of otitis externa . .Swahs are "rolled"
over the surface of a rnicroscopc slide ;vhich are then
:>lCtiJ1cu with a Rornanovsky-type stal n (Wrlgl1t's, Giemsa)
or G ram's stain . r.xamination of c;mp;irs will reveal ycasts
with charactcristic "bottle-shaped" or "shoe print" n1or-
phology (see Fig 45.2). lf the swabs are also streaked onto
the surface of a blood agar plntc, small colonics (ora grccn-
1sh colorat1on) will appcar withín Z4-48 hours after incu-
bation of the plate ~t 37ºC in air.
Thuugli 1\11. pachydennatis can be dernonstratcd on af-
fected arcas of the skin, <lermatologic;t.; rPcommend that
sorne measure of numbers be madc in order to gauge the
success of treatn1ent . Numbcrs are approximated by using
adhesive tape to remove yeasts from the skin and thcn
staining (Wright's, or Giemsa), followcd by a microscopic
examination o f the tape. For cxalnplc, the tape is placed
"yeast-s!de" tlovvn on a microscope slldc in a puddle of
Wright'c; stain . 'l'hP "prPparation" ic; Px:1mined throtrgh a
microscopc with the tape acting as a coverslip. Less than
two ycasts per lOOOX field is about the maximum to e,xpect
from prcparations madc from thc skin of a normal dog .
Molecular Techniques
Poly1nerase cha in reaction amplification of DNA encoding
elther thc large or the small rlbosomal subunit together
with the interna! transcribcd spacr.r rcgi.o n maybe used to
detect 1'vfalassez.ia spp. The san1e technology l1as been. ap-
plied to speciation of the yeast.
Culture
As a means of determining \vhether M . pacllydern1atis is a
contributor in otitis externa, culture is not worth the cf
fort, bccausc most samples caken from such conditions
contain bacteria (e.g., Pse11domo11ns) that quickly overgrow
the slower gro,vi11g yeasl. 1'he dele1111i 11alio11 Llial M.
pach,vdennatis is involved can be made quicker by micro-
scopic cxu1niT1ution of sa1nplcs.
Cultu re plays a role in assessing the microbiological
makeup of dermatological conditions, as well as formulat
lng a trcatment regim en. Either tape preparations (press
the tape on the surface of medium) or contact plates (petri
plates with agar mediu1n Lhal "IJul~t::>" alJuve the riln) .
Media that have proven useful include Dixon's (mo<1ifie<1
to contain lipid), Leeming Notman's, or Sabouraud's.
Plates are incubated at 37uc in air or an atmosphere con-
270 PART 11 Bacteria and Fu11gi
taining C0 2 . Greater than two colonies per 0.5 square .i nch
is considcrcd abnorn1al.
Treatment and Control
Corre<..-tion of tl1e underlyi11g co11ditio11 is tl1c i11osLil11po1-
tant aspect of t reatment of M. pachyderrnatis- associated otitis
externa or dcrn1atitis. Altnost ull conlillcrciully avuilublc top-
ical preparations that contain an antifungal agent (nystatin,
clortrimazole, or rniconazole) are e.ffective in treating the
fungal component of otitis externa. Meclicatect shampoos
(e.g., miconazole + cl1lorhexidille), along •vith systemic an-
lifu11gal adn1illisL1alion (keLoco11azole 01 il1aco11azole) a1e
effective in reducing the influence of M. pachydermatis upon
dern1atological diseases. Griseofulvin is not effective.
CANDI DA
Candidiasis is usually dueto tl1e parasitic yeast Candida al-
bicans, whicl1 iuhal.Jits u1ucous u1e1111.Jrd11es of 1110:.t 111<1111-
mals ano hiros. ()f t hP. mor<> than 150 othP.r spP.ciP.s of
Curul.ida lhal a1e associaled will1 i11a11y dive1se 11abilaLs,
few are associated with animal disease. Disease produced
by mcrnbcrs of thc gc11us Gandida usually occurs in a11 im-
munoco1nprom ised .host.
The subsequent discussio11 deals with G. albicans unless
ot.herwise indicated.
Descriptive Features
Cell Morphology, Anatomy, and Composition
On routine lahoratory mP.dia ano n111co11s mf'mhranf'S, r.
albícans typically grows as oval budding yeast cells (blasto-
con.idia), 3.5 µ111to6 µ111 by6 µn1 to lOµm in size. Undercer-
taiJ.1 conditions of tempernture, pH, nutrition, and ntmos-
phere, yeast cells sprout germ tubes (Fig 45.3) tl1at develop
into septate-b ranching myceliurn . "Pseudoh.yphae" is pro-
duced by elo11galion of Lhe blasloconiclia aud Lheir failure Lo
F 1G U RE 4 5. 3. Germ tube production (arrows) by Candida albicans in serum.
Ur:istained wet mount, 300X.
-
..
separa te. In vivo, mycelial (a collection of hyphae) or pseu-
domycclial growth is associated with active proliferation
and invasiveness.
The so-called chlamydospore (chlamydoconidium) is a
thick-walled sphere of unknown function, attached by a
suspensor cell to (pseudo)mycelium and essentially con-
fü1ed Lo ü1 viL10 giowlh (Fig 45.4) of G. albícans (1a1ely
other Candida spp.).
Thc ccll wall contains glycoprotcins; thc polysaccha-
ride portions are glucans and especially mannans. Lipids
and chitin are also present. Mannoproteins are found on
the cell surface. c:ellular products include peptidolytic en-
zymes, wl1ich may be virulence factors . Two major c.ross-
1eaclil1g se1og1oups are recogrlizcd. These are termed A
and B, and are identifiable with absorbed sera.
Gandida can be staincd with pcriodic acid Schiff (PAS),
Gomori methenamine silver (CiM::-i), and other fungal
stains, but is usually studied in culture unstained. Poly-
chrome stains (Wrlght's, Gle1nsa) are suitable for <.lemon-
strating it iI1 tissue or exudate. With Gr;:im st;:iin, <:andida
cell.s oflen appear gran1-po.sitivc.
Cellular Products of Medica! lnterest
Adhesins. Various cell wall components (chitin, manno-
proteins, a11d lipids) have been associated with adherence
to extracellular matrix proteins.
Mi:;c:ellaneuus Pruducls. Prute<1!>e."1 dIH.l i1eurauüuidases
have hP.P.n proposP.o to play a rolP. in pathogenesis. Cell
wall glycoproteins have endotoxin-like activity (see
Chapters 7 and 8).
Growth Characteristics
Gandida albicans, an obllgate aerobe, grows on ord.i nary
mf'di;:i nvf'r :1 widf' rangf' of pH and tf'mpf'rilti1rf'. At 25°(:,
creamy to pasty white colonies consisting predominantly
of yeast cells appear in 24 to 48 hours. Production of
(pscudo)mycclium is cnvironmcntally influenced, but thc
controllillg factors are disputed. lncubation temperatures
above 3SºC, a slightly alkaline pH, anda rich, carbohydrate-
free fluid 1uediu111 are ofLeu recouuueuded .
·..... ,
~·
 Chapter 45
F 1G U RE 4 5. 4 .Ccir1dido olbiloll~ culture on tfllarnydospore agar with trypan blue,
showinq pseudomycelium, yeast ce/Is (blastoconidia) (right), and chlamydoconidia (left);
1200X.
Differential ability to ferment or assimilate carbohy
drates is the basts of species identification..
Candida species are k.illed by heat above SOºC, ultravio-
leL ligl1t, <.Jlluriu<::, c.u1<.l yuater11ary aII1n1oniuu1-typ<:: <.lisir1-
fectants. They withstand frPP.7.ing ano SllíVÍVP WP.11 in thP
inartimate environ1n.e nt. They are susceptible to polyene
antimycotics, and usually to flucytosine and the azoles.
Reservo ir
Candida albicans is associated witl1 mucocutaneous areas,
particularly of tl1e alin1entary and lower genital tract, of
llldlllllléll::. clllU uil u::.. Eu VÍl Ullllll.:l 1Lal ::.uu11,,1.:::. dl t: Ílll¡JUl l clll l
especially for other Gandida spp.
Transmission
Yfost Gandida diseases arise from a.n endogenous source,
that is, they are caused by a commensal strain. The bovine
udder l.Jecu111e::. i11fecle<.l via tite Leat canal l.Jy way uf ad-
ministered medication, during milking, by cow-to-covv
sp rcad, or from thc environ1nent.
Pathogenesis
Mer.hanisms. r.hitin, 1n.annoprotei11, and lipids are possible
adhesins, and in hun1an car1didiasis; several exlracellula1
matrix proteins have been shown to be the receptor. Germ
tube forrnation is corrclated with experimental patho-
genicity, but the role of mycelium formation in virulence
is under dispute. Proteases a11d !1eu.raminidase may be vir-
ulence factors. Cell wall glycoprotelns have endotoxin-like
ac.tivity.
1~athology. Candidiasis n1ost freque11tly aJiecls lhe 1nu-
cous surfaces on which the igent is normally found, possi-
Yeasts- C:ryptococcus, Malassezia, and Candida 271
bly the anterior digestive tract from mouth to stomach; it
typically remains confined to areas of squamous epithe-
lium. 'fhe genital tract, skin, and cla"l<vs can be involved as
well. Occasional respiratory, Intestinal, and septicemic in-
fprtion.s orc11r_
On epithelial surfaces, candidiasis forn1s whitish to yel-
low or gray plaques, marking areas of ulceration with vary-
ing dcgrcc of inflammation. Diphthcritic membranes muy
form in the gut or respiratory tract, and abscesses may
form in the v íscera. Granulomatous lesions are rare.
lnflammatory responses are predominantly neutrophilic.
Disease Patterns
Birds. Avian candidiasis affects chickcns, turkeys, pigeons,
and other t)irds. lt resembles thrush ot humans involving
the anterior digestive tract (see Fig 68 .2). In th.e you11g, it
can be a stunting clisease and cause considerable rnortality.
Swine. ln the alimentary tract of pigs, candidlasls Is seen
as ulcerative lesions that n1ay lead to rupture.
Equine. ln tl1e alimentary tract of foals, candidiasis is seen
as ulcerative lesions that may lead to rupture. Equine gen-
ital infections cause infertility, metritis, and abortions.
Cattle. Pueurnuuic, <::11teric, anll gene.ralized candidiasis
affects calves on intensive antihiotic rPgimt>ns. Candida
mastitis in dairy cows is typically 1nild and self-limiting,
e.nding in spontaneous recovery within about a week.
Rovine abortions have been reported.
Dogs and Cats. In dogs, candidiasis produces ulcerative le-
sluus iu tl1e digestive anll genital tract. Rarely, dogs de-
velop septic:Pmía with lt>sions in muscle, bones, skin, and
272 PART 11 Bacteria and Fungí
F 1G U RE 4 5. 5. Candida albicans in nasal exudate from a dog. Note yeast ce/Is
(blasroconidia and pseudohypha. Gram srain, IOOOX.
lower urinary tract (especially those witl1 diabetes melli-
tus); feline pyotl1orax may rarelybe due to C. albicans.
Other. Lowcr primutcs und murinc mamrnals may acquirc
1nucocutaneous candidiasis.
Epidemiology
l'he common age.r1ts of car1didiasis are commensal '">i.tl1
n1ost warn1-blooded species. Visease is linked to immune
and hormonal inadequacies, reduced colonization resist-
ance (a rnea~ure oftlle "health" ofthe norrnal flora), or in -
tensive exposure of weakened hosts or vulnerable tissues.
·r11ese condilions accounl for susceplibilily o f i11fa11Ls, d ia-
betics, subjects on antibiotic and steroid regimes, patients
with i11dwelling catl1eters, and ma1nmary gla11ds of lactat-
ing cows.
lmmunologic Aspects
Immu11oincompetent lndividuals are preferred targets for
infection .
Poly111orpl1011uclcar neutrophil leukocytes (PMNs) and
activated 1nacrophages form the chief defense against
candidiases. 1'he role played by opsonins (antibody; coin-
p lemer1t) is to facilitate pl1agocytosis. Macrophages are ac-
tivated by gan1ma in terferon secreted by ·rHl cells stimu -
lated by ínterleukin 12 fro m macrophages actively
engaged in phagocytosis.
·rhere i:. r1u <1rliflt:ial ir11111 u11izalion.
laboratory Diagnosis
In exudate, Gandida appears a::; yeast cells (blastoconidia)
or (pseudo)hyphae. Ali forms are demonstrable in un-
stained wet mounts, or in fixed smears stained with
Grarn':; stain, RoII1<1uov:;ky-ly¡Je slai1 1s (Wrighl's, Gien1sa)
or f11ngal stains- e .g., periodic acid Schiff (PAS) and
Gomori methenamine silver (GMS) (Pig 45.5).
Candida albicans grows well on blood or Sabouraud's
agar, with or without inhibitors (scc Chapter 46). Othei
<:andida spp. rnay be inhibited by cycloheximide. Yeast
isolates producing (pseudo)mycelium can be considered
Gandida spp. lsolation of Gandida spp. fron1 mucous mem-
hr;inps (PvPn in J;i reP n 11mhPrs) s11ggf'sts a rli;ignosis of c<in -
didiasis only in the presence of compatible lesio11s, and
abundant (pseudo)hyphal forms in direct smears.
Incubation at 37°C for >2 hours of a lightly inoculated
tu be of seru m will p roduce ger1n t ubes if thc isolate is<:. al-
bicans (see Fig 45.3), wl1ich also producect chlamydospores
011 cornmeal-tween 80 agar (see fig 45.4). Yeast identifica-
tion ki ts are commerci<il ly availahlf'.
DNA probes and tests for circulat"ing antibody, antigen,
a11d metabolites have had n o adequate tria! in veterinary
medicine.
Treatment, Control, and Prevention
Correcting conditions underlying clinical candidiasis may
ü1 itself lead to recovery.
In poultry, copper sulfate in drinking water is a tradi-
tional treatment. Nystatin can be given in feed or water. lt is
also used topically in mucosal and cutaneous forms of can-
didiasis of 111a1I1111al:;, a:; are <'UHJJl 10Lericin B and n1icona-
1.nle. Fluc.ona7.ole (preferred) or flucytosine is useful for
treating dogs or cats vvith lower urinary tract candidiasis.
In disseminated forms, oral fluco11az0Je or tlucytosine
are drugs of choice. Susceptibility testing is advisable.
Con1bined flucytosine-an1photericin B is sometimes used
in humans and occasionally in animals.
 Derlllatophytes
ERNST L . B IBERSTEIN
\Vhether a fun¡_,rus is cateeori7ed as mold or yeast is b ased
upon th e n1icroscopic appearance in tissue or on routin e
culture media (the asexual stage). lf hyphal structures are
observed, the fungus is termed a mold; if a single-celled,
buddin g st ructure is observed, the fungu s is termed a yeast.
On routinc culture media, molds will have a "fuzzy" or
•VUUly ap pcararH.:C, l:tl\U a yeast ~vill lJe lJa<:teria-like in itS
colonial morphology and consistency. So1ne p at hogPnic
fungi will produce cither hyphal-likc structures or yeast-
.ike struch1res, depending upon the conditions in which
~hey are gro\~•ing. Such fungi are called din1orplzic fungi
Chapters 47, 48).
Dermatophytcs are molds capable of parasitizing only
~e1ali 11 izeu eµiuer111a 1 sl rucl ure:>: su µerficial skiu, hair,
:eathers, horn, hooves, claws, and nails. Those that havf' a
sexual reproductivc phasc bclong to the ascomycetes.
:;)ermatophytc infections are called ring~vorm ( tinea).
Descriptive Features
\1orphology
:n their nonparasitic state, including cultu re, d ermato -
phytes p roduce septate, branching hyphae collectively
called rnJ1,·c·li11m. The asexua l reproductive un its (conidia)
a1e fo u11d in l h e ae1ial 111yceliun1. ·r11ese u11ils 111ay be ei-
ther macroconidia: pl uricellular, podl ike structures up to
100 µm lo ng; or 1nicroconidia: uniccllular sphcrcs or rods
!ess tnan JU µm 1n any <.11mens1on. ~ nape, s1ze, structure,
arran gem ent, and abundance of conidia are diagnostic cri-
teria . Hyphal peculiarities-spirals, uod ules, "rackets,"
~rhan rlf'lif'rc;," ;:i nrl ch ll'11l1y<1ocon irl ia (rh lamydosporPs)-
are more common in sorne species than others, but th ey
are rarely diagnostic. Pigmcntation is useful in dermato-
phyte differentiation.
In the paras1ac state, only hyphae and arthrocon1dia
arthrospores), another asexual reproductive unit, are
seeu. Exceµt in size rauge::., wlúch uverlap among dermato-
phyte species, arthroconiclia arf' inclic;tinguish;:ihlt> from
:>pecies to spccics.
Sexual spores (ascospores) are absent in the parasitic
phase.
Thc distinguishing features of the three genera of
dermatophytes-Microsporurn, Trichophyton, and Epidermo-
pf1ylurt-<1rc sl1uwu iu 'l'al.Jle 46.l . Only 1\tlic.Tosporurn and Tri-
chophyton affect ;:inimaJc; consistPntly.
DWIGH1' C . HIH.SH
Growth Characteristics
Th c trudition al n1cdium fo r propagating dermatophytes
(and other pat hogenic fungi) is Sat)ouraud's d extrose agar,
a ?o/o agar containing 1% peptone and 4% glucose. Its acid-
ity (pI-I 5.6) renders lt m lldly bacteriostatic and selective.
Th f' sPIPctivity is enhanced by addition of cyclo hexirnide
(500 pg/ml), which inhibits oth cr fungi, and genla1nicin
and tetracycline (100 µg/ml of each) or chloramp h enicol
(SO pg/rnl), which inhibit bacteria. Dcrmatophytes are aer-
obes and nonfcrmenters. Sorne attack proteins aod dcam-
inate amino acids. They grow optimally at 25ºC to 30ºC
and re4uire severa! days ro weeks of lncubation.
SomP <lf'rmatophytpc; in c;ki n íl nd ha ir (but not in cul-
ture) produce a green fluorescence due to a tryptophan
inetabolite that is visible under ultraviolet light (A. =
366 nm), somctimcs rcfcrrcd to as a Wood's light. Of ani-
mal dermatophytes, only Microsporurn canis produces this
reaction.
Resistance
Dermatophytes are susceptible to common disinfect ants,
particularly thosc containing creso!, iodinc, or chlorine.
·rhcy survive tor years in the inanimate enviro11ment.
Variability
Th ere are a number of strains of each specles of M icro-
s¡H1n11n an rl 'f)·irhnphytnn, making ronstn1ction of pfff'ctivf'
immunizing products difficult .
Ecology
Reservoir
One speaks of geophilic, zoophilic, and anthropopllilic
dermatophytes when discussing dermatophytcs having a
soil, anlmal, or human reservoir, respectively. Table 46.2
shows the important dermatophytes affecting animals,
with rcscrvoi1 s a11d clinical ho:1l:>.
Rare causes of animal dermatophytosis are the anthro-
pophilic, globally prcvalcnt M. audouinii, T. rubn.11n, T. ton-
surans, and c.. floccosi11n, and the zoophilic i'vf. distorturn,
which has been recovcred from Iesions of dogs, cats,
horses, monkeys, and humans and appears to be restricted
to Australia, New Zcaland, and the Americas.
273
 Chapter 46 Dcrmatophytes 275

-able 46.3. lmportant Oermatophyte lnfections in Domestic body; protPin mnif'tif'~ stim11liltf' rf'll-mf'di!ltf'd responsf's) .
.:.'lima Is Antibody-mediated and cell-mediated hypersensitivi-
ties occur in the course of dermatophytoses. 'fheir onset
Host Agenl Ndlure of Lesions gcncra1ly coincides with that of thc inflammatory phasc
of infection and may contribute to its manifestations.
Horse T. equinum Dry, scaly usualtv noninflammatory Sterile inflammatory skin lesions (phytids) occur in
(unless se<ondarily infected) human rlngworm lnfccttons. 1'hcy are allcrgic reactlons to
M. gypseum Often suppurative under alopecic circ11lating f11ng;il ;intigPns
thickened areas
M. equinum Not more than míldly inflammatory,
resembling T. equinum lesions Recovery and Resistance
Cattle T. verrucosum Painless, thick, white, "asbestos• Antibodics play at bcst a limited role in resistance.
plaoues, local alopecia Evidencc favors ccll mcdiat<'d mechanisms as decisive in
Swine M. nanum Tanni1h, crusty, 1preading centrifugally protcctlon and rccovcry.
on trunk; painless, margins slightly Rf>rnvPrP<i indivich1i1l<: rP<;ist rPinfection, althcn1eh local
infl~med No hair loss. reactions n1ay be more acute and intense than on prin1ary
Dog M. canis Typlcally noninflammatory, scaly, exposure. i\cquired reslstance varíes in degree and dura-
alopecic patches, occasional kerion tion with host, dcrrnatophytc spccies, and possibly
T. mentagrophytes Often spreading, extensively scating to anatomical area.
intlammatory lesions, secondary
suppuration
M. gypseum As T. mentagrophyres Artificial lmmu nization
( ;¡t M. r;ini~ Oftpn 'uhrlini<;il in ;irlult\. CiPnPr;illy
MyceliCJI T. vern1cus111n va<.:<.:lnes, inCJ<.:tivated and live aviru-
noninflammatory except in young lent, are us<>d in F.11ropP. Thf>y !lrf> crf>ciitf>c1 with rf>c111cing
kittens; may become generalized in the number of infected hcrds and ncw infcctions. Mixtures
debilitated kittens. Occasional of ,\1icrospon1m and Trichophyton-either as a live, attenu-
mycetoma (Persian cats). ated vaccine ora killed product have been disappointing
T. mentagrophytes As in dogs
in protecting cats from developing dermatophytosis,
Chicken M. gal/inae Generally affecrs unfeathered portions. though they do appear to restrict spread upon individual
Whitish chalky scaling on comb and <.:CJts. Althuugh live va<.:clne!) appear to eli<..it a better im-
Wdtll~. 11onirtlldmmdlory
m11nf' (protf>rtivf') ri>,pon,f', thf' f)rf>Sf'11Cf' nf n11mf>ro11s
T. simii Superficially similar to M. gallinae but strains of Microsporu111 and Trichophyton make concocting
often inflammatory and even such products difficult.
necrotizing. A poultry problem only
in India.

Ringworm is rare in sheep. Agents are T. verrucosum, T. mentagrophytes, anó M. canis.

The same species affect goots.
~vith incrcascd prcvalcncc. hnprovc1ncnt in calves often
follows their release from damp, dark, crowded winter
quarters to the outdoors.
Infected indlvlduals of thc samc spcclcs pcrpetuate the
important der1natophytoses nf ¡¡nimal~ . Roclent sources of
sporadic T. r11e11tagrophytcs infections in dogs and cats seen1
:ikely. Sporadic occurrence of the soil-derived M. gypseu"1
~•fcctions contrasts with cndcmic to cpidcmic but bland
.nfections of swinc w1th geoph1llc M. nanurn.
Thc major agcnts of animal ringworm are globally dis-
rributed.
lmmunologic Aspects
lmmune Mechanisms of Disease
The majar antigens associated ~-vith dermatophyte infec-
tions are the keratinases (elicit cell-mediated responses)
and glycoproteins (carbohydrate moieties stimulate anti-
Laboratory Diagnosis
Direct Examination
In 50o/o to 70% of cases, hairs anti skin scales infected with
M. canis oc M. a11do11inii may emita bright greenish-yellovv
fluoresccnce u11dcr ult.raviolct light, c.g., a Wood's light
(/, = 366 i1m).
Microscopic Examination
Skin scrapings and hair are cxamincd microscopically tor
the prescnce of hyphac and arthroconidia. The scrapi11g
shoulc.l include material from the margins of any lesion
and the full thickness of the keratinized epidermis. The
hah is plucked, so as lo include Lile iuuafollicula1 po1 lio11.
The sample is placed on a slide, flooded '1-vith 10% to 20o/o
potassium hydroxide, covered ~vith a cover slip, and
heated gently. ·rhe treatment clears the sample (makes it
"transparent") whilc lcaving fungal structures and enough
of the hair and epidermis intact to rcvcal the agcnt in its
relation to the parasitized structures.
Mic1oscopiL t:.>.a111i11alio11 ~liuult.l l.Jt:gi1i u11ucr low
power (lOOX) and subdued light. Tnfected hairs are encasen
in an irregular sheath of arthrospores that may doublc thcir
normal thickness (Fig 46. 1). At higher magnification
276 1>A1tT 11 J3acteria and fungí

F 1G U RE 4 6. 1 . Cot hoirs infcctcd (ccntcr) ond uninfcctcd F 1G U RE 4 6. 2. Cot hoir infcctcd with Microsporum can is.
(right) with Mlcrosporum canis. 15% KOH mount. TOOX. Focus is on arthroconidla surrounding the hair (ectothrix). 15% KO~
mount. 400X.
•
• • '
•
•

.•
-
~
•
_,_ -
' ·
•

••
•1' •

••
' \·~
•
\.
•• I

• • • •

,,
,.
•
/

:;:::;

F1GU RE 4 6. 3 . Dog skin scraping infected with Tricophyton

mentagrophytes showing septate hyphae and chain~ nf arthrnroni-
dio. 15?i K011 mount. 400X.

(400X) of such hairs, lntlividual, spl1t'rical artluucu1litlia cyclul1cxi111itlc Dt:1111alupl1yte ·resl Mediun1 [DTM),
1

are recogni2able (Fig 46.2). In 11<ürless skin, branching hy-
phae a11d chai11s of ar throconidia occur (Fig 46.3).
Stains and penetrating and wetting agents (permanent
ink, lactophcnol cotton bluc, dimcthylsulfoxidc) improvc
visualization. Calcotluor white reagent imparts fluores-
cencc lo fungal structures and facilitates diagnosis \'l1l1ere a
fluorcsccnt microscope IS avallable.
Culture
Scrapings are plantcd onto and into thc surfacc of sclcc-
tivc media (::>abouraud's agar with chlorampl1enicol a11d
R;:ipicl Sporlll;:ition MPc1i11m íRSM]), which are incubated
at 25°C (room temperature) for up to 3 weeks. Samples
suspccted of containing T. verrucosun1 are incubated at
37ºC. On DTM and RSM, an alkaline reaction suggests
presencc of a dermatophyte. (Dermatophytes, if given a
choice of glucose and protein, will usually digest the pro-
tcin flrst, lead!ng to aJkali11t' pruuuct~; ~aprophylic fungí
will most oftPn 11tili7.P glucosc, leading to acid by-
products. Cautio11: Aftcr the preferred substrate is utilized,
the microorganism will make use of the other, shitting
thc pH in thc other direction.)
Susp1c1ous growth is examined microscopically. The ad-
 Cliapter 46 Dcrmatophytes 277

F1Gu RE 4 6. 4. Microsporum can is. Lactophenol cotton blue mount from patato flake
glucose agar. Note thick- and rough-walled spindle-shaped macroconidia, absence of
microconidia. 400X.
''
--- ·- ' ~
-
I
•••

• ..
'\
•
•
\
\~
llf
... . .... --._
•
~

- ..-
""

~.-

I
Ir , F1G u RE 4 6. s. Microsporum gypseum. Lactophenol cotton
blue mount from Sabouraud's agar culture. Macroconidia are cigar-
shaped and thin-walled; microconidia are absent. 400X

,.e side of a clear ccllophane tape strip is pressed gcn tly
·he suspect colony (takcn from RSM, or Sabouraud's
• \\'ith ch loramphenicol a11d cycloheximide-ctermato-
'es do not sporulate well on 'l)'l't-.1) and mounted in a
p of lactophenol cotton blue on a slidc and examined
-o~ropically (sec ·rabie 46.1, Figs 46.4- 46.6). With
· 1ophyto11 :;pp., in Ll1e abse11ce of tlia~11o:stic conidia,
trophic tests are used for speciation.
'lO\vlcdgc of sourcc (host spccies, type of lesion) aids
- ~1cantJy in provisional 1dentitication ot animal der-
phytes.
Molecular Techniques
·rhere are a number of DNA-based tccl111iques Lhal a1e
available for direct examination of clinical sarnples, or for
use in identification of an isolntcd dcrmato phyte. 'l'hese
tcchniques include dctcrmination of the sequcnce of DNA
encod ing specific products (c.g., chitin synthase 1 gene),
decermlnation of the sequencc of che interna! transcribed
~pacer regían, determination of the sequence of the DNA
encoding large riboso111aJ :.ulJu1lit (28S), and generatlon of
uniquc DNA fragments following arbitrary or randomly
primed polymcrasc chain rcactions.
278 PART II Bacteria and fungi

F1G u RE 4 6. 6. Trichophyton mentagrophytes. Lacrophenol cotton blue mount frorn

potato flake glucose aqar culture. Microconidia in single rows ("en thyrse," 1) and c/usters
("en grappe, • 2); thín-walled, long macroconidia (arrows). 400X

•• \

Treatment and Control Povidone-iodine (Betadine, Purdue Frecteric:k) anci

Cuu11.Ji11cd Lovical and syslemic treatn1ent is often prefer-
ahle. Of two systemic agents available, griseofulvin and ke-
toconazole, the latter is more costly and lcss preven. Both
drugs are given orally and are relatively 'vell tolerated.
Griseofulvin in small carnivores is given for at least a
month, or 2 weeks beyond cllnlcal recovery. Use in largc
anim;il" is ina<lPqnately <loc.umented. lt is not approved for
use in food animals. It is teratogenic in pregnant cats.
Terbinafine (an allylamlnc antifungal that inhibits ergos-
tcrol biosynthcsis, und concentrates in skin and nails) may
be a useful altcrnativc.
Antifungal orchard spray is effective on ringworm of
large and srnall anlmals (Captan 45o/o puwucr, 2 tal.Jle-
spoons/ga I). Affertecl arPas are first clipped. In large ani-
n1als, t wo applications at biweekly intervals are recom-
mended. With dogs, weekly dips can be repeated to ettect.
Contact with human sk.in should be avoided. Thiabenda-
zote (" rresaderm, Merck) LS used on small and large animals.
chlorhexidi11e (Nolva~a 11, Forl Dodge), available as lotio::5
and ointment'\, are general antiseptics with antifunga..
action.
A thorough cleanup of premises involving use of an io-
dinc, chJorinc, or phenol-containing disinfectant is essen-
tial. Utcns1ls and equ1pment are disinfected with Capta!:
or Bordeaux orchard spray.
Ic.lentiflcation of (;arricr~ i11 ke1111els a11d callerics can be
attempted hy culture of brushings. The Wood's light (ui-
traviolet light) is useful in population scrccning of ca¡
colonies v.1 here M. ca nis is the only concern. lnfected indi-
viduals should be isolated and treated. Exposed animals
are treated prophylactically.
Successful vaccinatlon is widely practiced on European
cattle. A livc atte11ualeu ~L1<ü11 (T. verrucosurn) appears to be
most imrnunogenic. Neither livc attenuated vaccines nor
killc<l products have been cffcctívc in prcvcnting dermato-
phytosis in cats.
 Agents of Subcutaneous
Mycoses
DWTGHT C. HIRSH
\'hcther a fungus is categori7.P<I a~ molci or yPast is based
_¡pon thc micro:icopic nppct1r1.1ncc in tissue or on routine
culture media (the ase>..'Ual stage). 11 hyphal structures are
>bserved, the fungus is termed a n·10/d; if a single celled,
..itkling structure is observed, the fungus is tcrmed a yeast.
~ routine culture media, molds vvill havc a "fuzzy" or
-ooly api;1::a1a11c1::, a1J<.l a ye11st will be hacterla-like in its
- '.onial morphology and consistPncy. SomP pathngPnir
a!lgi will produce cithcr hyphal-likc structures or yeast-
..Ae structurcs, depencling upon thc conditions in \vhich
ney are growing. Sucl1 fungí are called dirnorphic fi1ngi,
hich are dlscussed below and in Chapicr 48.
This chapter deals \.vith dimorphic fungí and fungus-
_.;e n1i.croorganisn1s af fecli11g ski11 a1i<.l sul.)(.:utis. l'o be d is-
_:;ssed are Sporothrix schenckii, wh ich i~ thf' cause of
.?Qrotrichosis in a variety of animal spccies, but n1ore fre-
..:ently in humans. horses. dogs, and cats; Histoplasma
.;;psulatun1 var. farciminosu1n 1 the cause of cpizootic lym-
'1angitis in eqwds (horses, donkeys, mules); the agents of
mycosis (AphnnonT}'ces, Lagenidi111n, Pythi11n1 and Sapro-
'fia), whlch cause a variety of dlseases in flsh and mam-
-al'; ;ind miscellaneous conditions involving the skin
"1d subcutis, i ncl udi ng chron1oblas1on1ycusi::i, pliat::ol1y-
~ '"lomycosis, and rnycetoma. Systemic mycoses, of which
i.ln lcsions may be onc manifcstation, are described in
_>:apter 48.
-
- ::oRO THRIX SCHENCKll
ufOtTiChOSÍS is a relative!y rare disea'e cau,P<I hy the
-?rophytic, dimorphic fungus, Sporothrix sclzenckii. l"his
-'<ase usually manitests as a chronic, ulcerative lymphan-
:::.~ of skin and subcutis. Systcmic (disseminated) discasc
.::urs occaslonally in imn1unocompromised human pa-
'lt~ (P .g ., alcoholics; individuals affccted with the
- -!llan in1n1unodeficiency virus). Ev<.:11 though dissemJ-
~áon co1nn1only occurs in cats, no assoc.iatínn hPtween
-Jerlying compromise and disseminatcd disease in vet-
..!:ary spccies has been de1nonstrated.
ERNST L. BIBERS'rEIN
Descriptive Features
Morphology and Staining
Sporothrix schenckii is a di111urµhic fungus, i.e., l t exhibits a
different 1norphology depending upon the conditions of
growth. At room temperaturc (2SºC, ou Sal.Juurau<.l':; agar),
S. sc/Jenckii gro"''S as a mold. This so-called saprophytic
phase consists of septatc hyphae, \vith oval or tcar-shapcd
conldla (Z to 3 ~un by 3 to 6 µm) in clusters on conidio-
phorf''\ and along hyphae (Fig 47.1). At 35- 37ºC (in tissue
or on rich 111cdia, e.g., blou<.l agar, i11cul.J11ted at that tem-
perature), it cxists as hudding plPomnrph ic ("cigar-
shapcd") yeasts measuring up to 10 µm in the longcst di-
mension (l·ig 47.2). 'fhe yeast phase stains with the Grarn's
stain, and either pl1aseaccepts Gicmsa or fungal stains (pc-
rlodtc acid-Schiff, Grocott methenam1nc silver, Gridley) .
Cellular Constituents and Products
Sporolhrix schenckii possesses a typical fungal cell ~vall con-
taining chitin, and ergosterol. Various glycoconjugates
found upon the \o\'all have adhesive propcrtics (scc ncxt
sectton, "Cellular Products of Medica! Intercst").
Cellular Products of Medica! lnterest
Adhcsins. The cell \.Yall of S. schcnckii contains glycoco11ju-
gates wíth attinity for extraccllular matrix proteins (fi-
bronectin, laminin, and Type II collagcn). l'his intcraction
does not involve an "Arg-Gly-Asp" (H.GU) sequence.
Ce// Wnll. The cell wall of S. schenckii contains severa!
subsla11ct:=> that rnay play a role in the virulence of this
microorganism . ThP'\P inchulP lipid, melanin, peptide-
rhamnomannan, and sialic acid:
1. Liµi<.l . 'fhe lipi<.I portion of the cell wall of s. schenckii
inhihits ph;igocytosis by monocytes and macro-
phages.
2. Melanin. Melanin found in the cell wall protP.cts S.
schenckii froin thc cffccts of reactive oxygen interme-
diates withín phagolysosomes ot phagocytic cells.
Melanin is a free radical scavcnger (reduces thc toxi-
clty uf hydroxy radicals, supcroXides, and singlet
oxygPn radicals found within the phagolysosome).
279
280 PART 11 Bacteria and Fungi

F1G U RE 4 7. 1. Sporothrix schencku. Hyphal rtrands on a

p/;itp of S;iho11ra11d'.~ agar incubated at 25ºC. Hyphae bear
conidiophores with daisy likc clustcrs of ov.?tc conidia. Wet
mount, about 400X.

•
, ..
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r.: .,,•
-~ª
o· ºº
•
.a,
.-\
• ~
..,
\:.:
a.-
'
F 1G U RE 4 7. 2 . Exudate from sporotrichosis lesion in a cat.
Wright-Giemsa stain. Note pleomorphism of dark-staining yeast
. - ce/Is, some of which are budding. 1000X.

3. Pcptidc-rhao1nomannan. Thc pcptidc-rhamno- Growth Characteristics

mannan traction ot the cell wall acts asan immuno-
suppressive substance by suppressing the liberation
of prolnflam1natory cytoklncs by phagocytic cells.
4. Sialic a cid s. Sialic acids fotn1d in the c:ell vvall in hibit
uplakc of S. schenckii by phagocytic cell:>. Sialic acid
directs complement protcins towards the degrada-
tivc patl1way, rathcr than generating effective op
son1z1ng fragments and anaphylotoxins needed to
gcncrate an effective inflammatory response.
J>roteases. Sporothrix schcnckii produces two protcascs, I
a~d 11. ·rhc signilicance ot these enzymes in the pathogen-
es1s of sporotrichosis is unclear.
After .se~~ral days un SalJourauú':; agar at rvv1u Le1upe1·a-
h1rP, 1n1t1ally moist, off-white to hlack colonies develop
that bccomc wrinkled and fuzzy. On blood agar at
35-37ºC, whitish, smooth yeast colonies appear within a
fcw doys.
Variability
Examination of mitochondrial DNA (by restriction Iength
polymorphism analysis) shows that there are at least 20
different types or strains of S. schenckii (1-20) .

Ecology
Reservoir
Sporothrix schenckzí is associated with plant material and
soil worldwide. Occasionally it is isolatcd &om mucous
men1b1a11c::s uf nur1ual auiinals an<.l animal products.
The disease occurs- rarcly-in humiinc:, horses, dogs,
and cats, and has been reported il1 n1ice, pigs, raLs, can1t:ls,
dolphins, foxes, mules, goats, and chickens.
Transmission
Thc agcnt cntcrs through skín contact, usually trauma.
Rare cases of interna! intection are dueto inhalation or in-
gestion. Discharging lesions may be contagious, especially
.i1 Lc1 t:. wl!e1e ll1e J1u1ulJt:rs uf nllcruurganlsms tn exudares
from this species are quite large. Tt has h PPn isol;;ited from
nails of affcctcd cats, and from clinically normal cats in
contact with in tected individuals.
, athogenesis
Proteases are possible virulence factors and protcase in-
h1bttors have been shown to suppress nodule formation.
':'ell wall constituents and adhesins d elay elimination of
·he 111icroocgauisni. Typically the first Ieslon Is an ulcerat-
ng c11tiineous nodule. 1'he infectious proccss follows sub-
cutaneous lymph cha11nels, produciug suppurating ulcers
::t intervals. These heal slowly but often re-erupt. Lympha-
:::ics bccomc thickcncd. Disscmination to viscera, joints,
bones, and the central nervous system is common in cats.
Lesions are pyogranulon1atous, having a purulent cen-
t.'f surrounded by epitheliotd cens, giant cells, and, p e-
:-:phera lly, lymphocytes and plasma cells.
T11e course is protracted. Observatlons on humans sug-
.:e'>t possihlP spont;;ini>o11c; rP<OVPriPc:
.: :iidemiology
porotrichosis is acquired from the no111iving envirou-
.....,ent, but transmission from suppurating lesions is possi-
.e, particularly from cats. Tn humans, disscmin.utcd dis-
~..:.se Is seen in the immunosuppressed (e.g., alcoholics;
:idividuals affected with the human immunodeficiency
-~). Sucl1 an associat1on has not been demonstrated in
-her animals.
::imunologic Aspects
e.1-mediated reactivity is significantly related to resist-
..,cc. No artificial immunization procedures exist.
. :boratory Diagnosis
- ~ect examination of cxudatcs is often unrewarding ex-
~pt with feline specimens, which gencrally contain abun-
....nc yeast cells (see Fig 47.2). Witb other hosts, fungal
11~ (see above) and lmmunofluorescence may help de-
....,_ yeast ci>lls.
CJiapter 17 Agents of Subcutancous Mycoses 281
The agent grows readily in culture. Idcntificalion 1e-
quires demonstration ofboth phases.
Serologic tests (ycast ccll und latex agglutination, agar
~el diffusion) have had limited use in animals.
Molecular techniques utilizing the polymerasc chain
11::al:liu11 wtth pr11ners ues1gned ro ampllty segments ot the
gene encoding thP chitin synth ase 1 have been used to
demonstrate the fungus in clínica! samplcs, as well as il.11::11-
t ification of isolates (uscful when an isolatc rl?.sists thP
mold-yeast convcrsion).
Treatment and Control
·rhe cutaneous for1111espo11d:; gt:11t:rcdly tu the oral admln-
istration of inorganic iodides. The azolc <ln1gs, f'SpPri;;illy
itraconazole, are effective. Tcrbinafinc (an ullylamine anti-
fungal agent which inhibits ergosterol biosynthesis) has
shown sorne success in treating human patients with the
culaneous ruru1 uf sporotrichosls. Amphotericin B and
flucytosine are 11sf'<l on <1eep and disseminated forms.
H ISTOPL ASMA CAPSULATUM VAR. FARCIMINOSUM
Histoplasma capsulatum is a dimorphic fungus existing as a
mold at 25 30ºC (saprophytic pilase) andas a yeast at 37ºC
(parasitic phase). Th1s fungus has three varieties: H. capsu-
latum v ar. caps11/at11m, H. capsulatun1 var. duboisii, and H.
capsulaturn var. [urc. imiriusum. Varietles capsulacun1 and
duboisii cause histoplasmosis, a c;y<;tf'mi< fungal dísease
thut will be discussed in Chapter 48. Variety farci111inosu1T1
causes epizootic lymphangitis (pseudoglanders), a cl1ronic
pyogranulomatous discasc of cquine (and rarely donkcys
and mules) skin. Adjacent tissues, regional lymph nodes,
and, rarely, interna! organs may be involved. Epizootic
ly111phangitis is dlscusscd below.
Descriptive Features
Morphology and Staining
Histoplasma capsulatum var. farcimí11n~u1n , ;;i di morphic h1n-
gus, produces budding yeasts (2 to 3 µm by 3 to 4 µn1) u1 lis-
sue, and usually sterile hyphae in its mycelial form when
grown at 25ºC or room tempcraturc. J::xccptionally, arthro-
conidia, chla1nydoconidia, and spherical, thick-walled
macroconidia (spheroidal cells, 8 to 14 µm in diameter,
sludded vvill1 ííugcrlike pro¡ections, scc Flg 48.4) are secn.
The yeast phasP. is hi>st demonstrated with a
Romanovsky-type slai11 (e.g., Wrigl1t '~ or Giemsa) or fun-
gal stains (periodic acid-Schiff, Groc:ott mi>th enamine sil-
vcr, Gridlcy, scc Fig 48.5).
l:lrowth Lharacterist1cs
Hístoplasrna capsulatum var. farcirninosum grows on com-
mon laboratory media (Sabouraud':s glul:u.se, ir1fusion, an<.l
blood-supplemented m edia with or without cyclohex-
imide and antibactcrial agents) over a broad temperaturc
28 2 PARr 11 Dactcria and fungi
range. The optimum for mycelial gr(n·vtl1 is 25~ to 3o~c,
tilking St>vf>riil wf>f>ks tn fnrm a cottony, white to hrown
colony. Pigmentation parallels abundance of macroconi-
dia. 'I'he yeast phase requires rich media (e.g., glucose cys-
tcinc blood agar; brain hcart infusion blood agar) and tem-
peratures of 34º to 37ºC, taking severa! days to form a
cream to tan, yeast-like colony. Co11version of mold to
yeast on blood-contalnlng agar requlres incubation at
37ºC under 1.So/o to 2.0º.1i C0 2 . Sever;i 1 p;.issagi>s may hf>
needed to convert tl1c n1old to the yeast phase.
Resistanc:e
Histoplas1na capsulaturn var. farciminosum is quite rcsistant
to physical and chen1ical agents. lt sur vives at amb ient
temperatures for months (severa! weeks in corrals and sta-
bles) a11d ar refrigerator temperature or in a desiccated
form for years.
Ecology
Reservo ir
The reservoit is unk11ovvn ; presumatJly infecteü el1uid~
(horses, donk.eys, m.ules).
Transmission
Infection is thought to occur t hrough skin wo1.111ds. Ar-
thropods may p lay a part. Rcspiratory, conjunctival, a11d
gastrointestinal infections are also reported .
Pathogenesis
A local skin noclule is founu, which l.Jecurnes al.J~ce~~ed
and u lceratf>ci . Thf> i1lcer may r ec11 r , f>n larging each time,
an cl ultimately heal by scarring. 'fypically, adjacent lyn1-
phatics develop nodules along their course, showing the
samc altcrnating activity. Adjacc11t lyn1pl1 nodcs develop
abscesses draining by sinus tract s to the outside. Hemato -
genous spread a.nd visceral involvem en t is possible. His-
tologically, the p rocess evolves from suppurative tu gra11u-
lom;.ito11s (pyngran11 lnmatou.s), marked hy Jymph ocytes,
n1ac::rophages, and giant cells, to eve11tual fibro5i5. Yeast
cells occur extracellularly and intracellularly, especially in
macrophages.
Skin Jesions, chiefly on head, neck, and li mbs, are the
predominant signs. ·rhe ge11eral condition is u sually u naf-
fected except in t l1e prln1ary respirato ry tract or dis~e1ni­
n;.itprl infc>ctions. Snmf> m ilci ca.sf>.s do n ot p rogress heyond
thc local 5tage.
Epidemiology
Endcmic arcas includc parts of Africa, much of Asia, an d
the Mediterranean littoral. 'f he epide1u iology of H . capsu-
latuni var. farci'minosun1 infection is un clear. Ma11ifesta-
tions vary with geographic area. Seasonal peaks ~ugge~t
arthropod transmission.
'fhe disease n1ainly involves horses, but mules and don-
keys 1nay also tJe affected. Yuur1g 11urse~ (les::. Ll1a1.1 6 yt.:a.cs
of age) are 1nost susceptible.
lmmunologic Factors
The role of immu ne mechanisms in pathogenesis or resist-
an ce is not known, but cell mediated immunity is proba-
bly t he key host defense . Skin sensitivity develops follow-
i11g expusure, 1::ve11 ilJ Lhe absence of d isease. Circulatil1g
antibodies, den1onstrahle by indirect fluorescent anti-
body, agar gel diffusion, or by the serurn aggluti nat ion
tests are indicative of infection.
Laboratory Diagnosis
Differential d iagnosis includes sporotricl1osis (see above,
Sporothrix schenckii) and ulcerativc lymphangitis produced
by Corynebacteri um pseudotuberculosis (see C:hapter 31). The
agent must b e demonstrated. Direct examination o f
stained exud ates (Wright 's or Giemsa stained) or biopsy
material (hematoxyUn -eosin, periodic acicl-Schiff, Grocott
rnethenamine silver) may reveal intracel\ular (1u acro-
phages) or extracellular ypasts.
Histoplasrna capsulaturn var. farcirninosu111 grows on
Sabouraud's glucose agar \o\1ith inhibitors (cycloheximide
and chloramphcn icol). Mycclia1 extracts contain genus
specitic antigens demonstrable by agar gel diffusion or by
the seru1n agglutination test. Growth patterns and in.icro-
scopic morpJ1ology d:ifferentiat e H . capsulatum var. farcimi-
nosum from H. capsulatum var. capsulaturn (Chapter 48).
Treatment and Control
Inlravenous iodides (witl1 or witbout griseofulvin) have
been relatively succcssful. A1nphotericin B has been used
with so1ne su cccss. ln noncndcmic arcas, dcstruction of
inlected animals is advisable.
ÜO MYCOSI S
Oomycosis is caused by a mem ber of a group ot eukaryotic
m icroorgan isms belonging to the Ki ngdom Stra1ne11opiles
(which also contains t he d iatoms and brown algae). They
are saprophyt ic microbes that usually live in water and
moist soil . Though they are n1orphologically similar tu
fn n gi (thPy p rnrhtrP hyphriP in t iss11f> an rl grow as mold-
like colonies on media in vitro), t hey are not mcmbcrs of
the Kingdom Fungí. Me1n bers of this group are the agents
of severa! plant and animal diseases. Historically, the most
r1otorious is Phytophthora, the cause of tl1e Jrish potato
famine, which at present is responsible for killing oak t rees
along the Pacific coast of North America. The following
genera are associated with. c.Usease in ;inima ls: Aphanomy-
Les (ulcerali ve disease of fisl1 and crustaceans), Lagenidiu111
(pyogranulomatous disease of dogs and cats identical clin -
ically to pyth iosis), I'ythiu111 (pyogranulomatous disease,

called pythiosis, of a variety of animal species), and Sapro-
legniu (sysLenlic disease o[ cullured saln1011ids). Tl1c discus-
sion that follows is focused on J>ythiurn insidiosurn, the
cause of pyogranulomatous conditions (pythiosis) of dogs
("swamp cancer"), horses ("Flori<la horse leeches"), cattle,
cats, and humans.
Cutaneous Pythiosis ("Swamp Cancer"-
"Florida Horse leeches")
1'he genus J>ythiu111 contains abou t 85 species, and only P.
tnsidtosum is assoclated wlth dlsease In animals. Pythiurn
;nsidiosum causes ulcerative pyogr::tnulomatous or fibro-
granulon1atous skin infections in horses, cattle, dogs, and
cats, mainly in tropical or subtropical areas (in Nortl1
i\ rncrica this discasc is sccn most frcqucntly along thc Gulf
c oast). uogs anc.t horses are the spec1es most con1mon1y af-
fected. The agent is an aquatic oomycete \.Vith wide (4 µm),
sparsely septate hyphc1e. Lesions in horses are large (~45
c:m) <iisch<i rgi ng swPll i ngs, 11s11a lly on PxtrPrn itiPs, vPntr<i 1
trunk, or head. The nasal n1ucosa may be involved .
Hyphae are demo11strablc within granulomatous coagula
tcrmcd "kun kcrs" or "lccchcs" in horscs) consisting of nc-
crotic macrophages, epithelioic.1 cells, and giant cells, with
eosinophilic admixtures.
Diagnostic methods incluc.le serology (an enzyrne-
rnked imm.unosorbant assay), molec:11lar technicp1es (uti-
lizing prin1ers designed to an1plify P. insidíosurn-specific
::>~A by means of the polymerase chain reaction [PCRJ),
•mmunohistochemical cxamination of cxudatcs, and cul-
,ure. Cultural techniques are tedious and time consuming
and e11tail growt h of a 1nold-like 1nicroorganism on
Sabouraud dextrose agar or brain heart infusion agar after
:4-48 hours at 30ºC. Identification requires den1onstra-
::!on of n1otile zoospores a11tl/or reacUou uf ex Lra<.:LS of U1e
..:solate with reference antisera. 'l'J1e PC:R-based assay men-
-~ned is used Lo eslablish Ll1e presence of P. insidiosurn in
,
•
"
~ ,_ ..
4(
..
~ . ' \
~· ~· ~•'--
Chapter 47 J\.gents of Subcutaneous Mycoses 283
clín ica l san1ples as well as for identification of isol;ites. The
PCR assay also successfully identifies members of the
genus Lagenidiurn (another oomycete), which produces a
similar clinical picturc in dogs and cats.
Early diagnosis is the key to successful treatment.
·rreatments include surgery and antifttngal therapy (am-
photericin B). lmmunotherapy utilizing killed whole or-
ganisms or cxtracts has shown promise.
(HROMOBLASTOMYCOSIS ANO PHAEOHYPHOMYCOSIS
Chro1noblastomycosis and phaeohyphomycosis are caused
by d::trk-pigmented (den1ati<iceous) fungí . Most of th.e
roughly 70 species implicated belong to the genera Alter-
naría, Cladosporiurn, Cladophialophora, Curvularia, Exsero-
hil un1, Exophiala, Fonsecaea, and l'hialophora. ln chromo-
blastomycos1s, the rungal eJements 1n tissue are large t<ll
µrn ), pig.mented, "sclerotic bodies" (Fig 47.3). lnfections in
which hyphae are presentare called phaeoflyphornycosis.
C:hromoblastomyc:osis is rare in nonhuman mammals,
but occurs in frogs and toads. Ph¿teohyphon1ycosis is see11
sporadically in cats, dogs, horses, cattle, and goats and
niay be systcmic. Cladophíalophora bantiana is the fur1gus
n1ost commonly see11 in dogs and cats, with central nerv-
ous system localization frequently observed.
'rtie agents, soil- and p.lant-associated saprophytes,
enter through skin ancJ n1u ltiply subcutaneously, c:ausing
pyogranulon1atous reactions. No tissue colonies or gran-
ules are seen. Nodular or larger swellings develop, which
may ulccratc and dischargc pus.
Diagnosis is t)y biopsy and culture. Sclerotic bodies
(chromoblastomycosis) and hypl1ae (phaeohyphomyco-
sis) are seen in stained (hen1atoxylln-eosln, perlodlc acld-
Schiff, Grocott n1ethenamine silver) biopsy sections.
Cullure, on Sabouraud's agar wilhoul il1hibilors, oflen re-
qui.res lengthy inc:ubation . ThP res11lting colonies r<ingP.
fron1 olive to bro\vn to black.
F1G U R E 4 7. 3. Chrnmnhla~tnmyrn~i~ in thP ~kin of ;¡ horse.
Pigmented bodies suggestive of yeast ce/Is (blastoconidia) are
surrounded by inf/ammatory reaction. Hematoxylin-eosin stain,
400X.
284 PART TI nac.te.ria and Fllngi
Lesions are excised, but may recur. Medica! treatment
(flucytosine, itraconazole, amphotericin B, ketoconazole)
has given mix.ed results.
(EUMYCOTIC) MYCETOMAS
Swelling, granule formation, and discharging sinus tracts
are characteristics of mycetoma. They may be associated
wit h bacteria, most notahly an actinomycetesuch as mem-
ber~ of the ger1era Nucardia or Aclinurnyces (actiuornycotic
1nyrPto1n;:i, sPe. C:haph~r 17) or ;:¡ fu ngus (eumyc.otic. n1yce-
toma). Mycetomas are occasionally reported in cattle,
horses, dogs, and cats. 'fhe cutaneous form is often nodu-
lar ánd is associated with similar nasal lcsions.
Fungí associated with eumycotic mycetoma fungí in-
clude i'Seudalfescherta boydii, c·ochltObOlUS Spici(erus (t'.:E
sexual forros of Scedos;1ori11m a;1ios;1ermum a11d Bipolar~
spicifera, respectively), ar1d Curvularia geniculata. All a.<0
sapropbytes that presu111ably enter via a wound. It is no.·
elcar what triggers this coursc of pathogcncsis, because th t:
same age11ts can cause other pathologic patterns.
Fungal colonies are surroundecl by suppu ration bo:-
dered by granulomatous reactions. Sinus tracts carry pus
ancl granules, consisting of microorganisms and inflam -
111a tory con1po11e11ts, to tl1e surface. The processes are
slowly progressive, involving adjacent tissues.
Treatment is cxcision if possiblc. Antifungal agen:s
(azoles, an1photericin 13) have been disappointing.
 Agents of Systelllic
Mycoses
DWIGHT C.
· \·hether a fungus Js categorlzed as mold or yeast is base<.!
1pon thc microscopic appp;ir;:inct> in tissue or on routine
cu!Lure 111edia (lhe asexual sLage). Microscopically, if l1y-
?hal structures are observed, the fungus is termed a mold; if
:íinglc-ccllcd, budding struct urc is obscrvcd, thc fungus is
termed a yeast. On routine cullure medía, n1olds will have a
-n1zzy" or wooly appearance, and a yeast vvUJ be bacteria-
llke i11 its colonial morphology a11d conslstency. Sorne
pathogenic fungí will produce e.ither byphol-likc? structures
tir yeast-like structures, depe11dil1g upon the conditio11s in
-,·hich they are growing. Such fungi are called dimorphic
i mgi. Thesc fu11gi are discusscd bclow and in Chaptcr 47.
'fhe agents oí most systemic tor "deep") mycoses are
saprophytic fungí. Morphologically and ecologically di-
~erse, they sllare disease-related features:
L Many are dimorphic, that is, their saprophytic and
parasitic phases differ morphologically. Coccidio-
ides, Histoptasma, Blastomyces, and Paracoccidioides
grow as molds in their inanimate habitat. In tissue,
Coccidioides pro<luces sporangta, whereas the others
grow as budding yeasts.
2. Infection is usually by inhalation.
3. Host factors are often the decisive disease determt-
nants. Sorne mycoses (aspergillosis, zygomycosis) are
seen primarily in tmn1unocompromised animals.
4. Lesions tend to be pyogranulomatous. After pri-
mary pulmonary infection, the course of disease is
determü1ed by the effe.ctiveness of cell-mediated
i rruuuue respu11st:s. If these are inaclequate, dissem-
ination may oc:c:ur to hone, skin, c'Pntr;il nervous
system, or abdominal víscera.
5. Systemic mycoses are no11contagious. AJthough the
agent is oftcn dcmonstrably shed, it fails to infect
indiv1duals on contact.
l'his chapter discusses the dimorphic fungi Coccidioides
~mmiti.s, Histoplasma capsulatu111, and Blastonzyccs der1nati-
¡fdis, togethcr vvith the mold Aspergil/us. '!'he fungus
Pneumocystis and the alga Prototheca are briefly mentioncd.
(OCCID/OIDES
~embers of the genus Coccídíoides are din1orphic fuugi,
existi11g as a mold in soU (saprophytic phase) and as a
HIRSH ERNST L. BIBEl\STEIN
spherical structure in tissue (parasitic phase) . 'fl1e ge11u:;
<:occidioides c:ontains two species, immitis and posadasíi.
The disease, coccidioidon1ycosis, i.s caused by both.
Coccidioides imm.itis and C. posadasii differ i11 their pre-
fcrrcd gcographic habitat:J. Both spccic5 occur only in thc
western hemisphere in the Lower Sonaran Life Zone, ap-
parently as a result of that area's peculiar soil properties
and temperan1re and ralnfall patterns. Coccidioídes immitis
is found in the Centr<ll Valley of California in the llnited
States (n1ai11ly tl1e San Joaquil1 Valley), whereas C.
posadasii is found in non-California locations (Texas, New
Mcxico, Arízona in thc Unitcd Statcs, and i.n South
America). Of domestic animals, dogs are most frequently
affected, although horses are occasionally affected as we!L
Jnfections also occur in cats, swine, sheep, cattle, human
and nonhuman primates, and sorne 30 species of nondo-
u1eslic u1an11nals.
Descriptive Features
Morphology, Structure, and Composition
In thc soil, Coccidíoidcs is a mold made up of slender sep-
tate 11ypllae that give rise, on thicker secondary branches,
to chains of infectious arthroconidia (arthrosporcs,
arthroaleuriospores, arthroaleurioconidia). These are
bulging, thick-waUed cells, 2.5 to 4.0 µm by 3.0 to 6.0
µ111, se¡;arale<l lJy ernpty cells (<lisjunctors), through
which hreaks or.cur whPn <irthroconidia are dispersed
(Fig 48.1).
In tissue, arthroconidia grov.i into sphericaJ sporangia
>vith birefringcnt walls, "spherules" (10 µm to 80 pm di-
ameter), vvhich by inter11al cleavage produce several hun-
dred "endospores" (2 µ1n to S µm dia1neter) (Fig 48.2).
'fhe \-vans disintegrate, allowing dissemination of en-
dospores, each of 1>Vhich may repeat the cycle or, on a
nonliviug su!Jstrate, give rlse to mycelial (a collection of
hyphae) growth. Thoueh only arthroconidia are natu-
rally infectious, endospores can experirue11tally initiate
disease. Sexual spores are not known.
"Coccidioidin" in supcrnatants of mycelial Coccidioides
broth cultures is largely polysaccharide, but co11tains sorne
a·m ino acid nitrogen. It is used in cutaneous hypersensitiv
ity an<l serologic tests. "Spherulin," a lysate of cultured
sphPr11 les, is olso used in skin tests.
285
186 PART 11 Ractf'ria an<l i:11ngi

F 1G U RE 4 8. 1. Coccidioides imn1itis. Teased preparation from a 5-day-old culture on

Sabouraud's agar. Lactophenof cotton blue mount. 1htel<-walfed barre!- or bríck-sriaped
;irthrnronidia a/ternate with empty ce/Is. On lower left an isolated arthroconidium with
fragments of ¡;¡dj<Jccnt ce/Is (disjunctors, hyalinc pegs) attached. 400X.

•
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. t~ F 1G U RE 4 8. 2. Coccidioides immit is. Transtracheal aspira te

. L-1 from a dog. Unstained. Top left: mature spherule (sporangium)

with endospores. Rnffnm right: immature spherule. 400X.

Cellular Products of Medica! lnterest

l . Beta-glucosidas<' ?.. Rgl2 (for heta-g/ucosidase 2) is
Adheslns. SOWgp (for spherule outer wal18ly1.:u¡;rulei11) is a <111 e11z.yn1e secreted by the e11dospores of Cocci-
prolinc-rich. glycoprotein ad he sin 011 th e su rfac·p of dioides that most likely plays a role in endospore
:sµlu.:ruh;:.. SOWg¡; has affiniLy for exlraccllular n1atrix pro- morphology. Antibodics (IgM isotypc) to Bgl2 are
tC'ins (laminin, fibronectin, collagcn). SOWgp stimulates a made early in the iniectious p rocess, and are useful
strong T H2. lymphocyte response, rcsulting in clcvatcd lcv- diagnostically (detected by t he p recipitin test) to
els of antibody and suppression ot the protecttve ccll- s1gnal that a reccnt lnfection has occurred.
mediated immu11e response. Mutants unable to produce 2. Chitinase l. Ctsl (for chilinase 1) is one of Sf'vf'ral
S()Wgp have greatly reduced v1rulence. chitinases tl1at are i11vulved will1 ll1e forn1ation of.
Miscellaneous Prod11cts: an<l release of, endospores from the spherule. An

important ta rget for the Cts enzymes is thc "sc-
gregation apparatus" (a weblike mesh of chitin-
contain ing structures within the sph erule).
A11liuullit::> (lgG i:>utypt:) to Ctsl are macle late in the
infectious process (in clissPminatPcl clisPasP), and are
useful diagnostically (detccted by the complen1ent
lixation test).
3. Beta-1, 3 glucanosyltransferase. Gel (for glucan-
elongating glucanosyltransferase) is Iocated on the
surface of endospores. Gel elicits a strong T H t
lyn1hocyle re~¡.¡011st:, rt::>ultiug iI1 elevated Ievels of
gamma interferon followed hy protPrtion against
disseminated disease (activatcs macrophages).
4. Serinc proteases. These enzymes play a role in the
stimulation of the inflammatory response clicitcd
by Cocctdlotdes. These enzymes digest elastin , colla-
gen, and immunoglobu!ins
5. Ureast:. Tliuugh lt:> rule in virulen ce Is u nknow n ,
the enzyme urease (l Jrf') Plirits a .~trone r rotPrti vP
ünmunc response followin g stimulation ofTHl lym-
phocytes (activates macrophages) .
Growth Characteristics
Coccidinides grows on simplP mPdia overa broad tempera-
~re rangc. On Sabouraud's or blood agar, growlh is u1y-
celial (mold). Over severa) days, initially dull gray colonies
develop with sparsc acrial mycclium, which gradually bc-
comcs more abundant. Arthroconidia are produced in 5 to
- days. Bovine blood agar is hemolyzed. Much colonial
-:-arlability existS.
Spherules (sporangia) can be demonstrated in vitro by
g¡owU1 al 40ºC in 111edia cuulai1li11g casei11 h ydrul ysate,
slucose, biotin, glutathione, ancla salt mixn1rP (spherule
::nedium).
Ecology
iteservoir
Cocddioides inhabits the soil in the Lower Sono ran r .ife
Zone, including parts of Cali fornia in North America (C.
anmitis) and the southwestern portion of the United
Statcs, Mexico, Central America, the northcrn and west-
ern rim states of South Ameríca, Argentina, and l'araguay
C. posadasi1). High prevalence is assoc..;ated with an an-
n ual rai11fall vf 5 tu 20 ll1cht::s anti mean summer and win-
¡:er temperatures of over 80ºF ancl 4."ºF, rPspPctively.
Arthroconidia resist drying and tolcrate heat and salin-
'.ry better than do competing soil organisms. In summer
~eat, Cocddioides survives in soil laycrs ncarcr thc surfacc
:han its competitors. When cond1t1ons favor gro\.vth again
after rains, Coccidioides repopulates the superficial soil lay-
ers fitsl, eu:.uriug i LS wiucspread dispersa l.
Transmission
rnfection is malnly by inhalation of arthroconidia.
Primary cutaneous infections are rare.
Chapter 18 Agents of Systcmic Mycoscs 287
Pathogenesis
Mecflanisms and Patflology. Following inhalation, the
barrel-shaped arthroconidia round into spherical-shaped
endospores, which-fvlluwi11g e1tlarge1nent and interna]
cleavage-differentiate into multinuc.lPatP sphPn1lPs ron-
taining hundrcds of endosporcs, a process that takcs
severa! days. Spherules rupture, releasing endospores, and
the cycle is repeated. Arthroconidia, endosporcs, and
spherules trtgger an lnflammatory response in the lung
(serine proteases are partly responsible). Arthroconidia
and endospo1es a1e eugulft::LI uut uot killecl by phagocytcs,
and are conveyed to the hilar Iymrh noclP, where another
inflammatory focus develops (as wcll as more spherulcs).
lnflammation .is stimulated in part by serine proteases.
which are liberated during the growth of t hc fungus.
Normally, cell-medtated ln1mune responses arrest the
process at this stage following stimulation ofT Hl lympho
cyLes ll1al ac.:tivate:: rna<.:rophages (following specific re-
sponse to Gel), permitting destruc.tion of thP endospores.
Antibodics to Bgl2 are made during this phase of the infec-
tious process. With inactequate cell-mediated iromunity
(caused by SOWgp directed immunomodulation, gcnctic
makcup, lnfect1ous dosc), dissemination can occur to
hones, skin, abdominal visccra, heart, genital tract, and
eye (and rarely in anin1ab lu uraiu ancl meninges). Gross
lesions are white granulomas varying from miliary ood-
ulcs to irregular masses. Peritoncal, pleural, and pericardial
effusions occur. Antibodies to Ctsl are made during this
phase of the infectious p rocess. Microscopically, the pre-
dominant response 1s pyogranulomatous: to arthroconi-
dia an d endospores, the response is suppurative; and to
spher ule~, Lhe re:::>µo11se Is proliferatlve, witl1 epithelioid
cells t he chief componPnt, acimixPcl with giant cells, lym-
phocytes, and neutrophils (Fig 48.3).
Disease Patterns
Dogs. Disseminated diseasc of dogs is Ll1e paLLeru :;ee11
most commonly by veterinarians. The complaints are Ias-
situde, anorexia, and loss of condition. Thcrc may be respi-
ratory signs (includ 1ng cough), fever, and tan1 eness dueto
bone involvement or arthriti.s, or discharging sinuses fro1n
ueep lesions.
Cats. Dissen1inated disease in cals p rese11 Ls witl1 les:; os-
seous and more visceral involvement.
Horses. As with cats, there is usually less osseous and more
visceral involvement.
Cattle, Sheep, and Swine. In these species, the disease is usu-
ally asymptomatic, li1niLed Lo luug:; a1H.I regional lymph
nodes, and undiagnosed until slaughtrr.
Epidemiology
In all species, overt disrasP is thP Pxreption. Highest preva-
lcnce of canine systemic coccidioidomycosis is obse1 ved i11
male dogs, 4 to 7 years of age, with peak occurrence of ill-
nesses January to March nnd May to July. 1"hese peaks may
288 P ART Il Bacteria and Fungi
F1G U RE 4 8 . 3 . Coccidioides immitis in lung tissue of a Bengal tiger. Hematoxylin·
eosin stain. Note prominence of neutrophils in exvdate. 400X. (Photograph
Dr. Roy Henrickson.)
reprcscnt seasonal stress and incrcased exposure, respec-
tivc!y. Geographic and climatic facto rs have been men-
tioncd. YoungBoxer dogs and Doberrnan pinschers appear
particularl y susceptible.
lmmunologic Aspects
Ct:U-u1cuialcu liypersensiLivity, as dctern1ined by skin
tests, develops •vithin one or more wccks of exposure and
can persist indefinitely. lts presencc is an indicator of rc-
sistance to progressive diseasc. lts absen ce is the rule in dis-
sem inated infections, and lts return is a favorable sign.
IgM antibody (specific for Bgl2), demonstrable by tubc
precipitin or latex particle test, appears temporarily after
inft:ctiou a11u u::;ually disappears agaln. JgG antibody titers
(spc>cific for Ctsl), which are detectable by complement
flxation anu immunodiffusion tests, risc in disscminated
discase and remain high (1:16) until the p rocess is brought
under control.
No vaccines are presently avallable.
Laboratory Diagnosis
Direct Examination of Specimens
Anin1al fluids and tissues are examined for sphcrulcs by
wet mount in saline containing lüo/o KOH. Spherules are
10 to 80 µm in dian1eter, have a thick wall (<2 µm), and
conta1n cndospores when maturc (see Fig 48.Z). Free cn -
dospores look indistinct in wet mounts but are recogni7a-
blc In flxe<.l smears staiI1t:u 1.Jy a fu11gal stai..11 (e.g., periodic
acid-Schiff [PAS], r.ridley, or Gomori methenamine silver
slaillS).
rn11rtP.~Y nf
..,
•
• ••
Stained tissue sections (hematoxylin an<.l eosi11 [H&t.1
Gomori metl1enamine silver, Gridley) show hoth thP
agent anll the characteristic lesion (see Fig 48.3).
Culture
Blood agar and Snbouraud's agar \.Vith or without antibi-
Otics are inoculated, tape-sealed, and incubated at 37ºC
and 25ºC, respectively. Ali processing of culhues is don"'
under a microbiological :.afety l1ood. Mycelial gro"'·tr.
should be evirlPnt within a wcck and is examined for pres-
cnce of arthroconidia in a lactophenol cotton bluc wet
mount. The isolate can be reconverted to thc sporangia,
phusc by nnilnnl inoculation or cultivatlon in a spherul<:
medium (sce above, "Growth Charactcristics").
A commercially availablc "exoantigen" test kit fur-
nlshes preparetl a11ti::;era Lu Cvlt.idioides, Histoplas111a caps11-
lat11m, and Rlastomyces dennatitidis to be tested against ex-
tracts of suspect cultures in an im munodiffusion agar
plate, where precipitation lines develop between extracts
und their homologous antisera.
lmmunodiagnosis
·rh P. con1plement fixation (CF) and immunodiffusion
tests detect disseminated d iscusc. Bcc<tuse it is quantifi
able, the CF test gauges prog ress and cure of infection
more accurately.
The coccidioidin skin test has becn use<.l in a11ilual
surveys.
Molecular Methods
Most molecular methods used to identify or ctetect Cocci-
dioidcs make use of primers spedfic for sequences con-

:ained within the DNA encoding nbosomal RNA. 111csc se-
~ences are amplified using the polymerase chain reaction.
:reatment and Control
\:nphotericin B J1as been the mainstay o( anticoccid-
oidal t herapy in human patients. Limitations are its
:uxicity and thc intravenous route of administration, re-
quir iug hospitaUz.atiou or fre4ueut visits. Liposorr1al pre-
parations of amphotericin R show promisP. hP.causP of
o•ver toxicity, which n1akes possible l1igl1er doses. Kelo-
::onazole and itraconazole are sometimes used in small
3Il.imals. Civcn orally ovcr u pcri<)d of m .onths, thcy havc
i!ffectcd permanent cures. Toxic effects are relatively
minor except during pregnancy, when fetal deaths may
..x.:cur. ·rhe corr1plernent fixation titer .is used to monitor
~he effec.t of trP.atmP.nt.
Vaccines are not available.
HJSTOPLASMA CAPSULATUM VAR. CAPSULATUM
."::fistoplasma capsulatum is a dimorphic fungus cxisting as a
m old at 25- 30ºC (saprophytíc phasc) and as a yeast at 31º<..:
parasitic phase). 1'his fungus has three varieties: H. capsu-
l.1tum var. capsulatum, H. capsulatum var. duboisii, and H.
cap s11/ah1m var. farcímínosum . Variety farciminosum causes
epizootic lynJphangitis (pseudoglanders), a cl1ronic pyo-
granulomatous disease of equine skin and is discussed in
Chaptcr 47. Varietics capsulatu1n and duboisii cause histo-
plasmosis, a systemic tunga1 disease o t ma1nmals. Variety
luboisíi is found only in Africa, whereas vad.e ty capsulaturn
!S found worldw:icle, ancl is the most commo11 cause of
F 1G U RE 4 8. 4. l listoplasma capsu latum, myce/ia/ phase. Teased preparatíon fron1 a
1-day culture on Sabouraud's agar at 25"C. Lactophenol cotton blue mount. "Tuberculate"
macroconídia and hyohae. The "tuberc/es" appear as projectíons from the thirk rell wallt
most obviously on the conidium in the center of the fie/d. 400X.
Chaprer 48 Agents of Systemic Mycoses 289
histoplasmosis. ln this chapter, histoplas1nosis will be dis-
cussed without regard to this varietal distinction, i.e., H.
capsulatu1n var. capsulatum, and H. capsu/aturn var. duboisii
will be regarded as H. capsulaturn.
Descriptive Features
Morphology, Structure, and Composition
The free-living form of ll. capsulatu111 consists of septate
hyphae bearing spherical to pyriform microconidia 2 to 4
µ1n in diamctcr, and "tubcrculatc" macroconidia, thick-
walled spheroidal cells, 8 to 14 µm iI1 diameter, studded
with fi11gerlike projections (Fig 48.4). In animal hosts or
appropüale culture, tl1e rnol<l !Jecom.es a yeast consisting
of ova l, singly bndding cells that rneast1re 2 to 3 µm by 3 to
4 µm. A sexual, ascon1ycetous state, Ajellotnyces cupsululus,
has bcen described.
Histoplasn1ins 1 vvhich are u::;cd in immunodiagnosis,
are obtaincd from mycelial culture fUtrates. ·rhey contain
polysaccharides, with variable admixtures of glycopro-
Lei11:; a11t.l cellular breakdown proclucts. Mycelial and yeast
phases differ in cellular c.onstituP.nts, somP of wh'ic:h (e.g.,
cell wall glucan) have been relatcd to virulence.
Cellular Products of Medical lnterest
Adhesins. Inhaled microconidia and yeast-phase parasitic
fonn:; a.re recognized by, and bin<l to, beta-2 integrins on
the surfacP of nc>11trophi Is anrl macrophages. It is un-
known what fur1gal structure is involved. Likewise, an u11-
knO\.Yn adhesin is responsible for tl1e adherence of Ji. cap-
sulatum. to fibronectin rcccptors on dcndritic cclls.
Adherence in this fashion to neutrophils, macrophages, or
290 PART 11 Bacteria and rungi
dendritic cells allows the tungus to enter the cell \.Y.i thout
triggering a11 effective oxidative burst and the generation
of reactive oxygen and nitrogen intermediates.
Miscellaneous Products. Histoplasma ca¡1sulaturn pro-
<.luces a varíe l y of producls thal n1ay play a role ü1 11isto-
plasmosis:
1. Calcium-b inlling protein (Cl>p). Tl1e yea:st (¡.¡ara-
sitic) ph:is<> of H. capsulaturn produces a calcium-
binding protein (Cbp) that pern1its the yeast phase
to grow in low-calcium environments (the phago-
lysosomc) by efficiently chelating calcium and de-
livering it to tl1e fUngus. In addition, since Cbp
chelates available calcium wit hin the phagolyso-
some, this impedes effectiveness of :severa! calciu111
requiring lysosomal enzymes. Normal acirlificiltion
of Ll1e pl1agolysoso1ue is also a calcitlm-depe11dent
event. Histoplasrna capsulatum mutants unable to
produce Cbp are avirulcnt.
2. H an tigen. Host immune responses to H antigen
were originally used as a diagnostic too! in the diag-
nosis of histoplasmosis. Subsequently, H antiger1
was found to be a beta-glucosidase that elicits a cell-
llH!Uiatc<.l (¡.¡rvtective) il11n1u11e response to the yeast
(parilsitic) phase of l-I. capsulatum.
3. !ron acquisition. Iron is an absolute growt11 requi rc-
ment for H. capsulatum (as it is tor al! lite forn1s).
Hístoplasma capsulatun-1 acq uires iron in several
ways: production of hydroxamate siderophores ca-
pable of ren1oving iron from host iron-binding pro-
teins (trar1sferrir1, lactvferri11); cx ¡.¡ re::.::.ior 1 of a
ht>min-hinding r<>r.t>ptor on the surface of yeast (par-
asitic) forms; glutatl1io11c-dependent ferríc reduc-
tase which reduces Fe(III) to Fe(II), thereby releasing
it from host iron binding proteins; and several
poorly aefineel iron reductases on the surtace ot tl1e
yeast phase.
4. M anligen. Hosl in1111une respo11se to M a11tigen vvas
originally used as a diagnostic too! in the diagnosis
of histoplasmosis. Subsequently, M antigen was
found t o be a catalase enzyn1e, >vhich plays a role in
the survival of the yeast phase within the phagoly
sosome.
S. }vfelanin. Mela11in is produced by H. capsulaturn.
Mela11i11 is a free rauical scaveuge.r (Jeducing lhe
toxicity of hydroxy radicals, superoxides, and sin -
glet oxygen radicals found within thc phagoly-
sosome).
6. Phagolysosome acidification . Normal phagolyso-
somes have a pH <5, a pH that optimizes the activ-
ity of many o f the digestive enzyn1es found in this
envirvnn1er1t. Hisluplusrrut cup:;ululurn raises lhe pH
of tht> phagolysosorne to 6-6.S, thereby reducing
the activity of these lysosomal cnzymcs. How the
fungus does this is un1<11o>vn.
Growth Characteristics
Histoplasrna capsulatum grows on co1nmon laboratory
i11cdia over a b road temperature range. The optirnum for
mycelial grov"th is 25" to 30"C. The cottony aerial my-
celium is white, brown, or intermediate. Pigmentatior:
parallels abundance of macroconldia. The yeast phase re-
quires ricl1er medía (e.g., glucose cysteine l>luu<.l agar) an.._
temperatures of 34º to 37°\.. r.rowt h may take a week o -
n1ore before characteristic colonies are seen .
Histoplasma capsulatu1n survives at ambient tempera-
turcs for rnonths and at rcfrigcrator tcmperature for years.
lt withstands freezing and thawing and tolerates heatin~
for more than an hour at 45ºC.
Variability
Histoplasma capsulatum exists as three varieties: capsula-
tum, duboisii, and farciminosurn. Varieties capsulaturn
(worldwide) and duboisii (Africa) produce hjstoplasmosis
and variety farcirninosum causes cpizvvl.ic ly1npha11giili
(pseudoglanders) of <>qni<ls (see Chapter 47). 'fhougl1 H.
capsulaturn var. capsulatu111 is found worldwide, its central
focus is in the Americas. Genetically, variety capsutatun1 is
dividcd into 6 Classes: Class 1 and Class 2 are found L-:
North Ameríca; Class 3 is found i11 Central and South
America; C!ass 4 is found in Florida (Nortl1 America); anC.
Class 5 and Class 6 are found in 11urna11 patieu ts wi ll,
acquired immunodeficiency syndrome (AJJ)S) frorn Ne,,-
York (NvrLIJ A1nerica) and J>a11a1ua (Central 11.merica),
respectively.
Ecology
Reservo ir
Although most concentrilted in the Mississippi and Ohio
River watersheds of North Amerlca, 11. capsu.latu1n oeeurs
sporadicallyworldwide. It is found in the topsoil layers, es-
pecially in the presence of b ird (m ainly starlings in North
Ameríca, and chicken.s in South America) a11d bat guano,
wl1icl1 provide both enrich1nent and inoculum. Birds are
mainJy passive carriers, 1.Yl1ereas uats u11<.lergo iliteslinal
infec:tious process<>s. Histnplasn-1a capsulaturn is favored by
neutral to alkaline soil environments with annual rainfall
between 35 and 50 inches and mean t emperatures be-
twcen 68º F and 90ºF.
Transmission
Tra11s111ission is mostly by inhalation o f microconidia or
hyphal fragments, possibly by ingestion, and, rareiy, by
wound infection.
Pathogenesis
Me.chunisrn:, uncl Puthology. Microco11idia, hyphal frag-
m ents (from the environment), or yeast cells (from an in-
traphagocytic cell environment) attach t o macrophages in
the lung by way of a beta-2 integrin. It is immediately
phagocytoscd, a minimal respiratory burst occurs, and a
pl1agolysosome results following fusion >vith lysosomes.
Microconidia and hyphal elements differentiate into
yeast. Survival within the pl1aguly:;u:;v111e is 1elaled t o

:nodulalion of phago1ysosome pi 1 (around 6 - 6 .5), secre-
- on ot M antigen (a catalase), secrelion of Cbp, meianin,
-id successful acquisition of iron from intraccllular iron
:ores. The yeast 1nultíplies '-vithin thc phagoJysosome, ul-
;;.;nately killing the cell and thc rclcase of yeast cells
hlch cuntinue the c:ycle). Tntrac:ellular multlpllcation
"'ntin111>~ 11nti 1 an pffprtive cell-mcdiatcd immune re-
:x>nse occurs resulting in activatcd n1acrophages, 1'\ hich1
mectively control the multiplication of the yeast.
Early events and lesions resemble those of tuberculosis.
_'loracic Jymph nodes become enlarged, and Jungs may
~untain grayish white nodules. "fhe histologic response
aries from suppurative to granulomatous inflammation.
~.aseation necrosis and calcification are c.:ommon.
In dlsscn1ina led disease, ly111pl1 uodes a1 1d pare11cl1y-
~atous organs are enlarged and may contain gross nodu-
iar Icsions. T11crc rnay be ulcerations of skin and mucous
membranes, abdominal and pleural effusions, and in-
olvement of the central nervous system (including eyes),
.:dn, and bone marrow. The ínflammatory exudate con-
sts of macrophage elements colonized by yeast cells
!'ig 48.5).
Disease Patterns. Histopl;ismo~is r;in oc:cur in almost
.J.."'ly nnimnl spccic.s, but dogs are the 1110.st con1n1only
:ected. Uogs may develoµ a primary pulmonary form with
coughing, fever, regional ly1nphadenopathy, and Iadi-
ograph!C abnormalities. Thc Inore common fonn in dogs
s disscminated d isease, marked by lcthargy, anorexja,
·,·eight loss, diarrhea, del1yd1aliu11, a11c.l cir1crnia. Hepato-
;:negaly, splenomegaly, mesenteric lymphadenitis, ;incl 11s-
citcs mny cause abdorninal distention.
Comparable pattems have rarely been observed in cats.
Disease patterns in human patients parallel thosc
descrlbed.
<.:hapter 415 Agents ot Systemic Mycoses 291
F1G u RE 4 8. 5. Histoplasma capsulatum, yeasr phase.
Sediment of peritoneal exudate from a dog. Wright-Giemsa stain.
Fovr y@ast c@lls within a macrophagl?. 1OOOX.
-
Epidemiology
m
Sul.Jclinical infections with hlstoplasmosis are comn1on in
clog.~ ;in<I r;its- and humans-in cndemic areas. Clinical
discasc is most prevaJent in dogs aged 2 to 7 years, in eally
autumn (September to November) and la ter winter to early
spring (f.ebruary to April). No scx prcdilcction is rcported,
but the po1nting breeds, weimaraners, and Hrittany
spaniels have the greatest risk. Disseminated histoplasmo
sis in human patients and dogs Is found in association
with immunosuppression .
lmmunologic Aspects
Recovery and resistance are governed by cell mediated im-
mune responses, while circulating antibodies have no ap-
parent protective function . Recovery from histoplasmosis
appears lo curúcr inuuunity.
No v;irrines are available.
Laboratory Diagnosis
clÍ-
Direct Examination
Smcars of buffy coat, scdiment of aspirates, and tissue im-
pressions are stained '-vith a Romanovsky-type (e.g.,
Giemsa, or Wright's) ora fungal stain (peri odie acid-Schiff
(PASJ, Grldley, or Gomori mcthcnamine silver) and exam-
ined for intraphagocytic yeast cells (see fig 48.5).
In sections slail1ed willi lic111atoxylin-eusin, H. capsula-
turn appears as tiny dots surrouncie>ci hy h;i loPs. A duplica te
fungal stain (e.g ., periodic acid-Schiff (PAS), Gridley, or
vomori methenamine silver) can be helpful.
292 PART IT Bacteria and Fungí
rmmunofluorescence has been used to identify thc
ycast in tissuc and cxudatcs.
Culture
Specimens are inoculated onto h lonc1 ;:ig;:ir ;:incl S;:iho11-
1aud's agar (will1 inlúbitors) and incubated in jars or plas-
tic bags at room tempcraturc for up to 3 months. Colonial
growth may look rcddish-wrinklcd bcforc the appcarance
ot cottony brown1sh to wh1te myceuum.
Microconidia and macroconidia are demonstrated in
lactophenol cotton blue wet 1uou11t~. Di111orpliisu1 n1usl
be proven by convi>rsion to t hC' ypast phase in culture or by
IV il1jc1.:tiv11 uf 11úce. Mice will die within a few weeks.
'f'hPir mac:rophages will contain the yeast forms.
A comn1ercially available "exoanJigcn" test kit fur-
nishcs prcpared antisera to c·occiclioicles, Histoplasma capsu-
latum, an<l Bl astornyces dermatitidis to be tested again st ex-
t racts of suspect cultures in an tmmunodiffusio11 agar
plate, where preclpltation lines develop between <:>xtr;icts
and tl1eir 11ornvlvgous a11 lise1 a.
lmmunodiagnosis
Histoplasmin skin and CF tests using antigens of cither
mycelial or yeast or1h11ns have not been reliable diagnostic
aids in anin1al infections. The position of the precipitin
band In lmm unodiffu sion tests dífft>reulialt:s t:arly and re-
cove.red hurnan c;isps (ni>;:ir seru m wcll) from active and
prog(cssive ones (near antigen well). Llmitcd use of these
tests on animals has given erratic rcsults.
A radioimmunoassay for antigcn dctection h as been
described.
Molecular Techniques
Molecular methods used to ídcntify or dctcct H. capsula-
tum make use of primers for spccific scquences contained
within thc DNA, c.g., genes cncoding ribosomal RNA, o r
those encodin g the M protein. ·rhese sequences are ampli-
fied using the polymerase cha in reaction.
Treatment and Control
Azolcs (keto conazole, itraconazoJc, fluco nazolc) and am-
photericin B have been used successtully in the treatment
of sorne cases of canine histoplasmosis.
Relapses are common. The progr1osls for u isse::111i11a1w
cases is grave.
8 LASTOMYCES DERMATITIDIS
Blastomyces dermatitidis is a dimorph ic fungus existing as a
mold in thc soil (saprophytic stage), andas a yeast in tíssuc
(parasitic stage). lt causes th e systemic fungal d isease
called "blastomycosis," "vhich occurs most cornrnon ly in
the eastern trurd of No rth Aruerit.:a. Cases of ll1e d isease
arise sporadically in Africa, Asia, a nd Europe. Affectcd
l 1osls are l1u1uan patients, dogs and, rarely, ho rses, cats.
and wildlife species.
Descriptive Features
Morphology, Structure, and Composition
ln the saprophytic phasc (in soll or on artifi<..ial inedia at
Z!>-:5UVC), the 11ypnae of ts. dermatitiáis produce coni<llo-
phores with spherical o r oval smooth-walled conidia, 2¡.Im
to 10 µm ln dlameter. In tissue or 011 l>lvou agar al 37ºC, lht!
agcnt isa thick-w;:i1Jpc1yi>ast,8 pm to lS µm in diam eter that
reproduces by single buds attached by a broad base (Fig
48.6). The fungus has a sexual form Ajellomyces dermatitidis.
Ccll wall extracts contain 3 to 10 parts of polysaccharide
to 1 partot p ro tein. JJrotein is highest in the mycelial phase,
whlle chltin is rughest i n the yeast phase. The lipid content
F 1Gu RE 4 8. 6 . Blastomyces dermatitidis, yeast phase, in
/ung of dog. Hematoxylin-eosin stoin. Note bud (lower right of
upper yeast ce//) arrached by broad base to mother ce//. 1ooox.

of B. dern1atitidis is highe.r than in othPr fungi . T.ipid, pro-
tein, and chitin lcvels vary directly with virulence.
Cellular Products of Medical lnterest
Adhesin. The yeast phase of B. dermatitidis produce.s a pro-
tcin adhc:;i n tcrmcd Bad 1 (for Blostomycc:s adhc:iin 1), for-
mally called WI- l. Badl has two functions related to viru-
lence of the fungus: 1) Bad 1 adheres to beta-2 integrins on
tlie surface of pl1agocytic cells, triggertng uptake of the
yea~t. invoking a mínima! rPspiratory burst, resulting in
little generation of reactive oxygen and nitrogen interme-
cliates; 2) Badl down regulates the production of proin-
flammatory cytokines (particularly tumor necrosis factor
:TNF]) by affected macrophages. The amino acid sequence
of Badl is homologous to the invasin protein produced by
Yersinia (see Chapler 10). Bluslurnyl·es <lermatilidis mutants
that do not prochJC'P R;icll ;ire ;ivirulent.
Miscellaneous Products. Blastonzyces der111atitidis pro-
duces a varicty ot extracellular enzymes and products t hat
may be involved with disease production. Thesc includc
;>rateases, phosphatases, esterases, and glycosidases.
Culture filtrates are leukotactic for human neutrophils.
-:ne role played by Lhese pruduLts iu the production of dls-
rase is unknown.
:irowth Characteristics
3/astomyces dermatitidis grows on most media at roon1 tem-
perature and 37ºC. Colonies develop from ~"lithin 2 to over
- uays. Mold colonles, formed at amblent temperatures
2.)-~0ºr.), :irP <'ottony white to tan depending on t he
abu11dance of conidia. Yeast colo11ies dcvelop 011 blood
~gar at 37ºC. They are opaque off-white to tan, with rough
surlacc anda pasty consistcncy.
.ariability
-:be two recognized serotypes are. re.Jateo to gpogr:iphic ori-
tin of the strains. Similar geographic rclationships are seen
::.-hen polymerase chain reaction-based fingerprinting
svstems are uscd.
• of infection. Cell-mediated immunity in mice is decisive
:cology
=-eservoir
:bere is 11mlted evidence of natural reservoirs. Moisture,
m" pH, animal wastes, and decaying vegetation appear to
:aYor coloniLalion. lluntidiLy !Jlv111vlt::> 1clcd:>t: vf cu1liLlia.
-ransmlsslon
3lastomycosis most commonly begins by respiratory expo-
~ure. Pcrcutancous infcction probably occurs in dogs.
~thogenesls
!icroconidia hyphal fragments are inhaled and c:onvf>rt to
::b.e ycast form within the alveolar spaces of the respiratory
;:ract. The yeast expresses Badl, which is followed by uptake
Chapter 48 Agen ts of SystP.mi<' Mycoc:Ps 293
(with minimal respiratory burst) by phagocytic cells. Down
regulalion of tu111or i1ecrosi:> factor prouuct:ion by affect:ed
macrophages delays stimulation of cell-mediaterl imm11-
nity (which is necessary to kill the yeast). In dogs, a11 in-
flammatory response involving macrophages and neu-
trophils and rcsulting in pyogrnnulomatous lcsions occurs
1n tne terminal broncl110Ies, foUowea t>y s1m1 1ar reactions in
satellite lymph nodes. Blastomycosis is more often progres-
sive than histoplasn1osi:; aJ 1Ll cuct.:idiuiuo1nycosis. Dissemi-
nation involves superficial lymph nod~!\, skin, honPs, hone
mnrrow, cyes, víscera, mammary glands, and urogenital
tract. ·rne nodular Jesions can be tubercle-like, with mono-
nuclear cell types predominating, or thcrc may be signifi-
cant suppuratlve admiXtures. L1quefacnon and caseation
occur, but calcification and encapsulation are cxceptional.
Signs include skin lesiu11s dud rc.spiratory distress wlth
fever, depression, anorPxi;i, ;ind weight loss. Locomotor
disturbances res ult from bone or joinl infection or-
rarely-central nervous system involvemcnt. Ocular dis-
easc is common in disseminated blastomycosis. Most dogs
wlth multiple organ system involvement die within
months. Evidence for a benign form is uncertain. Blasto-
myces induce 111ac1opl!ages lu produce elevated amounts
of calcitriol resulting in hypercalccmia.
Thc discasc in cats resembles that in dogs.
Epidemiology
Highest prevalence in dogs is from spring to fall. Malc dogs
Iess t:han 4 years of age are n1ost often affected. Differential
breed susceptibility has not been found. 'fhough generally
11onco11Lagious, 011e liurua11 il1fcctlou resulted from a dog
bite. Risk factors for infection includc proximity to watf'r-
ways and excavation.
lmmunologic Aspects
lmpaired cell-mediated responsiveness may explain ca-
nine disseminated blastomycosis.
Humoral and cell-mediated responses occur as a resu lt
in determlnlng resistance. Artificial lmmunization is not
availablc.
Laboratory Diagnosis
Direct Examination
Blastomyces dermarttidis can be dcmonstrated in wet
mo11nts of exudates and tissue smears as thick-walled yeast
cells with single buds allached l.Jy d 1.Jroac.l base. In section,
intracellular yeasts may be found (sr.c Fig 48.6). Smears of
aspiratcs, and tissue impressions are stained with a
Romanovsky-type (e.g., Giemsa, or Wright's) or a fungal
stain (periodic acid-Schiff [PAS], Gridlcy, or Comori
methenamlne silver) and examined for yeast cells.
In sections stained with hematoxylin-eosin, B dermati-
tidis appea1s as Lltick-wallt::d yea:;t cells wtth single buds at-
tached by a broad ha~e. A c1uplicatP fungal stain (e.g., peri-
294 PART JI Bacteria and Fungí

odie acid Schiff [PASJ, Gridley, or Gomori methenamine

silver) can be helpful (see Fig 48.6). f 1G U RE 4 8. 7. Anatorriy uf¡¡ 111e111úe1 uf lile yenus
Asperc¡illus (Asperc¡i ll us clavatus). Lactophenol cotton blue
mount from slide culture on Sabouraud's agar. 1 = foot celf.
Culture 2 = conidiophore, 3 = vesic/e, 4 = phialides, 5 = conidia. 400X.

Cultures on Sabouraud's agar (wit h o r without inhibitors)

are incubated at ambient temperature (25- 30ºC) for up to ,
3 wccks. It is difficult to obtain the yeast form on primary
isolatio11 but it is relat1vely easy to convert the mold to the
yeast phase by increasing the temperature to 37ºC..
A co1u111ercially available "exoa11tigen" test kit fur-
nishes prepared antisera to Cocddioides, Histoplasma capsu-
latu1n, and Blastonzyces derniatitidis to be tested against ex-
tracts of suspect cultures in an immunodiffusion agar
plate, vvhcrc prccipitation lincs dcvclop bctween extracts
and their 11on1ologous antisera.

lmmunodiagnosis
BlasL01nycü1 skil1 a11d con1plen1ent fixation tests lack ac- ..
ceptable sensitivity and specificity. ·rhe commercially
available agar gel do u ble diffusion test (making use of anti-
gens composed of a cell wall autolysate called "Antigen A")
appears to be specific (96%) and se11sitive (91 % ), bttt titers
remain after successful treatn1ent. Antibodies to Badl de-
tectecl by various mea11s (e.g., raclioimmunoassay [RlA])
iucn:'.dse duriug uisea~e. a1 id decline vvilh successful treat-
mP.nt, 1naking detection of antibodies with specificity to
Dad! more useful clinically.

Molecular Methods
Molecular methods used to identify or detect B. dermati-
tídi.s rnake use of prin1ers for sµecific se4 ue11ces cv11Laü.1ed
within DNA encoding rihosomal RNA (e.g., 28S RNA; the
internally transcribed spacer region). ·rhese sequences are
amplified using t he polymerase chain reaction.
Treatment and Control
Rl;istornycosis rP.sponds to amphotericin B and ketocona-
zole (or both togcthcr), and to itraconazolc. Fluconazolc is
mode¡ately ettective.
ASPERGILLUS SPP.
Me1nbers of the genus Aspergillus are u1Ji4uito u::. savro-
pl1ytic n1olds with opportunistic pathogenic patterns de-
pending on impaired, overwhelmed, <>r bypassed host de-
fenses. Of sorne 900 species. A . filmigatus is most frequei.1t
in animal and human infections.
Descriptive Features
Morphology and Co1nposition
Asperxiltus spp. are molds consisting of septate hyphae a11d
characteristic asexual fruiting structures that are borne on
,.
conidiopbores. Conidiophores are hyphal bra11ches orig.-
nati ng by a foot cell in the vegetatlve mycelium and e ne~
ing in an expancled vesicle.1'he vesicle is covered by ;:i laye:
or layers of flask-sl1aped pb.ialides, from which chains c.-
pigmented co11idia (the asexual reproductive units) ar'..5<:
(Fig 48. 7). They give the fungal colony its color.
In tissue, only hyphae are seen. In aerated caVlties (e.;;
nasal passages, air sacs, cavitary lesions), fruiting st.ru.:-
tures rndy !Je fuuud (:see Fig 73.8).
Fruiting hodies are i rnportant diagnostic features -
Aspergillus spp. by which species are identified.
Cellular Products of Medica! lnterest
Adhesins. Members of tbe genus Aspergi11us procluce a n u-
IJer of surfa<.:e µruLi:.ius (conidial and 11ypl1al) tl1at bine -
i>xtrac.P.llular n1at rix proteins (collage11, fibronectin, ff-
rinogen, and laminin).
Cell Wall. The cell wall oí A spergiflus clisplays _
"pathogen-associated molecular pattern" that is reco!f
nized by Toll-lil<e receptors (see Chapter 2) on the surf?.~
of host macrophages. Binding to these receptors leads -
t he secretion of prolnilam1natory cytuki11es.
E.xtrace11ular Enzyrnes. Aspr.rgill1L~ produces a nun1bfi

enzymes that have the potential to function in vivo to
areak down host tissue. TJ1ese incJude elastase, proteases,
m d phospholipases. In addition, members of this genus
produce catalases that reduce the effectiveness of perox-
_des ge11eraled l>y phagocyLic cells.
Iron Acquisition. Aspergi//us produces severa! hydroxam-
~te siderophorcs (a ferriChro1nc and a fusarinine) necdcd
<a acquire iron from host iron-binding proteins (transfer-
:ín, lactoferri.n).
Pigment (Melanin). Conidia of Aspergiflus are pigmented.
The pigment, melanin, is a free radical scavenger (reducing
the toxicity of hydroxy radicals, superoxides, and singlet
oxygen radicals found within the phagolysosome).
Growth Characteristics
-~pergilli grow 011 all con11nor1 lalJoratory rnedia over a
l'<ide range of temperatures (up to SOº\.). ThP.ir hioc:hi>n1i-
cal activities have not been clearly related to virulence or
utilized diagnostically.
Aspergilli thrive in the environrnent. Sorne are highly
:esistant to heat and drying. Most do not grow in cyclo-
hex.imide-containing fungal inedia.
Ecology
Reservoir
•.\spergilli are present in soil, vegetation, feed, and second-
a rily in air anrl wati>r anrl obji>rts exposec.1 to them . As-
pergillus fi1111í.gatus becon1es predon1inant over con1peting
microbiota in fermented plant material (e.g., hay, silage,
compost). Animal disease outbreaks are oftcn traced to
such sources.
F 1G U RE 4 8. 8. Aspergillosis in a waterfowl (grebe). The exposed air sac in the center
contains one /arge mold co/ony (arrow) anda smaller one on the upper /eft. The air sac on
the lower riqht (*)has hada portion removed(*), revea/inq /uxuriant mold qrowth within.
(PhotO[)fri(lh ro11rtP~Y nf f)r M11rrriy Fowler)
(;hapter 48 Agents of Systemtc Mycoses 295
Transmission
Aspi>rgillosis is acquirecl from environme11tal sources, gen-
erally by iuhalaliou or i11gesliou. Musl Aspergillus iuastitis
follo\>VS intramammary inoculation. Tntrauterine infec-
tion:> in cattlc rc:iult from di:;scmination of subclinical
lung or intestinal infections. In poultry, egg transmission
sometimes occurs (rare).
Pathogenesis
Mechanism. Follo\~1 ing their deposition in tiss11e or on a
surface (adhesins hinder removal) recognition (by way of
"pathogen-associated molecular pattern") by phagocytic
cells triggers an inflammatory response. Inflammation,
along with rclcase of fungal elastase, proteases, and phos-
pholípasi>s, res11lts in tissuc damage. Pigment and catalase
delay destruction by phagocytic cells.
Allergenic factors, which are recognized in l1uman asper-
gilloscs, are insufficiently documcnted in animal disease.
Patholo,zy. In pulmonary infection, ~uppuratíve exudate
accumulates in bronchioles and adjace11t parenchyma. It
surrounds colonles of m ycelial growth, which 1.nay extend
into hlood vessels ancl produce infected thrombi and vas-
culitis, leading to dissen1il1ation. Infeclion 1nay also
spread directly into adjacent air spaces. Granulomas de-
velop; tl1cy are grossly visible as grayish whitc nodules and
consist of mononuclear cells and fibroblasts. ln older le-
sions, colonies are fringed by acidoph.ilic clubs (asteroid
bodies), which resemble actinomycosis (see Fig 37.2).
Lesions in avían lungs are caseous nodu.les. On serous
n1enJbrancs 1 caseous foci are covered by n1acroscopic 111vld
colonies. accompanied by thickening of the 1nembranes
(e.g., air sacs) (Fig 48.8). Thc ccllular response is acute sup-
purative to chronic granulomatous.
296 PAKr II Bacteria anu Fu1igi
Movine abortion results from hematogenous seecting of
placentomes, which is possibly a response to a growth fac-
tor in placental tlssue. There Is hyphal invasion uf l>loou
vessels producing vasculitis anrl íl nPcrotizing, hemor-
rhagic placentitis. The fetus undergoes disseminated in fcc-
tion with signs of emaciation and dehydration. Lympl1
nodes, visccra, and b rain may be involvcd. Ringworn1-like
plaques on the tetaJ skin are often seen.
On mucosa] surfaces (e.g., nasal passage, trachea), mold
colonles form on top of necrotlc tlssue, which is sur-
rounded by a hemorrhagic zonc.
Disease Patterns
Pulmonary and disseminated intections, frequently in-
volving kidneys and the central ncrvous system, occur in
most species.
Avian. Avia11 aspergillusis, w h ich affecls 111a11y species of
hir<ls, sometimes in epidemics, reflects heavy exposure or
severe stress on dorncstic flocks or pct bird opcration~, or
the ettects ot oil spills on marine b1rds. The ctisease is usu-
ally a respiratory tract infection, sometimes "'rith hemato-
genous dissemination. 5igns are lnappetence, llstlessness,
weight loss, dyspnea, sometimes diarrhea, and abnormal
behavlor and posture. The eyes are oftt:u afft:clt:LI. Mo1 ta1-
ity may approrich :'iOºAi, PspPcially in young birds. In mild
cases, only gasping and hyperpnea may be seen. 1"11e
course varíes from a day to severa! v.1ceks.
Ruminant. Movine abortions usually occur late in preg-
nancy and resemble abortions duc to other causes. Fetal
Skin plaques occur also in other mycotlc abortions.
Mastitis due to A. f11migah1~ i<: rPportPrl atan increased
rale, especially from Europe. It is usually chronic progres-
sive, producing abscesses in the udder.
Dogs and Cats. Aspergillosis of mucous membranes occurs
in dogs, rarely in cats, in nasal passages or paranasal si-
nuses. It is manifested by snecztng and unilateral or bilat-
eral persistent 11aséll discl1arge th~t i<: 11nr<>sponsive to med-
ica! LJcaLtnent.
Aspergillus terreus and A. deflectus cause disseminated as-
pcrgillosis in dogs, particularly German shepherd dogs.
Osteomyelitis is a common feature.
Horses. Aspergillus infectlon of the cornea is the leauil1g
cause of keratomycosis.
Vague upper respiratory signs may point to an as-
pergiHosis of the equine guttural pouch.
Miscellaneous Species. 1I1testinal aspergillosis, presenting as
diarrhea, occurs in calves, foals, and cats.
Epidemiology
Intensity of exposure is a significant feature in animal as-
pergillosis. Bovinc abortion outbrcaks are often related to
moldy todder. Aspergillosis in ctucken flocks commonly
coincides with the use of heavily contaminated litter.
Stress aspects are usually recognizable in outbreaks.
Avían aspergillosis is seen under conditions of poor hus-
l><uJLlry. J11 oilt:u seabirds, Lhe1e is severely irnpaired ther-
mal regulation. In pregnant cattle, advanced gestation
combined with low-quality fccd and poor wcathcr and
housing add up to severc challenge.
Canine nasal aspergillosis occurs especially in young
dogs of dolichocephalic breeds. Sorne T lymphocyte defl-
ciency may exist.
lu keratu111ycusis uf ho1ses, lhe frequc11t history of top-
ical antihacterial and steroid treatment suggests immuno-
suppression and impaired colonization rcsistancc.
lmmunologic Aspects
Circulating antibody with no demonstrably protective
role may be present in dogs with nasal aspergillosis
(see under "Treatment, Control, and Ptever1tio11"). C~ll­
mcdiated immunity may be relat1><l to resistance.
1111111 u11il.dtion procedurcs nrc not available.
Laboratory Diagnosis
Direct Examination
Hyphae, fruiting heads, and conidia can often be demon-
strated in samples elther In wet mount~ iI1 10% KOH ur
with calcofluor white. Far flx.ed-stainPrl smears, fungal
:.taü1:. (pt:riouic acid -Schiff IPASJ, G ridley, or Gomori
methenamine silver) are best; Giemsa is satisfactory, and
Gram of limited use. Septate branching hyphac constitutc
strong evidence of aspergillosis. Other tungi (11enicill1um,
Pseudallescheria, Paecilon1yces) presenta similar picture but
rare. Conidia may occur in alr passages or other exposed
sites in the absence of infection (see Fig 73.8) .
lu :.tail1t:LI lis:>ue seclions, sepla le 11yphae dividing
dichotomously at acute angles are the only strt1cture~
sccn.
lsolation and ldentification
Aspergil/11s is readily cultured Rec;i11sP it is a 11hiqu itou~
cu11L<:1111inanl, inlerprclalion of positive cultures is often
problematic. Presence of the agent must always be corre-
lated with pathologic and clinical findings. Identification
of agents rests upon morphologic features and growth
characteristics of isolates.
lmmunodiagnosis
Serologic tests are useful a<lj11nct<: to the diagnosis of as-
¡.>t:1gillosis. Because tl1c tests that are available are species
specific, it is necessary to know whichAspergíllus to expect.
r:or example, A. fun1igatus is most comn1only seen in nasal
aspergillosis ot dogs, whereas dissemínated aspergillosis in
that spccies is either A. defiectus or A. terreus. An immunod-
lffusion kit for antibody detection i:; cuu1JJ1ercially avail-
able for detection of antibodiPS to A. fumigatus.

'\olecular Methods
-ri mf'rs rlPsigned to ¡¡mplify DNA encoding ribosomal
-:-"-A (including the "interna! tcanscribed space1" iegiou)
-·e available for demonstration or identification of mem-
-...:rs of thc gcn\.1S. Amplification of DNA is by the poly-
•erase chain reaction.
Treatment , Control, and Prevention
.spcrgillosis in birds is gcncrally not treated.
The nasal form in ctogs is treated topically with instilla-
::ion of clortrimazole or enilconazole into the nasal pas-
..ages and slnuses. Itraconazole (given orally) has been suc-
::essf11 lly 11c;f'ci to trcat nasal aspergillosis whcn topical
:::-eatmcnt was not possiblc.
Itraconazole has been beneficia! in treating dissemi-
""'ª ted aspergillos is.
There is no establishcd treatment lor rnammary as-
:--ergillosis.
Fur intestinal infectlons In pigs, foals, and calves, oral
~\"<itati n is rPcommPnrlPrl
•
Keratomycosis is treated topically wilh an Lil11yculic
.ntments and solutions.
/\voidance of inassivc cxposurc rcquircs cllmination of
..:attle feed, partícularly hay and silage that has undergone
-:oticeable deterioration. Aspergillus furnigatus only reaches
..igh concentrations under conditions of "biologic heat"
~... neration, after other microbiota are eliminated. With
:coullry lille1, p1ope1 slu1ag1:: a11<.l fn::4uc11t changes of litter
can prevcnt such buildup.
0 THER SAPROPHYTIC fUNGAL PATHOGENS
en1cilliun1 spp. are occasional causes of a nasal m ycosis in
_¡ogs.
Scedosporium apiospennun1 is prominent asan agent of
!!l)"Cetoma (see C:haptPr 47).
I'seudallcschcria boydii causes visceral myceton1a a11d
:'"'.leumonia in dogs, ocular discase and abortion in horses.
~nd mastitis and abortion in cattlc.
Paedlomyces spp. 1s associated with disseminated ca-
-1nc and feline paecilomycosis ,.,ith bone involvement
_.,<.la rt::>piiatory epidemtc In captlve turtles have been re-
~rted.
Rhizopus spp, Absidii1 spp, Mucor spp, <tnd Mortierellu spp
a use mycotic bovine abortion, gastrointestinal infections,
~arkcd by ulcerative lesions and mcscntcric lymphadeni-
:is, in ruminants, swine, and dogs, as well as respiratory
;.nd hcmatogenous infections affecting various víscera
... i<.l tite central nervous system. 'fhey are secondary to
..tress, such a-; rliPtary changes and inadequacies, antibi-
~ic suppression of the gastrointestinal flota, concurre11L
-'1tections, recent parturition, or traun1a.
Rhinosporidium seeberi en uses a granulomatous mucocu-
~neous i11fection affect1ng hurnans, horses, cattlc, 1nules,
.:.ogs, goats, and sorne wild waterfowl.
Chapter 48 Agents of Systemic Mycoses 297
Conidiobolus spp. and Basidiobolus spp. may cause nasal
gronulomos in hor3C5.
Dematiaceous fungi (See Chapter 4/, "l'haeol1ypho1ny-
cosis") have been associatcd with mycotic nasal granulo
111as in cattle.
PNEUMOCYST/S
Members of the genus Pneumocystis are fungi capable of
causing pneumonla In immunocompromised individuals.
'l'hPy have only been isolated from affected hosts (e.g., hu-
mans, dogs, cats, ho1ses, pigs, goal::;, ft:rrcti., n1ice, rats). At
present there are two species, jiroved (affecting human pa-
tients) and carinii (affccting all the rest). Pneurnocystis
carinii is composed of at least JO varieties or "special
forms,'' ,.vith each "special form" affecting a particular
!1ust. flor example, P. cartnii f. sp. ratti, affccts rats, while P.
cari11ii f . .sp. rn11ris, only mice. Thus, Pne111nocystis is a host-
spccific fungus (and it is nol zoonolic).
Sprcad is by way of aerosol. Affected hosts are almost al-
ways irnmunocompromiscd, though therc are reports of
animals (maínly foals) that have pneumocystis pneumo-
nia without an obvious underlying condition.
'file fungus has not been grown in a cc\1-free culture sys-
tem. Diagnosi~ is made by examination of material con-
taining alveolar macrophages (in whose cyluplas111 spheri-
cal to crescent shapcd cysts 4- 7 µm in diametPr arP
obscrvcd) staincd with a Romanovsl..)'·typc stain (e.g.,
Gicmsa, Wright's) ora silver stain (e.g., Gomori methena-
mine silver). DNA primers have also been designed to am-
pllfy speclflc segments of the fungal gcnome, so that <.le-
tc•c·tion and identification is possible using the polymerase
chain reaction.
Treatment incl11rl P~ one or more of the following:
trimethoprim-sulfonan1ides, dapsone, aLovaquo11e, ¡..>t.:H-
tamidine, clindamycin, or trimetrexate/leucovorin.
PROTOTHECOSIS
J'rototheca is an alga lacking chlorophyll. It 111ulliplies by
endosporulatioo, producing roughly sphcrical cells that
are 8 µm to 25 µm in diamctcr (Fig 48.?). l'rototheca zopfii
and P. •vickerha1nii (which probably belongs to the genus
Auxenochlorella) are occasionally pathogenic. They grow
011 fungal n1edia (wtthout cycloheximlde) at 25ºC and
~?ºC., respec:ti vPly, int(l white to tannish dull colonies in
less than a week and are differe11tialed se1ologically a11d l>y
carbohydrate assimilation tests.
Prototheca spp. are widcspread in nature. Exposure is by
1ngest1on, percutaneously, or, in dairy cows. by intramam-
mary injection.
Disease occurs l.n dogs, cats, cattle, deer, bats, snakes,
fish, ano humans. In dogs, it is usually disseminated and
accompanied hy hcn1011hagic d ia1rltca. Central nervous
systcm involvement and eye lesions are frP<J.11Pnt . In cats
and humans, cases to date have been cutaneous. In1n.1u110-
298 PART II Bacteria and Fungi

deficiency is suspected. In cattle, chronic progressive mas-

F1G U R E 4 8. 9 . Prototheca sp. in bovine mastitis. Wright titis develops. Tissue reactions are pyogranulomatous.
stain. The /ess intense/y stained organisms show evidence of The agent is easily cultured and can be demon strated in
endosporulation. IOOOX. unstained wet mounts from specimens or in fixed smears
stained with a Romanovsky-type (Wright's or Giemsa) or
fungal (per iod ic acid-Schiff [PAS], Gridley, or Gomar¡
met hena mine silver) sta in (see Figs 48.9 and 69 .7).
Amphotericin B and ketoconazole are used on humans.
The agents are susceptible to the aminoglycosides in vitro.
Treatment of an imals has not been effective, although ¡¡_
posomal formulations of amphotericin B show promise.
 Pathogenesis of Viral
Diseases
YUAN CHUNG Z EE
Virus-Host Relationsh ips
The outcorne of an y virus-host in leraction c:an c! ifff>r, c!f'-
pcnding on critica! facto rs such as virus-ccll interactions,
species oJ an imal host, ro ute of exposure, mode of v irus
dissemination, and host rcsistancc. Most virus-infected
.in1mals that present to veterinarians do so because thev
exhibit clinical signs of thei r infection. However, it is very
mpo1 lanl lo 1eLog11ize Llial 111i1,;rul.Jial i11fcctiuns of ani-
:nals often do not result in clinical discasC'. rn fact, thf> ma-
·ority of virus-animal in teractions result in asymptomatic
•r subclinical intection, and viruses, however virulent, will
::ot infect animals that are resistant to thcm. Thc potcn tial
consequences of virus-an imal relationships are shown in
Table 49.1.
There are severa! ma¡or routes of v lral entry lnto a host:
·hp rPspiratory, ;:i limf>ntary, ancl 11rogc>n ital routes and di-
:ect transmission (such as by an insect or anin1al bile) .
Successful establishment of viral infection dcpends on the
~~cscncc of susceptible ccll rcccptors and the physico-
~nem ical nature ot the viral agent. 1-'or example, viruses
:~at infcct animals through thc alimcntary tract typically
_re resistant to the lo'"' pH and potent enzymes that occur
~• the digestive tract.
~ odes of Uissemination of Viruses Within
~h e Hosts
· ·uuses cause two basic patterns of 1nfcct1on: localized and
;eneralizcd (Fig 49.1). In localized infections, viral m u lti-
.,,licaliu11 anti <.:cllular tlarnage remaín locallzed near the
-.te of entry (e.g., the skin or the 1n11co11~ mf>mhranes of
·he respiratory, gastrointestinal, or genital tract), so that
-,e intecting virus spreads only to neighboring cells im-
-ediately adjacent to the orih>inal sitc of infcction . For cx-
-':lple, rhinoviru s infection s of animals are often re-
~ictcd to the nasal epitheliun1 and do not even spread to
_.t: luwcr rcspiratory tract. Other respiratory viru ses, such
:;.,, parainfluenza a nd res pi ratory syncytia l viruses rcplicate
itltin the lungs of infccted animals, but tissue injury ü1-
_Jced by these viruses typically remains restrictecl to the
:.-spiratory tract. Generalizcd infcctions dcvclop through
e ·eral sequentlal steps: 1) the virus undergoes primary
•
N. ] AMES MACLACHLAN
replication at the site of entry and in regional lymph
11odcs, 2) progen y virus sprcads through b lood (primary
viremia) and lymp hat1cs to additional paren chymal o r-
gans, where 3) further viral rcplication takes place, 4) virus
is uis~c11tiI1a ted to t he oth er targct organs via a secondary
viremia, and S) it m u ltiplies further in these target organs
wherc it causes cellular dege11erativ11 a!ltl/or necrosis, tis-
sue injury, and clinical disease.
·rhe incubation period is thc asyrnptomatic pcriod aftcr
infection and prior to express1on of clinicat disease. In
generalizcd virus infections, overt discasc begins only after
ll1c viru~ l.Jecornes ~vitlely <.lissemlnatcd ln the body and
has attainP<l maxim11n1 titf>r'> . Tt is al this stage of t he infec-
tion that the veterinarian usually is firs t aler ted . Canine
distcinpcr illustrates a generalized virus infection of an i-
n1als. Thc caninc distcm pcr virus initiatcs th c in fection at
the s1te of entry, but then d isseminates through the blood
or the lymphatic syste1n to produce generalized in fec..Tion
with lnvolvement of a varlety of target o rgans (Fig 49.2).
The sequcncc of events during the incubation period and
devclopn1cnl of signs of disease i11 ex¡ie1 i111e11lal <.:a1li11c
distC'mper infec:tions in<licatf> th;it thP different cllitical
signs that occur in individual animals depend on which of
the various organ systems are infected by the virus. The
virus is disseminated to thcsc organs during vircmia,
which may be charactenzed by thc prcscnce of free virus
p<lrtir.lP.s in the b lo od o r, as 11Vith caninc d istemper v irus,
blood cclls also can serve as can icr~ lv tl i~scruiIIate virus to
target organs (cell-associated viremia). l.P ll-as<;oci;:itpcl
viremias typically involvc blood leukocytes hu t son1e
viruses, such as bluetongue virus, hog cholera virus, or
parvovirus, can associate \vith red blood cclls of thc in-
fected host. The dissemination ofv1rus to the central nerY-
ous system (CNS) can occur by viremia or, in the case of :a-
bie~, by l1a11~111i~~io11 aluIIg peripheral nerves.
Viral infcctions that occur \Vitho11t producing overt dis-
ease are vcry common and are potentially in1portan l in
the d issemination of viruses. Significantly, inapparent in-
fections can confcr protcctivc immunity against subse-
quent challenge with virulent strains ol the same agent.
Severa! factors are involvcd in producing inapparent infec
tivu~: 1) the nature of the virus (e.g., vtrulent or anenu-
atPct strain~), ?.) <lPgree of host immunity, 3) appearance of
viral interference, and 4) failure of lhe viru~ tu rca<.:l1 the
target organ (e.g., dueto the blood-brain harrier)_
301
302 PART llI Viruses

Tabl e 49 .1. PotPntir1l Con~f>quPn(e~ of Virus-Animal
Relationships
1. Animal is resistant to viral infettion-110 1eldliu1t)l1iµ ~slablished
2. Asymptomatic or subclinical infection- recovery or persistent infection
3. Acute viral infection-<leatb, cecovery, or persistent infection
4. Chronic viral infection-<ecurrent clinical disease, or persistent infection
S. Tumor formation
Host Responses to Viral lnfections
'rhe resistance of animals to virus infection is dependent
in part on factors that act inlliscrir11i11.alely vu 1uosl viiuses
and are thcrefore callf'ci nn11~peciftc or innate resistancc
faclol's. Tbe:;e include horn1onal factors, temperaturc, in-
hibitors other than antibody, and phagocytes. l'hagocyto-
sis is an important defense mechanism ü1 bacteria! infec-
tions. l-towever, many viral agents are capable of infecting
lymphcytes and/or monocytes/ macrophages, and thus
these cells actually can serve as a vt::l1.ü.:h:: tv :.vread virus
through the host.
lnterferon
Interferons are a group of cell protclns (cytokines) that can
modulate thc imtnune system, regulatc the differentiation
of certain cells, confer antiviral resistance o:n Sf'nsitivf'
ct::ll:s, alJU cxerl <u1Lica11cer effects. Many viruses will in-
ciuc:e interferon synthesis in infected cells, and many cell
types have the ability to synthcsizc intcrfcron aftcr appro-
priate stimulation. At least three different types of inter-
feron can be produced in the course of a viral infection:
itlpha, hf'ta, anci ga1nma. Two distinct mechanisms ha,·e
been identified in intcrfcron-treated cells (Fig 49.3). Thc
first involves the production in interteron-treated ceHs of a
protcin kinase (Pl/e!F2alpha kinase), which in the pres.
cnce of double-stranded RNA, blocks initiation of protelf'
synth esis by phosphorylating the p rotein synthesis initi-
atlng factor eIF-2. Tl1e othc..:r u1echanisn1 ü1volvcs an cn -
zyrne, ?.-SA synthf'tasf', which, in the presence of adeno-
siJ1e triphosphate (Al'P) and dsRNA, synthesizcs a group o:
oligoadenylates collectively known as Z-5A. Z-~A in tur:-:
activatcs a specific endonuclease that degrades viral an¿
cellular RNA and so inhibits protein synthesis.
Besides its action on viral replication, interferon f'xf'~
other effects on cells, iI1cluüi11g effecls 011 cell n1ultiplica-
tion and regulation of such cellular functions as phago-
cytosis, production of antibodies and lymphokincs b:
lymphocytes, expression of cell surtace antigens, and cyto-
toxicity of ccllular i1nmunity. lnterferon plays an impor-
tant role in host resistance to viral tnfection.
Humoral and Cellular lmmunity
Viruses are antigenic and typically induce a strong im-
mune response aftcr infcction. Humoral immune re-
sponses lead to the productlon of antibodies that can be
demonstratcd by the usual serologic procedures, such as
complemcnt-fixation, agglutlnatlon, precipitaliu11, auc
gel diffusion techniques. 'fhe basic principies governing
these tt:::.t:; are iden Lical wilh thosc used in bacteriology.
Sf'rological reactions like viral ncutralization are unique
to viruses and play an impo rtant role in tcrminating pri-
mary viral infection, limiting viremia, and preventing dis·
case and reinfection. When preparations of virus are
m1xed w1th appropriate antisera and the miXtures are ln-
oculated in susceptible ho<;t<;, inff'ction will not occur if

FIGURE 49 .1. Modes of viral dissemination within the host.

Virus

i
Disease +-- - - - - -- - - -- -- - Local site
(P..g.. rhinovirus)

Disease + - - - -- - - Target organs Peripheral nerve

(e.9 .. (e.g .. rabies)
poroinfluenza
Typ'=' 3)

Blood Di~co~e
(prlmary vlrem1a) central
nervous system

1
Target organs _ _ _ __ __,. Blood
i
1 (e .g .. canine distemper)
'4- (secondary viremial
Diseose
(e.g ., feline
panleukopenia)
 Chapter 4.9 Patl1ogenesis of Viral Diseases 303

FIGURE 49 . 2 . Schem;¿¡ti( rli;igr;im nf p;ithngPnP~i~ nf r;ininP rli~tPmpPr vir;i/ infP.rtinn in rlng~.

Day

o Exposed to viral aerosol.

1
Macropha~es in bronchial lymph nodes and tonsils: multiplication.

3 Lymph nodes, parenchymaf organs, eyelid, leukocytes: multiplication.

4

6 Blood stream: vlremla.

7

8
1.ymph norle". parenchymal organs, foot pad, leukocytes, a limentary tract, urogenital tract, conjunctiva, eyelid,
respiratory tract: multiplication.
9

10
Most dogs with serum neutralizing antibody titer 1: 100 or greater recover and virus rlisAppP.Ars.

Dogs with antibody titer 1: 10 or le~~. viru::o in vorious tissucs including CNS (meninges, cerebellum, cc:rebrurrl,
brain steni, spinal cord).

60

Figure 55.2. Schematic diagram of pathogenesis of caninc distcmpcr viral infection in dogs.

rhe antlsera contain virus-neutralizing antibody. 1'hree
classes ofimmunogloh11lins, lg<~. lgM, anrl lgA, can Sf'rVf'
as 11eutralizing antibodies. 1'he interaction of virus and
antibody, particularly antibodies specific to the v iral anti-
gcns responsiblc for attachmcnt to spccific ccll rcccptors,
results in a virus-antibody comp1ex formation that pre-
vents attachment of virus to cell receptors, and to a lesser
extent prevents the penetration ofvirus into the suscepti-
ble cell. It is possible to recover infectious virus from such
appan::1 1Ll y i11erl virus-a11libody n1ixlures by sin1ple dilu-
tion or centrifugation, suggesting that the virus and anti-
body rnay be linkcd h1 a loosc co1nbination in thc initial
stages ot reaction. ·rhe interaction between virus andan-
tibody <loes not physically alter viral structure; however,
the compleinen.t system and antiviral antibody can in-
duce lysis of enveloped viruses as well as destroy virus-
i11fct:teLl cells.
Cellular immunity in viral infections, discussed in
Chapter 2, is anoth.er irnportant factor in host resistan ce to
sorne viral i.níections. ·1ne destruction ot virus-intected
cells by immune lymphocytes can limit the dissemination
ot virus, particularly in instances where Virus is transmit-
ted from infected to noninfected cells through cell fusion..
Ret:e11L evide11ce also i11dicates ll1al 111acrupliage:; play a
role in host resistance to viral infections. l\1acrophages are
key participants in the inflammatory response, and they
can be activated either by interaction "vith viruses or by
the soluble products produced by virus reacting with lym
pllocytes. Activated macrophages have been sho,v11 to
participate in a wide range of host responses to viral infec-
lions, iJ1cl udi ng pl1agocytosis of virus-a11UlJud y coII1-
plexes, production of interferon, cytotoxicity for virus-
; nff'ct~rl <f'lls, anrl imm11norf'g11 latory f11nctions.
Viral lmmunosuppression
Severa! viruses of veterinary importance can infect lyrn-
phocytes, including canine distemper virus, feline pan-
leukopenia virus, feline leukemia virus, bovine viral diar-
rhea virus, 11og cl1olera vlcus, Newcastlc disease virus, and
infectious bursal disease virus of chickens. The destruction
of lymphocytcs and rcsultant atrophy of lymphoid tissucs
by the viruses can suppress or compromise immune re-
sponse, predisposing the affected host to other oppor-
tunistic bacterial or viral infections.
A variety of spontaneous primary immune deficiency
diseases occur iu clornestic a11i111als (equi11e, bovi11e, ovi11e,
porcine, canine, feline) that can predispose them to infec-
tious diseases. A good example is the fatal respiratory tract
intection oí Arabian toals with combined immunodefi-
ciency disorder (lack of production of functional 1' and n
lympl1ocytes) by equine adenovirus.
Persistent Viral lnfection
Persistent and latent virus infections are characterized by
the fact that virus is not eliminated from the host. Discase
may or may not occur in such chronically infected ani-
mals. Potential mechanisms of viral persistence include
r1oncytocida1 infection of host cells, destructlon of lm-
304 PART III Viruses

FIGURE 49.3. Mechanisms of interferon action on protein synthP.si.~.

lntcrfcron

l
Ccl l ~

~
~~ ~----- ~

:2' - '.:» synthetase Pl/elF-.7a k in ase

ATP l DsRNA
dsRNA

2'-5' A

l
./.\ct ivates cellular
Phosphorylates
iniLiaLiun faclur el F-2
endonuclease
(RNase L)

l
Degrades rnRNA
lnhibits init iution
of protein synthesis

lnhibits protcin synthcsis

mune effector cells or growth within these ceU types, eva-
sion of protective host responses including cytokines and
antibodles, and integration of the viral genome into that
of tl1e host cell. Persistc11t infections are those in which
virus continuously is prese11t, witl1 or will1oul expression
of disease. Uisease, when it does occur in persistently in-
fected anin1als, often is a result of immunopathologic
mechanisms. Latent infections are those in which virus is
demonstrated only when reactivation (recrudescence) oc-
curs; lhis is highly characterislic of her pesvirus infectio11s.
 Parvoviridae and
Circoviridae
YUAN CHUNG ZEE
PARVOVIRIDAE
Parvoviruses are small (18- 26 nm), nonenveloped icosahe-
dral viruses that contain a linear single-stranded DNA
geno111e (MW approxin1ately 6 x 106 dallons). Men1bers of
the family Parvoviridae, genus Parvovirus, are the causative
agcnt s of specific diseases (Table 50.1). A variety of other
an imal parvoviruses h.ave been identitied, including
ch icken parvovirus, mink enteritis virus, rnice minute
virus, mouse parvovtrus 1, anli raccoon parvovirus.
FELINE PANLEUKOPENIA
Disease
FE'l ine p::inlE'11kopeni::i (syn . feline infec.tious enteritis) is ;:i
h igl1ly co1llagious, acule v iral disease of cals characler-
ized hy high fever, anorexia, depression, vomiting fol-
lowed by dehydration, diarrhea, an.d deat h. Leukopenia
is characteristic of feline panleul<openia, and t11e severity
of clinícal dísease often m irrors the severity of leukope-
n ia. Cats of all ages are susceptible to infection, but mor-
tality is highest arnong kittens. Cats can be infected by ei-
th er Ll1e oral or respiralory routes, and tl1e incubatio11
p eriod after infec.tion is short. Intrauterine infection
'vi th feline panleukope11ia virus may lead to neonatal
death or congen itaI abnormalities of the central nervous
system (CNS) manifested by cerebellar ataxia in kittens
after birth; k.ittens up to 2 ,.,,eeks of age are susceptible to
t h e same teratogenic effects of feline panleukopenia
virus infeclion.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Felíne panleukopenia virus is a typical parvovirus. 'fhe
vírions are unenveloped (18- 26 nm in diamctcr) with
icosahedral symmetry. The genome is a single-stranded
DNA molecule that includes two open reading fram es, one
uf whic!1 encode:; at least four proteins that mediate the
fu nc.tions require<l for tr::insrription ::in<l DNA repHcation,
N . JAMES MACLACHLAN
and the otl1er, whicl1 en codes tl1e capsid proleins of lhe
virus. VP3 constitutes the major capsid protein, and it ap-
pcars to control tíssuc/ ccll tropism of thc virus. Virus rcpli-
cation occurs within t he nucleus of host ce lis and, because
the viru s lacks its own DNA polymerase, replication of par-
voviruses requires cells that are cycling (late S phase or
early G 2. phase of the cell cyc!e) so that they can l.ttilize
host cell enzyn1es for tJ1eir ow n replicatio11.
Feline panleukopenia virus is closely relat ed to canine
parvovirus and to mink enteritis virus, althougb thc thrcc
viruses can be distinguished by sequence analysis.
Resistance to Physical and Chemical Agents
Feline pa11leukopenia virus is very resisla11L lo environ-
mental factors and many commercial disinfectants. A
0.175')-Q sodiutn hypochloritc solution (Clorox 1 :30) is the
most effective and practica! virucidal disintectant.
lnfectivity for Other Species and Culture Systems
All u1eu1l..H:!r:; uf tlle fau11ly Felidue are likely to !Je :;u:;ceµtl-
hle to infec.tion wi th feline panleukopenia virus. A very
closely related virus that .is antigenically indistiuguishable
from feline panleukopenia virus causes enteritis in ranch
mink, and the same virus can produce disease in raccoons
and in coatimundi. Canine parvovirus Type 2 has emerged
relatively recently, and it is ancestrally closely related to fe-
line panleukopenia v irus.
Feline prinleukopeni::i vin1s grovvs in pr.imary or con-
tinuous feline kidney cell cultures but not in canin:e cell
cultures.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Feline panleukopenia occurs worluwi<.le, auu iufecteu cats
are the principal reservoir. Both infected c.ats suffering
from acute disease and t hose having clinically inapparent
intection excrete virus in their urine, teces, and various se-
cretions. The infection spreads rapidly by contact wit h
contaminated utensils, cages, ar1d bedding, and the virus
is highly stable in the environment.
305
306 PA1u 111 Viruse:,
Ta b 1e 5 O. 1 . Diseases Caused by Pa rvoviruses
Vernacular Name Disease
Fclinc panlcukopenia virus Enteritis, ataxia
Mmk entent1s virus Enteritis
únine parvovirus Type 2 Enteritis, myocarditis
Por<lne parvovirus Stillbirth, mummification emblyonic
death. infertility
Bovine parvovirus Non e
Minute virus of caninesª Non e; dia.rrhea?
Minute virus of micc Dcvclopmcntal anomalies
Aleut1an m1nk d11ease Aleut1an disease
Kilham rat virus Developmental anomalies
Goose parvovirus Hepatitis
0 Also known as canine parvovirus Type l.
Pathogenesis and Pathology
Cell-free viren1ia occurs for severa! days in kittens experi-
mentally infected intranasally or orally with feline pan-
leul<open.la virus, durlng whi<.:h the virus is tlis,:seulil1ateu
throughout thP hndy ;incl inff>cts Cf>lls with the necessary
receptors. The virus then replicates in those cells that are
in S phase of the cell cycle, particularly hcmatopoietic cells
within thc bonc marrow and lymphoid tissues (thymus,
spteen, ano 1ymph nodes), lead1ng to severe ano pro-
tracted leukopenia that affects all white blood cell types
anu atruphy uf lyu1phuiu ti:,::.uc.:s. Tl1e rapiuly ui vidi11g ct'll:.
of the intestinal crypts ;il~o ;irP highly snsci>ptihli> to inff>r-
lion, which leads to n1arked destruction of the intestinal
epithelium with resultant malabsorption diarrhea. Histo-
logic Jesions are charactcrizcd by necrosis of the epithe-
lium of the intestinal crypts and marked destructlon and
depletion of lyn1phocytcs in tl1e lympl1 nodes, thyrnus,
and spleen. Regeneratlve lymphoid hyperplasia may be
present in tl1e later ph¡¡se of the d isf>asi>.
111 lalc tern1 fetuses and very young kittens, the virus iI1-
fects and ctestroys the cell s within the external granular
!ayer of thc ccrcbcllum, lending to cerebellar hypoplasia
and atrophy as a conscqucn<.:c of fail ure of the interna!
granular !ayer to dcvclop, and to degeneration and loss of
Purkinje cells. Similar congcnttal lesions can be pruuuceu
hy in-ntf>ro infPrtions of rats, hamsters, ferrets, and rnicc
after infection al critica! stages of gestation with the ap-
propriate species of parvovin1s.
Host Responses to lnfection
Neutralizing antlbodles flrst appear in cats at appruxi-
mately 1 week after infcction, and high titf>rs of antihody
gene1ally are present after 10 to 12 days. I Iemagglutination-
inhibiting antibodies rise from the fourth day after infec-
tion, rcaching a peak on thc scvcnth day. These antibodies
can persist in cats for severa! ycars. Maternal antibody with
neutralizing titer of 30 or grcatcr against feline panleukope-
nia virus protects klttcns agalnst viral infectiun, but it alsu
Natural Host
Cats
Mink
Dogs
Swine
Cattle
Oogs
Mice
Mink
Rats
Goose
interferes with active immunization by modified live or in-
act1vated feline panleu.kopcnia viral vaccines.
Laboratory Diagnosis
Clinical signs, the presence of lcukopenia, and histopatho-
lohTical cxamination are uscful for the presumptive diag
nosis ot teline panteukopenia. The diagnosis can be con-
firmed by one of the following laboratory methods:
J) isolatlon of fellne panleukopenia virus fru1u tlle fece:. u;
urine on feline kidney cells, 2) detection of viral antigen ir.
i11f1:cled lissu1:::s by caplure .ELISA or i..n1.n1unofluorescence
staining with a felinc panleukopenia virus-specific antis-
era conjuga te, 3) polymerase chain reaction (PCR) amplifi
cat1on ofviral nucleic acid, 4) serologic diagnosis is acco1n-
pllshed by enzyme-linked immunosorbent assay (ELISA
o r indirect tmmunofluorescence, altl1ough paireu sera are
required to confirm the diagnosis.
Treatment and Control
'rhere is no effectivc trcatrnent for ff>l inf> panleukopenia
l11us conlrol ls achicvcd by vaccination, quarantine of cau
that survivc infcction. and rigorous sterilization of prem-
iscs that havc housed affected cats. Higl1ly effective inaeti-
vated and modifted Uve feline panJcukopenia viral vacctne
are commercially availablc, although maternal antibodi3
can interferc wlth thc tmmunizatiun uf yuu11g kitte11s.
(AN INE PARVOVIRUS 0 1SEASE
Disease
Parvoviral dlsease in dogs ts characterized lJy tl1e suudd.
onset of diarrhea, vomiting, anorexia, ff>vi>r, cit>pre.ssior
ly1r1phvv1:::11ia, and del1ydration. Mortality is higl1er ;;:.

!JUppies than adults. Very young puppies sometin1es de-
eluµ r1.1yocar<.liti:s vvill1oul cli11ical signs of enletilis.
The disease occurs worldwide. It vvas first recognized in
Xorth America in 1978, but retrospective serological stud-
:es indicate that the virus rapidly spread around the >'llorld
:..'1 the early 1970s to cause. a subsequent global pande.míe.
canine parvoviral disease is caused by canine parvovirus
-:-y'Pe 2, which is a variaot of feline paoleukopenia virus.
C<t.! Lir1e varvoviru~ Type 211a~ co11U11ueli Lo evulve :si11ce il
~merged in dogs, vvith the appearance of newvariants that
have been designated as canine parvovirus Type-2a and
•ype-2b. ·rhe Ininute virus ot carllncs that does not pro-
duce disease in dogs h.as be.en desigoated as canine par
,-ovirus Type l ..
ftiologic Agent
:ihysical, Chemical, and Antigenic Properties
Type 2 is very sin1ilar to fr~line pnnlf'11-
.Canine. parvovirus
~open1a
.
virus.
~esistance to Physical and Chemical Agents
Parvovirus is very resistant to environmental factors, such
as extremes of temperature, pH, and sorne disinfectants.
Th P vir11s r;:in pP r<;i<;t fnr long pPrincls in prPmi<;P<; whPrP in -
fected dogs are kept and can be trans1nitted to other areas
by fomites. It can be inactivated by comrnon bleach such
as Clorox (1:30).
nfectivity for Other Species and Culture Systems
Canine parvovirus 1'ype 2 infects dogs of all breeds and
ut lrer Iue1u!Jer::. uf Ll1e íarILily Cuniúue, :>uch a:- vvvlve:>,
foxes, and r.oyotes. Do1nestic cats without antibodies to
the virus are susceptible to experin1ental infection but re-
DJain asyn1pto1natic.
Canine parvovirus 1'ype 2 can be propagated in primary
cell cultures of canine or feline fetal lung and l<idney, as
';\·en as continuous cell lines such as canine cell line A72
au<.l fcliue ccll liue:s NLFK alH.l CRFK. 1'1Je virus al::.o gruw:s
in sorne r.ells frorn son1e other animal species.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Type 2 parvovirus infection of dogs and other members of
rbe family Cantdae is prevaJent in many areas of the worlct.
Parvovirus-infected dogs continue to excrete infectious
virus in LheiJ feces for up Lo 10 days afler Lhe onsel of infec-
tion, and the virus is readily transmitted bet,.veen dogs by
rhc fcco-oral routc.
~athogenesis and Pathology
~he patl1ogenesis of Type 2 parvovirus infection of dogs is
~n1ila1 lo Lhal of panleukopenia v irus infeclion of cals, al-
though cerebellar hypoplasia and atrophy is not recog-
Chapter 50 Parvoviridae and Circoviridae 307
nized as a consequence of in-utero infection of dogs with
canine parvovirus. Sinlilarly, n1yocardilis is a poleILlial COIL-
sequence of parvovirus infection ofyoung pups, whereas it
is not described in panleukopenía-virus infec'ted kittens.
Cell-free viremia precedes infection of the intestinal ep-
itheliun1 and lymphoid tissues, including thymus, tonsils,
retropharyngeal and mesenteric lymph nodes, and spleen
of infected puppies, and '"'idespread infection of the intes-
Liual u1ucu:sa uccur:s ur1 a!JouL Ll1e :sixll1 <.lay a(Ler experi-
mental inoculation. Fecal excretion of virus begins as soon
as the third day after infection and peaks soon thereafter.
Most infected dogs stop excreting virus by the n..,elfth day.
The 1nost striking lesion of parvovirus enteritis in dogs
is hemorrhage '"ithin the lumen of the small bO\..,el and
accompanyi og cnlargement and edema of the mesenteric
1yrnph i rode:s. MuLlle<.l w lllle :streaks withi11 the n1yucarc.liurr1
are indicative of cardiac involvement in young puppics.
Microscopic lesions associated with can.ine parvoviral
in1ection are confined to o rgans \·vith large populations of
rapidly proliferating cells, such as the small intestine,
.lympl1 nades, and bone marrow. rhe most frequent find-
ings in the small intestine are necrosis of crypt epithelii1m
aud alrophy of epillielial villi. Reger1erC1tiur1 uf ir1testinC1l
epithelium occurs in dogs surviving the acutc~ phase of en-
teric infection. Lymphocytolysis in the thyn1jc cortex and
ger1uü1al centers of lymph nodes is common and results in
cellular dcplction. Jn thc n1yocardial form of p arvoviral in-
rect1on, the ventricular myocard1um shows myof1ber de-
generation and 11ecrosis that is accompanied by infiltra-
tiuu lJy 1Ilü11U11U<..:leC!I <..:ells.
Host Response to lnfection
Dogs infected vvltl1 parvovtrus rapidly dcvelop hlgl1, long-
\;:i<;ting titPr<; nf vir11<; nPntr;:ili7.ing ;:intihncliP<; ThP irnmn -
nity that develops after natural infection appears to be life-
long in dogs that survive. Cellular imm une responses also
are gcncratcd during infcction, and likcly are important in
hmitü1g virus replicatio11 during acutc il1fection.
Laboratory Diagnosis
CJinical signs, history, contrast radiography, a11d histo-
pathological exan1inatio11s are useful il1 a presun1ptive di-
agnosis of canine parvoviral disease. Laboratory proce-
durcs uscd to confirm thc diagnosis includc the following:
l. lsolation ot canine parvovirus ·rype 2 from the teces
or tissues of infected animals on susceptible ce.Il cul
tures, or by identification of parvoviral nucleic acid
in infected tissues by PCR.
2. DeLecliur1 uf JJC1rvuvirC1l C1Htiger1 ir1 the histological
sections of intestine hy the imm11nof111orf'S<f'nt or
immunohistochemical staining with virus-specific
antibodies.
3. Dem.onstration of parvovirus in feces or infcctcd
tissue by electron rnicroscopy or immunoelectron
in1croscopy.
4. Jc.leutification of parvovirus in teces by hcmaggluti-
nntion test 11sing S\~>inf' or rht•sus tnonkey red blood
308 l'ART 111 Viruses
cells and t h e specific hemagglutination-inhibition
by nnticnninc pnrvov irus nnti:icrum.
5. Detection of parvovirus in feces by ELfSA using
monoclonal antibodies to the canine parvovirus
Type 2 bemagglutl11atlng protein.
6. Demonstration of anticanine parvovirus antibody
i11 :;eru111 l.Jy sucl1 serulugic t est:; as l1eu1agglutiua-
tion inhihit ion, virus n eutralization, or ELTSA. The
IgM-cnpturc .CLISA cnn. be u3cd t o co1úirm rcccnt in-
tection ot dogs.
Treatment and Control
Severe cases of canine parvoviral rlisPasP arP c.harac.terize<l
by n1arked dehydration and n1etabolic acidosis, thus sup-
portive treatment relies on replaling lost body fluids and
corrccting disturbcd clcctrolytc balance nnd acidosis.
Hroad-spectrum antibiotics are used to prevent secondary
bacteria! iI1fections. Vacci11ation of susceptible canine
populations remai11s tbe best prophylaxis for canine par-
voviral infection. An tibody levels correlate directly with
lhe deg1ee oí p1otecliort. Effective inactivated and modi-
fied live parvoviral vaccines are available, although the
presence of mater11al antibodies illterferes with active im-
munization ot puppies. Canine parvovirus is very resistant
to environ1nc11tal fact ors and can persist under adverse
conditions for a long period, thus prompt disinfection of
premises where infected animals are being kept and vacci-
uatior 1 of .LJU.LJpies vrior lo lheir inlroducllon lo such
premises are important in preventing this disease.
PORCINE PARVOVIRUS INFECTION
Disease
Jnfection of swine wit h porcine parvovirus occurs world-
wide and sometimes leads to reproductive failure in swine
and cutaneous lesions in piglets. Transplacental infection
of fPt11sPs IP:irls to stillhirth, m11mmiñration, Pmhryonic.
death and infertility, the so-called SMEDI syndrome. 'fhe
porci11e reproductive and respiratory syndrome virus is
now considered to be a more important cause of SMEDI
syndrome tha11 porcine parvovirus.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Porcine parvovirus resembles feline and ca11ine parvovi-
ruses. 1'here is only one serotype, and porcine parvovirus is
antigenically different than other parvoviruses.
Resistance to Physical and Chemical Agents
Pon..:iue parvoviru:; is very resislaul lo heal, e11¿yrnes, ar1d
most commercial disinfectants. The virus is inactivated by
heat at 73ºC for 30 minutes or at 70°C for 1 hour, or by ex-
posure to 0. ~o/o SOdium hypochlorite tor ~ minutes, to
0.06% potassium dichloroisocyanurate for 5 minutes, or
to 3'){, formaldchydc for 1 hour.
lnfectivity for Other Species and Culture Systems
Porcine parvovirus apparently infects only swine. 'l'he
viru:; <.:a11 !Je .LJIU.LJagated 011 vri111ary or secor1dary cultures
of fetal porcine kidney cells and swine testicle cells.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Parvovirus infection is endemic in. many hPr<ls, anrl in -
fecled swine serve as tl1e reservoir of i11fection. Infected
swine develop a viremia and shed virus in their oral secre-
tions and feces. Sincc thc porcinc parvovirus can pcrsist in
the environment for long periods of time, contaminated
premises serve as a major reservoir of virus that is respon -
sible for its transmlssion to susceptible anirnals. Cdirier
boars also can disseIDÍI1ate the virus in their se1nen. Al-
ll1ough rals n1ay be h1fected experin1entally with porcin e
parvovirus, they are unlikely to serve as natural reservoirs
of the virus.
Pathogenesis and Pathology
Swine infected with porcine parvovirus produce antibod-
íes without developir1g clirlical <.lisease or obvious lesions.
Reproductive disPasP orrnrs when seronegative sows are
exposed to the virus during gestation, and the conse-
quences (fetal death with or without mummification, em-
bryonic dcath, or infcrtility) of transplacental transmis-
sion of the virus are reflective of the gestational stage of
the sow at lnfection. Lesions within stillborn fetuses are
ofte11 i1011svecific, bul can include foci of necrosis anc
mononuclear cell infiltration in organs such as the liver.
heart, kidney, and cerebrum.
Host Responses to lnfection
Piglets and no.npregnant adult swine infected wit~
porcine parvovirus tlevelop virernia witl1uut olJviuus clini-
ral <lisease. 'fhese animals develop a strong humoral im-
mune response and are resistant to subsequent reinfectior.
\vith the virus. Piglets acquire high titers of virus-specific
antibodies tl1rough the colostrum of immune sows.
Laboratory Diagnosis
Parvovirus antigen can be detected in the tissues of affected
fetuses by lm1nunofluorescent or immunohisto<.:henli<.:al
staining or by capture ELISA. A PC~.R assay has ;i lso hPen rle-
veloped for tl1e detectio11 of porcine parvovirus.
Treatment and Control
There is no treatment for reproductivc failurc produccd
by porcine parvovirus, and vaccinat1on of breed1ng g1lts

remains the best method to ensure tbat gilts develop ac-
tive immunity prior to being bred. Both inactivated and
rr1otlifietl live v<1cciJ1e~ are <1vailable, bul vaccina lion n1usl
be carefully timed to ensurP that anirnals a rP i mm 11 n i7.P.cl
afte1 passive antibodies are lost and before the animals
are bred.
ALEUTIAN DISEASE IN MINK
Di sea se
.<Ueulian clisease (AD) in n1ink is a cl1ronic progresslve dis-
easP. c.harac.tP.rizerl hy anorexia, polydipsia, severe anemia,
and hemorr.h ages. The disease has a long incubation pe-
riod. Characteristic features of the disease in adult minks
are dissemianted plasmacytosis (aggregates of plasma cells
in many tissues), uveitis, vascular damage, hypergamma-
globulinemia, and glomerulonephritis, the last of \.Yhich is
caused by deposition of circulating imn1une con1plexes. In
newborn mink kits, Aleutian disease virus (ADV) causes a
fatal, acutc intcrstitial pncumonitis. A gcnctic prcdisposi-
tion for the disease exists in Aleutian mink. lt appears that
mink homozygous for the recessive Aleutian gene have a
genetic defect in preventtng the clearance of ctrculating
irnmune complexcs.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Aleutian mink virus resembles tb.e other parvoviruses that
p rcviously wcrc dcscribed. Tl1e Aleutian disease virus is
antigenically unrelated to mink enteritis virus, which is
closely related to feline panleukopenia virus.
Resistance to Physical and Chemical Agents
Aleutian mink virus is in::ictivatPcl hy socli11m hypoc.h lo-
rite, iodophor, glutaraldehyde, formalin, and phenols.
lnfectivity for Other Species and Cell Systems
Alcutian discasc virus infccts mink of all typcs, although
disease is more prevalent in the Aleutian. m.ink. Ferrets can
be infected wit h ADV, but they do not develop clinical
signs of the disease. 'fhe virus can be propagated in fetal
mín k kiclnPy cPI 1 c11 lt11rPs or in fpJ i nP c.ell l i nP.s.
Host-Virus Relationship
Distribution, Reservoir, and Transrnission
Aleutian disease of mink is present in many mink ranches
worldwide. The virus is found in the blood, saliva, feces,
and urine. Mink with overt cllnical signs or with inappar-
ent infection are the reservoirs of infection. 'I'he disease is
transn1itled by fecal-oral or respiratory roules.
CltuJ1ler 50 Pur voviriclae and (;ircoviridae 309
Pathogenesis and Pathology
In mink experimentally infected with ADV, viral antigen is
first detected in the intestine and kidney and subsequently
in the spleen, liver, kidney, lymph nodes, and bone mar-
row. 1'here h: a significant increase in the amounts of
gamma globulin and anti-DNA antibody in the sera of in-
fected rnink. 'rhe serum garnma globulin does not neutral-
ize Al)V, a11d i111111une con1plexes appea1 in circulatlon as
early as 2 weeks after infection. The glomerular deposition
of iinmunc complcxcs rcsults in glomcruloncphritis.
Host Responses to lnfection
Hypergammaglobulinemia (predominantly IgC;) is the
u1ust proullnent fedture of ulink infected with ADV, l>ut
the prcsencc of t his antihody <loes not fac.ilitate virus
clcarancc.
Laboratory Diagnosis
Solid-phase radioilnn1une assay, direcl ilnn1u11oíluores-
cence, and immunoperoxidase staining techniques ca11 be
uscd to dcmonstratc AD viral antigcn in tissucs. Thc cou11-
terimmunoelectrophoresis ((JE) t est for AIJ viral antibody
can be used to identify infected mink.
Treatment and Control
No effective vaccines have been developed for the AD of
mink. Control is achieved through the isolation and/or
elimination of affected animals.
CIRCOVIRIDAE
Circoviruses recently have been shown to be important
pathogens of animals. Like parvoviruses they are small
no11enveloped icosahedral viruses (approximately 1?.- ?.6
11111) witl1 a ge110111e of si11gle-stranded DNA. In1portant
viruses witl1in the family Circoviridae include beak and
feather disease virus, chicken infcctious anemia virus, and
porcine circovirus. A pathogenic circovirus also has been
idc11tified in pigeons, and sirnilar viruses infect humans as
well as o the r bird species.
BEAK ANO fEATHER DISEASE
Disease
Beak and feather disease (BFD) is a disease of psitaccine
birds. Thc scvcrity of BfD varíes with the age and species of
the affected bird. 'J'he characteristic changes associated
with BFD are the appearance of necrotic and abnormal
feathers (feathers that are bent, contain hemorrhages, or
are prematurely shed), although the pattern of abnormal
feall1erin,g varjes wiLl1 ll1e age of tl1e l>ird at iI1fec"tion. Beak
310 PAKr 111 Viruses
and nail deformities occur in sorne chronically affected
birds. Severe, acute disease characterized by pneumonia,
Ciltt'.ritis, i:aµid weigl1t loss, ano U<;:ath is µartiLularly t:o1n-
mon in neonatal cockatoos and Africa11 grey par.rots, an,d
death niay occur in these birds before feather abnorn1ali-
ties occur.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Virions of BFD virus ure upproximately 19 26 nm, nonen
veloped, with a ge11on1e of circular single-stranded DNA
that is transcribed to produce a single, polycistronic m RNA
that encodes at least threc proteins.
Resistance to Physical and Chemical Agents
A1tl1ough tl1c spccific properties of BFD virus ren1uin to
be determined, other circoviruses are resistant to low pH
(pH 3), high temperature (>70ºC), as well as ma11y chemi-
cals, such as chlorofor1n, ether, and alcohol.
lnfectivity for Other Species and Culture Systems
The DPD virus appears to infect only psittacine birds, and
it has yet to be propagated in cell culture systems.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Psittacine BFL1 has been described among birds in North
An1erica, Europe, Asia, a11d Australia. Tl1c virus likcly 11as
been disserninated by the moven1ent of infected Aus-
tralian psittucinc birds, bccausc BFD virus is cndcmic i11
tree-ranging populations of severa! psittacine species in
Australia, notably cockatoos. A wide variety of cockatoos,
parrots, lovebirds, and parakeets are susceptible to BFD.
The virus is sp read directly beti'\Teen infected and suscepti-
ble birds by aerosol or by fo1niles. ·rhe fea ll1er dusl, excre-
tions, and secretions from infected birds all contain BFD
virus, a11d th.c virus is vcry stablc and resistant in thc cnvi-
ronment. Vertical tra11sm ission of the virus from infected
birds to their chicks also n1ay contribute to disse1nination
of the virus.
Pathogenesis and Pathology
'I'he majority of free-ranging and captivc birds cxposed to
BFD virus develop only subcJinical infections and are re-
sistant to reinfection . The age of the bird at initial expo-
sure, the presence and levels of p rotection provided by rna-
ternal antibody, a.nd the route and titer of the infecting
viru!) all likcly iufluence Ll1e oulcon1e. 1'he incubalio11 pe-
riod of BFD virus infection is often p rolonged, and signs of
disease including death n1ay not occur until long aftcr ini-
tial infection. Acu tc or peracute death occurs in very
young birds that lack maternal im.munity. Affected young
birds n1ay exhibit inappetance, lethargy, crop-stasis, pro-
gressive feather abnormalities, and eventual death. Death
in sorne birds likely is a conseque11ce of secondary iI1fec-
tion!), perl1aps a!)sociated witl1 iluu1uue supµression.
Histologic Jesions are dependent on the (h1ration ancl
severity of the disease in individual birds, but characteris-
tic intranuclear and intracytoplasmic inclusion bodies
may be prcscnt in the epithelial cells lining thc fcuthcr
shaft, and in macrophages in the thymus, bursa, and other
ly1nphoid tissues.
Host Responses to lnfection
Birds that previously were exposed to BFD virus are resist-
ant to reinfcction . Maternal antibodies likcly provide pas-
sive protection of chicks against BFD virus infection for
several >veeks after birth.
Laboratory Diagnosis
Dicfgnosis of DF.D currently is dependent on idcntifi cation
of tl1e clinical signs and characteristic gross and h istologic
changcs in tl1e tissues of affect ed birds. Botl1 11ucleic acid
hybridization and imn1u11ohistocbemical staining assays
can be used to detect BFD ' 'irus in the tissues of affected
birds.
Treatment and Control
Control of BFD currently is dependent on the elilnination
or qu aran tine of carrier birds to preve11t transn1ission of
BFD virus to susceptible cohorts. Severa! vaccines have
bee11 devcloped to prcvent BFD virus infection, but they
currently are not •videly avaUable.
(HJCKEN INFECTIOUS ANEMIA
Chicken infectious ane1nia (CIA) is a d isease of young
chickcrts (2-4 weeks of age), characterized by aplastic ane-
mia, generalized lymphoid atrophy, a11d profound im.-
mune supprcssion that leads to secondary (opportunistic)
viral, bacteria!, and fungal infections. Bin.1s become resist-
ant to CIA disease after approximately 3 wee.ks of age, but
the disease causes serious economic losses to tl1<::: µoultry
industry vvorldwide. Genetically clistinc:t strains of CTA
virus are recognized, although these are a11tigenically sim-
ilar or identical and tl1ere is little difference in the viru-
lence of individuul struins. C IA virus is highly resistant to
n1ost treat1nents. The virus is infectious only to chicl<ens,
although related viruses may infect otl1er bird specif'S. The
ClA virus cau !Je µropagaled in chicken en1bryos, day-old
chicks, and in avi~in T ;:incl R lymphoc:yte lines. Diagnosis is
by virus isolation, PCR detection of the virus, or immuno-
histoche1nical staining of the tissues of affected birds,
using ClA virus-spcclfic antiboclies. Serological assays
(ELISA, indirect immunot1uorecsence, and vir11s neutral-
ization) have been developed for serological detection of
ClA virus infection of birds. 1'he virus is Ui!)se1uü1aled bolh

:Corizontally and vertically, and vaccination of breeding
:':ocks wi th modified live virus is used to prevent vertical
u a11sn1ission of ll1e virus. No specific lrealn1e11l is avail-
able for chickens infected with CIA virus.
PORCINE PosTWEANING MULTISYSTEMIC WASTING
SYNDROME
Two distinct types of porcine circovirus (Types 1 and 2)
h avc been idcntificd in pigs. Typc 1 porcinc circovirus ori-
ginally was identitied as a celJ cu.Lture contaminant and is
not known to cause any disease in pigs. In contrast, Type 2
porcine circovirus recently vvas incriminated as t he cause
of postweaning multisystemic wasting syndrome (PMWS)
Chupler 50 Purvuviridue a1H.l Circuviridue 311
in swine. PMWS ts an economJcally important disease of
young pigs that is characterized by generalized lymph
node e11largerne11l, cl1rorlic pneurno1lia, aucl weighL loss.
Characteristic histologic lesions include generalized lym-
phoid depletion and granulomatous inflammation. Ma-
crophages in affected lymphoid tissues may contain ba-
sophilic inclusion bodies. Whereas Type 2 circovirus
infection is ubiquitous in swine herds, PWMS occurs only
sporadically in susceptible svvine, suggesting that other
fac tors may potent iate development of PWMS in
circovirns-infecterl pigs. The porcine circovirus TypP ?. may
be detected in the tissues of pigs by immunohistochem-
istry, in situ hybridization, or by PCR assay. The virus can
be propagnted in ccll culture, and ELISA assays havc bccn
developed for serological detection of ·1ype 2 circovirus in-
fection of swine.
 Asfarviridae and
Iridoviridae
N. ] AMES MACLACHLAN
ASFARVIRIDAE
Thf' Asfarviridae includes just one genus, the gen us Asfi-
virus, of whi<.:h African swine fcvcr virus is t hc typc
specics.
AFRICAN SWIN E fEVER V IRUS
Disease
African swine fever (ASF) is a highly contagious disease of
domestic and somc spcclcs of wiltl swine. Visease iI1 ASF
virus-infected pigs ranges from per.icutf' to chronic .ind in-
appa1enl¡ incursions of the virus into native populations
of do1nestic swine typically result in extensive outbreaks of
acutc ASr, and thc subacutc and chronic forros appear
alter the virus becomes established m the p1g popuJatíon.
Acute, severe AS!-' has a high mortality, often approaching
lOOo/o, and dcath may occur prior to tleveloprnent uf clirli-
cal signs. Thc discasc is ch;ir;ictf'ri7.f'd hy high fever and
leukopenia, oftcn followed by the appearance of erytl1ema
(red arcas on the skin), weakness, accelerated respiration
and pulse.:, vomiting, bloody diarrl1ca, a11d nasal and con-
junctival tlischarges. Subacute ASF is charactertzed by less
dramatic clinical signs and dcath or recovery in 3 to 4
weeks. Affectcd plgs typlcally experience a lligl1 fever;
abortion is common ;.ind n1;iy hf' thf' on ly .sign of illness.
Pigs wilh chronic ASF fail to thrive (sh111ting and emacia-
tion), and cxhibit swollen joints and lameness, skin ulcer-
ations, and pncumonia.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Afr1can swine fever virus is an enveloped DNA virus with a
nucleoprotein core of 70-100 nm that is Sl.urounded by
lipid layers anti an icu~al1etlral LatJ:>id oC 170-190 nn1 in di-
ametf'r. Thf' virion is .surrounded by a lipid-containing en-
velope (fig 51.1). Thc genome is a single largc (170- 190 kilo
bases) molecule of double-strandcd UNA that includes ap-
proximatcly 150 open reading frames. Virions contain
more t11an 50 d1fferent proteins, including a Jarge number
312
) EfFREY L. STOTI
of enzymes and proteiI1:; reyuirell Coi ieplicalio11. The viral
eenomf' ;i lso Pncod~s proteins that appear to modulate the
protective host ant iviral response.
Severa! gcnetically distinct groups of ASF virus have
bccn idcntificd by rcstriction endc>nuclease analysis of the
gcno1nes of ASF viruses 1solatcd in different regions of t he
world. Strains of ASf virus can vary markedly in their viru-
lencc in swlne.
Resistance to Physic;il and Chemical Agents
Afriuiu ~wi 11e feve1 vi1 us is stable iI1 tissues and excretions.
The virus can withstand a considerable range in pH (pH
4 to 13). The ASP virus is inactivatcd by hcating to 60ºC for
20 minutes and by lipid solvents and some disinfectants
(paraphcnylphenolic disinfectants are very effective
against thc virus).
lnfectivity for Other Species and Culture Systems
African swine fevcr viru~ u1ft:cl~ do111eslic and wild pigs
(incl11ding r.11ropP.an wild hoars), warthogs, giant forest
hogs and bush pigs. Soft tick::; of the genus Ornithodoros
tra11smit thc virus as biological vectors. 011ly domestic and
wild pigs (feral pigs and European ~vild boars) express clin-
ícal disease, whereas African wild p.igs do not.
African swine fever virus replicates in pig macrop.h ages,
and can be ¡Jrv¡.¡agateu i11 vít10 on cultures of swine bone
rnarrow cclls, monocytes, and alveolar macrophages. 1'he
virus can also be adapted to various cstablishcd ccll lines
(pig kidney, VERC), and 13HK).
Host-Virus Relationship
Distribution, Reservoir, and Transmission
African swinc fcver was first described in European domes-
tic pigs In Kenya In the early 1900s, a111l Lhe disease regu-
larly has emcrg<>rl in to othf'r rf'gions of the world since that
tiI11e. The disease spread outside of Africa for the first time
in 1957 when it appeared on the Iberian Península, aná
subscqucntly has occurred in Mediterranean Europe
(Spain, Francc, Ttaly, Malta, SardiI1ia), northern Europe
(Belgium and the Netherlands), tl1e Caribbean (Cuba, the
Dominlcan Rcpubllc, anti Haiti), a111l Soull1 Arneric.a

f 1G U RE 5 1. 1. A frican swine fever virus in thin section of
infected tissue culture ce/Is. 58,000X. (Courtesy of te Pang.)
(Brazil). African $\<Vine fever has been described through-
out much of Africa.
'f'he ASF v irus e.xists in tvvo distinct cycles of infection:
first, a sylvatic cycle in ticks and "vild pigs in Africa;
and, second, epidemic a11d endem ic cycles in domestic
swinc. '!'he rcscrvoirs of the sylvatic cycle of ASF virus in-
fection in Africa are perststent or inapparent infections in
.'\frican \.vild pigs ('"'arthogs in particular) and the soft
tick vector. Vertical trans1nissiun uf ASP virus iI1 the tick
ve.ctor makes them an e.specially efficient reservoir of the
virus. 'fhe virus sprcads in to dornestic swine through the
bites of infected ticks, or by ingestion of tissues from car-
rier swinc. Tl1e virus the11 is readily transmitted to suscep-
tible pigs by di rect contact, including aerosols and
fo1nites . The virus is easily transmitted over long dis-
tances because uf its stability in infectell tissues, inclu<.1-
ing uncooked and sorne cu red pork products. 1mpor-
tantly, soft ticks become infected '-vith t he virus after they
feed on viremic swine, and thus they can beco1ne reser-
voirs of the virus after it incurs into previously nor1iI1-
fected re¡.,>ions. Pigs that survive infection with r'\SF virus
beco1ne carriers of the virus.
Chapter 51 Asfarviridae and Iridoviridae 313
Pathogenesis and Pathology
Following oral ur r1a:;al ex.pu:;urt:: uf llu1n1::stic pig:; to ASF
v irus, virus replication initially occurs in the upper respira-
tory Lracl '1'Vitl1 subsequenl dissen1il1atio11 to adjacent
lyn1ph nodes and then systemic spread via leukocytcs, ery-
throcytcs, or both ir1 thc lymph ond blood; this occurs
within 3 days postinfecti<.n1 and corresponds closely vvith
the onset of pyrexia. TI1e virus replicates in macroph ages;
thus highest titers uf virus uccur in those tissues in w!licl1
macroph;:iges <lrf' most ;:ihunclant.
Ac.ute severe ASF is c.haracterized by edema and hemor-
rhage vtithin internal organs, particularly the lymph
nodcs and splccn, whic.h can be vcry largc and intc11scly
11emorrhagic. Pulmo11ary edema and intestinal congestio11
and he1norrhage also are com¡non. The lesions of sul1acute
ASF are similar t>ut less p ronounce<.l, whereas anin1als with
chronic ASF rriay shO\<V fihrino11s peric;:irciitis ;:ind plP11ritis,
lobular consolidation of the lungs, swollen joints, and
patchy necrosis of the skin. Lesions in aborted. piglets are
rclati.v cly nonspecific, but may il1clude disseminated pe-
tecl1ial hemorrhages.
Microscop ic lesions are most pronounced in the Jy1n-
p hoid tissues, and in elude extensive necrosis of both lym-
phocyte<; ;:inci monon11cle;:ir phagocytic cells. F.nrlothelial
cell necrosis and thrombosis of the puln1onaryvasculature
is common in fulminant cases of acute ASF.
Host Responses to lnfection
!'igs that survivc infection witl1 ASF virus develop a strong
humoral immune (antibody) response; hovvever, this re-
sponse is largely ineffectual in neutralizi.ng the virus. Wliy
the antibody responsf' of swine to ASF is largely ineffectn;:il
ren1ail1s u11certain, but it clearly is a reflectio.n of the inher-
ent properties of ASF virus itself. Nevertheless, efforts at
developing an effective vaccinc are complicatcd by thc in-
ability ot swine to produce !1igll titers of neutralizing anti-
bodies to the virus.
Laboratory Diagnosis
Laboratory tests are requirecl to d istinguish between hog
cholera and .l\SF, beca u se the two diseases cause very similar
signs ancl Jesions in susceplible pigs, inclucling fever, high
mortality, and hemorrhages within interna! organs. 11ssues
submitted should lnclude spleen, liver, lympl1 nodes, and
blood. Virus isolation ca11 be used to identity ASF virus, and
then confirmed with hemadsorption. In1.munofluorescent
or immunoperoxidase stalntng o.f sections of rtssue fro1n af-
fected pigs using ASFvirus-specific antisera provides a rapid
1ueLI1od oJ lli<lguusi~. Tecl1uiques l.Jdsed 011 puly1nerase
chain reaction (PC.R) can also he used to rapidly identify the
presence of ASP virus genomic material.
Treatment and Control
There is no effective vaccine or treatment for ASL". Eradica-
tion of the dísease is accomplished by slaugh.ter and dis-
314 PART III Viruses
posal of all exposed pigs after the virus incurs into new
i:lreas. These dri:lstic IIH:'dsures, however, rndy not prevent
the virus fron1 spreading to the local populations of soft
t icks and wild pigs. Prcmiscs t h .a t un.dergo cradication pro-
cedures must not onJy slaughter all. pigs but also must be
treated with insecticides and disinfectant containing O -
phenylpl1enol wíth surfactants and must remain free of
livestock for at least a montl1. Prior to restocking, suscepti-
ble sentinel anlmals should be placed on the premises to
confinn eradication of the virus.
IRIDOVIRIDAE
The family /ridoviridae includes four genera (genus lridovi-
rus, Chloriridovirus, Ranavirus, and Lyrnphocystísvirus) that
are serologically distinct. Viruses in the genera Ranavirus
and Lyrnµhu<.yslisvirus ci:luse ilui>OrlaIJL disease:> of fish .
I ridoviruses are enveloped viruses that ha ve icosahedral
symmetry vvith a virion d iameter of 120- 200 nm but occa-
sionally up to 350 nm. The geno me is a single molecule ot
double-stranded DNA of between 110 and 300 kilo base
pairs. Iridoviruses, like asfarviruses, are structurally com-
plex with large ¡1umbers of virus-specific proteins (at least
36) e11coded by the genome. A single p rotein constitutes
the majority of the outer capsid. The double-strande:.
DNA genome is circular dntl terrnir1ally re<.lur1ddnt \.\i::.
many of the interna! cytosine residues heing h iglL
mcthylated. Dcfinitivc diagnosis is bcst madc by v iral iK-
latiOn an.d/or characterization of the genome by molec.:-
lar biology techniques.
Lymphocystis virus infects a wide variety of fish ~-i-:
causes unsightly "''art-like c-utaneous lesions. 'fhese lesia__
con.sist of benign proliferations of l1ypertruphied, vi~
infectf'cl cells (fihrohlasts and osteohlasts) on the s~
perito11eum, and inesentery. The lesion.s typically resol.-.e
with mini1nal mortality. The virus spreads by direct co::-
tact through abrasi.o ns, especially when fish are crowdec..
thus disease caused by lymphocystis virus is especially ~­
port ant in fish raised in aquaria and in sorne commerd.:...
aguaculture operations. ·rwo st rC1ir1s uf ly111phucystis tlb-
ease vi rus (T.CDV) h;:ivp hef'n clescrihed, with LCDV-1 heir.::
associated with flounder and LCDV-2 being associateé.
wit !1 dabs. In contrast, the Ranavirus genus has been as-
sociated with high mortality in both farmed and fret;-
ra11ging fish . These pathogenic iridoviruses causing fara¡
systemic disease are closely related to frog virus 3 (F\'3
the latter servln.g as the prototype ran¡-¡virus. Meu1lJers c.:
this genus l1ave been reporteti to causf' Ppizootir hematr-
poietic necrosis a11d syslcn1ic hen1orrhagic disease in fisJ-"-
 Papillomaviridae and
Polyomaviridae
YUAN CHUNG ZEE
PAP/LLOMAVIRIDAE
The papillomaviruses are widespread among marnmals,
!laving been identified in cattle, sh<::'.cp, goats, dccr, clk,
!lorses, rabbits, dogs, monkeys, pigs, opossums, mice, ele-
~hants, and several species of birds. Cottontail rabbit pa-
pillomavirus is the type species. Virus-induced papillomas
~varts) are benign, hyperplastic epithelial proliferations of
•.h e ski11 or 111ucous n1eu1branes Ll1al n1ay u11dergo n1alig-
=1ant transformation in certain circumstances. Sorne papil-
_o maviruses also cause proliferatio11s of mcscnchymal tis-
S'..ies in the skin, with or without associated epithelial
z¡roliferations. Those proliferations with exclusively ep-
::helial proliferation are papillomas, whereas those \.Yith
:;'T-Oliferatlon ofboth mesenchymal (fibrous tissue) and ep-
Lhelial tissue are termed fibropdpilloruC:Js. Papillonra-
""iruses are highly species-specific, and thus are gener::illy
,_;JUtC:Jgiou:; ouly Lo Lbe ani1nal species iI1 whicl1 they natu-
-ally occur. Virus-induced et1taneous papillomas (warts)
:L~ con1mon in horscs und cattlc and infrcquent in dogs,
·'heep, and goats. Not all papillomas are caused by viruscs,
:I'ld p apillomaviruses have yet to be identified in cats.
:=:iologic Agents
~pillomaviruses havc i1akcd icosahcdral capsids approxi-
=:ately SS nrn in diarneter (Fig 52.1). ·rhe viral genome is a
...::-!glc circular 1nolccule of double stranded DNA that en-
.xles betwee11 8 and 10 proteins; 2 are structural (Ll and
:...: an d the remainder are nonstructural proteins that are
~n Lial for virus replicalion.
,apilloma Types
Papillomaviruses are highly restricted in l)Oth their host
specificity, tissue and cell tropis.m, and sequence related-
ness. 'fhey are distinhruished also 011 the l>asis uf tl1e lesiou:;
dley induce, including skin papillomas (warts), prolifera-
4ion of nonstratified squamous cpithclium (polyps), and
Sl1bcutaneous fibromas with o r \'Vithout associated cuta-
aeous papillomas (fibropapillomas).
N. JAMES MACLACHLAN
BOVINE PAPILLOMAVIRUSES
At least six types of bovine papilloma virus are distin-
guishcd on thc basis of thcir antigcnic and nuclcotidc sc-
quence homologies. They can be further distinguished on
the basis of the nature of the lesions that they cause in cat-
tle. Cutaneous fibropaplllomas (wart s) caused by paplllo-
maviruses Types l , ?., and .5 o r.c11r c'ommonly in c::ilves lP.ss
tl1an two years old. They appear n1ost frequently on the
head, especially in tl1e skin around the eyes. 'fhey may also
appcar on thc sidcs of thc neck and less commo11ly on
other parts of the body. They begin as sma.11, nodular
growths that then grow rapidly into dry, hor11y, whitish,
cauliflower-like masses tl1at event ually regress sponta-
neously. ·rhe histological appearance is a vari.a ble mi.xturP
of proliferaling dern1al fibrous tissue and overlying epithe-
li11m. lnfP.c.tious papillomas (without a fibrous tissue com-
ponent) that occur on the skin and teats of dairy cattle are
also associated >-vith intection by bovine papillomaviruses,
as are sorne epithelial proliferations (polyps) that occur in
the bladder and gastrointestinal tract, particularly those
that affect the esophagus, forestomachs, and intestines.
Fibropapillo1na is a ¡.iapillo111avirus-i11duced Lun1or thal
occ11rs on the penis of young bulls and the vagina and
vulva of young heifers. 'rhese are fleshy, raised multinodu-
lar proliferations that consist ot abundant tibrous tissue
covered by epithelium ofvariable thickness.
rlost immune responses eventually control papillo-
mavirus infections because most warts persist for variable
periods and usually spontaneously regress. The host Is
then imrn11nP to rPinfP.rtion with the samic virus, hut not
to other types of bovine papillomavirus. Treatment of
bovine papillomatosis with finely ground wart tissue sus-
pended in a 0.4o/o formalin !;olution has been used for
many years to com.bat outbreaks ofthe disease, but it is dif-
ficult to evaluate the efficacy of this procedure since the
disease ls self-llmiting and its duration varies IJetween in-
dividual animals. A significant proportion of vacciI1ated
aJ1irnal~ appareuLly fail Lo re ject Lheir warls afler vaccina-
tion with autologous tumor preparatio11s. Cattle vacci-
natcd w ith thc L2 structural protein of bovine papillo-
mavirus Type 4 did not develop alímentary papillomas
when challenged w ith that virus type.
315
316 PARr Ill Viruses

F 1G U RE 5 2. 1 . Negatively stained preparation of equine papillomaviruses.

75,000X. (Reproduced with permission from Sundberg JP, O'Banion MK. C/oning
and characterization of an equine papillomavirus. Viro/oqv 1986; 152:100.)

11'
ll.._---
EQUINE PAPILLOMAVIRUS AN O EQUINE SARCOIDS (ANINE ÜRAL PAPILLOMAV IRUS

Skln warts of horses are notas comn1on as thnsf> ;iffpcting
cattle. TI1ey develop n1ost often on the nose and around
the lips of you11g horses, appearing as small, elevated, pap-
illary (horny) masscs. Thcy atso occur in t hc inncr aspccts
of ear (auraJ p laques). ·rhe causative virus is spread by di-
rect contact of infectious material through ivounds and
cutaneous abraslons. ·rhe virus ca11 be experilne11tally
h·ans1nittecl to horsf>s by intrarlermal inoc11lation of a sus-
pension of >vart tissue, but not to other ani1nal species.
Equine papillomas are usually self-limiting and disappear
spontancously in 4 to 8 wceks, although they can progress
to squamous cell carcinoma in rare instances. Natural in-
fection provides solid immunity.
Sarcoids are com1non skl11 tumors ofl1orscs tl1at grossly
a nd histologica lly resen1 ble fibropapi llom;:is of c;:ittlf>.
Interestingly, the genon1e o f bovine papillon1avirus (Types
1 or 2) is present \Nithin sorne of these tumors, whereas
that of cquine papillomavirus is not. Sarcoids havc been
reproduced by direct inoculation of bovine papillomavirus
into suscepti ble horses. The turnors range from being
largely epithelial in nature to ll1tcnsely flbroblastic, ancl
the histological diagnosis is dependent on df'monstra ting
both epithelial and n1esenchymal components to the
tu mor. Sarcoids are frequently multiple in affected horses,
and thcy comn1only are ulccratcd. Rccurrcncc aftcr surgi-
caJ removal is common, but metastasis has not been de-
scribed¡ thus t hey are not malignant tu mors despit e tl1eir
Jocally aggressive behavior.
(~a ninf> p;:ipillomavirus induces warts in the n1ouths of
dogs. ·rtie warts genera lly develop 011 the lips and spread to
the buccal mucosa, tongue, palate, and pharynx. Tl1e
\~'arts are usually bcnign and disappear spontancously
after severa! months. Uogs recovered from the infection
develop immunity to reinfection. The i.nfection is highly
contaglous, ofte11 spreadll1g tl1rougb aJJ tbe dogs in a ken-
nel. Wa rts have been experimentally tr;:insmittf'd hy n1h-
bing pieces of \<Vart tissue on scarified m ucous membranes
of susceptible dogs. Under such conditions the incubation
p eriod was from 4 to 6 wccks. Infectious vc11crcal papillo-
rnas (vvarts) also h ave been described in dogs.
POLYOMAVIRIDAE
Polyomaviruses have not been associated with diseases of
domcstic animal:; with thc notable exccption of un avian
polyon1avirus that causes an acute generalized infection in
fledgling budgerigars. Polyomaviruses are i1onenveloped
40 nm in d iameter, wlth an icosahe<.lral capsi<.I ar1<.l a
ge11ome of a single molec.ule of circ11lar <1011hlP.-stranded
DNA. The genon1e encodes at least tl1ree structural and
five nonstructural proteins.
 Adenoviridae
YUAN CHUNG ZEE
Adenoviruses have been isolated from many species of an-
.mals, but it is likcly thnt ndditionnl animal ndcnoviruscs
cxist that have not yet been identified . ·111e host range of
~•dividua! adenoviruses frequen tly is highly restricted.
.\lthough adenovirus Infectlons of animals are often
asymptomatic or subclinical, sorne adenoviruses are path-
ogenic a11d cause respira Lory and/or syslen1ic diseases.
~able 53.1 lists the diseascs of domcstic animals caused by
:tdenoviruscs.
The fam1Iy Adenoviridae is div1ded into two genera:
~Jastadenovirus, which includes adenoviruses that infect
m an1rnals, antl Aviadenuvirus, which includes aden-
o•iruse_~ th;it infect hircl~. Memhers of these t"''º genPra cio
1ot share a common group antigen. Adenovirus virions
.:re nonenveloped icosahedrons that are 70 run to 90 nm
.n diamctcr nnd nrc composcd of 252 capsomcrs (Fig 53.1).
E.xtended fibers project from the virion surface. ·rhe
;enome of adenoviruses is a Iarge molecule (26- 45 kilo
:,ases) of double-stranded DNA. ApproXimately 40 differ-
.::it proteins are encoded by the adenovirus genome.
:\tlenoviruses replicate i11 the uucleus uf ir1fectetl cells.
INFECTI OUS (ANINE HEPATITIS (CAN INE ADENOVIRUS 1)
Disease
:Ufectious canine hepatitis (TCH) is a disease of dogs
caused by canine adenovirus 1 (CAV-1). Although oncean
:mportant disease of dogs, ICH is increasingly rare in
:nuch of the world, perhaps as a result of widespread vacci-
::?.ation. The maio rity of infections are asymptomatic, but
tlle disease in susceptible dogs is characterized by fever, he-
patic necrosis, anti witlt:s¡.¡rt:atl l1t:rnurrhage as a cunse-
quence of vascular injury. Affected dogs may exhihit in-
creased thirst, anorexia, tonsillitis, petechial hemorrhages
on the mucous membranes, and diarrhea, and be reluctant
ro move. During the acute phase of illness, dogs may also
develop conjunctivitis and photophobia. Severe ICH is
:nost likely to occur in pups that are not immune to the
disea:st:.
Most dogs that survive acute JCH recover uneventfully,
out transient cornea! edema may occur in sorne convales-
..:ent animals after acute signs disappear. The CAV-1 also
h as been irnplicated as a cause of chronic progressive hep-
atitis and interstitial nephritis, but its role in spontaneous
occurrence of these disorders in dogs is highly conjectural
N. ] AMES M ACL ACHLAl'J
(CAV-1 is most unlikely to be a significant cause of either
of thcsc two common discascs of dogs) .
Etiologic Agent
Physical, Chernical, and Antigenic Properties
can1ne adenov1rus 1ype l lCAV -1 ) is antigenically related
but dístinct from canine adenovirus 2. Canin.e adenovirus
1 i:s 111urµli o logically ~irnllar tu uther atlenoviruses, but
antigenically distinct.
Resistance to Physical and Chemical Agents
CAV-1 is resistant to ether, alcohols, and chloroform. It is
stable for at least 30 minutes ata wide range of pH (3 to 9),
a11tl is alsu staule in solle<..l material at room temperature
for ' f'Vt>ral ci;iy~ . Vir;il inff'ctivity i~ lo~t ;iftf'r hf'!!ting for 10
minutes at 50 to úOºC. Steam cleaning and treatment with
iodine, phenol, sodium hydroxide, or lysol are effective
mcans of disinfcction.
lnfectivity for Other Species and Culture Systems
Canine adenovirus 1 causes clínica! disease in dogs and
oth.c r Canids (wolvcs, foxcs, and coyotes). Skunks ai1d
bears also are susceptible. Infection in foxes can manifest
ns cnccphnlitis. Cnninc ndcnovirus 1 rcplicntcs ;vcll in ca-
nine kidney cells.
Although sorne strains of CAV-1 and CAV-2 are onco-
genic in inoculated hamsters, these viruses have not been
associated with neoplastic disease in dogs.
Host-Virus Relationship
Distribution. Reservoir, and Transmission
Infectious canínc hepatitis has a worldwidc distribution,
although clinical disease is increasingty rare. ·rhe intection
is sprcad through the urine of infected dogs. Dogs may re
tain virus In thetr kldneys and shed it in urine for months
;iftf'r inff'rtion.
Pathogenesis and Pathology
Following aerosol infection, the virus localizes in the ton-
sils and spreads to regional lymph nodes and then to the
317
318 PART 111 Viruses

Table 53.1. Diseases of Domestic Animals Caused by

J\denoviruses F 1G U R E 5 3 . 1 - Ncg<1tivcly st<1incd prcp<1r<1tions of <1vi<1n <1dcn
ovirus. 204,000X. (Courtesy of R. Nordhausen.)
Virus Type of Disease

Mastadenovirus
Bovine adenovirus, Types 1-10
Canine adenovirus Type 1
(CAV-1; iní~tiou~ tdnin~
hepatitis)
Canine adenovirus Type 2
Equine adenovirus Types 1-2
Ovine adenoviruses Types 1-6
Porcine adenoviruses 1ypes 1-4
Deer adenovlrus
Aviadenovirus
Chicken adenoviruses Types 1-12 Respiratory disease, enteric disease,
Turkey adenoviruses Types 1-4
Conjunctivitis, pneumonia, diarrhea.
polyarthritis
Hemorrhagic and hepatic
Respir¡¡tory
Respiratory
Respiratory and enteric
Uiarrhea or meningoencephalitis, or
both
Systemic vasculitis, with hemorihage
and pulmonary edema
egg-drop syndrome, aplastic anemia,
atrophy of the bursa -0f ~abricius
Respiratory disease, enteritis, marble
spleen disease

Deer adenovirus, egg-drop syndrome viru) of poullly, bovine •de1111vi1U> 4, am! dtJ<..l
adenovtrus 1 are proposed to be membeB ot a new genus Atadenov1rus; simílarly,
hemorrhagic enteritis of l\l!keys virus and marbte spleen disease of pneasants ,;rus are
proposed to be membeB of a new oenus Siadenovirus.

systemic circulation. Viremia results in rapid dissemi11a-

tion of virus to nll body tissucs and sccrctions, including
saliva, ur1nc, and fcccs. Thc virus has a particular trop1sm
for hepatocytes and endothelial cells, which produces the
characteristic slgns uf tlle tlisease. Virus-incluceú injury to
énciothPl i;:i 1 <'Plls Jp;icl'\ to cnnsumptivP cnag11 lnpathy (<lis-
seminated intravascular coagulation) and a generalized
bleeding tenclency (hemorrhagic diathesis) that is re-
flected by abnormal clotting parameters.
Vogs that die during the acute phase generally have
edema and h emorrhage of superficial lyni.ph nodes and
cervical subcutaneous tlssue. Tt1e abclominal cavit y ofter1
cont;iins f111icl , which m::iy v;iry in color from c:Je;ir to
bright red. 1Ten1orrhages are present on all serosa! surfaces.
A fibrinous exudate may cover the liver, which can be
swollcn and congcstcd. Thc gall bladdcr chaiacteristically
is edematous. Large characterist1c intranuclear 1nclus1on
bodies may be prcscn t in hcpatocytes, vascular endothe-
lium, and macrophages.
Thc ocular IP<;inn<: th;:it <lPVPlop in sorne clogs that re-
cover fron1 JCI-J are the result of deposition of immune
complexcs within the ciliary body of the eye.
Host Response to lnfections
Recovery from ICH, rcgardless of the severity uf illr1t:!ss, rt:!-
sults i11 lo11g-lasting imm11nity th;it likely is life.long_
Recovercd a11ln1als have high titers of neutralizing anti-
body to CAV-1.
Laboratory Diagnosis
The d iagnosis of TC ll can be confirmed by serologic test-
ing (complement tixation, hemagglutination inl1ibition
enzyme-linked immunosorbent assay [ELISA]) to dernon-
strate rislng titcrs of antlbody to CAV-1, polymt:!rast:! chair'.
reaction (PC R) detection of viral nucleic acid or virus isola-
lio1 1 f1on1 afft::cled lissues, or in1111u11ol1istochen1ical stain-
ing of tissues with CAV-1-specific antibodies.
Treatment and Control
Therapy for dogs that develop ICH involves supporti\>'-
a11d sy1npto1nallc treatn1enL Control is by vaccinatio::
and strict sanitation of affected p remises '.vith quarantir.e
of exposed dogs. Availablc vaccincs includc both inacti-
vated and modilicd livc virus varieties, including CA\ --
vaccines that induce heterologous protection against CA\ -
l. CAV-2 vacclnes do not induce immune complex uveit:!:
in dogs as do so me other modified livc virus vaccinf>s. C;i·F
u1usl be t::xe1cised lo ensure that ni.aternal antibodies e_
not intcrfcrc with active immunization of pups, because
vaccination succcss is di rcctly rclatcd to the lcvel of n eu-
tralizing antibody.

- ~E ADENOVIRUS T YPE 2 tuchc111 ic<1l staining of infected tissucs. Adenovirus nu-
'1e adcnovirus ·rype 2 (CAV-2) has been isolated from
_ \vith acute cough and is one of severa! intectious
_:.-ts implicatcd in infcctious tracheobronchitis (kennel
-;'1J. 1::xperin1ental infection produces mild pharyngi-
: nsillitis, and trachcobronchitis, and virus persists in
_ =esplratory tract for up to 28 tlay:.. Uulike CAV-1, CAV-
...<s not ptoduce gf>nf>Ta\\1.E>ct cti~<'a~e. i~ not exc1eted in
. ..irine, and does not produce renal and ocular lesions.
-- ·-2 is antigenically related to CAV-1, and CAV-2 vac-
- ....., havc bccn dcvcloped as ICH vaccine since they do
- IlTOduce postvaccina1 ocular les1ons.
! J/IN E ADENOVIRUS ES
-.., bovine adcnoviruses (BAV) are <.."urrently classified into
~ serotypes (BAV 1- 10), of which BAV 3 anti BAV 5 ap-
..ir to be more patl1ogenic than the other serotypes. Ade-
iru:.e:s tyµically µruuuct:: µ11t::u111u11ia ur t::11teriti:> \Jut
-..y in vcry young or imn1unosupressed cattle. Adeno-
.:-uses are frcqucntly isolatcd from appurcntly normal cat-
e. and serologic surveys indicate that asymptomatic or
... bclinical BAV infection of cattle occurs worldwide. Bo-
_.,e adenovtrus 3 can produce mild pneumonia in suscep-
'-le calves, with necrosis of the epithelium lining termi-
.,J airways in the lung:;, cau:;iug necrutiziug \Jrunclüuliti:;
~th c:-haractPrist i< intran1111Par in1 l11 <:i()n h()cliPS in the af-
.cted epithelial cells.
ln1munocompetent calves inoculated with bovine ade-
--;~wcf o'crr/opneum?/@/,~f'c?.t7d~/C'f/n .?~ro.?#o'v/:J;
_-:?unity after natural infection is long-lasting. Uiag-
-~u\1e<E. v\ra\ \<;o\at\on ot <E.<!to\o'b)'. \'!.ov\n~ ad~no'»\
_,e, can be isolated from rectal, nasal, or con\unctiva\
..bs. Most typcs do not produce cbaracteristic cytopath-
: enic cffccts until after scveral bl\nd passagcs.
Although no vaccines are li<Pn<:P<I fnr 11sP in the TJnited
5ta tes, there is JinJited use of vacciI1es against BAV 1, 3, and
~ in Europe.
t QUIN E ADENOV IRUS
•denovirus infection rarely results in respiratory tract dis-
ase In hcalthy horses, and urununocomperent foals nor-
-i;i!ly dcvclop cither subclinical or asymptomatic infec-
:!Ons. Howcver, Arabia11 foals wil h sevcre con1bined
.:nmunodcficiency (SClD) are highl y susceptible to infec-
~on \vith cquinc adcnovirus l. Thc rcspiratory disease in-
~uced by cqui ne adenovirus in SCll) Arabian toals is pro-
:::acted and characterized by coughing, dyspnea, and fever.
riese foals also develop generalized adenovirus infections,
ith involve1nent of a variety of organs and tissues.
'I'Jiere are twu :;crutyµc:. uf cquiuc <1uenovirus, as deter-
mined by serum neutralization. For laboratory diagnosis,
irus may be isolatcd from infcclivc tissue and nasal and
.cu lar swab material in equine letal kidney or equine fetal
....ermis cell cultures. Thc virus can be identified by electron
Chapter 53 Adenoviridac 319
microscopy or by immunofluoresccnt (JF) nr imm11nohi<:-
c:leic acid also can be detccted by PCR assay. Viral neutral-
ization and hemagglutination-inhibition test are uscd to
detect the presence and rise ot antibody titers for serologi-
cal diagnosis of adenovirus infections.
There is no commercially avallable vaccine.
Ü VINE ADENO VIRUSES
Adenoviruses have been isolated fro m thc fcccs of appar-
ently normal sl1ccp and from \ambs with rcspiratory dis-
ease. Six serotypes have becn identiticd. ·rhe pathogenic
role of 1nost of the ovine adenoviruses is uncertain because
tl1ey typ'l cally produce only mild or tnapparent infection of
the respiratory or gastrointestinal tracts. However, appar-
en t outb1eaks of adenovirus-i11duced pneun1011ia a11d en-
teritis have been described in lambs, as have sporadic cases
of generalized (systemic) infections of very young lambs .
DE ER AD ENOVIRUS
A unlque adenovirus recently was ldentified as the cause of
a fatal systemic hemorrhagic disease of mule deer (includ-
ing black-lailed <leer). ·r11e vi1us 0 1igi nal ly was idenlified as
thc' c;i 11s<' nf PXtPnsivP 011thrPilk<: ()f f;:ital disease in rnule
deer in North A111erica (Califo1nia), bu l s ubsequently ha:.
been recognized to havc a much wider distribution. A sim-
ilar discnsc hns ruso bccn idcntifi cd in moose. TJ1e causa-
tive aáenovirus is geneác:a«y unique and is sero(og{caffy
relatcd to sorne adenoviruses of cattle (BAV) and goats. lt is
µro-pose<l t'nat <lee.r aue.nov\1us 'be \nc\u<.ic<l m a ne.w ~,en.us
Atndcnovirus, along "'' ith BA.V 4, duck adcnovirus l, and
ccrtaln othcr adenoviruscs. ihc virus causes either local-
i7ed or systemic vascular injury as a consequence of infcc-
tion of endothelial cells and subsequent thron1bosis, lead-
ing to severe pulmonary eden1a, ulceratio n of the o ral
cavity and gastrointestinal tract, and widcsp rcad hcmor-
rhagcs. The vir us can be propagated in p ri ma ry deer respi-
ratory epithelial cell cultures, but diagnosis is based on the
characterlstic gross and histologlc Iesions, immunohis-
tochcmical staining with antisera to B.A.V, and electron
microscopy.
AVIAN ADENOVI RUSES
Adenoviruses infect poultry and other bird species world-
wide. Although adenoviruses are oftcn isolated from ap-
parcntly normal birds, spccific discascs also are associatcd
with adc novirus infections. 'J'hese include egg-drop syn-
drome, a disease of both wild and domestic birds that is
characterized by production of cggs that lack shells or have
;ihn()rmally soft shells, and hemorrhagic enteritis of
turkeys and marble-spleen discasc of pheasanl:>, whicl! ar1;:
similar diseases characterized by intestinal hemorrhage
and cnlurgcmcnt of thc spleen of affected birds.
 H erpesviridae
ALEX A. ARDANS
Thc family Hcrpcsviridac consists of viruses that have
been isolated frorn a wide ra11ge of animal species, hu-
mans, fish, and invertebrates sucl1 as oysters. Herpesvi-
ruses !1ave been found In vJrtually every species that has
been investigated . Witb.in tbis group of vin1si>s, thi>ri> is
wide variatio11 il1 biological properties including patho-
ge11icity, a propensity to form latent infectio11s, a11d 011-
cogcnic potcntial. Hcrpcsviruscs are morphologically
sin1ilar, with a double-stranded DNA core andan icosahe-
dral capsid consisting of 162 capsomeres, surrounded by
a granular zo11e composed of globular protelns (tegu-
ment) and encompassed by a lipid envelope (Fig 54.1) .
Tl1e ge11vu1e oí lJerpesviruses is large, 125-135 kilobases,
and encodes many clifferent proteins; functions of the
proteins encoded by the viral genon1e includc virus rcpli-
cation, virus structural proteins, anda variety ot proteins
that regulate cell growth and i11odulate tl1e host's antivi-
ral response.
·r11e fa1nily Herpesviridae co11sists of three n1ajor subfam-
ilit::., dl !.Jhd- 1 l.Jt Ld- 1 d llU ¡)dlUUJdlt t:l )-'t::> vil i 11 dt:, vv lii t. l 1 vvt: 1e
initially distinguished by host range, duratioo of repro-
ductivc cyclc, cytopathology, und lutcnt infcction churac-
teristics. ·rhe alphaherpesvirinae have a variably-restricted
host range, are generally 11ighly cytopathic in cell culture,
have a relatively short replicative cycle (24 hours), and fre-
quently cause la tent viral infections ir1 sensory ganglia .
l3elal1er pesvlrinae 11ave a variable l1ost range and a long
replicative cycle; infected cells often become enlarged (cy-
tomcglia, thus thcir dcsignation as cytomcguloviruscs).
Latency can be established in numerous tissues. Ga1nma-
herpesvirinae, with sorne exceptions, tend to be tropic fo r
B or T lymphocytes (lyrnphotropic), replicate in lympho-
blastoid cells, and 1nay cause lytic infections ln certaln
type~ of epitl1elial a1H.l filJrulJla!>tic cell:;. luit::cl.io11 is [re-
q11<>ntly arreste<l at a prclytic stage with persistent and
minimu1n expression o f viral genome in the cell. Latency
frequently is established in lymphoid tissue. Host range is
narrow with experimental 11osts usually lln1ited to the
order of tI1e natural host.
EQUINE HERPESVIRUSES
Disease
There are nine proposed equine herpesviruses, five th.;:it in-
[ecl horses (EHV 1 lhrough 5), lhree tl1at infect do11keys
320
(EHV 6 8; asinine herpesvlruses 1-3), and one that likely is
l1eríved from zebras (EHV-9). EHV-1, EHV-3, EHV-4, EHV-6
(asinine herpesvirus 1), EHV-8 (asinine herpesvirus 3), and
EHV-9 are typical alphal1erve::;viruses ancl EHV-Z, EHV-5
and EHV-7 (asinine herpesvirus 2) are classified as gan1ma-
l1erpesviruses. EHV-1 has been associatcd with abortions,
upper respiratory disease, and neurologic disease. EH V-~
has been isolated from cases of chronic pharyngitis, mild
respiratory disease in young horses, severe respiratory dis-
ease in foal s 3 to 4 months old, and from clinically norma:
horses, and its pathogenic sig1lifica11ce re1uai11s uncerlai1..
Venereal. disease (coital <>xanthe1na) is a well-recognizerl
clinical n1a11ifestation of EIIV-3. EIIV-4 sharcs a close anti-
genic relationship with EHV-1 and has been associatee
vvith rcspiratory discase and sporadic abortions. EHV-;1
like EHV-2, has been isolated from horses with upper re-
spiratory diseases, but its pathogenic sig11ificance still is
u.ncertain.
Etiologic Agent
Physical, Chemical, and Antigen Properties
·rhe equine herpesviruses have typical morpholo~ry anc
cannot be distinguished from each other based on thei:
morphology. They are, however, antigenically distinct aI:c
can be distinguishi>cl hy s<>rological assays.
Resistance to Physical and Chemical Agents
Thc equine herpesviruses are inactivated by etl1er, aéc.
(pH 3), and exposure to heat of s6•c for 30 minutes.
lnfectivity for Other Species and Culture Systems
Equine herpesviruses 1-5 usually infect only horses
Ocular disease dueto EHV-1 chuructcrizcd by vitritis, re-
tinitis, and optic neuritis leading to l)lindness has been ct-
scribed in new-world camelids (alpacas, llamas). Limite::.
serologic surveys have not demonstrated widespread Jnfe.:-
tion in these species. EHV-1 can be propagated in equú:
fetal kid11ey, rabbil kidney, a11d L (n1ouse fibroblast) cefu
resulting in formation of cytopathic effect and intra:r:~
clear inclusio11 bodics.
 (;hapter S4 Herpesviridae 321

F 1G U RE 5 4. 1 . Negatívely staíned preparatíon of infectíous bovine rhínotracheitis

virus. n = nuc/eocapsid, ev = enve/ope, rv = enve/oped virus, tn = twin nuc/eocapsids.
=
17,000X. lnset: Minute projections on the enve/ope of a matured virus. sp virus spikes,
cd = virus core, ve =virus envelope. 100,000X. (Reproduced with permission from
Tatens LT, Zee YC. Purification and buoyant density of infectious bovine rhinotracheitis
virus. Proc Exp Biol Med 1976;151:132.)

m--P.
n
~

EQUINE HERPESVIRUS-1 Pathogenesis and Pathology

Di sea se
Equine herpesvirus-1 (EHV-1) causes abortion in mares,
respiratory tract disease in young 11orses, and occasio11al
n eurologic disease. Wl1ile abortion in 111arcs n1ay occur as
early as 4 months of gestation, it n1ost frequently occurs
between the seventh and eleventh months of gestation
and usually without any premonitory signs. roals infected
in i.1tero may be born alive but are usually weak and die
within z to :~ aays. Mye1oencepnalitis with signs of ataxia
and posterior paresis has been associated with certain
slrains of cHV-1.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
EHV- 1 is prevalent in horscs worldwide. The vi rus appears
to be maintaincd in thc horsc, but it i.s possiblc that dogs,
foxes, and carrion birds rnay carry infection with frag-
m en ts of aborted fetuses from one farm to another.
Respiratory disease is transmitted by droplet infection. In
a<1<1ition, transmission may also occur by direct contact
with virus-laden aborted fetuses or placentas. Tl1e annual
occurrence of respiratory disease in young 11orses suggests
the existence of carriers and latent infecti ons.
1'he pathogenesis of EHV-1 abortions remains an enign1a.
Viremia in the presence of neutralizing antibodies has
been detected in experimentally induced abortions and
virus cultured from the buffy coat, but not from the cell-
free plasma or from washed red-cell suspensions. 'l'he cell-
associated virus may escape neutralization by ar1tibolly,
h11t the. me.c.h<inism hywhic.h the. virus re.<ic.he.s the. fe.tus is
unknown.
Macroscopically, the inost prominent lesions of the fetus
are jaund.icc, n1ucous mcmbranc pctcchiation, subcutanc-
ous and pleural edema, splenic enlargement with promi-
nent lymphoid follicles, and focal hepatic necrosis. Histo-
logicalJy there Is bronchlolitis, pneumonltls, severe ne<.1osls
of the splenic wbite pulp, and focal hepatic necrosis, all ac-
con1parlied by intrar1uclear inclusion bodies. The early fetus
( <3 months) shows little or no response to the viral infec-
tion. Howcvcr, thc fctus in its last 4 months of gcstation
shows a marked ability to react in a specific manner to the
presence of the virus. Lesions in the fetus less than 7
months of age differ from those in older fetuses, suggesting
that the lesions represent a fetal response to the virus.
Host Responses to lnfection
Experiu1e11tally, within 24 hours after exposure to EHV-1,
foals an<l mare.s <lemonstriltf> ;i IP11kopPni;i nf ?.4 to 48
hours' duration. Virus persists in circulating leukocytes for
322 PART IJJ Viruses
as long as 9 days following infection. Abortion in mares
may occur as long as 90 to 120 <.lay:> fulluwu1g iiút'<.tiu1i.
Roth complPmf'nt-fixing anct vi rus-neutralizing anti-
bodies appear in tl1e sera of infcctcd hor:;cs. In general,
complcmcnt-fixi11g antibodies are den1onstrable for 6
months <.lftcr i11fcction, with virus neutralizing antibodies
persisting longer. lgG ant1bod1es aga1 nst the viral envelope
neutralize virus, wlúle those against the nucleocapsid
do not. Experlmentally pregnant mares with antibully
to EHV-1 may abort when ch;1llPngi>cl with EHV-1. ·rhe
lesions fro1n which virus cannot be isolated suggest an
antigcn-antibody complex pathogenesis.
Laboratory Diagnosis
In case of abortions, diagnosis is based on cbaractP.ristic li>-
siuu~ wil li i 11 l1a1 1uclear inclusions prcscn ti 11 the fetal liver,
splccn, lung, and thymus. ·rhe same tissues provide a good
sourcc of virus, v.1hich can be demonstrated by irnmuno-
tluorescence or immunohistochemical staining of tissue
sections or by virus isolation. The virus is usually relatively
easy to isolate. ·rhc mares' sera may, but does not invari-
ably, demonstrate a rise in titer. Diagnosis of EHV-1-
lnduced myeluenct'pllaliti~ i:> liifficull a11le11101 len1 becausc
~Prology may hP c.onfusing and EHV-1 is rarely isolatecL
T!i.:1topnthologic11lly thcrc is (1 v(13C\.1Jiti3 prc::;cnt nl::;o ::;ug-
gesting an a ntigcn-antil,ody complex mechan1sm. On sev-
era! occasio11s, n1yeloencephalitis has occurred in North.
American (California) horses exposect to mules. With aspe-
cific enzyme-linked imm unosorbent assay (ELlSr'\.) i1ow
avatlable, tt is possible to <.listinguish .EHV-1 fru1u EHV-4
sernlogic;illy, anct a polymPrasP chain rPaction (P~R) assay
has been described far the diagnosis of CI IV-1 abortion.
Treatment and Control
Llmltlng traffic in and out of brood-n1are 1.Ja11ll:; a1i<.l wt'au-
ling fields ¡¡nd n1inirnizing stri>ss to prPgnant rnares have
bccn suggested to help prevent abortion d i:scasc. Vaccincs,
although widely used, havc not bccn cornpletely effective
in clinúnating abortion loss. J\ hamster adapted live viral
vaccme that was fully capable of causing disease in young
animals and pregnant mares is no longer used. A modificd
live viral vaccme of cell culture origin, altl1uugh ca paule uf
evoking antibody responsi>s, has not provPn effective. An
inaclivaled cell culture vaccine, given at the fifth, seventh,
and ninth months of gestation, was shown to be effective
in ficld trials, but abortions have been seen in mares vacci
natect with tl1is product. More recently, combined EHV-
1/EHV-4 inactivatcd vaccines are being used.
EQ UINE HERPESVIRUS-2
Disease
Equlne herpesvirus-2 (EHV-2) Is prevalent in l1ursc~ wurltl-
widc and has been isolatcd from hoth clinically healthy
and ill horses. It has bcen isolated from cases of superficial
a11tl c1Jru11ic .IJilary11geal ly111phoid follicular hyperplasia,
mild respiratory disease in young anirnals, and foal pneu-
monia and from foals with kcratoco njunctivitis. 1' hcrc is
considerable skcpticism by sorne concerning its role in
diseasc.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
EHV-2 has been isolated trom borscs worldwide. Hoth
"hcalthy" and dinically ill horses act as a reservoir far the
virus. 111 ponies experirnentally lnoculated with EHV-2,
thc virus was recovered up to 118 days postinoculation
EHV-2 has been associatt'd witli :>ig11~ ~,r upper respiralory
t r;:ict di~f'i!Sf', with thf' virus pe rsisting for 418 days.
Pathogenesis and Pathology
Little i~ k11uw11 aboul lhe palhogenesis of EllV-2. Nincty-
seven percent of horsPs IP~s than 1 year old have antibod-
ies lo EHV-2, and the virus could be isolated from the
leukocytes of 88.7% of normal horscs. Similarly, virus was
isolated from 68 of 69 fonls samplcd once ben,•een 1 and 8
months ot agc. lt is suggested that 1nfcct1on is initiated in
tonsils, with replica tion in other sitcs based on an ob-
served vlremi.a and it.s cell-associatt'tl uCJlure.
Laboratory Diagnosis
·rhPrP arP no specific diagnostic features associated with
Cl!V-2 infection. The virus can be isolated from nasal and
pharyngeal swabs and trom blood butty coats ancl iclenti-
fied by PCR assay.
Treatment and Control
Cuotrul 111etliuds l1ave 11ol beei1 eslablishcd, anda vaccine
is not available for EHV-2.
EQ UJNE HERPESV IRUS- 3 ( EQUINE COITAL EXANT HEMA)
Disease
Equine cuita! t'XCJI1tl1e111a (ECE) is a11 acute, :;exuall;-
transm ittPd disease characterized by the forrnation o :
papules, vcsicles, pustules, and ulccrs on thc pcnis anc'..
prepuce of stallions and on the externa! genitalia anc'.
perineal skin of tnarcs. Thc lcsions usually heal after ap
proximately 14 days, leaving dcp1~mented patches or
the vulva and erosions and ulcers on the prepuce a r.e.
penis of the male. Leslons bave uccurretl, ilúreguenll.
around the lips, extern<il narPs, nasal mucosa, and co~­
juncUva.
A unique characteristic of equinc herpesvirus-3 (EH \'-j

is its inability to grov.r in cell cultures other than those of
~uine origin.
J istribution, Reservoir. and Transmission
EHV-3 wns first isolatcd in 1968 concurrently iI1 North
America and Australia. The only known reservoir for the
•irus is the horse and the usual mode of transmission is
>enereal, but EHV-3 can be spread without coitus. 1'he
n.1lva and vagina need not be damaged far infection to
occur. The ü1fectiol11uay also spread via fonlites, or insects
may actas mechanical carüers. It is suggested t hat in sorne
infected stallions and mares, EHV-3 becomes latent in the
nonbreeding season an.d is reactivated during t he breeding
~ason.
í'athogenesis and Pathology
Li ttle is known about the disease pathogenesis. but virus
and an antibody increase can usually be demonstrated
during development of the lesions. Perivascular and. peri-
glandular lymphocytic accumulations suggest that an
i111111u11e-1nedialed reacLiou 1uay play a role i11 palltuge11e:;i:;.
Microscopic examination ofvulvar tissue demonstrates
sballovv erosions along with occasional typical intranu-
clear inclusions scattered in germinal epithelium or in nu-
clear remnants in necrotic areas.
Host Responses to lnfection
~ n ti horl i f>S 11sn;illy ;ippP;ir <lnring thf> flC11tf> st;:¡gp of thf>
disease.
Laboratory Diagnosis
Clinical signs, serologic tests, histopathologic exa1nina-
tion, and viral isolation can be used in diagnosing ECE.
Treatment and Control
There i:; uu EHV-3 vaccilH:: availal>le. Treat1ue11t:; iut:lutle
use of topical ointmeots to reduce pain and sexual rest for
at lcast 3 wccks.
f QUINE HERPESVIRUS-4 (fQUINE RHINOPNEUMONITIS)
Clinical manifestations of equine herpesvirus-4 (EHV-4) are
seen principally in foals and younger horses, with a seasonal
peak of disease in fall a11d winler. Signs oflen L11clude
malaise and elevated temperature up to 105ºF, which may
pcrsist for 2 to 5 days; watery nasal discharge, which be-
comes mucopurulent in the Jater stages; congested conjunc-
tiva; and, infrequently, enlarged submandibular nodes.
'I'he agent appears to have worldwide distribution; how-
ever, it is infrequently isolated. Both inactivated and mod-
ified live vaccines are used bul resulls are noL u11jfo11uly
favorable.
Chapter 54 Herpesviridae 323
EQ UINE HERPESVIRUS-5
In a study of multiplc cquinc hcrpcsvirus-5 (EHV-5) iso-
lates, severa! were íou11d to diíter signiticantly genomically
and in their protein co1nposition. EHV-5 has been pro-
posed for this group of Viruses that were isolated from
eql.line respiratory tracts. The pathogenic significance of
EHV-5 has nol been adequaLely defined.
RUMIN AN T HERPES VIRUSES
Herpes viruses, representing alpha- and gammaher
pesviruses, are responsible for a wide range of conditions
in rumina11ts, including neurologic, genital, fet al, and
res pi raLory ui:;ea:;e:;. Iufet:tiuu:; 111ay range frorn inap-
parent to fatal. Severa! herpes v iruses infe<.t <.flt t le
(bovine herpes viruses [13IIVJ): infectious bovine rhino-
tracl1eitis virus (BHV-1), bovine mammilitis virus (BHV-
2), and bovine encephalitis virus (BHV-5) are alphaher
pesviruses. BHV-1 causes both respiratory (infectious
bovine rhinotracheitis) and genital d isease (infectious
pustular vulvovagiliitls) in catlle. BHV-4 is a gan1n1aher-
pesvirus, but its role in causing disease is uncertain. ·r,-vo
gammahcrpcsviruscs th.at prcviously wcrc designated as
BHV-3 have now been named .1\lcelaphine herpesvirus
(AlHV): AlHV-1 is the cause of African malignant ca-
tarrhal fever (MCF) associated with wildebeest and AIHV-
2 is the cause of atypical MCF associated with the harte-
beesl. There are Lwo sheep gan1n1aheJpes viruses (ovine
l1erpes viruses [OHVl 1, 2): OHV-1 is associated w ith p ul-
monary adenomatosis, and OHV-2 is the cause of the
sheep-associated torm oí MCf. Alphaviruses also have
been isolated from goats (CpHV 1) and deer (CerHV 1
and CerHV-2), and the CpHV- 1 is the cause of reproduc-
tive disease in goats.
BOVINE HERPESVIRUSES
BovtNE HERPESVJRUS -1
Disease
Bovinc hcrpcsvirus-1 (BHV-1) infcction in cattle may pres-
ent as ocular, genital, respiratory, and, intrequently, neu-
rologic disease. Respiratory disease typically presents as
rhinotracheitis (infectious bovine rhinotracheitiS [IBR]),
which may lead to severe and often fatal bronchopneumo-
nia. Conjunclivilis is conHno11. Occa:;iu11ally the <.:ur11ea iS
involved anda panophthalmitis 1nay occur. BHV-1 ca11 in-
fect genitalia, resultir1g in abortion, balanoposthiti s, and
vulvovaginitis (lntectious pustular vulvovaginitis llP\lJ).
Meningoencephalitis in young calves occurs infrequently
with BHV-1, and herpes virus isolates from cattle with
neurologic disease are most commonJy designated BHV-5.
BHV-1 has bee11 isolaLed fruu1 ve:;lcular lesiuns un the
udder and teats of a cow.
324 P ART III Viruses
Etiologic Agent
lnfectivity for Other Species and Culture Systems
Altho ugh cattle appear to be thc major species affected by
BHV-1, the virus has been lncriminatcd in swine vaginitis
and balanitis, itTHl has beell i::.UléJtCU ÍfUlll ::.lill1Ju.r11 élltd
n<>whnrn pig<;. Apprnximiltf'ly 11 OA1 of swi ne tested in parts
of North An1erica (Io'" 'ª and ·1exas) had antibodies to TilR.
The virus has been isolated from red deer with ocular dis-
casc, and could be activated in Malaysian buffalo by
steroid administration. It docs not appear that BHV-1 virus
is a signiflcan t pathogen of goats. Only 3o/o of 1,146 serun1
samples of captive ruminants in Unlte<l States zuus 11a<l a11-
tibodies to lBR virus.
BIIV-1 virus can be grown in a wide variety of cells, in-
cluding bovine, cani11e, fclinc, equine, ovine, rabbit, mon-
key, and hun1an, whcrc it produces a cl1uractcristic cyto
pathic ettect.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
fhc u1sease occurs \"70rldwidc. During the late 1950s, an
apparently i<lentical virus to IBR virus was isolated from
tnfcctlous pustular vulvuv<1gi11ilb (TPV).
Tt h::is ht>f'n <;11gge.sted t hat wildli fe may play a role in dis-
case transn1ission, but in ligh.t of demonstrated viral re-
crudescer1ce, cattle must be considered the primary reser-
voir. Virus is transmitted by respiratory, genital, and
conjuncttval sccret1ons of infected cattle.
Pathogenesis and Pathology
Thc virus replicates in the upper respiratory tract and
spreads via the lacrimal ducts. Virus can be recovered from
nasal secretions for alrnost 2 wccks following infection.
Although viremia is difficult to demonstrate, experimen-
tal infe<.:tiur1s 11av1:: yiel<leu vi ru:. ÍHHU va1ious orga11s, per-
h::tps as a conscquence of a leukocyte-associated viremia.
Genital infections are most likcly venercally transmit-
tcd. Lcsions consisting ot postules and later fibronecrotic
plaques are usualiy lunitcd to 1h e vulva and posterior
vagina 1n the female. Similar lesions are seen on the pre-
puce of affected bulis. Rcspiratory disease has been pro-
duced with genit<1l isulatc~. a11u ge11ilal lesio11s have been
prnc111cf'd \'Vith rf'_<;piratory isolates. Viral shedding "''ªS
observcd in cows 14 <lays after experimental genital infcc-
tion and in males up to 19 days tollowing infection. Virus
was she<l following prednisolonc treatinent in these cattle
2 and 7 months, respectively, fo ll owing initial lnfectlon.
'fhcsc observations suggest tl1at the virus may be rnain-
talncd by perioc..lic she<ltlir1g wl!cu <.111i111als aJe subjecled
to stress. Although the frcquency is low, occasional bulls
a re encountered that shed BHV-1 intcrmittcntly in semen.
lnscmination ot cattle with such semen may result in en-
dornetritis, reduced conception, and shortened estrus pe-
riods. Experimentally, semen lsolares can induce sevcre
rhinotrachcitis and vulvovaginitis in c;ittlf'. N;ih1ral out-
brca.ks of simultaneous respiratory and genital ~
are rare.
Abortion is often sccn in prcgnant c;ittlt> with TRR r-p
casio11ally followü1g vaccination with n1odified Jive ,-:.-
vaccine. Tl1e il1cubation period between infection of
da1n and fetal dcath varíes from 15 to 60 days. Sincc ,
dea th occurs severa! days befo re abortion, the fetus is o.
severely autolyzed. Fetal edema, especially of the ;-.,.
membranes, occurs along Wlth cxtensive hemorrhz_
edema in the peri.renal tissue F.xtPnsiv<> hf'mnrrh
111:c1osis of ll1e ren.al cortcx i.s scen along with a foca! ::-
crosis in the liver and usually in the lymph nades. !>c,-
nccrosls n1ay be observed in placentomes, which are u
ally good sources of vi rus for isolat1on attempts. Nec -
lesions occur in ovaries of cattle cxperimentally infec _
by nongenital routes.
Conjunctivitis is common in RT-fV-1 inff'cti~­
Ty1-1il.:ally il p1esen ls wilh profu:;e lacrimat ion a11d occ_
sionally extends into thc cornea, resulting in a keratitis.
sorne cuttlc, ¡¡ inultifocal lymphoid hyperplasia ma~
seen in the palpebral conjunctiva.
Meningoencephalitis has been recorded in natural a:- _
experimental dlscase. Histopélthulugic lesious i11c1 __
those of a nonsuppurative meningoencephalitis, neuron
necrosis, focal malélcia, autl uíle11 i11 l1anudear ü1clusion _
astrocytp<; and nf'urones associated with lesions.
Latent infection.s 11avc been de monstrated in thc tri-:.
minal ganglia of clinically normal cattle. Kecrudescer
shcdding of virus has been observed naturally and in : e
spon.se to corticosteroid adtninistration.
Host Response to lnfection
The i1un1u11e respo11sc Lo BHV-1 involves many factors !::-
addition to the stimulation of neutralizing antibodie--
most of which are dircctcd toward surface glycoprotein_
lg(j and lgM antibodies appear I days follo'-vi.ng exposur~
The lgG response during this primary phase is restrictec.
primarily to the IgGl subclass. Scconctary respun.ses set..,
mainly in the IgG class are due to incrf'il<;f' in Tgc;.2 anr-
body. Seco11dary int ranasal exposure did not produceª'"'
increase in IgM antibody, whereas an increase in IgM le\··
cls was sccn following nbortio n. The neutralization of ex
tracellular virus prevent s extracellular spread of viru
stressing the importance of an tibody at mucosa! surfaces
Antibody can also play a role in the cu111µleweol- 111ediat~
lysis of infected cells an<I in ilntihody dcpendent-cell cyto-
Loxiclly. TI1e induction of cytokines can activate effecto:
cells that <lestroy infected cclls directly or through anti-
body intcraction. ·rhcsc actions occur in concert with each
other, n1aklng it dífficult to ascribe a lcvcl of in1portance te
each. Por vaccine development, it is important that con-
Slderallon be given to él protlu\:t':> alJiliLy lo boll1 induce
antibody and ceJl-mPrli;itpcl immunity.
Laboratory Diagnosis
·rhe fibrinonccrotic plaques commonly prP<;f'nt in the. f'X-
ter11al 11élrc::. autl u11 Lhe nasal scplun1 of cattle with infcc-
tious hovine rhinotracheitis are good sources of material

:Or viral isolation. The conjunctival form can be tent a-
tively diagnosed by ll1e observation of nlul tifocal white le-
sio11s in the palpebral conjunctiva. In their abse11ce, viral
isolati.on is nccdcd. Thc virus can be rcadily isolatcd from.
:he conjunctiva! swabs. Abortion may be (1iíticult to diag-
nose as the fetus is often presented in an autolysed condi-
:ion. Tf placenta is available and relatively fresh, isolation
a ttempts can be macle from the placentomes. While im-
rnu11ofluoresce11l slai11ü1g lecl1niques 11ave been used 011
~tal tissue '"'ith varying degrees of success, immu11operox-
idasc staining that has bccn positivc in IFA ncgativc tissucs
h as improved abortion diagnosis associated with HHV- 1.
Diagnosis based on serology may be difficult because ani-
m als often have high titers at tlle time of abortiOn, regard-
-ess of cause, making it difficult to demonstrate rising
:iters. Detection of BHV-1 ü1 sen1en is difficult by c.onven-
tional methods. A PCR-based technique has been used to
detect .BHV-1 in semen.
Treatment and Control
•.\ 1nodified vaccine used intramuscularly was associated
·v-it.h reduction in disease but could cause abortiot1 in preg-
uaut cdttle. Au iI1trC:1nC:1sC:1! vaccine was shc.n-vn to be safe for
use in r.attle. This var.cine r.ontaining a tPmpPratHre-
sen sitive n1ula11l virus lhal will replicale only al lhe lowt:r
remperature found only in the upper air\\>ays of cattle is
? romotcd as snfc and cffcctivc for use in prcgnant animal~>.
Corticosteroid treatment of anirnals vaccinatecl \-vith mod-
L'led Jive vaccines has resulted in recrudesence of virus.
9 espite these uncertain t ies, modtfied live BHV-1 vaccines
are widely used because of the potentially heavy economic
1oss resulling f10111 infeclion.
Inactivated vaccines have not heen consistently effica-
cious. Subunit vaccines have been suggested as an alter-
native to modified live vaccines, as have gene-deletion
:nutan.ts.
Eradication
Countries >vith relatively small cattle populations have
used serology to guide eradication efforts¡ hovvever, co1n-
:non prattices in larger countries prevent thi.s approach.
·"ppropriatc scrology uscd \-vith genctically cngincercd
-.-acc1nes may nold prom1se.
BOVINE HERPESVIRU S-2 {BOVINE MAMMILLITIS)
Bovine herpesvirus-2 (RHV-2) has hef'n isol;ited from cat-
rle with generalized skin disease (pseudolun1py skin dis-
ease), mamin.illitis, and stomatitis.
BHV 2 will replicate in a wide tange of cells, but bovinc
:.;1dney cell culture is most widely used. c::attle appear to be
p rimari.ly illfected, with mild experimenta.! disease pro-
d uceu i11 sl1eep, goats, C:111d pigs.
BHV-2 has heen isolaterl from rattlf' skin and 1nucosal
~nfection in the ·united States, Africa, Europe, and Auslra-
:ia. Ori¡,'inally virus >.vas isolated from South African cattle
Chapter S4 Herpesviridae 325
\.Yith genera1ized skin disease, subsequently termed pseudo-
lurrzpy skin disease. Man1n1illilis due Lo BrIV-2 was clescribed
ill Africa and England, and subsequently in the United
States. Stomatitis in bovine and buffalo calves has been de-
scribed in association with calves nursing co\-vS wit h mam-
millitis. Suggested modes of infection include transmission
at milking, by insects, o r activation of Jatent virus.
Intravenous exposure produces generalized skin le-
sions, which a1e cl1araclerizet.l 1.Jy a severe iI1tercellular
edema in the epidermis along with syncytia with int ranu-
clear inclusions. An epidermal mononuclear cell and neu-
t rophll in1iltrate is present along wit h mononuclear and
lymphocytic dermal perivascular infiltration.
The diagnosis of pseudolumpy skin disease and mam-
mill itis can be based on clinical signs and viral isolation in
cell cullu1e. Serulugy uu paired sau1ples will de1nonstrate
a11 increase in antibody.
BOVINE HERPESVIRUS-4
Bovine herpesvirus-4 (BHV-4) consists of a group ofviruses
that have bee.n isolated from diffcrcnt clinical syndro1nes
and normal cattle. Their importance as pathogens is un-
clear. ()nly strain DN-599 has been reported to produce
conjunctivitis ancl respiratory disease. Viruses related to.
this group have been repeatedly .i solated from cases of
n1elrilis ü1 son1e Nor Lh A1nerica11 (Cali fornia) cattle and
are suspected of causing vaginitis in heifers. 'fhe North
American a.nd Europcan strains appcar to be closely re-
lated. Latency has been suggested for this group as tbere
appears to be reactivation in response to other inflamma-
tory processes.
BOVINE HERPESVIRUS·5
Nonsuppurative meningoencephalitls has been assoclat.ed
with hovinf' hPrpPsvin1s-.5 (RHV-.5) infection of cattl.e. Iso-
latcs have been obtained in Argentina, Australia, lfungary,
Ja pan, and the United Sta tes from cattle with sporadic cases
of encephalitis. BHV-5 strongly cross-rcacts \vith BHV-1
an<i currently the two cannot be distinguished serologically
but can be differentiated by their restriction en.donuclease
parrerns. It appears that BttV-5 nas neen circulating in cat-
tle for at least 20 years, witl1 only sporadic disease occur-
rences. Mortality is usually reporled Lo be 100%. Lesious
vary as to severity but usually co11sist of pertvascular infil-
trates throughout thc brain, witl1 neuronal necrosis, dis-
ruption of the neuropil, and gliosis. Experimental infec-
tions produce similar sign.s and lesions as those naturaJJy
occurring and BHV-5 can be reisolated from the brain.
ALCEPHALINE HERPESVIRUS · 1 ANO -2 {MALIGNANT
(ATARRHAL f EVER)
Malig11aul catarrl1al fever (MCF), an often fatal infection
of many species of hovi<iaf' :incl cervidae, occurs as t'"'' º
326 PART lll Viruses
epidemiologically distinct entities: wildebeest-associated
a11J :>l1ee.[J-a:>:>uciatetl MCF. Alct!.[Jlialiue l1erpesvirus-l
(AlHV-1) is responsihle for '"'ildeheest-derived (or Afri-
can) MCP, which occurs when cattle and wildebeest graze
together. A virus isolated from clinical MCF in cat tle in
North America (Minnesot a) is closely related to J\JHV-1.
An etiologic agent for sheep-associated (or European
and North American) M(~F has not been isolated; how-
ever, evidence basec.l on competitive-inhibition ELISA
;:inri ;:i PCR ;:iss;:iy strongly implic;:itp ovinf> hPrpPsvinis-2
(OIIV-2) as the cause of sheep-associated MCF. AlllV-2,
which is associated with the hartebeest, has not been in-
criminated in naturally occurring MCF disease of domes-
tic cattle.
AlHV-1 is a typical gammaherpesvirus. Infectivity is lost
by freeze-tl1awiug a11d sorlicatiuu of iufecteJ cell:.. Tl1t:
virion is reported to range from 98 nm to 240 nm. Virus
has been propagated ir1 bovine thyroid and testicle cell
culture.
The clinical disease is limited to cattle, buffalo, and sev
eral species of wild exotic ungulates (Pere David deer, ban-
teng, gaurs). Disease is associated \.vith mixing wildebeest
a11d cattle during periods when the wildebeest are calving.
Newborn wildebeest calves shed virus in nasal and ocular
secrt:tiu11s uµ Lo 3 t11u11Lh:. o[ age. Viral :>hedding also oc-
curs in adult wildebeest given corticosteroid. ·rhere is no
evidence for congenital infection of sheep with OHV-2;
rather, lambs appear to become infected during the first
year of life. While sheep associated MCF in cattle has been
associated with lambing, it appears that the newborn
la1nb does 11ot play tl1e same role as the newborn wilde-
beest. lt also appears tl1at all domestic United States sl1eep
c.arry OHV-2.
Diagnosis of MCf is made based on hi.stopathology and
a recent ly developed PCR test. Affected animals present
with mucopurulent nasal and ocular discharge and
corneal opaclty. Oral Iesions may be present, consisting of
multiple erosions preceded by a diffuse hyperemia and
µrofust! :>alivatio1i. Ce11tral r1ervuus disturl>ances are fre-
quent and diarrhea is c.ommon.
Tiistologic lesions are those of a lym.p l1oproliferative
disorder characterized by perivasc-ular mononuclear infil-
tration, necrotizing vasct1litis, and tissue lymphoid infil
tration. Deposition of immunoglobulin and con1plement
has been described in the glomeruli of affected cattle, sug-
gesting an irnrnune-n1ediated disease. Viral a11tige11 was
rarely detec.terl, however, and virus-spec.ific. antihodies
were not detected.
(APRINE HERPESVIRUS-1
A herpesvirus (previously designated BHV-6) was isolated
from young goats (1 week) dying in North An1erica
(California) with enteric sig11s and necrosis and ulceration
in thc rume11, cecu1n, and colon. Signs of conjunctivitis
and rh1n1ns were observed in 5Wiss goat l<ids. Althougn in-
fection in adults usua!ly is apparent, genital diseases 1nay
occur as vulvilis or balanoposlhilis. A 11erpes virus tl1at
was isol;:itPrl from a l.a lifornia shePp fP.tus was determ i ned
by DNA analysis to be caprin.e herpesv.irus (CpHV-1). Tu-=
California isolate of CpHV-1 replicates in canine, rabbit
fel ine, equine, hovine, and lamh cel ls.
Virus was isolated from vaginal secretions of does ex-
posed intranasally, intramuscularly, and intravenous~:­
with CpHV- 1. Pregnant does abort, but no virus was iso-
lated from the tetuses. Infections were severe in kids, ~ié
signs of conjunctivitis, rhinitis, and diarrhea. FÍbronecro-
tic lesions on the nasal septum were rernarkalJly sirnilar LG
thosf> of cattlf> \Vith TRH.
Gross lesions are 1i1nited to the gastrointestinal tracr
with necrosis and uJceration in rumen, cecum, and colon
Intranuclear inclusions are present in epithelial cells nea;
the 11ecrotic areas.
The disease can be diagnosed by viral isolation fron:
r1a:>al ~ecrelio11:. aud fecal n1alerial. fl appears lo be u11com-
mon, and no vaccine is available.
OVINE HERPESVIRUSES
ÜVINE HERPESVIRUS · 1
Ovine herpesvirus-1 (OV-H-1) '"'ªs lsolated from the lungs
of sheep with pnlmonary adenomatosis (j;:iagsiPkte)
Alll1ougl1 tl1e virus causes experin1ental pneumonia ir;
lambs, pulmonary adenomatosis is caused by a retrovir us
and the pathogenic role of OVH-1 is u11ccrtain.
OVINE HERPESVIRUS - 2
OVine herpesvirus-2 (OHV-2) is proposed to cause the
sheep-associated form of malignant catarrhal fever in cat-
Lle. ·r11e virus has never been isolaled¡ however, OHV-2
J)NA has hPen dPtec.tecl hy Pl.R in cattle with MC:F. Tt
shares a close genomic relatio11ship with AlIIV-1.
PSEUDORABIES (AUJESZKY'S DISEASE) VI RUS
Disease
Pseudorahies in swine is rn ost severe in younger animals.
The virus commonly affects the nervous system and the
mortality rate varies from 5o/o to 10011/o. lntection of so'"'s
during mid to lat e pregnancy can resu lt in abortion, fetal
death, mummification, or stillbirths. In adult pigs, severe
n ervous disorders are rare, and pseudorabies usually pre-
:>e11L:. a:> a 1all 1e1 vague illness of Lra.11sie11l pyrcxia, dull-
ness, inappetence, incoordination, a11d ataxia. Respiratory
discasc can also be sccn in pigs of various ages but is most
con1mon in grower and finishing pigs. l11apparent or mild
disease may be missed or 1nisdjagnosed in older swine.
Pseudorabtes also occurs In a number of otht!r :>pt!cle$, 111-
cluding cattle, sheep, dogs, cats, ancl rac.c.oons, in which
t11e clinical signs are usually neurologic and manifested by
an intense pruritis.

Etiologic Agent
?liysical, Chemical, and Antigenic Propertics
'!'he pseudorabies virus (PRV) is au alphal1erpesviru~
tl1at
is designated Suid herpesvirus 1 (SuHV-1). ()nly one
serotype has been identified; however, strain variability
:las been shovvn by restriction endonuclease digestion of
tjruses from different geograpbi.c a reas. Attenuated strains
have been demonstrated to have a deletion. in their ge-
nome, suggesting that specific regions are associated with
. irul1::11<.:<.:.
5.ensitivity to Physical and Chemical Agents
The PRV is fairly sensitive to high temperatures and is sta-
ble i11 cell cultur<:: lluiu lJ1::tw1::en <1 pH of 6 to 8 at cooler
temperatures. Virus has heen ohserved to s11rvivP in
u ncl1lorinatcd water for 7 days and for 2 days in an anaer-
ob1c lagoon. Chemicals that cleave chlorine appear to be
:he most effective disinfectants.
.ofectivity for Other Species and Culture Systems
The disease occurs naturally in cattle, sheep, dogs, cats,
and rats. In all b ut adult swine, tbe disease is almost always
fatal; hence, other animals are essentially "dead-end"
hosts. Although one report exists of hi.1man infection, PRV
is 11ol readily Lransnlilled Lo hurnans.
'l"he virus replicates readily in cell cultures from m;iny
species and tissues, including cat, dog, cattle, badger, coy-
ote, deer, buzzard, chicken. an.d goose.
Host-Virus Relationship
)istribution, Reservoir, and Transmission
Pseudorabies is recognized as asevere, highly fatal disease of
newborr1 J>igs in Europe ar1d tI1e United Sta tes, and has been
reported in other rarts of the world. The princip;il resPrvoir
of PRV appears to be the pig and transmission is frequently
pig to pig. The virus is transmitted by ingestion and inhala-
tion, and during coitus the virus can be transmitted from
ooar to sovv or vice versa. Transmission can occur ín a con-
taminated environment under crowded conditions.
Feral swine c<u1 tr<1nsrnit tl1e virus to domestic svv1ne
and among wild animals, the r;icr.oon h;is hPPn the niost
studied. Infected raccoons may t ransmit by close contact
" ·ith swine and swine may be exposed by consuming
infected raccoon carcasses. l'he pig is the primary sourcc
of viral sprea(l to other species. Cases in dogs have been
linked to consumption of feral swi11e tissues. 'fhe cat
appt:ar~ to !Je rnore se11sitive, and infection .in cats VI.ras
observed in SlºA1 of f>RV-infer.te<l farros \<VhPrP cats were
o resent.
Pathogenesis and Pathology
The virus replicates prirn;irily in thP i1pper respiratory ep-
itheliu1n including the tonsillar tissue. Virus can be iso-
Iated from the brain 24 hours following infection, which
Chapte:r 54 Herpesviridae 327
suggests that the route of infectioi1 is via the axoplasm.
Vir1::r11 ia is uifficult to dernonstrate; however, V.ira! shed-
ding may persist in nas;il serrPtions for 11p to 14 days.
Lower airway infection often results, and cardiac and
splanchnic ganglia beco me involved.
The virus produces a nonsuppurativc mcningocn-
cephalomyelitis with exter1sive damage to neurons, wide-
spread perivascular cuffing, and gliosis. 'fhe brain stem is
particularly <1ffected, but lesions also occur tl1roughout the
cerehral cortex and r.erehPllnm. There may be il1tra11uclear
inclusion bodies in all types of cells. In the respiratory
form of the disease, a necrotizing tracheitis and pneumo-
nia occur that result in loss of epithelium in airways and
necrosis ot alveolar cells.
Microscopic Iesions ln aborted fetuses include necrosis
of many organs, but primariJy liver, spleen, visceral Iymph
nodes, and adrenal glands. Intranuclear inclusion bodies
are often. prcsent in degeneraling he!J<'tlo1..yt1:::>, 1..1::11:> uf Lhe
adrenal cortex, and occasionally mononuclear phagocytic
cells of the spleen and lymph nodcs. Placenta] lesions are
cl1aracterized by degeneration and necrosis ot tl1e tro-
phoblasts and mesenchymal cells of the chorion. "
l
Host Response to lnfection
lgM antibodies are first detectable about the fiftl1 day after
infection followed by xncasurablc IgG antibodics about tl1e
seventh day, reaching maximum levels by the t\velfth to
fourteenth day.
Laboratory Diagnosis
.Becausc signs of thc discasc in swinc vary widely with the
age of t he animal, the dose of virus received, the strain of
virus, and the route of exposure, clínica! diagnosis is often
difficult.
In the laboratory, a definitive diagnosis of pseudorabies
can be n1ade by viral isolalio11. II11u1u110fluures<.:ent stairling
of frozen tonsil or brain tissue can provide a rapid diagnosis.
Scrologic tests for pseudorabies antibodies include solid-
phase radioilrununoassay, im1nunodiffusion tests, enzyme-
linked i.mm.unosorbent assay (ELIS1\), con1plement-fixation
test, serum virus-neutralizing test (SVN), counterneuronal
immunoelectrophoresis,. and indirect hemagglutination.
ELISA Lesls are U$<:!U to <lifferentiate antibody respon.se to
gene-deleted vaccines and field infPction. Tn an acute out-
break, serology may not be helpful because of the tin1e
11eeded for antibodies to develop. In the United States, the
rnost commonly used tests ure latex agglutination (LAT),
ELISA, and SVN. rn eradícation efforts, the sensitive LA'l',
which is quick and easy to perform, is commonly used as a
~creenir1g <1ssay. For conftrmation, the SVN and ELISA are
used '"'ith specific ET .TSA tPsts, which are especially useful in
detecting anirnals vaccinated >vith ge11e-deleled vacci11es.
Prevention and Control
ln an effort to avold tl1e disease in a breeding herd, a pro-
d11cPr should J.) purchase animals from sources free of PRV,
328 PART 111 Viruses
2) rcquirc testing prior to purchase, 3) isolate 11ew arrivals
and test for antibodies a mir1irnu111uf12 uay:, after receipl
and isol;.ition, 4) restrict human traffic among the swine
and practice hygienic measurcs, and S) make efforts to
restrict contact of the swine with other animals. fecd is a
potcntial sourcc of virus and appropriate measures should
be used.
ln infected herds, quarantine is the most urgent obliga-
tlOn and it is recommendcd that the muveruer1t uf :;wil1e
be limited for slaughtcr only. Porcinf' origin antiserum
with Lilers of at least 1:256 has proven effective il1 reducing
death losses if adn1inistered to neonatal pigs. However,
none is con1mcrcially availablc.
Attcnuatcd live vaccines are avatlablc and have been
successful in reducing death losses in endcmic areas. 1'hese
vaccines do not prevent reinfectlon Witl1 virulent field
vi rus or the shedding of virulent virus for v;i ri;.iblt> perio<ls.
Latently íuf1;;cleu a11d vaccinated aJ1in1als 111ay shed thc
virus for indeterminate periods while asyrnptomatic.
Inactivated vaccines are commcrcially available. Their
principal use has been in susceptible SO\.\'S in endemic
arcas lo provide antibodies to colostrum for protection of
ncwborn p1gs <.Iuring the first fcw "veeks of lile. Genctically
enginecrcd (gene-deletion) vaccincs are currently used in
deslgnated ~<ates in the U11itl'U Sldle:, :,i11ce co11uol pro-
grams utili7f' <iifferential serolo¡,ry as part of a federal erad-
ic<1tion progran1.
(ANIN E HERPESVIRUS
Di sea se
Canine hcrpcsvirus (CHV) causes neonatal dcaths, abor-
tions. and mun1mification as well as fatal systemic infec-
tion in ncwborn pups and rclativcly mild infections in
oldcr dogs. ·rhe virltS induces a genera!Jzed, often lethal in-
fection within the first 2 ,.vccks of life. Pups are either still-
born or die after a short illness.
CHV has been isolated frorn <lo~~ with re~p iratory ctis-
eases, a11d, '"'ilh other viruses and bacteria, may be in-
volved in thc "kennel cough" syndromc.
CHV can induce gc11ital lcsions in n1ale and female
dogs. Altected animals appear hcalthy but often presenta
history of infertility.
Etiologic Agent
Physical. Chemical. and Antigenic PropertiP.s
Caninc hcrpcsvirus is a typical alphaherpesvirus. 'fhere is
no cross-neutrallzation between C.HV and the viruses of
herpes simplex, pscudorabies virus, or infectious bovine
rhinotracheltls. However, CHV aµµear:> lu lJt:: a11 ligenically
rPl;.itf'<1 to hPrpes simplex virus.
lnfectivity for Other Species and Culture Systems
A coyote herpe:;viru:., :>liu\v11 Lu be a11ligcn.ically related to
CHV, was found in coyote p11p~ helieve<l to he infected by
indirect contact with dogs No othPr animals have heen r.·-
po1 Led lo sl1ow susceptibil ity to Cl IV. Limited grov.'1:h oc-
curs in human lung cells and calf, monkcy, pig, rahbit, an'-
hamstcr kidncy cclls.
Host-Virus Relationship
Distribution. Reservoir, and Transmlsslon
C:anine herpesvírus has been isolated in Europe, Japan
Australia, New Zealand, and thc Unitcd States. The onl•
known reservoir oí CH V in ali geograph.ic a reas is the do,
with the possible exccption of coyotes in tl1e United Stato
Modes of tra11smission of CliV may incluc.le transpl--
cental, congenital, oral, and airborne transmission . ·rhe~­
IS abo su1111;; evidt::11<.:t:: uf L1 ansnllssioi1 by indirect contac-
via an anin1al handler.
Pathogenesis and Pathology
Can1ne herpesvirus has been associatcd with respirator
disease in adult dogs, but the severe acute necroti7.ing ari
hemorrhagic disease uccur:, u11ly i11 puppies infected at lt
than approximately 2 we<>ks of age. 1-he virus can be i5<-
latcd fron1 the nose and pharynx of young dogs cxposc...
intranasally. It can multiply in the respiratory and tema,-.
genital tructs of oldcr dogs and can be isolatcd for up to -
days l-ron1 the vagina of bitches inoculated i11travagina ll~
lt has also been isolated fro1n pharyngeal mucosa. The
virus is disseminated hematogcnously, resultíug in y
nccrotizing vasculitis in many organs. (; ros~ly, th<' ki<lnry-
111ay appea1· 1uoltled a11d thcrc n1ay be pulmonary conges-
tion and edema, splenomcgaly, lymphadenitis, and non-
suppurative meiúngocnccphalitis.
Widespread toci ot necrosis and hemorrhages chara\.-
terizc the histologic lesions in affectcd organs such as kid-
ney, livcr, and lung. Intranuclear lnclusions may !Je pre -
ent il1 areas adjacent to necrotic les.ions.
Host Response to lnfection
Nculralizing antibOl1ies dcvclop 1n ctogs inoculatcd witl
CHV, but the duration of imrnunity is not known. CHV in-
duces serum-neutralizlng anrltJuuíe:> iu iilft::<.:Lt::d puppi~
Reactivation of CHV in expPrimf'ntally infected pups an1.-
uu~s has been den1onstrated with administration of corti-
costeroids.
Laboratory Diagnosis
C linical signs and histopathology can be useful in diagn0-
SiS. IJefinitive diagnosis is by viral isolation and 1nore rap-
i<lly by immunofluorescent staining of affected tissues.
Prevention and Control
Commercial CHV vaccincs are not available. Hyperim
mune globulin may be useful but ditlicult to obtain be-

cause the virus is poorly immunogcnic. Rcmoval or scpara-
tion of infected animals should be consldered.
FELINE HERPESVIRUS - 1 (FELINE VIRAL
RHI NOTRACH EITI S)
Disease
Fcline herpesvirus-1 (FHV-1) is the cause of feline viral
rhü1ouacheiLis (fVR). Alo11g will1 u¡Jµt:r rc:.¡Jiréitory dis-
ease, the virus is also associated with conjunc:tivitis, 111-
ccrative kcratitis, ulcerative stomatitis, abortions, and
pneumonia.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Fcline herpcsvirus is a typical alphaherpesvirus. Serologic
comparisons suggest that FHV from around the world are
similar; howevcr, diffcrences in the clinical manifestations
caused by varlous isolates have been observed.
Sensitivity to Physical and Chemic¡¡I Agents
fhe i11fc<.:tivity is reduced or eliminéitec.I by ether, chloro-
form ancl sodi11m hypochloritP. Vir11.~ in cPll cu lture fluid
loses 90% of its viabilíty -..vithin 6 hours at 37°C, 6 days at
75ºC, and 1 month at 4ºC. ·rhe virus is most stahle at pH 6,
and complete activity is lost in 3 hours at pH 3 and pll 9.
t-HV can be recovered tor up to 18 hours in a moist envi-
~onmcnt at lSºC, but for less than 12 hours in a dry room.
1nfectivity tor Other Species and Culture Systems
'\atural infcctions with FHV have been observed onlv , in
the cat faniily. fHV-1 i.n vitro growtl1 is lin1iled 1naiI1ly Lo
cells of feline origin . The virus propagatcs to high titers
'·"ith dcmonstratcd cytopathic cffcct in primary cell cul-
tures of feline testicle, lung, and renal cclls.
Host-Virus Relationships
)istribution, Reservoir, and Transmission
~atural cases of the disease have been reported through-
out the United States, Canada, Europe, Australia, and New
Zealand. Cats serve as reservoirs. Healthy cats that are la-
tently infectec.1 with FHV may shed virus when stressed, .for
~xa1nple, by corticosteroid administration, and viral tran-
scripts have been detected li1 Lrigeuti 11al ga11glia uf latently
_,_fectecl cats.
The major avcnuc for spread of FHV-1 is by direct cat-to-
cat contact through intec."tious discharges and aerosolized
::1icrodroplets. Indirect or fomite transmission via a con-
~mlnated environment, personnel, or feedíng and clean-
'lg utensils appears important only in catteries.
C11apter 51 Hcrpcsviridac 329
Pathogenesis and Pathology
The pathogenesis of the infectíon d1ffers w1 th thc route of
inoculation. As FHV-1 infection is often manífested in the
uµµcr rc~piratory tréict, experimental studies have been
donP. hy nasal ancl ocular ro11 tP~ . When introduced in-
tranasally, the virus produces rapid, cytolytlc li1fection of
epithelial cells of the nasal passages. The virus generally
persists in the upper respiratory tract for 2 wccks. Although
many cars aeve1op con¡uncnv1ns dunng the pr1mary a1s-
ease, very few develop cornea! disease. Experimentally, sup-
pression of local ilnmw1e iespo11st:::. µt::rrnitted viral access
to the cornea. 1'he resulting keratitis appears to hP cl11P to
thc immune response to the virus.
In the respiratory tract, changes occur in both stratified
squamous and pseudostratified columnar epithelial cells.
Lung changes consist of intcrstitial pneumonitís with
foc11l nccrotic lesions in the bronchi, bronchioles, and
alveolar sepla wilh alveolar accurnulations of inflamma-
tory cells and fibrinous exuclate.
Intranuclcar inclusion bodies are numcrous in the strat-
ífiea squamous epithelium of the conjunctiva w ith con-
junctival and cornea! lesions. Histologically, cornea} ulcers
reveal dlsorientation and degeneration of the ep1thelial
cells, sorne of lvhich contain nuclear inclusions.
Host Response to lnfection
'l'he primary immune response of cats to intranasal infec-
tion as rneasured by scrum ncutralizing antibodies is not
impress1ve. Antibodies usually persist tor 1 to J 1nonths, al-
though titers havc been observed to fluctuate in a cat bc-
tween 1 :48 and l :256 overa 12-month period. Correlation
between presence of antibodics and resistance to infection
is noL absolute.
Laboratory Diagnosis
Immunofluorescent staining can dcmonstrate viral anti-
gcns in thc tissucs of cxpcrimcntally infected cats. Early in
thc course of fVl{, the conjunctival and nasal mucosa
contain sufficient numbers of infected cells for antigen
detectlon.
fclinc herpesvin1s is isolated from tissue samples and
fron1 swabs of tl1e ocula1, 11a:.aJ, vr oroµltaryugeal mucosa.
The virus can be cultuied from nasal or pharynge.al swah-
bings for 14 to 21 days after infection, but most consis-
tently during the fust week.
Virus-neutralizing antibodies can be detected in thc
serum of convalescent cats. Paired samples, the fírst col-
lected during acute illness, the other collected 2 or 3 weeks
lale1, can be used Ior tl1e serologlc c.lléigiiosis of FVR.
Prevention and Control
Care should be taken to avoid intro<lucing cats with devel-
oping, subclinical, or latent infections to a colony. A nlodi-
fied live teline viral rhinotracheitis vaccine is available.
Protectivc immunity appears to be rclativcly short, and re-
330 PAHT III Viruses
vaccinatio11 evcry 6 to 12 months is rcco1n1ncndcd. An in-
tranasal vaccine is also available. A rccornbinant vaccine is
rcported experimentally to result in a reduced latency load.
S IMIAN HERPESVIRUS 8 {CERCOPITHECINE
HERPESVIRUS -1)
Hcrpcsvirus 13 cnuscs a .natural infection in Old World
rnonkeys (rhesus and cynomolgus specics). The infection
in monkeys is characterized by oral vcsicles similar to the
cold sores in humans caused !Jy herpe:. :.i111plex virus. The
disease in monkeys i<; not fatal and the virus may rcmain
lale11L u1 li1feclcd anin1als. Direct contact is the most com-
mon mea11s of viral spread in monk~ys, since virus can be
rccovered from saliva and fro1n the central nervous tiss ues
of clinically asymptomatic, pers1stently infected arun1als.
1'he virus can cause fatal central nervous infection in
hu1nans and laboratory animals such a:. ral.Jl.JiLs and un-
\veancd mice. Most hu1nan infPctions ílr<' caused by a bite
fro1u a Luo11l-.ey ~ec rew1g infectious virus, although han-
dling virus-infected prilnary monkey kidney cell cultures
can also lead to infection . Thc incubation pcriod in hu
mans is JO to 20 clays. Usually there is local inflammation
ot the bite site followcd bv , vesicle formation and necrosis
of thc a rea. Virus reachcs the central nervous system uy pe-
ripheral nerves, and death may occur duf' to :in1tf' Pn-
ccphalitis or en<.:l'phalu111yelitis.
HerpP<;vír11s B i<; morphologically similar to other al-
phaherpesviruscs. lt is readily inactivatcd by dctcrgcnts.
The virus can be cultivated on thc chorioatlantoic mem-
branc of embryonated chick eggs and in rabbit, monkey,
and human cell cultures that produce lntranuclear lnclu-
sion bodies and syncytia forn1ation. Strong cross-reactivity
exist~ !Jetwl'L'H liu1111u1 he1pes si1nplex viruses and her-
pP~vin1s B.
llerpes süniac ,·irus infection can be diagnoscd by iso-
lating virus from the central ncrvous tissues of fatal
human cases. /\ PCR based assay has been developed for
viral identification; however, diagnostlc dete.ction of her-
pes B specific antibodics is difficult. 'fhere is no treatn1Pnt
for herpesviru~ B i11 fecliuu in 11u111ans, nor ha::; an effective
vaccine heen developed. Human gamma globulin tha t
contains virus-specific antibodics should be uscd imn1cdi
ately after cxposure to monke.y bites. c_:aution and \'Vear1ng
of protcctive gear for handling monkeys remain thc best
way to avoicl infection.
MAREK'S DISEASE
Di sea se
Marek's disease (MD) is a lymphoproliferative disease of
chlckcns that may involvt: 11u111e1ous LlsslJcs. Most fre-
cp 1Pntly periphPr;il nPrves are affectcd. Prior to vaccine dc-
velopment, MD was responsible for hcavy losscs, and in-
creased losses to MD in vaccinated tlocks have suggestcd
an cvolution toward greater virulcnce. There are two
species of Marck's d1sease virus (MDV): gallid herpesvirus-
2 (GaHV-2), which is synonymous with MDV type 1, in-
cludes isolates that cause mild to severe signs of dlscaSt
gallid herpesvirus 3 (GaHV-3), which is sy11onyn1011s witl-
MDV type Z, i11clulle~ suains lhal are nononcogenic .....
third species is meleagrid herpesvirus-1 (MeHV-1), whict
includcs viruses from turkeys.
Progressive paralysis ot one or more extremities, 1ncoo --
diI1ation1 d rooping \-vings, and lowered 11ead position a;.
tl1e most common sigr1s ofMD. Mortality varles from 10'
with. 111ild MD to over SOO;ó in unvaccinated bircls.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
GaHV 1 and 2 a11d MeHV-1 are alphaherpesviruscs.
Resistance to Physical and Chemical Agents
Cell-free virus is readily inactivated at temperah.1res grcat
than 37ºC and is only relatively :.-ra!Jle al 25ºC (4 days) af'
4ºC (2 weeks). MDV ca11 be maint;iin<'d for long periods
27ºC. Viru:. i:. iuaclivaled by pl-1 3 and pll 11. lnfectivity
ciriecl MDV-infected feathers is destroycd by chlorine, :-
ganic iodine, a q uaternary ammonium con1pound, ere
sylic acid. synthetic phenol, and sodiu1n hydroxide .
lnfectivity for Other Species and Culture Systems
The chicken ls the p rimary 11atural liusl for ll1e MDV, a:-
disease is rare in othPr spPc:ies except for quail. Marek's d
ease virus has not been sl1owo to affect any no11avian ar
mals. No etio logic link has b een demonstrated bet\>\'et
MDV and human cancer. The virus is lnost <>ften cul
vatect on chicken or duck embryo fibroblast cells. Chicke
kidney cells have also been used.
Host-Virus Relationships
Distribution, Rescrvoir, and Transm ission
Marek's Liisease is a n1ajor disease of domestic chickc-
flocks worldwide. The virus can persist in excreta, litter
and poultry housc dust, and horizontal infection '"
aerosols ot chickens appears to be the main method __
transmission. Egg transmission is doubtful. Marek's d.~
ease virus matures to its enveloped lnfeL"tiuu:s furr u 0111) 1-
the feather fo llicles a11d can thf'n ht> srread to the environ-
111e11 Lvía Liesquan1atcd cells. Wholc live cells from blood o-
tumo r material, or infected whole cell cultures, are intec-
tious expcrimcntally. Ccll-frce fluid does not appear to l>é
infectious. Certain chicken llnes are geneticaJly resistan-
to MDV, and resistan ce in most bi rds is associated with th
development of serum-neutralizing autil.Juuy.
Pathogenesis and Pathology
The incidence of MD is variable, depending on the stra·
of the virus and the strain and age of the cliicke11. TLu:.-
ally occurs in chickens ben-veen?. ;ind .'i months old and
commonly felt I1ol Lo l.Je seen ü1 birds older than 22 week.

howf'Vf>r, <liSP.flSf> h.as hPen ohsf>rvf><I in hirrlc: .ac: yo11ng as 3
to 4 wccks and in 60-week-old laying hens. 'fhe virus pri-
marily affects the nervous system although visceral organs
and other tissues may also be involvcd. Lcsions are present
in the nervous system and il1volvc pcriphcral nerves and
spinal roots. The principal ncrvc trunk involved shows
gross le::.iur 1:-. cur isi::.Li r tg uf a glaybl 1-wl 1i le :;wclliug, which
histologically are characterizecl by extensive lymphocytic
infiltratlons. Edema may be present and myelin degenera-
tion ot nervc sheaths may be apparent.
Ocular lymphomatosis is another possiblc outcome of
YIDV infection, with blindness resultlng due to iris in-
"-olvement. Histologically, a similar infiltration oflympho-
cylcs is prcscnl, wllich cair also ulcu1 i11 Ll1e uptic I1crve.
In thc visceral form, lymphoid tumors of varying de-
grccs of scvcrity infiltra te thc gonads, livcr, lung, and skin.
Affected chick.ens have enlarged visceral organs with white
nodular or miliary foci. Oclusive atherosclerosis has been
ub~ervec.i expcrl mcntally.
Host Response to lnfection
Thc immunc response to MDV is complex, in that both
humoral and ceLJ-mediated immunity (CMl) develop in
normal birds. MDV infection can be immunosuppressive.
In addltlon, the i1nmune response may be involved in
t11mor form<1tion. Bursectomized birds survive experimen-
tal infcclion, suggesling tl1al CM! is i111po1 La1 1L. Trr chicks,
passivcly acquired antibody is thought to limit the r.xtPnt
of infection rather than prevent it or clear thc virus. Viral-
specific antiboclies appear within 1 to 3 wecks following
infection and neutralizing antibodies persisl for tl1c lifc of
the bird. rollowing infection, transient CM l suppression is
common; it may persist in birds that develop ncoplasms.
Both B and T lymphocytes have bccn idcnrified in tumors,
and thymectomy has been shown to reduce the level of
ly111plro111<1s i11 affecte<.l bir<.ls. It l1as ueen suggestec.i that
YID might have an autoimmune compon<'nt hasf>cl on an-
tibody responses to myelin and periphcral ncrves.
Laboratory Diagnosis
011 111;:1.1vv::.y, g1u:>:> 11;::-ivu:> al\.'. 1.u111111u11 i11 pe1ipl1eral
nerves, root ganglia, and tl1c spinal roots. Lyrnphomatous
lcsions are charactcristically composcd of smaH lympho-
cytcs, lymphoblasts, and reticulum cells. Arterial lcsions of
atherosclerosis are often present. Confirmatory diagnosis
is made by viral isolat ion or by antigen detecnon usiog flu-
orP<>cPnt antihody, immunoperoxid<1se, or ELTSA on
ieather follicle cells. Antibodies can be deLecLed by agar gel
immunodiffusion, indirect imm11nofh1orf>scPnce, and
,·iral neutralization and ELISA assays.
Prevention and Control
I.xpcrimentally, MDV-free flocks can be 1nai11Lai11eu by
strict isolation. constant surveillance, and frequent moni-
toring far virus and antibody, but these techniqucs have
been of limited commercial use. Commercial vaccincs are
available and have been effectivc in reducing the il1ci-
Chapter S4 Herpesviridae 331
den ce of ML). 'fhe three live virus serotypes have been used
for vaccincs resulth1g h1 inore vacci11e~ beco111i11g avail-
able. ITVT vaccine (serotype 3) is the most economical to
produce and is most cffcctivc whcn cxposurc is not hcavy.
vacc1nat1on does not prevent infection or shedding ot vir-
ulent ~!fDV, but it does prevent tumor formatio n. Bivalcnt
anti polyvalent vaccincs llave been used successfully
wh PTP m on ov;:i 1Pn t v¡¡ cci n es h<1vc bcen ineffective. Em bryo
vaccination is currently used in at least 55% of broiler ern-
bryos. Gcnctically resistant lines of chickens havc been
maintained experimcntally.
INFECTIOUS lARYNGOTRACHEITIS VIRUS (GALLID
HERPESVlRUS-1)
Di sea se
111fecliuu~ laryugutracheitis virus (Tl:TV) usually occurs as
an acute clisPasP in chickPns and represents a serious prob-
lem in arcas of intense poultry husbandry. 1·11e vil us p1u-
duces signs of respiratory distress and coughing that ofter1
produce a bloody dischargc. Mild cnzootic forms of JI.:fV
infection may result in reduced cgg production, conjunc-
tivitis, and pcrsistent nasal clischarge.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
¡¡; rv is a typical alphaherpP_<>vin1<> that is al<>o <IP<;ignatPd as
gallid herpesvirus 1 (GaIIV-1).
Sensitivity to Physical and Chemical Agents
TLTV is inactivatcd by 3% crcsol, 1% sodium hydroxidc,
and 1% lye, 24 11ours' exposurc to et her, and 10 to 15 min-
utes at SSºC:. The virus can be stored for lengthy periods by
lyoplllli:¿ation anti freezing.
lnf Prtivity fnr Other Species and Culture Systems
IL'fV is primarily a disease of chickcns, but thc discasc has
also been reported in pheasants and peafowl. Young
turkeys havc bcen infected experimentally, and the virus
replicates In cmbryonated turkey eggs. Starlings, sparrows,
crowc:, <IOVf>'> , d11rk<:, pigPon<;, anrl g11ine;i fowl h;ive been
found to be resistant to lLfV.
C~hicken kidney monolayer cell cultures, embryonated
chickcn cggs, and cl1icken embryo (kidncy, liver, and lung)
cell cultures have been used to culture TLTV.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
ILTV has bcc11 idenLified in al111o~t cvery country in the
world; it occurs primarily in arcas with high concentra-
tions of chickcns and occasionalJy occurs in pheasants.
332 PART Ill Viruses
Chickens are assumed to be the primary reservoir and
mode of tran!i1ni!i!iio11, wllicl1 vccu1s by direcl co11Lacl
through droplet in fection of the ocular and respiratory se-
cretions. Mechanical transmission can occur via contami-
natcd equipment and litter. Egg transmission of !Ll'V has
not b ccn demonstrated. A carrier statc can develop in birds
with sublethal disease, and ILTV has been isolated from
chickens 2 years after infection. Unvaccinated birds are
susceptlble to infection fron1 vC1cciualc::u 1.Ji1d:s, and vacci-
nated birds mi"ly hP<nmP c:arriers. Acutely infected birds
representa greater source of virus than clinically recovered
carrier birds.
Pathogenesis and Pathology
lLTV in natural conditions enters through tht: uµµc::r n:::.¡;i-
ratory tract and ocular tract. Tn thP ni"lh1ral d isease, the
gn.:alt:=>l concenlralio11 of TLTV is found in the trachea,
since the virus repllcates only in the nasal cavity, trachea,
and lower respiratory tract. Latcnt virus has been demon-
strated in the trigeminal ganglion. Viremia has not been
reported.
In lethal lnfections of ILTV, thick1;:11i11g of Lracl1eal mu-
cosa, hemorrhage, congPstivP ht>art failure, and severe
Lu11geslio11 of all interna! organs have b een found.
rlistopathology demonstrates fibrinous tracheobronchi-
tis, with detachment of the trachcal cpithclium and large
intranuclear inclusion bodies in detached cells that are the
basis fo r a strong p resumptive d iagnosis.
Host Response to lnfection
ThP fi r~t signs of TLTV infection usually appear 6 to 12 days
following natural exposure. Resistance to discasc following
the infection or vaccination usuaUy persists tor approXi-
mately 1 year. lnfected birds develop precipitating and
serum-neutraliZing antibodies; however, the cell-medlated
response appears to be important in resistance. ful! immu-
nity can be dernonstri"ltPci in hursectomized chicken~
thc absence of a humoral response.
Laboratory Diagnosis
Vlrus ca11 be lsolate<.l frorn tra1.:h1;:C1l a11d lung lissue in e•. -
bryoni"lted chi<kPn Pggs, c.ell culture, and ILl'V DNA can_
den1onstrated by PCR. lmmunofluorescent staining of ~
chea can demonstrate presence of viral antigen up to __
days postinfcction. ELIS/\ based serology is widely used
Prevention and Control
Use of live ILTV vaccine results in carriers, which can sr '-'-
to nonvaccinated susceptible birds. Since the virus can s-_-
vive for 10 days at temperatures of 13ºC to 23ºC, t he cli:r
ing of infected premises is very important. Complete c.
population and disinfection of premises has been used ·
control the disease.
Varrination has been successful via the cloaca, infra -
bital sinuses, intranasal instillation, fcathcr follicles, a!:
drinking water. Birds youn ger than Z weeks of age do ~
rcspond as wcll as older birds. Water administration
1Ll'V vaccine does not give as complete or 1ong-Iast111g l~
munity as otl1er methods. For water vaccine to be effecti"'""¿,
th e virus has to penetrate tnto the nasal cavity or tracl!t:_
Cloaca! administration of the vaccine lei"!cls to ri"lpid a'°l-
sorption uf t he viru:. iulo L11e bursa of Fabricius and resu,-
in ;¡n immune response. This route is undesi"rable, h o' -
ever, since ILTV da1nages the bursa of young bird
Adequate lLl'V vaccination occurs vía aerosol procedure<
Modificd live TLTV vaccines have been associated with d?s-
ease and chicken embryo origin vaccines have bee:-
demonstrated to increase in virulence after in vivo pa .
sage. The develop1uent uf genc::lically engu1eered vaccint'"
holci~ promise for improved control strategies.
 Poxviridae
N. J AMES MACLACHLAN
Poxviruses infect many vertebrate and invertebrate
5pecics. Discascs caused by poxvhuses often affect Lhe
skin, although sorne cause systemic infcctions in which
clinical signs of discasc may or may not be apparcnt.
Poxvi rus infections often cause proiiferative epithelial le-
sions in birds, '"'hereas papular and/or pustular epithelial
lesions are characteristic of poxvirus-lnfected mamm als
an<l on ly in ~omP infPctions do these become proliferative.
Poxviruses are large and con1plex and rcplicale u1 lhe
cell cytoplasm. Of the two subfamilies of poxviruses,
:nembcrs of thc Chordopoxvirinnc infcct vcrtcbratcs,
-,·hereas v1ruses w1thin the Ento1nopoxv1nnae infect insects.
There are eigh t distinct genera in thc Chordopoxvirinae
Table 55.1 ), and there is considerable antlgenic cross-
·p;ictivity between viruses in tbe various genera. Chordopox-
" irinae have plcon1orpl1ic, roughly brick-shaped v irions
approximatcly 220- 450 nm lon g x 140-260 nm w ide) t hat
in elude a Ji poprotein surface membrane anda biconcave or
cylindrical core. 'l'he gen ome is enclosed in the core and
consists of a single, very large (up to 130 kilobases) mole
cule of double-stranded DNA (dsDNA). Tl1e viral genome
encodes sorne 150- 300 proteins, and approximately 100 of
tl1ese are contained wíthir1 virior1s. lncluded are manv, en-
"'}'111P~ that arP involvP<I in virus rPplic<ition .
ORTHOPOXVIRUS
Disease
Orthopoxvirus infections cause a variety of papular diseases
of humans and animals. Papules are raised epithelial pro-
!iferatio ns thnt oftcn ulcernte rnpidly. Lcsion dcvclopmcnt
:nay be conñned to speciftc areas of thc skin or the infec-
tion may be generalized. Skin Iesions often appear first as a
ra:.li or papule followed by pustule formatlon that rapidly
•s followed by crusting and scab formation.
Eti ologic Agent
Orthopoxviruses include vaccinia (and rclated bu ffalopox
dl tu ralJIJitµox ), cowpox (Fig 55.1), camelpox, monkeypox,
.:>etromelia, and variola (human smallpox). They are mor-
phologically iJ1disliJ1guisl1able, a11d Lltt:lt: b CUll:Sideral>Je
-erological cross-reactivity between the viruses within this
:;enus. Orthopoxviruscs contnin a hcmagglutinin. 'I'he
]EFFREY L. S'fOTT
various orthopoxviruses can be distinguished on the basis
uf ll 1t:i r ge11ornic DNA :se4uence, and by pock formatlon
on chick em hryo chorioallantoir mPmhr<inPs (pork ;ip-
pearance and t he highest tcn1pcrature at which they de-
vclop ). ·rhe an imal h ost-species of origin is sometimes pre-
dictive of the type of orthopoxvirus, but not invariably so.
Host-Virus Re lationship
T1ansn1ission of ortl!uµoxviru=>t::. occur:; by contact,
aerosol, arthropod bites, or exposure to fomites. l"hp pnx-
viruscs tcnd to be very stable in the environmcnt.
Hoth cellular and humoral responses are critica! to im-
munity against poxviruses. Cellular immunity facilitates
destruction of vir us-infected cells and is important in con-
fin ing the virus to a localized area. Defects in cellular im-
11 1u1 1ily µenui t witlt:spread distrilJutlon uf viru s, resultl ng
in generalized disease. Ne11tr;¡lizing ;:intihn<liPs <irP impnr-
tant in recovery from i nfection. Immunity appears to be of
long duration.
Laboratory Diagnosis
Diagnosis of Orthopoxvirus infection can be madc by clcc-
tron m1croscopy of virus extractcd from lesions or by v iral
isolation. Isolation m ay be achieved by inoculation (scari-
flcatlon) of Iat>oratory antm als (especlally rabblts), of cell
c11 lt11rPs, nr nf thP chorio~1ll::into i c membrane of embry-
onated chicken eggs. The latter procedurc 11as been widely
u sed and may differentiate viral typcs.
V ACCINIA
Vaccinia virus is the genus prototype and was used to vac-
ciIJC:tte humans against smallpox (varlola), which was a
<lPv<istating rlisease prior to its eradication from the world
in the second half of t l1e 20lh cenlu1y. Vaccinalion o í liu-
mans with vaccin ia was responsiblc for the global elin1ina-
tion of smallpox, using the principlc devclopcd by Edward
jenner prior to 1800. Vaccinia was oriKJnally believed to be
a cowpox, but its origin is uncertain since it is quite dis
tinct from lts purported ancestor. Buffalopox and rabbit
pox (<in infPrtinn of l;ihor;itory r¡¡bbits) are caused by
viruses that are closely related or identical lo vacciJ1a.
333
334 PART JJJ Viruses

Ta b 1e 5 5 . 1. Genera (Subfamily· Chnrrlnpn1<virinriP) nf

Significance in Veterinary Medicine F 1G U RE 5 5. 1 . Cowpox virus in skin lcsions undcr thin scc
tioning. BOOOX. (Courtesy of A. Castro.)
Genus Members

Orthopoxvirus Vaccinia (and highly related buffalopox and rabbitpox

viruses)
Cowpox (rodents, cats, cattle, and humans)
Camelpox
Mouse pox (ectromelia)
Monkeypox
Racoonpox
Parapoxvirus 8ovine papular stomatitis
Contagious ecthyma of sheep (Orf)
Pseudocowpox virus of cattle (Milker's nodule ot
humJns)
.
Squirrel parapoxvirus
' -~ .'
Sealpox vir1.1s
•
Avipoxvirus Fowlµox
Turkeypox
Junopox (peacocks)
Ouailpox
Sparrowpox
Starlingpox
Canarypox
Pigeonpox (AM ELPOX
Capripoxvirus ShPPflf)ílX
Gudl¡;ux Camelpox is ;1 c;PvPrP, gc>neralized pustular disease of t ...
lumpy skin disease (Neethling virus) skin of affec"ted carnets. lt is of economic importancc
Suipoxvirus Swinepox countries with an indigenous carne! population, includi" ~
leporipoxviru5 Myxoma, rabbit, and squ1rre1t1llroma thosc of thc Middlc East, Africa, and p:lrts of Asia. Dise:i
Molluscipoxvirus Molluscum contagiosum occurs pr1nc1pal ly in younger anJmals and is charactcriZ<.-
unnamed vlruses of horses and donkeys by fever, gcncralized rash, and scquential development
Yatapoxvirus Yaba monkey tumor viru1 papular, pustular, anli sc¡-¡l.JlJing k:~ iu11::. vu lhe lin1bs, ncc
and head.

MOUSEPOX (ECTROMEL IA )

Vaccinia n1ay infect cattle vía handling (milking) by in-
fected personnel; infected c:attle can also intcct personnel.
Vaccinia irlfcction of cattle can produce lcsions that are in-
distinguishable from those causcd by cowpox virus; thcsc
occur on the teats and udder and appear as small papul<'s
that progres.s to pu~tuh:'~ a111J, fi11ally, exudalivc scabs.
Buffalopox is a clic;p;ic;p of milking \Vater buffalo in Egypt,
India, a11d Southeast Asia that also resembles cowpox. Likc
vacci na, buffalopox virus can infect humans.
(OWPOX
Cowpox virus is closely reJated to vaccinia, hut antigeni-
l'Cilly <.li~Li11ct. c:owpox ü1 cattle is characterizcd by papular,
pustular, and crusty cxudative lesions on the udder and
adjacent areas of skin . Rodcnts serve as reservoir hosts of
the virus, which also is contag1ous to humans, cats (do-
mestic and large wild cats), and a variet)r of othcr animal
specics. Cowpox appears to be a disease tl1at i:> cu11fi11ed to
Europe incl11ding th<' Rritish Isles.
Mousepox occurs in laboratory mice, although the vi r~
readily is excludcd from appropriately n1anaged facl.
ties. Mousepox is asevere, rapidly fatal cl ist>asP charaC"tf'--
izcu l>y ex l e11:>i ve uecrosis of thc Ji ver of affectcd m ice.
more chronic form also occurs, charactcrized by necro
of the distal extrcmitics (fcct, tail, and snout) of affect.,._
m1ce.
PARAPOXVIRUS
P;ir;iroxviruses infect a wide variety of anin1als, prin .. -
pally ungulatcs and domcstic livcstock, although seve:--
are also contagious to humans (zoonoses). Parapoxviru~
typically produce localized papular and proliferative cut..-
neous lcs1ons¡ examples in.elude t>ovine papular stornati-;:.
virus, orf virus (contagious ecthyma), ancl psr.11docowr-
virus (milkt:r'::. nullule) .
P;irapoxviruses are rnorpl1ologically unique in that.:
organized tubular, threadlikc structurc forms a crisscn..
pattern on the virion surtace. 1h e v1r uscs are ali anr1ger:
 Chaptcr SS Poxviridae 335

cally relatcd, but the envelope contains antigenically dis-

tinct epltopes. The viruses are stable for long periods of f 1G U R E 5 5 . 2 . Negatively sta1ned preparation ot contagious
time at ambient tem peratures. ecthyma viru~. 4(},000X. (íourtesy of R. Nordhausen.)

BOVINE PAPULAR STOMATIT IS

Disease
Bovine papula1 slon1alilis is cliara~tt:rizcd uy the presence
of papulcs, which are raised epithelial prolift>rations, on
thc mu.zzlc, nares, lips, bucea! cavity, dental pad, palate,
anct tongue. Hyperemic papules, with central necrosis and
concentric colored rings, are highly characteristic. Thc dis-
easc Is generally o f minor i1uportance, although it occa-
~ion;illy can mimic important vesicular diseases of cattlc
like foot-and-n1outh disease ancl vesicular slu1uatitis.

Host-Virus Relationship
Bovinc papular stomatltls virus lnfcction of cattle occurs
lvorlrlwirle; humans sometimes are infccted with the virus.
lt is believed to persisl i11 a lalelll :.tale iu cattle. Virus is
present in oral and nasal seaetions and transmission m;iy
occur by dircct contact.
Laboratory Diagnosis and Control
Buviuc ¡;a¡;ular stomatitis must be dlfferentlated from im-
portant vt>sic11la r clist>ast>s. Vir11s may be isolated on cell
cultures or directly visualized in skin scrapi11gs 01 biopsy
material by electron microscopy. Vaccines have not been
dcvclopcd.
Host-Virus Relationship
In addítion to sheep and goats, contagious ecthyma virus
may also infect humans (zoonoses), deer, and pcrhaps
other spcc!es. Related vin1ses il1fcct chamois and seals. ·rhc
vir11<; is clístributed worldwide and is maintained in naturc
by persistenl infeclions of sl1eeµ, a~ wcll as survival of the
virus for prolonged periods in dri~<i scah~. 'fhP virus is
rcadily transmitted on rough feed that causes erosions and
ulccrs in the oral cavity of affectcd sheep.

CONTAGIOUS ECTHYMA
Disease
Contagious ecthyma virus (fig 55 .2) is the etiologic agent
of contagious ecthyma of shccp and goats (synonyms in-
clude scabby mouth, contagious pustular dermatitis ot
sheep, soremouth, infectious labial dermatitis, and orf).
Q1f i:. llie le! 111 u:.etl LU tle:.l:ri!Jc ti IC Ulsease In human s.
Contagious ecthyma of sheep and goats initially is char-
actcrizcd by the appearance of papules and vesicles on
the lips, mouth, interdigital skin, genitalia, and udder.
Papules and vesicles ra pidly progress to pustulcs, fol-
lowed by scab formation. Lesions on thc lips interfere
with suckling or grazing, and thus to loss of condition of
affccled anin1als. Youug a11i111als are rnost likely to be af-
fected, and the virus rapidly spreads among .~11sceptible
animal s.
A related Parapoxvirus causes ulcerative dermatosis of
sheep, a disease characterized by the prcscncc of ulccratcd
papules on rhc lips, face, legs, feet, and externa! genitalia.
Genital infections are transmitted by sexual contact.
Laboratory Diagnosis and Control
Presum ptive diagnosis is hast>rl 11pon clíni.cal observations
and pathology (e.g., epilhelial p1ulifcralio11 witl1 intral:el-
lular edema leading to cytoplasmic vacuolation oftf'n with
thc prcscncc of cytoplasmic inclusion bodies). Virus can
be identified by electron microscopy and isolated in em-
bryonic sheep skin or testicular cells.
Vacclnation is used to control the disease in endemic
a reas, ancl imm11nity i.s considered to be long term in dura-
tion. Virulent virus is used in vaccinalion by :s1.:arification
of thc skin in areas not affccted by the disease; a potential
adversc impact of this approach is that it does ensure per-
petuat1on of the virus in nature.
PsEuoocowPox
Pseudocowpox is a mil<i disp;a~p of cattle that particularly
affccts lactating animals. Teat lesions develop as papules
with an ulcerated center that becomes cncrusted. 'fhis re-
sults in a pathognomonic scab with a ring or horseshoe
336 PAHT III Viruses
appcarancc. The causative l'arapoxvirus ls closcly rclatcd to
bovinc papular stomatitis virus, and can intect humans vía
dircct contact, producing so called milker's nodules.
Pseudoco\<Vpox occurs in most countries but 11as llttle
cconomic signiflcance because m ost infections are either
asympturnatic or vt::ry iuilu. !111111un iLy is of sl1ort duration
and rPcnrrPnt infPctions are common. Cattle occasionally
dcvelop chronic infections.
AVIPOXVIRUS
Avipoxviruses infect many avlan speLies (see TalJle 55.1).
Fowlpox virus is the genus prototypP.
Disease
Pox is a common diseasc of commcrci<il chickens and tur-
kcys, and of many ditterent species of pet and wild birds.
Fowlpox causes decreased egg production and increascd
mortal1ty on affected commerclal premlses. The lesions iI1
affectcd birds are designated as either cutaneous or <liph-
thcrltlc. Cutaneous lcsious <11e cha1aclcrlz:ed by nodular,
wart-likP p roliferations of hyperplastic cpithelium tha t in-
volve the skin of the head (con1b, wattlcs, corncrs of the
n1outh, nostrils, and eyes) . 'J'he d ipl1theritic form is char-
actcrizcd by proliferat ive lesions on the m ucous 1nem-
IJranes and 1uay extend into the slnuses; involvement uf
the larynx and trachea results in dyspnea ;incl ril lPs. The le-
sions are characterizec.I by ir1fla111 111aliu11, epidern1al 11ypcr-
plasia, an<i <iPvPlopmPnt of eosinophilic intracytoplasmic
inclusion bodies.
Mortality in affected commercial chic1'ens and turkeys,
pigcons, and psittadnes is typically fo,v, \vhereas infection
in canar1cs is almost always fatal.
Etiologic Agent
Th<' :ivipoxviruses are antigenically related but are differ-
entiated by ho:;t rungc, scrologic tc:;t~, plaque formation
in cell cultures, and pock formatron on the chorioallantoic
membrane of embryonated chicken eggs. 1'he viruses are
also distinguished by analysis of thclr genomic D:-iA. Li ke
other poJ<.-viruses, avipoxviruscs are highly resistant to
desiccatton.
Host-Viru s Relationship
Avipoxviruscs .bave a worldwidc distríbution. Viral t rans-
mission can occur by direct contact o r 1nechanical trans-
mission. Virus can survive for long periods in scabs, lead-
ing to cutancous or respiratory infection. The viruses also
can be transn1itted by lJitir1g i e1s1::cl:., especially n1osquitoes
and mitP~. c:hronic infections of individuals may con-
tribute to viral persistence on a given prcmisc.
Humoral and cellular immune responses develop fol-
lowing lnfection and apparcntly confer long-term im-
munity. Maternal antibody docs not confer protectlon to
h atched chicks.
Laboratory Diagnosis
Diagnosis of avían poxvirus infcctions is based on clínica!
signs and histopathology and electron microscopy. Virus
can be isolated by lnoculating susceptible birds, the chori-
oallantoic membrane of embryonated chicken eggs, ;111ll
cell cultures (chicken and duck cmbryo fihrohlasts).
Treatment and Control
Control of avian poxvirus infcctions can be aided by pro
viding adeq uate nutrition, housing, a nd insect control
among birds. Livc virus vaccines are used to i1n1nunize
bi rds against pox; tl1ese vaccines induce a milll lllstase Lhal
leads to protective immunization. RPcomhinant vaccines
rccently have lJeeo ueveloped and provide a vector for
the incorpor;ition of heterologous genes for protective im-
n1un ization.
CAPRIPOXVIRUS
Tl1e ge11us Capripoxvirus lnclutle~ i;ht:t:]J]JOX, goatpox, and
lumpy skin disease (Nef'th li ng) viruses; sheeppox virus is
the genus prototy¡;e;:. TI 1e vi1 uses are n1ore elongated than
othPr poxviruses and m easure about 115 nm x 194 nm:
hemagglutinin is absent. 'l"hc viruscs are closcly related but
differ antigenically. Iníection with any virus produces
cross protection to hetcrologous virus, witl1 the exception
of certain strains of goatpox virus. ·rhe viruses are pret10111-
inantly host-specific, though sorne strains prod11<'P Jp-;ionc;
in sheep, goat~, aud/or hu1nans.
SH EEPPOX ANO GOATPOX
Disease
Sheeppox and goatpox are important poxvirus diseases of
livestock, allhougl1 il1fection with these viruses can pro-
duce clinical signs that range in scvcrity from inapparcnt
to a severe gencralizcd condition. Animals of ali ages are
susceptible but disease is more severe in younger animals.
especially in endemic arcas. Breed and im mune status also
affcct disease severlty. Both shet:ppux a11u goalpox vicuses
cause system ic infections of .~ll~<'Pptihle h osts, and vircm ia
occurs soo1 e afler infection. Virus is dlsseminated to thc
skin, lymph nodes, and multiple organs, including the
spleen, kidneys, and lungs. ·rhc first clinical signs of dis-
ease include pyrexia, rhin1t1s, an<l con¡unctiVitis, follO\.'\'ed
by varylng degrees of lesion development on extern<>1
nares, lips, tongue, gums, and skin (cspe<.:ially wl1ere woc

or hair is minimaJ). Skin lesions are initially papules and
vesicles that rapidly bec:ome pustular and necroti.c: with
scab forn1ation. Infected epithelial cells 111ay contain eosi-
nophilic cytoplasrnic inclusion bodies. Lesions may also
d cvclop in thc rcspiratory and alimcntary tracts, livcr, kid-
n eys, and other organs. A nodular form of the disease has
been referred to as stone pox. Increased mortality rates are
associated wíth secondary bacterlal infectlons and disse1n-
in ated in te.rnal lesions.
Host-Virus Relationship
Infections occur predominantly in Africa, the Middle East,
11nd the lndian subcontinent. Viral transmission is by
aerosol, direct contact, and possibly ar tl1ropod vectors.
Viral persistence is probably dueto the sur viva! of virus io
scabs and its transmission to susceptible anin1als within an
endemic area.
Antibodies, including those with viral-neutralizing ac-
tivity, develop within 1 week of Iesion deve.lopment.
Tmrnunity is considered to be lifelong.
laboratory Diagnosis
Histupatl1olugy, fluurescent antil.Joc.ly staining, electron
m ic.rosc.opy, ;:incl serology c:an help c.onfirm clinical di.ag-
n osis. Virus can be isolated in cell cultures of ovinc, bo-
vine, and caprine origin. The virus grows relatively poorly
in embryonated chicken eggs as compared to orthopoxvi-
ruses and parapoxvíruses.
Treatment and Control
Prcvcntativc mcasurcs such as ilnport rcstrictions are prac-
ticed by countries free of the virus. Attcnuated and inacti-
vated viral vaccines are used in endemic areas.
lUMPY SKIN DISEASE
Disease
Lumpy skin disease o f cattle (and buffalo) is caused by a
virus (Neethling virus) closely related to shccppox and
goatpox viruses. Infection is followed by v1rem1a and fever,
witl1 tl1e subsequent formation of nodular lesions on the
skin (predorninantly on the neck, tace, muzzle, brisket,
flank, IP.gs, prrinr111TI, ;ind scroh1m) and internally in the
respiralory, digeslive, and reproduclive tract:;. Tlie <.:uta-
neous lesions are firm, raised and circumscribed, and they
oftcn undcrgo central nccro:¡j:; and ul ccratio11. Similor lc-
sions occur within the epithelium lining the upper respi-
ratory and alimentary tracts. 'fhe virus causes a dissemi-
11atec.J infe<.:tion that results in vasculitls, lymphangitis,
ancl lymphaclrnopathy.
Chupter 55 Puxviridue 337
Host-Virus Relationship
Neethling virus is confined to ,'\frica ancl Madagascar. Viral
transmission probably occurs by dircct contact, aerosol
and insect vectors. Persistence of Virus iI1 nature is proba-
hly similar to t hat desc(ibed for sheeppox and goatpox, but
it is proposed that buffalo n1ay serve as viral reservoirs in
sorne areas.
Laboratory Diagnosis
f)efinitive diagnosis requires fluotescent ant ibody staining
or electron microscopy of tissues, viral isolation in cell cul-
tures (lamb and calf kidney) or embryonated chicken eggs
(developn1e11l of pocks on lhe chorioalla11lui<.: u1e111-
brane), or serology.
Treatment and Control
Treatment is confined to supportive therapy. Vaccination
resul ts i.o lo11g-lern1 i111n1u11ily. Cou11trie::; free uf. lu111py
skin disease have irnposed import restrictions on stock
from infcctcd countrics.
SUIPOXVIRUS
Swlnepox vir us is lhe only n1e1nber of Llie ge11u::; Suipux-
virus. It is the cause of swiI1epox, a disease characterized hy
dissc1Tiinatcd cutancous pox lesion s.
Disease
Swiuepux u<.:<.:urs ir1 pigs uf all ages, a.lthuugh yuunger ani-
mals are more commonly affec.tecl. ·rhe disrasP. ns11ally i.~
very mi.Id, '"'i th virtually 110 mortality. Lesions typically
develop on the abdomen and inner thighs and sornetimes
on other areas of the skin. Lesion development progrcsscs
from papular through pustular and scabbing stages. Micro-
scopic lesions are similar to other poxvirus lesion and
include hyperplasia of epidermal cells with hydropic de-
genrr;=ition and thr formation of eosin ophilic intracyto-
plasmic inclusion bodies. Inflan1n1atory cells are fou11d it1
tl1e dermis. Vaccinia can cause an identical disease in pigs.
Host-Virus Relationship
Swin.epox has a worldwide distributi.on. Virus n1ay b e
svrea<l IJy dire<.:t <.:untact ur mechanically by Uce. Transpla-
cental transmission may also oc.cur. Thr virus persists for
long pcriods in dric<l scabs.
Swine develop immunity in the absence of detectable
neutralizing antibody, suggesting that local humoral im-
rnunity or cell-rnediated immunity is important in viral
clearance and protcction against reinfection.
338 PART 111 Viruses

Laboratory Diagnosis Control

The skin lesions of swinepox are highly characteristic, and Control ot swinepox is best rca11zea by e11m1nanon of ex-
oftcn are associated with !ice infestation. Laboratory con- terna! parasites. Vaccination is not commonly used.
firmat1on may be required to rule out other important
vesicular diseases. Definitive diagnosis can be obtained by
fluorescent antibody staining, electron 1nlcroscopic evalu-
ation of lesions for the presence of poxviruses, or virus iso-
latiou iu purcl11t: ct:ll cul Lu1 es.
 Picornaviridae
N . }AMES M
]EFFREY L. STOTT
"'tcornavlruses comprise a large family of small RNA
·"irnses. 'l'he fa mily Picornavíridae recen tly was proposed to
~'1cl ude nin e genera: Aphthovirus, Erlle1ovir us, Curdiuvirus,
Rlrinovirus, I Iepatuvirus, Parechovin 1s, Erbovirus, Kobusvirus,
:nd Tcschovirus, most of which contain members that are
_'TIporta nt in veterinary medicine(" l'able 56.1 ). A variety of
m an picornaviruses including duck hepatitis viruses l
"-rid 3 and turkcy hepatitis virus are not yet classified into
'.')ffifir gPnPr;i
GENERAL FAMILY CHARACTERISTICS
ne viruscs includcd within t11c various genera of the fam -
.Jv Ficornav1nclae nave sim ilar general properties. ·rhe
-iruses lack an envelope and exhibit a ct1bic symmetry
~ ith diameters of approXimately 30 nm and densities in
;::esium rh loriciP of 1 ..~3 to 1.4.5 g/cm3. The capsid, which is
.cosahedral, is composed of 60 subunits, cacl1 consisling of
:hree surface proteins (VPl, VP2. and VP3 or, respectively,
:o, 1 B, and lC) and (gcncrally) an interna! protein (VP4 or
: ...\) that is closcly associated with the genomic RNA. Each
0: these proteins is derived by systematic cleavage of a sin-
: le precursor protein, fron1 which 11 or 12 viral proteins
,·cntually are prod1.1ced by post-translational cleavage.
Thc vi1al ge110111e consisLs of a si11~lt: iJit:1.:t: uf :siI1gle-
rranded RNA of 7-8.S kilobases. 'fhe genome is p ositive-
'SC'n sc RNA, thus lt serves as messenger RNA and it also
J.S infectious. A small viral-specilied protein, VPg, is linked
to the S' terminus of the geno1ne and may play a role in
.nltlatlng RNA synthesis and in viral maturation (RNA
:-ackaging).
APTHOVIRUS
-:ne gcnus J\phthovirus incl.u des foot-0J1d-mouth discasc
and equínc rhinitis A v_ i ruses. Thesc are typical p icor-
naviruses with the notable exception that they are labile
".lelow pH 7 (acid-lal>ilt:) a11<.l they havt' a leac.ter protein
:~at is cncodcd immediately prior to thC' r;ipsici proteins.
.oot-and-mouth disease virus is thc type species. The VPl
:::=otein ol aphthoviruses is responsiblc both for cellular at-
::lchmcnt and for viral neutraliwtion.
ACL ACHLAN YUAN C HUNG ZEE
FOOT- AND - M OUTH D tSEAS E
Although foot-an d-mo11t h cl i sPil~P (f.Mn) is n·o t as widely
d istributed as it once was, particularly in iI1dusl1ialized
countries, it remains an enormously important disease of
food animals. The economic impact of FMD reflects not
only direct losses associated with disease in attected ani-
mals but also interference with international and regional
'
n1ovt:111t:11L uf a11ilr1étls. The vlrus lnfects all wild and do-
mestic cloven-hoofed anim;il~, h11t th<:> disease typically is
most dramatic in cattle and sv.•ine; sheep a11d goals are
usually not as severely affected. Economic \osses due to
FMD con be vast; t hc 2001 Unitcd Kingdom outbreak, for
example, is estin1ated to have cost producers severa] b illion
dollars in Jost revenue, and tbe loss of revenu e from re
uuccd tourism that resulted from quarantine m easures im-
pose<i 011 ri ng thP out bre;i k was estima ted to have cost a
similar amou11t.
Disease
FMD In cattle and swine is characterized by fever, depres-
sion, excessivc salivation, lameness, and formation ofvesi-
cles on the n1ucous n1en1branes of Lht: vral 1.:avity (tungue,
dental pad, and b>ums) and muzzle, epidermis of t h e co ro-
nary band a n d intcrdigital spaces, uclder, and teats.
Vesicles may also develop i.n the epithelium of th e p h ar-
ynx, larynx, trachea, esopl1agus, and rumen. Necrosis of
the heart muscle occurs in some young animals. The vesi-
clcs rapidly erode to leave ulcers that result in reduced food
consun1plion, weighL lu:.:., a11<.l ernaciation. Secondary
hacterial infPrtions ;ilso occur in thc ulcers of sorne af-
fected animals. While mortality is gc11e1ally less LJ1a11 3%,
morbidity is very high and economic losses reflect de-
creased productivity and protractcd convalescence of af-
fectea an1ma1s. Mortallty is notably 1ncreaseá in young
pigs and somctimes calves.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
FMD viruses are Lypical picon 1aviru:.c:. (Fig 56.1 ). There are
seven distinct serotypes, desi&rnatPci í!<; O, A,\., SATl, SAT2,
339
340 PAKT 111 Viruses

Table 56. 1. l'icornaviridae ot Veterinary Significance

F 1G U RE 5 6. 1 . Negatively stained preparation of foot-and-
Genus Member Disease mouth discasc virus (A 12). 108,000X. (Courtesy of B. Baxt.)

t.phthovirus Foot and mouth disease Foot and mouth disease of

virus
fquinP. rhinitis A
Enterovirus Porcine enteroviruses
A and D(formerly
enter-0viruses 8-10)
Swine vesicular disease virus
Bovine enteroviruses 1 & 2
Simian enteroviruses
cardiovirus Encephalomyocarditis virus
Theilovirus
Rhinovirus Bovine rhinoviruses
1, 2, & 3
Heparovlrus Avían encephalomyelitis-
lilc:P. virus
Erúoviiu) Equine rhinitis Bvirus
Teschovirus Porcine teschoviruses
1-7, 11-13 (formerly
porcine enteroviruses
1-7, 11-13)
Unassigned Duele hepatitis virus
Turkey hepatitis virus
ctoven-hoofed anlmals
Systemic infectíon with
respiratory signs
Reproductive (SMEDI) and
dermatitis (skin)
Vesicular disease
Uncertain
Uncertain
My1X.ardilb
Polioencephalomyelitis
-Theilers murine
encephalomyelítis
- Rat encephalomyelitis
Upper respiratory (mild)
Neurologic disease
Respiratory
Neurologic disease
(poliocnccpnJlomyclitis)
Hepatic necrosis
HPpatir necrosis

SA'f3, and Asia l . There also is extensive genetic hetcro-

gencity \Vithin individual serotypes, with many distinct
virus subtypcs occurring within cach serotype.
'l'he V Pl protein (lLJ) is respons1ble for virus neutraliza-
tion and contains a conservcd RGD integrin-binding
motlf that is responsible for the blndlng of virus to inte-
grin <'Pl111l;ir rpc.e.ptors.
Resistance to Physical and Chemical Agents
FMD virus can survive for extended periods in animal se-
crctions and products. The virus is inactivated by heating
abovc 50ºC and is sensitive to uotl1 <11.:itl (IJH less lhan 6.5)
and alkaline (pH greater than 11.0) treatmf'nts. FMD virus
re::.i:.b i11aclivalio11 by lipid solvents. Sodium hydroxidc
(lo/o) is recommended for disinfecting premiscs following
outbrcaks.
lnfectivity for Other Species and Culture Systems
A widc varicty of animals are susceptible to inff'ction ;inct
tlisea.se i.:<tu::.etl by FMD virus, lncluding cattle, domestic
anct wild pigs, sheep, goats, ccrtain wild ruminants, buf-
falo, camels, South American camclids, and humans
(rare). J\mong domestic livestock, cattlc and swine are usu-
ally most scvcrely affected. E.xperimcntal infections of
dogs, cats, rabbits, and chinchHlas have been reporteo.
Susceptible laboratory animals include guinea pigs, suc!.-
ling Inii.:e, rat:;, rauuil:., cllld han1slers. Son1.c viral strain
have heen propagated in chick embryos, day-old chick
and severa! other avian spccics. FMD virus rcplicatcs in ::
variety oí ceH cultures ot l)Ovine and ovine onh>in, bab·
hamster kidney (BHK) cells, and rabbit and mouse cells.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
The worl<.lwt<.le <lt.strll.>utlo11 vf P1v1D vi1u:. lat~t:ly 111i11 u1
th;it of tht> gloh;i 1e.c.onom ic situation, thus the d isease OI..·
cur:; principally in dcvcloping countries; most industrial-
i7.cd countries are free. fMD is endemic in Africa, Sout•
America, and parts oí Europc und Asia. North and Centra
America, the United Kingdom and Ircland, Japan, Au -
tralla, New Zealand, Scandinavia, and the Caribbcan re-
gion are currently free of FMD, altl1ougl1 u1a11y of Lhe
countries have previously suffPrPct outhreaks, sorne ver
rei.:e11 LI y.

The epizootiology of FMD differs somewhat depending
-?On whether or not fMD virus is endemic iI1 a country/
~on . Outbreaks of fMD iil preViously free regions spread
· ery rapidly, as a consequence of the highly infectious na-
::re uf the virus a1H.l its sl1ort i11cuuatio11 perio tl, tl1e large
~n tities of v in1s exc.retect from the respiratorytrac.t of af-
~ed animals, and the relativc stability of t he virus in the
~\ironment. Virus may be transmitted by direct cont act,
:3?rosol, and fomites and possibly arthropod vectors. Virus
=:¡y be recovered f rom all body secretions/discharges
:ears, nasal, saliva, urine, teces, .m ilk, vagi.na.l, semen, and
;.:;e p laceula of abor Led feluses) . The survival of virus i11
......:h excretions depends upon temperature, pH, an d hu-
- dity. FMD can be transported over long distan ces via i n-
=cted animals or their products and is v iable for up to 3
-on ths in frozen meat and up to 2 month.s Ln ha1n, bacon,
::.rd certain sausages. When discarded as garbage, contam-
-ated pork products constitute a source of FMD virus in-
;a:tion for s~vint: ill ¡Jarlicular. A11 irnal hides ca11 serve as a
-Jrce of virus for extended p eriods of tirne. Hurnans may
.::sseminate virus mechanically (fomites) or biologically
- t>ecoming- intected. Long-d - istance airborne spread of
-=us 11as also been implicated as the source of som e out
:_eaks. It ls to be stressed that epidemics of FMD can occur
~ en demic areas whe11 new serotypes or strains of the
-..!S a re u1troduct:d a11d tl1at the severity of a11y outhreak
-;::'lects the inherent biological properties of tl1e infecting
--:_-us strain.
;:~ogenesis and Pathology
:--:e predominant route of FMD virus infection is respira-
!)', altl1ough i11gestio11 uf co11ta111inated food or direct
~lation also are hoth highly effec.tive in transmitting
:.fection. Viren1ia precedes developn1enl of lesions and
-i::.Ll disease, and virus c:an he isolated from hody fluid s
=.nü seciet\ons at t\\\s t\rn e_ \T\iu s ia-p\<i\-y mo\les ~iom t\\e
':>!ood during viremia to intect the epith elium ot the oral
cavity and feet, where lesions develop. ·rhe v irus can per-
:>Jst in the oral cavity of infected animals for long periods
"ter the acute infection.
The cbaracteristic epitl1elial lesio11 of FMD is tl1e vesi-
:~e, which forms as a consequence of both int racellular
spongiosis) and intcrccllular edema. Thcse vcsicles rap-
dly rupture to leave erosions and ulcers in the attected ep-
thelium. In young animals, FMD virus can infect the
::eart n1usculature, causing degeneration and necrosis; the
-naracteristic yellow-wl1ite strcaks of myocardial necrosis
a:!e reft:rred Lo a~ L(s;er h eurl.
-uist RP.sponsP. to lnf P.rtion
5erum IgG develops about 2 weeks postinfection and is
::pe-specific. Colostral antibody in newborn calves l1as
'"IE'f'n reported to interfcre with vaccination. The relative
"IDportance of local :secretory, syslen1ic, and cell-111edialed
-:rununity is not well defined. Duration of im1nunity is
_'Qngcr in cattlc than in swinc but apparently persists for
Jllly about a year. Serum-n eutralizing antibody, actively
-i: passively acquired, appears to correlate wit h p rotecti.o n
.:: p!gs.
Chapter 56 l'icornavi ridae 341
Laboratory Diagnosis
Due to the econ on1ic and political significance of FMl)
and its sin1ilarity to otl1er vesicular diseases- vesicular
stomatitis (VS), swin e vesicular disease (SVD), and vesicu-
lar cxan thcma of swine (VES)- a rapid definitive diagnosis
is essential. Polymerase ch ain reaction (PCR) techI1iques
are increasingly used for rapid identification of f.MD virus,
and sequence analysis of any PCR-positive material t hen
can be used to type t he ü1fecting strain of FM11 virus and to
undertake molecular epiden1iologic studies. FMD vicus
can also be isolated from clinjcal samples (vesicular fluid
and othcrs) by propagation in ccll cultures or laborato ry
anirnals followed by physicochemical characterization
and serology using viral neutralization, enzy.m e-li.nked im-
m u.n osorbent assay (ELISA), fluorescent antibody (FA), or
agar gel i1n 1n11nodi.ffusion (AGID). Electron microscopy
(EM) and immuno-EM 111icroscopy also can be used for
rapid diagnosis.
Serologic diagnosis of FMD virus infection can be made
by EL1SA, AGID, and viral neutralization (either in cell cul-
tures or suckling m ice). The AGID test can be used to iden-
tify antibody to group-reactive an.tigen (viral infection-
associated antigen); such antibody is typically found only
i n a1li1J1als Lh al 11ave experienced an act ive infection and
not in animals vaccinated with i nac.tivatrcl virn.~ . Rec()m-
binant FMD virus nonstructural proteins (ZC and 3AI31)
can also be used in serology to distin guish vaccinated from
naturally infected animals as vaccinates do not make anti
body to these proteins.
Treatment and Control
No s9ec.i.fi.c. treatment exi.sts for FMD . tlO\l\lever, \)IO\)E:I an-
i.ma\ h\.\'O.band1y -p1ac.ti.c.e':> an<i t1eatment o1 seconO.a1'j 'oac.-
terial intections reduce Josses.
Control of FMD is difficult dueto its highly contagious
nature, multiple hosts, viral stability, multiple antigenic
types an d subtypes, and transient in1munity. Countries
free of FMD in1pose stricl in1porl regulalions on anirn a.ls,
animal products, a11d potentially contaminated materia.Is
from FMD countries. Previously unaffected countries have
often resor ted to massive slaughter and quarantin.e pro-
grams to control outbreaks; for instance, more than 1 mil-
lion ruminants were killed in order to control the recent
out break of FMD in the United Kingdom.
Quarantine and vaccination programs are aiso used to
c.ontrol 011thrraks and to prevent spread of the disease_
Cou11tries with enden1ic FMD rely 11eavily on vaccines
to control t he disease; however, steps still need to be taken
to avoid importing additional v iral strains sincc cross-
protective immunity betvveen strains is not complete. •
Ina<..'tivated vaccines of tissue cuJture origin that are ad-
1ninislered i11 adju va11t are rnost commonly uscd in FMD-
endemic countries_ The safr~ty of livr attenuated vaccin es
is questionable, so their use is not widespread. Vaccil1ation
is conducted one to three times a year since immunity is
short term. Vaccincs m u st be of thc appropriate type/ sub-
type. c:urrent research is d irect ed at developing subunit,
342 PART 111 Viruses
synthetic peptide, and recombinant-typc vaccines. The
lnttcr two nppronchcs hnve been directed at VPl \.Yith en
couraging results.
EQU INE RHINITIS A VIRUS
The equine rhinitis A virus is an economicatt y important
pathogen of horses that is closely related to FNfD virus.
Equine rhinitis /\virus produces a systemlc infection wlth
respiratory signs in affccted horses, and outbreaks of dis-
ea:ot:! t1avt:! l.Jeeu th::>tril.Jeu. Di:.li11cl sl1ain s of eq uine rl1ini-
ti~ A vin1s havc hc>c•n identified by sequence analysis. Sero-
lo¿)ical surveys indicatc -.v;dcsprcnd infcction of hor:;c:;,
although the true impact of equine rl1initís A virus re-
mains uncertain. Thc vi rus is difficult to isolate in cell cul-
ture, thus it is best dctected using polymerase chain reac-
tion (PCR). Tl1e vi rus can be detcctcd in the feces of
infected horses, as Wt:!ll a:; in tl1eir re:.¡.¡iratv1 y sec1elions.
ENTEROVIRUS ANO TESCHOVIRUS
The genus Enleroviru:; e11<.:v1uµa:>:>e:> a la1ge nun1ber of di f-
ft>n>nt vin1sE>s that typically are species-specific ü1 their
host range, including viruses that infect humans (polio,
coxsackie, entero, a nd echoviruses), cattle (bovine en-
tcroviruses 1 and 2), pigs (porcine enteroviruses A. and B
ffo rmerly porc1nc c nteroviruses 8, 9, and 10] and S\>Vine
vesicular disease virus), and monkeys (simia11 enterovi-
ruses). 1'he porclne Enterovirus serotyp1;:::. 1-7 auu 11-13
now are included in;¡ prnpnst><1 gPn 11s 7P.schovirus. Entero-
viruses are thern1o labile (infectivity destroyed quickly at
SOºC), insensitive to detergent treatme nt, and stable at low
pH (can survivc pH of 3).
PORCINE ENTEROV IRUSES ANO TESCHOVIRUSES
Disease
A variely of cntcroviruses and teschoviruscs have been de-
scribed in S\>vine, including S\>vine vesicular disease virus
(closely relatcd to human coxsackie B virus), ten se.rotypes
of porcine teschoviruses and tvvo of porc1ne enteroviruses
(/\ and .8). T.h ese viruses are the cause of severa! important
diseascs of swine. Teschoviruses (especlally serotype 1) are
t he cause of encepl1<ilomyelitis in pi g.~, .~o-called Teschen
ar1d Talfa11 discasc. Tnfections with porcinc enteroviruses
are generally asy1nptomatic, but they also have been asso-
ciated V.' itl1 infcrtility (so-callcd SMEDI, refle<.."ting still
birth, mummitication , embryoruc death, and infertility),
d ermatitis, and pneumonia. Swinc vesicular disease virus,
which is caused by a distinct porclne F,nterovirus tilat b
closely related to human coxsackie R virus, is thP cause of
:swlue ve:sicula1 discasc, which n1ust be distinguished from
foot-and-mouth disease. The various disease entities
caused by these viruscs in swine are discussed separately
below.
Host-Virus Relationship
Porcine enterovln1sf's oc<11r in mo~t swi ne-raising areac;
These viruses generally cause asymptomatic enteric infec-
tions of swine. Viral persistence is probably facilitated b·
the continucd introduction of susceptible pigs and tho:
presence of carrier animals. Transm1ss1on occurs by diret..~
contact and exposure to viral-contaminated feces.
Porcine enterovlruses rypically lnfect the l1vst vi<i iugc)-
tion, inl1alation, or hoth . Snnn aftPr i nfection, virus can lx
isolated fron1 the respiratory system, blood, and intestina..
contents. The virus has a tropism for the intestinal trae:
and may also be isolated from mesenteric lymph nod~
liver, spleen, kidney, tonsils, turbinates, lung, diaphragrr
and sometimes the central nervous system (brain af'I
cor<.!) an<.l/or fctuses vf µreg 11a11L a11in1als. Tl1e properties c.
individual virLIS stra ins arP rlParly v~r i ahle.
SMEDl infections result in fetal mumn1ificat ion, stiL-
births, and reduced litter size, although porcine reproduc
tivc and rc:;piratory syndrome virus and porcine pa •-
vovirus are more important causes of tlús syndrome. Th ~
consequences (infertility or fetal death/ n1ummificatiol'
reflect the stage of gestativ11 wlien :-.uscepLible so"vs are in-
fected . Sows generally are infectecl with porcinP entf'rc~
vir use:> µrivr Lv :sexual n1alurity, thus they are typically im-
mune hefore they become pregnant, although ir11rnunit:
is serotype-specific.
Laboratory Diagnosis
f)pfi n itive
diagnosis requires fluoresccnt antibody stainin .
of the tissucs of affected pigs (or fctuscs), virnl isolation, or
PCR. Viral isolation can be atternpteCI on primary pig kJd-
ney cell cultures. Serologic diagnosis can be done usin:::
paired serum samples to demonstrate seroconverslon or ª"
increase in antibody titer.
Control
Control of porctnc En terovirus !J1fectivr1 is uifficull dut l
the multiplicity of sPrntypPs an<l thcir ubiquitous naturt
in sv.'ine opcrations.
TESCHEN ANO TALFAN DlSEA SES
Disease
Teschen and Talfan are the na mes given, respectivPly, tn ~.. -
vere and milo forrns of pvlive11tephalo111yelitis in pif
caused by i nfPrtinn with teschoviruses. The diseases ari::
nan1ed after towns in Europe where they first wcrc d .. -
scribed in detail. ·rhe 1nild d isease ("faltan) also is referra.
to ns bcnign cnzootic paresis or polion·1yelitis suum. Sucklin .
or weaner pigs are most susceptible. Acute dlscase is cha;--
acterized by fever, anorexia, weakness, and variable rf.--
grees of central nervous systcu1 uy:.fu11clio11. As the disea .e:

:¡:irogresses, anlmals may have trouble standing or walklng
and PxpPriPn<P trPmors, convulsions, ;¡n<l nystt'tgm11s with
eventual development of paralysis and death.
Host-Virus Relationship
Teschen dtsease, first described tn the Teschen region of
r7Prhn~lflv!iki::i, ~ti ll flrrnr~ in (~Pntr::il f..nropP Mild~r
forms of E11terovirus-induced poliomyclitis in young swine
analogous to Talfan disease) have been described
throughout the world. There are no characteristic gross lc-
sions in pigs with polioencephalomyehtJs. Microscopi-
cally, poliomyeJitis is most evident in thc spinal cord and
....crc!Jcllurn. Lesions are characterlzed by neuronal degen-
eration, gl iosis, and lymphoc:ytic infl;imm;ition. Thf' p;ith-
ogenesis of polioencephalomyelitis in pigs is analogous to
that ot human polio (also caused by an En tcrovirus), with
initial virus rcplication in the intestine and then dissemi-
nated to the central nervous system in immunologically
naive animals.
Laboratory Diagnosis
The clínical signs described and the histopathologic obser-
vation of neuronal necrosis and Iymphocytic polioen-
cephalomyelitis cellular aggregations are suggestive, but
defi 11il.iv1;: Jiaguusis re4uires de111uustration of virus in tis-
<;ues hy irnm11nohistochPmical stt't ining, vir:il isolation,
o r PCR.
Treatment and Control
Treatment of t:nrerovirus-induced polioencephalomyelitJs
is usually futile, since most affected pigs die. Inactivated
\ dLLÍl 1e~ llave 1Jee11 u~eLl i11 Eurupc willt varia!Jle succt:s~
but have not been used else\·vhere. Strict quarantine is often
used to prevent introduction of the most highly path-
ogenic lorms ot t11c virus to regions free ot such viruses.
SWINE VESIC ULAR DISEASE
Disea se
Swine vesicular diseasc (SVD) rcscmblcs foot-and-mouth
disease (FMD), vesicular stomat1t1s (VS), and vesicular ex-
anthema of swine (VES). Affected swine exhibit signs sim-
ila1 Lo ll1u~e uf PMD, willt fever, la111 c111::s~. a111.J veslcles In
and around their feet (coronary hands, snlcs, and 'ntt>:rdig-
ital arcas) and, less commonly, the oral cavity and nares.
Host-Virus Relationship
SVD was fi1sl desc1ibed i11 ILaly auu :.lill ut:curssporailically
in Europe and Asia. 'fhe causativc virus i<; closPly rPlt'tted to
human coxsackie virus 135, and SVD is a zoonoses. The
Cflupler 56 Piiurnuviridue 343
virus Is highly contagious to swtne and is spread by direct
conttlct and by ingestion of contaminated foods. The virus
is very stable in pork producls such as sausage. Followiug
oral infection , the virus first rcplicates in the intestinal
tract and thcn is disse1ninated throughout the body and
can be isolated trom a varicty ot tissues and from feces.
Encephalitis occurs in sorne infcctcd pigs. Mortality usu-
ally is Jow in uncompl1cated 1nfections.
Laboratory Diagnosis
Kapid diagnosis ot SVlJ is critical because it must be distin-
guished from FMD and other vesicular diseases like VS and
VES. ·rhe diagnosis is best done by PCR, and sequence
analysis can be used to distinguish SVD virus from other
enteroviruscs. ·rhe vir us 01 vi1al a11 lige1t:. also can !Je iuen-
tified with vesicles by ilnmunohistochemical staining or
clcctron microscopy, or by virus isolation on primary
porcine cell cultl1res or in suckling mice. Serological diag-
nosis can be accomplished in convalescent animals by
ELTSA or viral neutralization.
Treatment and Control
No specific treatrnent for SVL> is available, and only exper-
imental vaccines have been described. 'fhe real economic
importance of SVD is that lt readlly can be confused with
FMD, so control requires strict quarantine and import re-
slriclions in counl1ies free of ui::.ea~t:. Strict 4uara11tir1e anti
animal depopulation are also used to control inc:ursions of
the virus into previously unaffected regions.
B OVINE f NTEROVIRUS
Two scrotypcs ofbovinc cntcroviruses (1 and 2) have been
dcscribed. ·rhese viruses are ot uncertain si&>nificancc be-
cause they have been isolated from both healthy and dis
cascd cattle.
HEPATOVIRUS
'fhe genus Hepatovirus 1ncludes human hepatitis A virus as
wcll as avian encephalomyocarditis like virus.
AVIAN ENCEPHALOMYE LITIS
Di sea se
Avian cnccphalomyclitis virus (AEV) causes neurologic
discase and high rnortality in young chickens (1 to 2 weeks
of age). Mortality can be 25<Jlo oc higher in susceptible
young blrds, depending on the virulence of the infectrng
virus strain and flock immunity. lnfection of adult birds is
usually subclinical.
344 PA l{T 111 Viruses
Etiologic Agent
Although l\.f.V is a typical picornavtrus in terms of its
virion structure, its nucleotide sequence is dissimilar to
those of other picornav1ruses. The virus is stable and resist-
ant to acid pH.
Be:.iue:. l;l1il.:k~11:. a11u lu1k~ys,Japru1ese quail ru1d pl1eas-
ants are susc.eptihle to AEV infection. Ducks, pigeons, and
guinea fowl can be experimentally infected. Chick em-
bryos can be infected (the yolk sac route is the most com-
mon), but rapid passage of field isolates is required before
clinical s1gns (muscular dystrophy) are observed in the em-
bryo. Cell culture systems for viral propagation include
chicl<en neuroglia! cells, e1nl.Jryu filJrulJl asLS, brai11 cells,
and pancreatic 1ells.
Host-Virus Relationship
Avia11 encephalomyelitis occurs wherever poultry are
raised commercially. 'fhe virus replicates in t he intestine
and thcn sprcads to thc central nervou s systern in suscep-
tible young birds. 1he virus is transmitted via the feco-
oral route, although vertical transmission hen-to-egg also
occurs.
Microscopic lesions occur in the hrain and spinal cord
of affecled birds and are those of viral encephalomyelitis,
characterized by neuronal degeneration, perivascular cuff-
ing, and gliosis. Visceral lcsions, charactcrized by hyper-
plasia ot lymphoid tollicles, occur in the proventriculus,
pancreas, and myocardium.
·rhc humoral immune response to AEV results in viral
clearance and protection. Neutralizing antibodies develop
witlii11 2 w~eks of infectio11. 111101unity is apparently of
long duration as exposcd flocks rarely have recurrent out-
breaks. Maternal antibody protccts young chicks for sev-
era! weel<s, thus mortal1ty is n11n1m1zed 1n 1mmune nocl<s.
Laboratory Diagnosis
/\vian encephalomyelitis must be distinguished from
other neurolog1c d1scases, especially other viral diseases
like Newcastle diseasc. Histopathology provides a pre-
sumptive dlagnosls, whlch then can IJe l;U11fir111eu uy virus
isolation or by immunohisto1hen1i1::il staining of the tis-
sues of affecLed blrds.
Treatment and Control
No tre::itment i<; availahle for affected birds. Attenuated
and inactivated vaccirles have been successfully used to
control the disease. Birds in high AEV incidence areas may
be vaccinatcd prior to reaching breeding age with live viral
vaccine strains. Only part of the flock need be vaccinated
sirlce the virus will spread througl1out the flock. Inacti-
vated vacclnes sl1uulu l.Je u:.1;:11 if ureediug flocks are to be
immunize<i . Chi1kens horn to immune hens are resistant
Lo AEV-induced disease.
CARO/O VIRUS
ENCEPHALOMYOC ARDITIS V I RUS
Disease
Encephalomyocarditis (EMC) virus naturally infects ro-
dents. The virus Is transmltted from rats to humans anc!
domcstic animals, in which it can cause myo1aniitis. in
uuuie~lic a11in1als, Lhe disease most common ly occurs in
young pigs. The disease in swine is typically manifested by
sudden death '.vith few, if any, prcccding clínica! signs.
Mortality can be high, especially in young pigs.
Etiologic Agent
There are a number of strains of EMC virus, including
mengovirus, Maus .Elberfcld virus, an d Columbia SK virus.
Host-Virus Relationship
Encephalomyocaruitis virus µriuc ipally ü1fects rodents.
but it al~o can infP<t nonhuman primates, humans, ele-
phants, squirrels, calves, horses, and pigs. Lack of pig-to-
pig transmission following experimental in!ections sug-
gcsts that infcction usually derives from rodents.
l·oci of myocardial necrosis in affected pigs manifest as
white trac."ts of coagulation necrosis with associated lym-
phocytic inflarnmatton and calclflcation.
Laboratory Diagnosis
Viral isotation is advised since im1nune responses require
about S days and may not develop prior to d eat h . PCR
techniques have also tJeen succe:;:;fully used for diagnosis
of i11fection.
MUR INE ANO RAT EN CEPHA LOMYE LITIS
A varicty of related strains of cardioviruses cause enteric
infections in rodents. Like human p olioviru scs an d por-
cine tcschoviruses, these viruses can spread to the ce11tral
nervous system to cause polioencephalomyelitis in im-
munologi.caUy naive animals. These viruses can be <.ie-
tected by serological screening, and control is hy qllaran-
til1~. t~:.tiug, c111u sa1tilalion.
RH/NOVIRUS ANO ERBOV/RUS
Rhinovlruses are characterizell uy acid sensiUvity (pH of
less th;in:; to 6). ThP gPnus Rl7inovirus includes human and
bovine viruses, whereas equine rhirloviruscs 2 and 3 are
now tentativcly classified together as equine rhinitis B
virus in thc gcnus Erbovirus. All of these viru ses share a tro-

¡nsm for the upper resp1ratory tract, best exem phfied by
human rhinoviruses that cause the majority of common
colds.
oovine Rh inovirus
Bovine rhinov1ruscs 1nfect the upper respiratory tract of
susceptible cattle. The majority of infections are asympto-
inatic ur v«:!ry uül<.l, re:;ultiug i11 1uiui111al seruus nasal tlis-
charge, mild fever, and c:oughing. Morhidity is ve.ry high,
as determined by serologic studies, but the sig11ificance of
Rhinovirus as a pathogen ot cattle is uncertain. R11inovi-
:uses have been implicated in the pathogenesis of the bo
vine shipping fever complex.
Transmission of the virus is probably by direct contact,
aerosol, and co11tan1inated n1a terials.
Protective immunity is best correlated to n eutralizing
antibody in scrum nnd nnsnl sccrctions. 1"itcrs of seru1n an-
tíbody to Rhinovtrus tend to increase with age in cattle,
probably dueto reinfection with homologou s an d h eterol-
ogous viral stralns.
Diagnosis of cl inical R~1i11ovirus infection is difficult due
to the n1ultiplicity of viruses associated will1 respiralo1y
disease. Viral isolation is often difficult and requires spe-
cific bovine cell cultures; n sandwich ELISA has bcen de-
scribed for confirmation of bovine J<hinovirus identity. LJe-
monstration of a rising neutralizing titer on paired serum
samples is somettmes useful.
Equine Rhinovi ruses and Erboviruses
Equine rhinitis A virus is an Aphthovirus that causes a sys-
temic iiúection in horses that is sometimes characterized
by respiratory discasc, whereas equ1ne rhinitis B virus
causes inapparent or 1nild upper respiratory tract in fect ion
of susceptible horscs. Although rhinltis B v irus has been
isolatcd from horses with acute respiratory disease, its true
pathogen ic sign ifica11ce is poorly defined. Diagnosis is b est
acco1nplished using PCR assay, because virus isolatio n
rarcly is successful.
UNASS IGNED PICORNAVIRUSES
D ucK ANO T URKEY H EPATITIS
Disease
Duck hepatitis is a contagious and highly fatal disease of
young ducl<Jings. t\ffcctcd ducklings are dcpresscd and
death follows qu1ckly. Morb1dity is often high and mortaJ-
ity may approach 90o/o. A similar disease caused by a re-
lale<l viru:; uccurs 111 young turkeys.
Chapter 56 I'icornaviridae 345
Etiologic Agent
At least 2 serotypes of duck hepatitis virus (DHV) currently
are listed as unnssigned membcrs of thc family l'icorna-
vináae, along with turkey hepatitis virus. ·rhese viruses ap-
parently can infect other avían species, and experimental
infcction of young chickens, pigeons, geese, pheasants,
and guinea fowl has been reported, sometimes with high
11101 Lalily.
The viruses can be propagated hy allantoic inoculation
of embryonated chicken eggs (ECEs). Infected chick em-
brvos die 5 to 6 days postinoculation and appear stunted
and edematous. Various chick, duck, and goose embryonic
cell lines support viral replicarion. Developmen t of cyto-
pathic cffect is variable.
Host-Virus Relationship
Duck 11epatitis virus appears to have a worldwide d istribu
tion. Transmlsslon of virus occurs by direct and indirect
contact with contaminated feces. The virus apparently
replicates in the intestinal tracl of infecLed uir<l::,. Tlie cllC!I-
acteristic gross lesion is an enlarged and hemorrhagic liver.
Additional lesions may includc an enlarged and mottled
spleen and congested, swollen kidncys. Microscopic le-
sions include hepatocellular necrosis, bile duct hyperpla
sia, and lnflammatory responses in various organs.
Imrnunity develops following infection, and maternal
lra11sfer of anlibody p10Lecls duckli11g::. fur :;everal weeks.
Laboratory Di agnosis
Diagnosis is suggested by the rapid spread and peracute
naturc of the discasc. Hemorrhagic lesions in the Iiver of
aff«:!<.:t«:!<.l 1Jiru::. are very characteristic, but m u st be distin-
guished from oth0.r ra11Sf'S of hPpat i< nf'rrosi<;. Df'finiti vP
diagnosis may be n1ade by demonstration of viral antigen
in the affected tissues by immun ohistochemical stainin g
or by virus isolation in ECF. or cell cultures.
Treatment and Control
1·here is no effective treatment for clinically affected biJds.
Attenuated and inactivated DHV vaccines are available,
and immunization of brecding birds facilitates maternal
transfer of an t1body. Ducklings mayal so be immunized di-
rectly but successful vaccination of ducklings requires that
they be free of marernal antlbody.
 Caliciviridae
YuAN C11uNc1 ZEE
Callc1virtL~es are smaU (27-40 nm in diamcter), nonen-
veloped, icosahedral viruses with a genome of singlP-
stranded, positive-:.e11:.c RNA (Fig 57.1). Tl1e viral RNA
serves as mRNA é!ncl is infectious. ·r11e namc calicivirus is
dccived from thc chalice-~haped sphcrcs on thc surfacc of
negatively staíncd viral particles. ·r11ere are four d1st1nct
groups in thc fa mi ly: Lagovirus, Norwalk-like viruses,
~apporo-like viruses, and Vesivirus. Severa! members-
specifically vesicular exanthema of S\~ine virus, San
Miguel sea llon virus, feline calici vir u:., European brown
hare syndrome, and r;ihhit hPrnorrhagic disease viruses-
a11:'. ilnporlanl anin1al pathogens.
GENERAL PROPERTIES
The genome of caUciviruses has only tv"v vr three open
reading frames. Virions are comprised of a singlP major
capsid proteiu. Tlic 11v11slruclural ptolcins of caliciviruses
share features with those of picor11aviruses. Replication of
caliciviruses occurs in thc cytoplasm, altl1ough both cyto
plasmic and intranuclcar i nclusions occur m infected ce lis.
VESIVIRUSES
Vesicul;:ir P)(élnthf'n1a and feline calicivirus are classified to-
gether in the genus Vesivirus. Viruses within this gcnus are
readily propagated in cell culture, in <11st1nct contrast to
caliciviruses in the other genera.
VESICULAR EXANTHEMA OF SWINE
Disease
Vesicular exanthema of swine (VES) IS an acute viral dts-
ease characterized by the formation of vesicles in thP ora 1
cavity, interdigltal spaces, cu1tl coru11a1 y ba.nd of the foot.
VES clinically is inclistinguishahle from foot-and-mouth
disease (FMD), swi ne vesicular disease, a11d vesicular
stomatitis. Thc incubation period of the disease is ap-
proximatcly 24 to 72 hours and the cou rse is about 1 to 2
weeks. ·rhe discasc has a high rnorbidlty but a lo'"' mortal-
ity. It is of sorne economic importance as a disease in pi3s;
however, 1ts rual11 i111v<tLl i:. Ll1al il 111i11ücs .t'MD, from
346
N. }AMES MACLACHL/\N
wl1icl1 il 111usl be distioguished. Thc last occurrence
this disease occurrcd in the Unitcd States in 1956. ~u
sequently, the U.S. Dcpartmcnt of J\griculture in 1959 d
clared VES an cxotic disease; howcver, viruses capable
causing VES are endemic in marine mamn1als, wher_
they also cause vesicular dtseases C:111tl revrvductive fa_
uxe. A wide variety of se;i 1"· si>a 1ions, \~alrus, and do
pl1i.u:. a1e infecled with these viruses. Outbreaks of \ t-
occur >vhen thcse marine mammal caliciviruses sprcad ·
swine, likely as a rcsult of fccding of dead marine mac:
mals to swine.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
VES virus (VESV) is a typical calicivirus and viral particl
are associC:1tetl \<VitlJ cytvvlasn1ic cislcrnae in infected S\~·ir.e
ri>lls (Fig S7.2) and in crystallinc arrays in the cytoplasr::
(Fig 57.3). VESV is stable at low pH (pl-J S) . A largc numb..-
of antigenícally distinct typcs 0 1 Vi-:~v have been idcnt1-
fied (at least 13), and a nu1nber of antigenically <listinc<
v1ruses that originally were isolated from species othe-
than S\vine are capable of causing VES and so ;ire cl;:i-;<;ifir
as Vf..'> viru:.e:., i11clutliug bov~ne calicivirus, Cetacean ca!.-
rivin1s, primate calicivirus, and a nun1ber of so-called Sa:-
Miguel sea !ion viruses (17 typcs). Similar viruscs ha·~
been isolated fro1n fish, birds, reptiles, and othcr rnarr-
mals, including skunks. These viruses are distinguished b·
sero1ogica1 reses, usually serum ucucralJzaliou, cu11.l ll11:: ';..-
túence of thr<;c> vir11sPs to pigs varíes significantly.
Resistance lo Physical and Chemical Agents
VES virus can persist in thc environment and in contam.-
nated meat products for very long time periods. "fhe vin......
1s completely i nacrivate(1 by 2o/o sodl um hytlroxltlc or 0.1
sodiu1n hypochlorite.
lnfectivity for Other Species or Culture Systems
Naturally occurring VES disease is conflned only to S\,·i..c..
of all ages and breeds. Experimentally, VESV causes \"es,
eles at inoculated si res in seals. Veslcles are al~v prvtluLt:-
at the sites of inoculation in horsi>s ;:inrt hamsters. Virus
i~u!C:1tc<.l i11 low Lilers fron1 son1e sites of inoculation an-
clraining lymph nodcs. VES virus can be propagated in et
lines of swinc kidncy or Vero monkcy kidney.
 Chapter 57 Caliciviridae 347

Host-Virus Relationship
f 1G U RE S 7 . 1 . NP.gativP./y stainP.d prP.paration of fP./inP r:ali-
civirus. 240,000X. (Courtesy of A. Castro.)
Distribution, Reservoir, and Transmission
VES was first described in North A.m erica (California) in
1932, and outbreaks were reported every year in California
between 1932 and 1951 (with the exception of 1937- 1938).
Tl1e disease firsl appeared ouLside of Califorula ü1 1951,
and from 1952 to 1953 it spread to a total of 42 states in the
Unitcd Statcs. Thc discasc had ncvcr becn reported else-
where in the world except Jceland and. H.awaii, and these
two incidents resulted from shipping contaminated pork
products from California.
Marine marnmals serve as reservoirs for VESV infection .
.A calicivirus was firsl isolaLed iu 1972fru111 :sea liuu:s 011 San
Miguel IsJand off the coast of southern California. This
calicivirus was nam.ed San Miguel sea lion virus (SMSV),
which was indistinguishahle tro1n VESV by morphologic,
biophysical, and biochemical criteria. Experime11tal SMSV
infectlon of swine produces a disease indistingtlishable
from. VES. SMSV also has been isolated from asymptomatic
don1e.stic swh1e. Serun1-11eu L1aliz:iug <111 Lil.Jullit:.s to .several
serotypes of SMSV and VESV have been demonstrated in
marine man1mal.s and both wild and domestic S'.Yine in
California. Earlier epidemiOIOgic studies during the out-
breaks of VES confirmed the relationship between feeding
of raw garbage and outbreaks of the disease, and dead sea
lions are knoi.vn to have been utilized as a food source for
swine.
Although outbreaks of VES li.kely originated from feed-
ing SMSV-infected marine animal parts to swine, the infec-
tion subsequently spread rapidly within affected herds by
direct contact.

Pathogenesis and Pathology

VES is characterized hy the appearance of fluid-filled vesi-
cles on the snout, coronary band, and tongue of infected

F 1G U RE 5 7. 2. Para/le/ ruw~ uf VESV particles in cytop/asrnic cisternae. 72,000X. (Reproduced

with permission from lee YC, Hackett AJ, Talens LT. Electron microscopic studies on the vesicular
exanthema of swine virus. //. Morphogenesis of VESV Type H54 in pig kidney ce/Is. Virology 1968;34:596.)

....
•

-
\ ..
•

•
348 PART 111 Viruses

F 1G U RE 5 7. 3 . Seetion of a viral crystal ín VFSV-infP.cted

ce/Is. 64,000X. (Reproduced with permission from Zee YC,
Hackett AJ, Talens LT. Efedron microscop1c studies on rhe
vesicular exanthema of swine vini~. ti. Morphogenesis of VESV
Type H54 in pig kidney cells. Virology 1968;34:596.)

swine. ·r11ese samc lesions ueveluµ iu ::.wine lliaL are inocu- chain reaction. Although the vesicular discascs ali product
lated intraderrnillly with either VF.<;V or SMSV. lnfectcd similar signs in swine, there are 1najor differences: whereas
anin1als are febrile, and virus is prcsent in blood and nasal- VES and swine vesicular disease are almost exclusively dis-
oral secretions for severa! days aftcr infection. Vesicles ap- eases of sw1ne, vesicular stomatitis frequcntly affeLt.
pear on the eoronary band and interdigital space of the horses as well as ruminants, and FM11 also affeC"t<: r11m·-
feet at 3 to 4 ctays after infection. Thc vesicles rapidly rup- nants (Table 57.1).
ture a11d healing takes place unless complicated by sec.:ond-
ary bacteria! infcctlon. High titer:> uf viru::. i:tre presenL
within the fluid in vesicles, which may also contaminare
ll 1e enviror1n1ent. Mild encephalitis occurs in sorne swinc Ta b 1e 5 7 . 1 . Susceptibility of Domestic Animals to Four Viruses
infected ''\fith VESV, and virus also may be recovercd trom That Cause Vesides in Swine
brain tissue of swinc infcctcd with SMSV.
Animal Spedes SVD VES FMD vsv
Host Response to lnfection Cattle ++ ++
Swine ++ 11 +f +
Neutralizing antibodies to VF.'\V ;inct SMSV appear in the
::.era uf auirnals infccted with viruscs soon after inocula- Sheep + -·
! ion and titers peak within 7 to 10 days after inlection.
Horse
Guinea pig
-·_.. +
++
+
Suckling mice + + +
Laboratory Diagnosis Humans + -· +

• Occasíonal le1ioru produced by spe<ific virus strain1

VES must rapidly be differentiatecl from othcr vesicular svo = Swine vesicular dlsease
di:;ca:;1:::; uf :-.wi111::, such as FMD, swinc vesicular diseasc, VES = Vesicular exanthema ot swine
;¡nci vesicular stomatitis. Laboratory diagnosis is accom- fMD = Foot·and·mouth disease
plished by virus isolation in ccll cultures, direct elcctron VS = Vesiculor <tom~titis
microscopic exanunation ot ves1c1c nu1a, or polymerase

Treatment and Control
There is no treatment for VES and there are no vaccines tor
control of thc discasc. Jt now is considered to be eradicated
in the United States. l:.nforcement of laws requiring cook-
ing of garbage beforc fccding it to swine was the most im-
portant factor In cllmtnatlng the c..li::;ea::;e.
fELINE (AL ICIVIRUS
Di sea se
Feline c<1 l i civirn~ (i::(~V) in fpc.ts the oral cavity and upper
respiratory tracts of cats to produce fever, sneezing, and
nasal and ocular discharges. Clinical signs in elude rhini tis,
conjunctivitis, oral ulcerations, and, in severe cases, pneu-
monia. Joint or muscle sorcncss, hyperesthesia, and
c hronic oral ulceration also have been attributed to FCV
infection, anc.J a c.Ji::;:,c1t1i11alcll liiglily virulenl and fatal
~y~tPmic cli~PasP associt1te.rl with specific strains of FCV re-
cently has bccn dcscribed. The incubation period of the
disease is 2 to 3 days and infected cats usually recover in
7 to 10 days in the absence of secondary bacteria! infec-
tions. V1r ulent systemic FCV infection is characterized by
alopecia, cutaneous ulcers, subcutancous edema, and high
mortaUty.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
There are multiple strains of FCV, and thc virulcncc of in-
dividual strains varics substantially.
Resistance to Physical and Chemical Agents
Feline calicivirus is resistant to rnany con1mon disinfec-
tants. lt is rcadily inactivated by a 0.175o/o sodi11m hypo-
chlurilc :;ul ulio11 (Clo1ox), whi.ch is the disinfectant of
choice. The virus is stablc at a pI-I of 4 to 5 and is inacti-
vated at 50ºC within 30 minutes.
lnfectivity for Other Species and Culture Systems
FCV is a ubiquitous pathogen that has been isolated from
cars all over rhe worlc.J. There 1::; uo cvtuc11cc tl1at FCV pro-
duces di~Ptl~P in lahoratory animals. The virlL~ can readily
be propagated in fclinc ccll lines. Sorne strains have been
grown in Vero monkey kidncy and dolphin kidney cells.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
The diseasc occurs worldwide, and ali species of cats likely
are susceptible. Tnfer.tion ;ind dise;ise ;1re m ost r.ommon in
young cals, and oldcr cats usually are in1n1une. Infected
Clrapter 57 Calicivirida.e 349
cats recoverell íru111 Llie uisea:se n1ay carry virus in their
oropharynx for long periods of time and serve as reservoirs
of infcction. 'T"he virus is transmitted by horizontal aerosol
infection and by contaminated tomites.
Pathogenesis and Pathology
Cats acquire FCV iI1fcLtiu11 via lile;: rc:.piraLory 1oule, eilher
hy aProsol or from fomites. The primary sites of viral rc-
plication are epithelial cells of thc oral cavity and rcspira-
tory tract and in the tonsils. Viremia occurs during acute
infection.
The characteristic lesions in typical cases of FCV infec-
tion in susceptible kittens and young cats are vesicles
within rile oral cavlty (tuuguc, harll pahtlc) aud OH lile
narPs. ThP VP.sic.les rapidly rupture, leaving erosions and
ulccrs. llighly virulent strains can cause pneumonia in kit-
tens. Regeneration of the o ral mucosa occurs rapidly in un-
complicated cases.
Virulent systemic strains of FCV cause epidemics of
fatal disease in susceptible cats. Affected animals may ex-
hibir severe oral ulccratiuu, cxtcu::;ivc ::;ulJcuLa11e;:ous
P<IPma, ancl variahl<' 11lcC'ration of the pinnae, Pª"'' pads,
nares and skin. Sorne affected cats also have pneumonia as
well as liver and splenic necrosis, and fCV antigen is de-
tected by immunohistochemical staining in both epithe-
lial and endothelial cells.
Host Response to lnfection
Cats infected with fCV or vaccinatcd "\Tith inactivated or
live modified fCV vaccines develop serum-neutralizing
antiboclics. Kittcns born to cats that are immune to FCV
acquire maternal serum-neutrahz1ng antibody to fCV vía
colostrum.
Laboratory Diagnosis
Laboratory tests are requircd to distinguish FCV infection
fron1 other agcnts t hat produce similar respiratory signs in
cats, particularly fcli ne viral rhinotracheitis (her pesvirus).
·111ese lnclude the 1so1ation of FCV in feline cell cLJltures
from nasal sccretions, throat swabs, or conjunctival scrap-
ings and the Jdenttflcatlon of FCV antigen in cunjunctival
scrapings of tonsillar hiop<;ipo; hy im n111nohistoc.h Pmical
:staining. The characteristic appearance of the virus by
electron microscopy can also be used for rapid diagnosis.
Treatment and Control
Treatment for Fl.V infPction in cat<; is mainly s11pportivP
and sympton1atic. Broad-spectrum antibiotics help pre-
vent secondary bacteria! infections, and fluid therapy is
uscful in thc cvcnt of dchydration. /\ll strains of FCV are
considcred variants of a smgle serotype because there is
considerable serologic cross reactivity among viruses.
Furthermore, cats immunized with onc variant of FCV are
protected against other strains. Both inactivated and mod-
ified live fCV vaccines are co111n1ercially available and af-
350 PAHT 111 V1ruses
ford reasonable protection against FCV infection. The FCV
vaccincs are usually combincd with fcline rhinotracheitis
(a herpesvirus) and feline panleul<openia (a parvovirus)
and administered either intranasally or intramuscularly.
FCV infection Is controlled primartly by isulatiI1g <.:at:.
that show respiratory signs ano oisinft>rting c:ages and
pre111ises wilh Clorox before susceptible animals are in-
troduced.
LAGOVIRUSES
RABBIT HEMORRHAGIC DI SEASE ANO EUROPEAN
8ROWN HARE SVND ROM E
Disease
Rabbit hen1orrhagic disease (RJID) and European Brow11
T-Iare Syndromc CEBHS) are similar diseases t hat are caused
by rclatcd but antigenically distinct caliciviruses. RHD is
an acute infectlous disease of the European rabbit, Orycto-
lagus cunniculus, and frequently has a very high mortality
rate in susceptible rabblt populations. A novel feature of
RI-ID is that the disease is only fata l to rahhits over 2
montl1:. uf agt::. TI 1t:: disease is cl1aracterized by a short incu-
hation period, followed by fever, disseminated hemor-
rhage in ali body tissues, and rapid dcath. Thc disease "vas
first described in China in 1Y84 and it then rapidly spread
throughout much of the rest of the world. EBHS occurs in
the European hare, Lepus europaeus.
Etio logic Agent
Physical, Chemical and Antigenic Properties
Various strains of R.HO anc1 EBHS v iruses are recognized
a11d distinguished serologically.
lnfectivity for Other Species and Culture Systems
Neither RHD nor EBHS viruses are readily propagat ed in
cell culture, whlch 1uc<111~ Ll1al Ll 1e viruses have been
J;.1 re<'ly rh;:ir;:ictt>ri7.Pcl usi ng homogenates of tl1e livers of af-
fected animals. The viruscs appcar to be highly species-
specific.
Host-Virus Relationship
Distribution. Reservoir. and Transmission
J\lthough RHD was first reported in China, EBHS had bee11
recognized earller ln Europe. It is posslble that a mutatiu11
ot the EBHS calicivirus led to the emergence of RHD\-
causing the lcthal pandemic of rabbits. The disease is
transmitted by the oral-fecal route.
Pathogenesis and Pathology
Rabbits with RHD havc an cnlargcd splcen, swollen live::.
and disseminated hemorrhages . .Extensive liver necrosis is
highly characteristic and potentially explains the dissem!-
nated intravascular coagulatlo11 (DIC) that u<.:cur$ i11 af-
fectcd animals. The DIC induced hy R.HDV is not charac-
tcri:.lic uf uthe1 calicivirus infections, but does occur ir
such flavivirus-induced diseases as yellow fever anc
de ngue in humans.
Laboratory Diagnosis
Iuuuu11ufluu1escence a11d ELISA tests have been developec
for the rapid diagnosis of RI-ID. ·rhe genome of RHDV has
bccn complctcly scqucnccd, so polymera se chain reactior:
(I'Cl{) readily can be developed and used for rapid diagno-
sis of the infection.
Treatment and Control
There is no treatmcnt for the acutc discasc. A formalir.-
inactivated vaccine that incorporates in.tected rabbit tissuc
providcs cffcctivc immunization against tbe disease
Control also can be ach1eved through strict quaran-
tine and isolation to prevent transportation of RHD\·.
contamlnated materlals lnto comm~r<.:ial ral>l>iLrie:.. Il .:.
interesting to note th<Jt, althn11gh most c:ountries have fo-
cused on lhc control and prevention of RI-ID, RHDV ha...
becn used as a biologic weapon to control rabbit numbers
in othcr countrics.
UNASSIGNED CALICIVIRUSES
Euleiic callcivi ruscs have been described in cattle, dogs
chickens, and pigs, among others. At least sorne ot these
appcar to cause clinical signs of intestinal disease anal~
gous to that caused by the Norwa11<-111<e v1ruses in humans.
 Togaviridae and
Flaviviridae
N. JAMES MACLJ\CHLAN
TOGAVIRIDAE
The family Togaviridae derives its name from "toga," the
T.atin word for gown. or cloak, which refers to the envelope
possessed by all me1nbers of the fan1ily. The fan1ily iI1-
cludes two genera, Alphavirus and Rubivirus (the cause of
human rubella). Viruses in the Alphavirus genus are ar-
bov1ruses that are transm1ttea by mosqu1toes; thus tney
have the capacity to replicate sequentially in insects and
vertebrales.
ALPHAVIRUS
Sindbis virus is the prototype of the genus Alpliavírus. AJ-
phaviruses of veterinary significance include eastern
equine encephalitis (EEE), western equine encephalitis
(WEE), anct Venezuelan equine encephalitis (VEE, incluc!-
ing subtype 11 Everglades) viruses, along with severa! other
viruses (Getah, Highlands J, and Semliki Forest). The three
equine encephalitis viruses are also zoonoses, and they are
described in detail below.
Disease
EEE, WEE, and VEE all cause encephalitls in horses, but the
sig:ns can vary from inapparen.t to fatal disease. Mild and/or
i.napparent infections are n1ore con1111011 witl1 WEE,
whereas EEE and VEE are typically more virulent. Death
from VEE n1ay occur in the abser1ce of neurologic signs.
Central nervous system (CNS) involvement of .h orses fol-
lowing encephalitis virus infection is characterize.d by aim-
less walklng, followed by severe depression and behavioral
changes (dummy), central blindn.ess, paralysis, and death
soon after the 011set of dinical signs. Mortalily ca11 be very
high (up to 90º/o), especially with VEE and EEE.. Young
horses are more susceptible to scvcre disease.
EEE and W.EE may also cause significant disease in do-
mestic birds; EEE is more common and mortality can be
very high. Clinical disease is especlally common in pheas-
;:ints an<1 riltites (emus, etc.) EEE also occurs in swine and
cattle. Equine encephalitis viJus disease i!1 birds is char-
acterized by encephalitis with leg paralysis, torticollis,
]EFFREY L. STOIT
and tren1ors. WUd birds 111ay also be infecled bul rarely
experience disease, and they serve as vertebrate reservoirs
of virus.
Etiologic Agent
Physlcal, Chemlcal, and Antigenic Properties
Alphavirus virions are spherical, enveloped, and approxi-
mately 70 nm in dia1neter. "fl1cenvclopc is derived from thc
plasma membranes of host cells through which Virions bud
as they mature. The envelope encloses an icosahedral nu-
cleocapsid that is approximately 40 run in d!ameter and
consists of ;:i single capsid protein as well as the genome of
linear single-stranded positive-sense .RNA. Tl1e e11velope
contains a heterodimer of 2 viral glycoproteins (El and EZ),
and some alphaviruses (Semliki Forcst virus) havc a third
glycoprotein (E3). At least four different nonstructural viral
proteins are produced in infected cells. The alphaviruses ali
are antigenically related as determined by seroiogicaJ as-
says, and are grouped into di.stinc..i: antigenic complexes
wilh nun1erous sublypes or slraiI1s ,.,,ilhin each. The exlen-
sive genetic and antigenic variation that occurs \.Yithin the
various subtypes and variants of each antigenic complex is
reílected by ditferences in their v irulence, biochemical
characteristics such as elcctropl1oretic mobility of protein
and RNA digests, physicochemical characteristics, host
range, geographic distribution, and vector/host tropism.
Resistance to Physical and Chemical Agents
Alphaviruses are sensitive to lipid solvents, chlorine, phe-
nol, acid pH, and heating to 60ºC for 30 minutes.
lnfectivity for Other Species and Culture Systems
The equine encf'ph;:ilitis viruses can infect a wide host
range, including humans, horses, rodc11ls, repliles, an1phí-
bians, 1nonkeys, dogs, cats, foxes, sk11nks, cattle, pigs, birds,
and mosquitoes. Alphaviruscs can be propagatcd in a vari-
ety of cell cultures, including chick anc! duck fibrobfasts,
Vero, L cells, and mosquito cells; cytopathology is often ab-
sent in the latter. A variety oflaboratory animalscan be ex-
pPrimenta.Uy infected, with suckling mice being the. rnost
351
352 l'ART 111 Viruses
c.:0111Iuu1i. E1ulJryoIJdtell clücken eggs dlld young chicks
may also he susceptihle to infection .
Host-Virus Relationship
Distribution, Reservoir, and Transmissíon
·rhf' f'Cp1inf' f'nct>ph<ilitis vir11ses occur in the Western
IIen1isphere, although related viru:ses occur elsewhere in
the world. EEE occurs in eastern North An1erica (predon1i-
nantly cast of thc Mississippi Rivcr and thc i\tlantic
Seaboard region in particular), the Caribbean Hasin, and
Central and South An1erica. WEE occurs throughout
much of North America, particularly in areas west of the
Mississippi River, and South America. VEE is confined to
Soulh aud Cenl1al Arnerica, alll1ough incursio11s inlo
North America have occurred periodically. The U11ited
States considers VEE to be a forcign animal discasc; how-
ever, an avirulent VE.E virus ('l"ype 11, Everglades) is en-
de1nic in portions of Florida.
1'he equine encephalitis vtruses are transmitted by mos-
quitoes and, despite their names, horses and humans are
llead-eull hu~t~ that are uuilupurtaut tu tltt: 11alural c.:ycle
of EEF. and WF.F. virus infecrions. Th e viruses ali persist in
similar but distinct natural cycles of infection that include
mosquitoes and birds or rodents that function as the verte-
brate reservoirs of each virus. Except in tropical areas
\.Yhere infection occurs year-round, the peak incidence of
these diseases typically is in late summer and declines
when cün1atic conditions are less favorable for the mos-
q11ito vPctors. Mosqnitoes are biological vectors of these
viruses, which requires that the n1osquito actually becon1e
infected with the virus rather than simply transmitting it
mechanically. For a biological vector to bccomc infcctcd it
must obtain a blood meal from a viremic vertebrare host.
1'he leve! of viremia required to infect the vector is dictated
tJy viral strain, species of mosquito vector, or both. Upon
ingestion, thf> virus infects thf> insPct g11t ;.ind thf'n sprt>;.ids
to the salivary glands, where replication p rovides a ready
source of virus to infect additional vertebra te hosts during
insect feeding . The time requircd for this proccss is the cx-
trinsic incubation perioct. l)nce infected, the vector re-
mains infected for life.
EE-E virus iIJfectiun cduses clínica! disedse in huu1aus,
horsPs, and hircis, Pspecially ring-n Pcked pherisants,
ratites, and sorne commercial poultry (Pekín ducks, etc.).
EEE also has been described in cattle and pigs, sometimes
\.Yith high 1nortality. EEE virus cxists in two distinct ccosys-
te1ns; North An1erica11 (eastern Un1ted States and Canada)
and Caribbean strains of the virus are transmitted by
Culiselu melunuru rnosy_uitoe:;. 'l'ht: viru~ i:; u1<JiutaiI1t:ll ir1
;in Pnzootic cyrlP of infection that inclucies these or-
nithophilic mosquitoes ru1d the passerine and wading
birds that serve as vertebrate virus reservoirs in coastal and
inland S\-vamp env ironn1ents. Periodic spillover of the
virus occurs in al1jacent horses, humans, birds, and otl1er
animals. A variety of mosquito species can transmit the
virus cluri11g epitle111ics, aud cli1ec l horiLonlal spread oc-
curs among b irds vvhen they peck viremic birds. Another
n1osquito (Cu/ex n1elanoconio11) is responsible for transrnis-
sion of EEE ln Central and South America, where smal1
m;im mals an.d birds serve as the vertebrate reservoirs of the
virus.
'fhe host-virus relationship of WEE is similar to that of
EEE, v. ith thc virus bcing 1naintaincd in a transmission
1
cycle between inosquitoes (Culex tarsalis) and do1nestic
and passerine birds, with periodic spillover into humans.
horses, and domestlc birds. Other species of mosqultoes
can transmit the virus durine outbreaks.
The lifecycle of VEE i:; n1ore con1plex. There are severai
distinct clusters of VEE viruses (1ºypes I-Vl), most o~
which do not cause disease in horscs (1'ype 1, varietics D
through f, lypes 11-V l). ºlhese viruses are ende1ruc
throughout tropical and subtropical regions of the
Americas (including Florida, where VEE Type II [Ever-
glades] occurs). These endernic VEE viruses are main-
tai11cd il1a11atural cycle of infection between Cu/ex mos-
quitoes and small rodents in tropical swamps. Only VEE
Types 1 AB und 1 C are virulent to horscs, ¡u1d tl1ese are
only isolated during the epidemics of VEE that regular!~
occur in northern South America. lt is believed that thes.e
epidemic strains emerge after mutatlon of the E2 enve-
lope glycoprotein of endemic ·rype lD strains of VEE that
cousla11lly are circulaling in enden1ic areas but are 11ot
pathogenic to horses. Once strains of VEE e1nerge that
are virulent to horses and humans, infected horses are e
major source ofvirus because, unlike EEE and W.E.E, VEE
replicates to high titers in horses. 'fhus, the endemic anc
epidemic (epizootic) VEE viruses have very different cy-
cles of infection.
Pathogenesis and Pathology
Alphavirus infections can range from in.apparent to severe.
fatal discasc. Infcction follows thc bite of an infcctcd mos-
quito. Primary viral replication occurs i11 regional lymph
nodes adjaccnt to the site of the bite and is followed by
generalized infectlon and viremia in whlch thc virus repl.:-
c ;ites in the lyn1phoid tissues throughout the body, bone
n1arrow, and certain other tissues. The encephalitis viruses
likely gain entry into the CNS after replication in the en-
dothclial cclls lining blood vcsscls iI1 thc brain, and pas-
sage ofvirus to the brain occurs after the acute phase of i n ·
fection and virernia. Lesions occur throughout the gray
IIldtter uf the tJrain and ir1clude pt:rivast:uldr t:uffiug with
inflammatory cPlls, infiltration of neutrophils into the
gray matter 1-vith neuronal and parenchymal necrosis, a11d
vasculitis, thrombosis, and hemorrhage. Neutrophils are
especially characteristic of the early cerebral lesions of EEE
and VEE.
Host Response to lnfection
The dcvelopment of neutralizing antibody to encephalitis
viruses appears to be important in limiting viral replica-
tion and spread and is clearly important in preventing re-
lnfection. Antibodies develop to all viral proteins, and
peak titers of neutralizing antibody typically develop
wilhi11 2 Vl'eeks o[ infeclio11 h1 anin1als that survive. 'fhis
antibody may be effective in neutralizi..ng virus, enhancing
viral clearance, and lysil1g infected cells via complement
 Chapter 58 'J(Jgaviridae and Flaviviridae 353

u r Natural Kill<::r c<::lls. C<::ll-1ut:diatt:Ll ilnu1urllty alsu ap-
pears to contrihute to viral clearance and protective in1m u-
:iity. Cytotoxic T lymphocytes can be identified as early as
3 to 4 days postinfection.
La boratory Diagnosis
Alphavirus infection may he suspected hased on the occur-
rence of neurologic disease among susceptible animal
species in endemic areas, thus prior history and seasonal
occurrcncc ca11 be important. Dcfinitivc diagnosis rcquircs
laboratory confirmation, which usually is done by serol-·
ogy with an IgM-capture enzyme-linked immu nosorbent
assay (ELISA) tu Lletect virus-specific antiouuies in the
serum of acutely affected animals. The diagnosis is u n -
equivocally confir1ned by virus isolation from blood or
CNS tissue, the latter beü1g preterable due to t he variable
occurrence of viremia by t he time signs of encephalitis are
manifest in infected anin1als. Virus may be isolated in a va-
riety of systems, including cell cultures, chick embryos,
and sucklir1g mice.
Treatment and Control
·rhere is no treatment for clinically ill antmals. Control of
Alphavírus infections can be achieved through vaccil1ation
and pest111a11age1neJ1L prograrus. V1::ctor cuutrol cC1r1 ue Clp-
proachi>d through eliminating mosquito hreed ing site.~ hy
water control or spraying programs. In the case of domes-
tic bird farms, the use of tightly screened (insect-p roof)
reCJring pens CJnd locating such pens avvay from freshwater
s\o\ra1nps is also a possihilit·y . Atten11atP.rl (VEE) anrl inacti-
vated vacci.nes h.ave been developed for the equine en-
cephalitis viruses. Re&>ular vaccination of susceptible ani-
m.als is reguircd to inaintain immunity if inactivntcd
vaccines are usect.
Other Togaviruses
A nun1ber of other togaviruses can infect animals, includ-
ing Sindbis, Scmliki Forest, Higl1lands J, and C1etah
viruses. Highlands .l virus has a sirnilar distribution in
North America as EEE virus, b ut only rarely causes en-
cephalitis in horses. It ls, however, an important patho-
gen o f h 1rkt>ys, pht>as;:ints, c:hukrir partrjdges, ducks, emus,
and whooping cranes. Getah virus infect5 horses a11d
swine in Asia and Southeast Asia . Infection in horses is
sometimes characterized by fcvcr, rash, and limb edema
(but not encephalitis), and Getah virus also can cause
abortion in p regnant si;.vine. Getah virus is included
vvit hin the Semliki Forest virus complex of alphaviruses,
and Semliki Forest virus also can cause a febrile disease of
ho (.:;c;.:; in Africa .
FLA V/VIRIDAE
The family Flaviviridae contains a large number of viruses
within three antigenically distinct geneia (Table 58.1):
.Flavivirus, Pestivirus, and Hepacivirus (human hepatitis C
virus). Altl1ougl1 not dlscussed here, the family Flaviviridae

Tab 1e 5 8 . 1 . Flaviviruses ot Veterinary lmporta nce

Genus SerogroupfAgent Vector Affected s~cies Di:Stribiliion.

Ffavivirus
Tick-borne encephalitis group 1 icks Humans turope, Asia
Louping 111 T.icks Sheep
PUWilS~on Tícks Horses North America
Japanese enceflhalitis group Mosr¡uitoe.~
Japanese encephalitis virus Swine, humans, birds Asid
Murray Va11ey encephalitis Humans, birds Australia
'
virus
West N1le VlfUS (Kun¡in) Humans, horse-S, birds Americas, Europe,
.
Asía, Australia,
Africa
St. Louis encephalitis virus Humans, birds Americas
Yellowfever group Mosquitoes
Wes.selsbron virus Sheep Africa
Pestivi~us .Nonc
Sorder disease virus Sheep Worldwide
Bovine virus diarrhea virus Cattle \JIJorldwide
1and2
Classical ~wine fever virus Swine Wodrlwide*

· • Eradic¡¡tedin,a substantial number of countriés

354 PART 111 VLruses
includes a substantlal number uf very importar1t l1U111cu1
pathogens
fLAV IVIRUS
MPmhPrs of thc gcnus Flavivirus include arboviruses that
are transmittcd by cithcr ticks or mosquitoes. The tick
borne tlaviviruses include tick-borne encephalitls (Euro-
pean, Far Eastern, and Siberian), Powassan, and Louping-ill
viruses. T11osc that are mosquito-transmitted include the
Dengue virus group (the cause of human Dengue hemor-
rhaglc fevcr), Japanese encepl1nlitl:. viru:. group (i11cludiJ.1g
Japanese encepht1litis M11rrt1y Val ley encephalitis, St. Louis
1 responsPs arP <l i rPltf'd at the nonstructural viral proteins.
encepl1alltis1 and West Nile and I<unjin viruses), and Yellow
Fever virus group (including Yellow Fever and Wesselsbron
viruscs). Thcsc viruses cause either encephalitis or systemic
hemorrhagic-septicem1a 10 animals and/or humans.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
The namc Flavivirus is derived from thc Latin word flavus,
which 1neans yellow, since Yellow Fever virus is the famíly
prototype. Members of the genus consist of spherical (50
nm in diameter) enveloped particles with small surface
projection (peplomers). A single nucleocapsid protein en-
capsidates the geno111e 1 a11u l l1e e11velope co11lains two
viril! mPmhranP prot<>ins (F. and M). Severa! nonstructural
viral proteins are also produced in virus-infected cells
(NSl-5). ·rhe viral genome is a single linear strand ot
positive-sense RNJ\. The genomic RNA is infectious and
encades a single large polyprotein that is co- and post-
translationally cleaved into the various viral str1.tctural and
nonstructural µrulei11:>.
ThP flt1viviruscs are a ll serologically related as deter-
mined by group-spccjfic assays such as ELISA and hemag-
glutination inhibition. 'l'hey are distinp;uisl1ed by neu-
tralization assays, although there is considerable cross-
rcactivity among vlruses wlthln tl1e same serogroup (sero-
complex). ·rhe envelopc E protf'in rnn t;iins the major de-
terminants uf viru:> llt::Ulralizalion.
Resistance to Physical and Chemical Agents
Virions are stoble ata pH of 7 to 9, but are inactivated by
acid pli, temperatures above 40ºC, lipid solvents, ultravio-
let light, ionic and nonionic detergents, and trypsin.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
The t1aviviruscs ot veterinary importance are arboviruses
that are transmitted by either ticks or mosquitoes; thus
they share the ability for sequential replicntiu11 i11 verle-
brate and invertebrate hosts. ThPy infPc:t a range of verte-
urate svecies, including 1nan1mals and birds.
Pathogenesis and Pathology
}' /avivirus intection can produce inapparent to fatal dis-
ease. Encephalitis and hemorrhagic fever are characteristic
of Flavivirus-lnduced diseases, depending ur1 tl1c spccific
virus.
Host Response to lnfection
Both humoral and cell-mediated il11mune responses de-
velop in anima Is following Flavivirus infection. Antibodies
(HI and viral-neutralizing) develop within a few days after
infection. Neutralizing antibodies largely are directt>d ilt
the E envelope prutciI1, wl 1ereas cyloloxic T lymphocyte
Hun1oral responses appear to play a11 im.portant role in
both recovery and long-term protection against reinfec-
tion. Ccll-rnediated immune responses (cytotoxic T .ly1n-
phocytes) hkely contribute to viral clearance. Immunity is
probably lifelong after natural infectio11, b11t only to the
homologous viru:s.
DISEASES
JAPANESE ENCEPHALITIS
Japanese encephaliti:> virus OEV) i:. tl1e prololype of theJE
antigenic complPx within the Flavívirus genus. The JE
con1plex includes St. Louis encephalitis virus, Murray
Valley encephalltis virus, and West Nile virus (and related
Kunjin virus), in addition toJEV itseli. Theseare allhuman
pathogens, butJEV and west Nile virus are important vet-
erinary pathogens as well.
JEV infectlon Is typlcally inapparent l.Jut ca11 cause clin-
ical disease in hl1m;:ins, horsPs, ;inci swine. Tnfection of
dogs and do111eslic poultry also occurs. ·rhe major adverse
impact of JEV infection of swine is the occurrence ot abor-
tions ond neonatal deaths after infection of nonimmune
pregnant swine, whereas infected horses can develop sc-
vere neurologic disease that resembles eastern equinP Pn-
cephalitis (EEE), altl1uu~h u1orlality is lower.JEV infection
currently is confinP<l to temperate and tropical areas of
Asia, where serological survey:; indicate that infcction of
horses, cattle, and swine is widespread. ·rhe virus occurs
throughout much of Asia, fro1n the Indian Subcontinent
to the west to the l'aciúc Islands in the east. Most infec-
tions are inapparent or mild. Horses and humans are
unimportant to the epidemiulugy ufJEV i11feclio11 because
viremias arP. vPry low-titered in these species, insufficient
to serve as a virus reservoir for susceptible mosquito vec-
tors. In contrast, infected swine and birds serve as ampltfy-
ing hosts for the virus because they have high-titered
viremias. Cu/ex mosqu1toes are the principal vectors of
JEV. In swine, virus may also be transmitted from the in-
fected dam to the fetus and frorn l.Joar tu sow via insen1ina-
tion ofviral-cont;:imint1tP<1 semen. The virus is maiI1tained
il1 tropical regions probably by continua! transmission be-
tween mosquitoes, birds, and swine.
Diagnosis of JEV is accomplisl1ed by isolating virus

from the tissues or blood ot attcctcd animals using cell cul-
tures (vcrtebrate or insect) or suckling mice. Viral antigen
may also be demonstratcd dlrectly In braln tissue sections
by immunohistochemical staining A variety of serological
assays also are available, but den1on stration of virus-
specific lgM (indicativc of recent infection) is generally re-
quircd as IgG assays often detect antibodies to rclatcd fla-
viviruses, which complicates interpretation.
Vaccination is uscd for control of JEV infection in
endemlc a reas, and both attenuated and killed vaccines
are available. Vector con trol measures are another possi-
ble app1oacl1.
WEST NILE ANO KUNJIN VIRUSES
Wcst Nilc virus (WNV), a rncn1bcr of thc JEV antigenic
complex, occurs througl1out Africa as well as portions ot
Europe, the Middle East, Asia, and Australia (where it is
called Kunjln virus). '!"he virus recently emerged in the
Western Hemisphere, precipitating a massive epidemic of
discasc iJ1 hu111a11s, hotscs, birds, andan eno1111ou::. varieLy
of other animals (alligators, squirrcls, mountain goats,
cte.). Thc virus is maintaincd in a mosquito-bird cycle of in-
fection, and humans and horses are "dead-end" hosts be-
cause viremia is not sufficient to infect susceptible mosqui-
toes that feed on infected individuals. There are two
distinct genetic lineages of WNV, so-called "lin eage l" and
uli11eage 2." Li11t:age 2 viruses are e11dt:111ic soutl1 uf tl1e
eq11ator in Afrira, whC'rC' th<'y ca11st- littl<~ if any diseasP in
horscs. In disti nct contrast, li neage 1 strains of ~ havc
been associated with outbreaks of disease in Mediterranean
and castern Europc, North Africa, Asia, and North A1nerica.
Whereas many bird species undergo subctinical or
asympton1atic infection and have viremias of sufficient
111agnitude to serve as ampllfylng reservolr hosts of the
virus, other species of birds suffcr high 1nortality similar to
horses. ·rhe 11eu1ological discase thal occurs ir1 son1e
WNV-infected horscs is characterized by ataxia, weakness,
rccumbcncy, musclc fasiculations, and high fatality ratcs.
Among birds, raptors and corvids have been especially
hard hit by the WNV epidemic in Nortl1 An1erica .
Speclfic specJcs of mosqultoes transmlt WNV in en-
de1nic areas, whercas a wide varicty of mosquito species
have been ü1crin1inaled in thc trans111ission of WN\T il1fec-
tion in North Arnerica.
Diagnosis of WNV is best done by polymcrasc chain re-
action (i'CK), which is rapid and very sensitive. Virus isola-
tion in appropriate cell cultures also is possible. WNV-spe-
cillc IgM capture ELJSA Is very usefu1 for the serological
identification of animals that are acutely infected with
VVNV. Au iI1altivated vaccine currently is availa!Jle for pro-
tective immunization of horses.
WESSELSBRON
Wesselsbron v1rus is a membcr of the Yellov.r rever virus
antigenic group and causes disease of sheep in sub-
Saharan Afrlca. Subclln tcal lnfcctlon of cattle, horses, and
Cfzapter 58 Togaviridae and Flaviviridae 355
swine also occur, and the virus is a zoonotic pathogen. The
virus causes extcnsivc livcr necrosis, jaundice, subcuta
neous edema, gastrointestinal hcmorrhage, and fever in
infected sheep; mortality in lambs can be high, and abor-
tio11 is con1n1011 in prcgnanl cwcs. Wesselsbro11 vi1us is
transnlitted by Aedes mosquitoes.
Diagnosis may be based upon viral isolation using a va-
riety of cell cultures (13HK and lamb kidney), chick em-
bryos, or suckling mice. Vaccination and/or prior exposure
to other flavlvlruses compllcate the interpretation of sero-
logical assays. Attenuated vaccines are used to prevent the
tli~easl.! i111;:11dl.!111il.: arcas.
• r
LOUPING - ILL •
Louping-ill virus is a tick-transmitted navivirus that natu-
rally infects many anim.al species, including humans,
sheep, horses, deer, and birds. Thc virus causes encephalo- i
myelitis in sheep, and high mortality may resultwhen sus-
:i: 1
ceptible slieep art: i11Lrudu<.:l.!U intu ar1 enuemic area.
Louping-ill is characterized hy neurologic signs s11ch as hy-
pcrexcitability, cerebellar ataxia, and progressive paralysis.
·rhe ctisease derives its name trom the Ieaping gait some-
w,
<( •
times observed in ataxic animals. Louping ill is a zoo
noses, and dlsease also somctimcs occurs in cattle, horses
and goats.
::> •
Loupi11g-ill is cu11fi11ed Lo ll 11:: Britisli Isles a11d purtio11s
of continental Europe. Txndes rincinu~ ticks arP thP princi-
pal vector. Diagnosis of louping-ill is done by viral isola-
tion, immunohistochemical staining of CNS tissue sec-
'
•
tions from affcctcd animals, or scrology. Virus can be
isolated usinp; vertebrate or insect cell cultures and intrac-
erebr al inoculation of suckling rnice. Propagated virus can
!Je uefinitively itlt!Iltlfit!tl IJy viral neutralization.
l .011p ing-ill rfln hP controllrrl thro11gh tick control hy
dipping and spraying of sheep. A forn1alin-inactivated cell
culture-origin vaccine is available and effective. Lambs
born. to immunc dnms are protcctcd by colostral antibody
for approxirnatcly 4 months.
POWASSAN ANO RELATEO TICK-BORNE
ENCEPHALITIOES
Powas<;fln virus is the cause of a tick-transmitted en-
cephalitis of hun1ans and horscs in Norll1 An1erica. 'l'l1e
virus infects a wide variety of animal species, both lVild
and domestic, as well as :;everal species of ticks. lt causes
encephalomyelitis in horses that mimics that caused by
other flaviviruses like WNV, frorn which it m u st be <listín
guished by elther PCR assay or by virus isolation. Similar
tick-borne enccphalidities of humans are caused by related
viruses in Europe and Asia, including On1sk hernor1hagic
fever virus, Tick-borne cnccphalitis viruses (European, Far
Eastcrn, and Sibc.rian), and Kyasanur forest disease virus.
rhese viruses are maintaincd in a cycle of infection that in-
cludes ticks (which vcrtically transmit the viruses) and ver
tebrates (livesrock and dogs) that serve as amplifying hosts
356 PAKr 111 Viruses

of the virus. Encephalitis occurs sporadically among ani- caused severe, fatal mucosa! disease-like signs (see
mals in areas where these viruses are endemic. below) in calves when they first emerged, often ac-
co.111va11ied l>y wide:svread he111orrhages as a conse-
quence of thrombocytopenia . However, Type 11
strains of BVDV are now increasingly associatcd
PESTIV/RUS with mild disease i11 calves, similar to that produced
by most Typc I strains.
'fhe genus l'estivirus ineludes the etiologic agents of bovine 3. Mucosal disease is characterized by low morbidity
viral diarrhea (BVUV; ·rypes 1and2), border disease (BUV), but high morta]ity in cattle of several months to
and hog cholera (also referred to as classlcal swine fever severa! years of age. Tt is characterized lJy severe,
[CSFV]), all of whicl1 are important pathogens of livestock. fata l ciis<"ase with f'Xtt>nsive.11lc:e.ration of the upper
In contrast to the gen.us Flavívirus, members of the genus gastrointestinal tract, diarrhea, and lymphopenia.
Pe:sliviru:s ar1:: 11ul arllirovud-bor11e. They a1e ieadily inacti- Mucosal disease occurs only i.n persistently infected
vated hy low pH, heat, organic solvents, and detergents. animals t hat were originally infected with BVDV in
Virio11s are spberical to pleomorphic (40- 60 nm in diame- u tero, thus it 11as a 11ighly distinctive patl1oger1esis.
ter) a11d enveloped wi.th small surface projections (spikes)
cmanating fro1n the viral envelope. The virions consist of BVDV also causes reproductive losses in cattle from
an envelope and nucleocapsid and include four structural abortion and fetal tcratogenesis.
proteins- a nucleocapsid protein (C) and three envelope
glycoprotein~ (Ern•, El, anti E2). Su1ue seve11 ur eigJ·1t viral
nonstr11ct11 ral p rote.ins also are produced in infected cells. Etiologic Agent
The genome is a single molecule of positive-sense, single-
stranded RNA that contains a single, large open reading Physical, Chemical, and Antigenic Properties
frrunc that cncodcs a largc polyprotein that is co- and post- BVDV is the type species of the Pestivirus genus (Fig 58.1);
translationally cleaved into the various structural and thus it is serologically related to hog cholera and border
nonstructural viral proteins.
111e pestlvlruses are all antigenically related and in-
clude cross-reactive epitopes. Several other pe.stivin1st>s
have bee11 isolated that are ge11etically distinct from BVDV
(fypes 1 a11d 2), BDV, and CSFV, and it is p roposed that F 1G u R E 5 8 . 1 . Negatively stained preparation of bovíne virus
pestiviruses isolated from a giraffc and from rcindccr, for diarrhea virus. 250,000X. (Reproduced with permissíon from Chu HJ,
Zee YC. Morphology of bovinc viral diarrhca virus. Am J Vet Res
example, be identitied as new species. Neutralizing anti-
1984;45:845.)
bodies are directed against the Erns and E2 envelope glyco-
proteins. Infected animals also mount a strong immune re-
sponse against the NS3 protein, whereas the antibody
respo11se to otl1er viral proteil1s is ge11erally weak.
All three viruses are i1nportant food-animal pathogens
and will be dcscribcd scqucntialJy. Thcy are vcry closcly rc-
lated and can only be distinguished by application of
monoclonal a11tibodies and/or molecular biology tech-
niques; however, they tend to be host-specific.

BOVINE VIRUS DIARRHEA VIRUS

Di sea se
BoviI1e virus diarrl1ea vir us (BVDV) is responsible for bovine
virus diarrhea-rnucosal disease (BVD-MD). Tl1is disease com-
plcx actually includcs t hrcc distinct disease syndromes:

1. Type 1 viral diarrhea is a usually rnild disease of young

calves characterlzed by leukopenia, fever, an<.l ero-
sions and ulcers of the upper gastrointestinal tract,
oflen wilh accon1pa11yiI1g diarrl1ea. T11e il1fection is
typically characterized by hlgh morbidity and low
mortality.
Z. ·1ype .U viral diarrhea is the consequence of infection
of calves with genetically distinct strains of BVDV
(so-called Type JI BVDV). Tl1ese viruses 1nit1ally

diSease vtruses. ·rhe two types of BVDV (fyv1::s I a11Ll TI) aie
distinguishcd by genetic ancl antigenic methods (using
m onoclonal antibodies in particular). It also is clear that
there is cnormous genetic variation among field strains ot
BVDV of bolh typcs, which likely influences their biologic
;>ropertics.
'l'here also are two distinct biotypes of BVDV; one is cy-
:opathogenic for cell cultures anc.I the other is I1ut. TIJe cy-
ropathic strai ns apparently ari.se. hy mutational or recombi-
n ational events in noncytopathic strains (deletion or
~eduplication of the viral genome or insertion ot host cell
scquences into the genome of the noncytopathogenic
virus).
Resistance to Physical and Chemical Agents
BVDV ts sensitive tu lipi<.l :sulvt:11ls such as elhe1 and cl1lo-
ro form anrl is inactivated by treatment with trypsin. The
,;rus is most stable in the pH rangc from 5.7 to 9.3, with
maximum stability at pH 7.4. 'fhc vi rus is readily main-
rained in a lyophilized or frozen state for many years.
nfectivity for Other Species and Culture Systems
Pestiviruscs infect cattle, sheep, goats, pigs, and wild rumi-
nants, l>ut BVDV vri11cipally is a11 infeclious pathogen of
c;ittlP an<I sometimes sheep. Isolates of BVDV, whether cy-
topathogenic or noncytopathogenic, can be grown in ccll
culture systems, il1cluding various bovine embryonic cell
cu ltures. lmrnunofluorcscence or other immunohisto-
chemical staining methods are used to detect noncy-
topathogenic strains of BVDV, which contaminate many
commonly used cell Unes. Cytopatt1ogt::uiL straius vru-
d uce plaques and can be userl for ac-c-11rate viral titrations.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
BVDV has a worldwide distribution, and inlection ot cattle
is vcry common, as determined by serologic surveys.
Transmission is by contact and horizontal transfer of
virus, particularly frorn persistcntly i nfected carriers of the
vtrus that shed high titt::rs of virus i11 a 11 of lheir body sec1e-
tions ancl exc-retions, and transmit the virus vertically to
t heir progeny.
Pathogenesis and Pathology
The conscquences of infection of cattle with BVDV vary
from an tnappart::nt infeLtiu11 tu scv1::rt:: (atal disease. The
pathogenesi~ ;.in<I conse.que.nc.cs of BVDV infection of
cattle are dependent on thc age and immune status of
cattle at infcction, as '1-vell as the biological properties
of the infecting virus strain. 'I'ype l and 11 BVDV infection
1n calves results in systemic infcction of variable severity.
l esions in affected calves range from mild erosion and ul-
cerat1on of the upper J:laStroi11tc.sti1111I tr11ct to .severe ul-
ceration thro11gho11t the gastroi nte.sti nal tract and di.s-
.sen1 i nated hemorrhages (characteristic ofType II DVDV).
Cliapter 58 Togaviridac nnd Flaviviridac 357
Highly virulent strains of DVDV can produce severe le-
sions in susceptible calves that mimic mucosa! discase
(sec bclow).
Fetal infections are important to both the epidemiology
of BVDV infection of cattle and to the pathogenesis of mu-
cosa! dtsease. BVDV lnfectlon ofbovtne fetu:se:s witl1a11011-
cytopathogenic strain of Bvn vir11~ prior to micl-ge.station
oflen leads to tl1e birth of calves that are persistently in-
fccted with BVDV (if they survivc infection). These ani-
mals subscqucntly serve as a reservoir of virus in the herd,
which they transmit both hor1zontally and vertically (to
their own progeny); furthermore, they develop weak orno
obvlous humoral lmmune response to tl1t:: virus witli
which they are persistently infecte<t . M11cosal <lisease man-
ifesls when a cylopatbogenic strain of BVD virus emerges
in thc animal. Interestingly, the noncytopathic and cyto-
pathic strnins of BVD virus isolated from cases of mucosa!
disease are extremely similar gcnctically. Recent evictence
clearly índicates that the cytopathic virus arises from
changes (genome deletlon or reduplication or insertion of
ccllular gene sequences into the viral genome) in tl1e orig-
iual vcr:.istc11t 11u11cytupalliic :.llai1i. Mucosal disease is
characterized by the presence of erosions and ulcers on thc
muzzlc, throughout thc ornl cnvity, esophagus, and small
intestine (ulcers over Peyers patches are characteristic).
There also is widespread necrosis of lymphocytes. The
mechanlsm of tissue destruction tn this fulrninar1t <.liseast::
currcntly is uncharacterized h11t might involve. apoptosis
of cells.
BVDV is capable of crossing the placenta of immuno-
logically naive pregnant cattlc, and BVDV infcction of
the developing tetus can result in fetal death, developmen-
tal abnormalities, or persistent infection in fetuses prior
to mid-gestation. Ocular leslons (retlnal degeneration
and hypoplasia, optic neuritis) and cerebellar atrophy/
hyvoplasia art: Ll1aracLe1islic of fetal BVDV infection.
Host Responses to lnfection
BVDV infection of cattle usually results in the production
of high tlters of neutralizing antibodics in serum, and cat-
tle that recover from infection have long-lasting immu-
ntty. BVDV infectiuu uf aniiual:; i11 ule10 can 1esull in pe1-
sistent po~t-natal infe.c.tion, and these animals have no or
very Iow titers ofvirus-spccific antibodies.
Laboratory Diagnosis
CUnlcal dlagnosls may be d!fflcult. Mucosa! dise11st:: 111u:st
be differentiated from other rlisea~e<: that cause nlc.e.ration
of lhe gastroil1testi11al tract, including malignant ca-
tarrhal fever and rindcrpest. Rapid diagnosis is best ac-
complishcd by immmunohistochcmical staining of the
tissues ot attectect anirnals with ~VUV-specific antibodics,
or by poly1nerase chain reaction to detect viral nucleic
acld. Virus isolation also can be uscd, as can serology;
however, the widespread use ofvaccines complicates sero-
lugicaJ diaguosis of BVDV infec 1io11, as does ll1e fact that
persistently infected cattle have little or no obvious titers
to DVDV.
358 PART 111 Viru:;e:;
Treatment and Control
Control of BVDV infcction of cattle is generally achieved
by vaccination wlth cithcr attcnuatcd (live) or k.illed BVDV
vaccines, and by the identification anc.1 removal ot persist-
entlv , infected carriers of the virus. Concerns have been
.ratsed about the use of Uve attenuated BVDV vaccines,
including their potential reversion to virulence, dissemi-
rtaliu11 ir r callle pupulaliu11:,, a11d pole11 lial lo cause i111-
munosuppression as vvell as fetal infections. ln the past, in-
advertent contamination of bov ine cell cultures used for
vaccine p roduction wit h virulent but noncytopathogenic
BVDV strains has also occurred. Vaccination of persistently
infected cattle vvith attenuated vaccines can son1etilnes
produce mucosa! disease.
Si11ce colosl1al anlibodies n1ay persisl for 6 n1onlhs or
more in calves, vaccination prior to this time may not be
effective and repeated re.vaccination muy be ncccssury.
BoRDER D1sEASE V1Rus
Disease
Border disease (BD) is a congenital Pestivirus disease of
shccp charactcrizcd by thc birth of lambs with ubnormal
("hairy") \.YOOI and tremors that ret1ect abnormal n1yelina-
tion in the central nervous system (so-called "hairy-
shaker" lambs). Expression of disease varies depending on
gestational age of the lamb at infection as well as the in-
fecli11g viral slraln. I11feclio11 of lhe developing fe lus n1ay
result in fetal death, rnumrnification, abortion, stillbirth,
or birth of ahnormal lambs, whcrcas infcct ion of adult
sheep is subclinical.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
13order disease virus (I3DV) is a Pestivirus that is genetica1ly
a11d antigenically distinct from bovine virus diarrhea
(J3VDV) and hog cholera viruses. Multiple strains of BDV
I1ave been isolated, n1ost of which are noncytopathic i11
ccll cultures. Sorne viruses isolated during outbreaks of RD
are in fact BVDV.
Resistance to Physical and Chemical Agents
Border disease virus exhibits physicochemical properties
similar to BVDV. The virus is inactivated by chloroform,
ether, trypsin, and heating to 56ºC for 20 minutes.
lnfectivity for Host Species and Culture Systems
While Bl)V is classically associated with infection of sheep,
it also can infect cattle and goati:, and pregnant sowf: that
\.Yere experimentally infected with BDV gave birth to
piglets with cerebellar hypoplasia. ·rhe virus can be propa-
galed in prin1ary and seco11dary c.ell cultures of bovl11e a11d
ovine or1g1n and in established cell lines, including :::_:
kidney, fetal lamb muscle, and bovine turbinate.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
BDV was initially described in thc late 1940s as a congcni-
tal disease ot sheep in .England and \!Vales. ·rhe disease has
since been rccognized in many European countries, Au:;-
tralia, New Zealand, Canada, and the United States. "fhe
virus persists in shef>p, likf>ly in ;i m;innf>r simil;ir to that of
BVDV in cattle, a11d extensive outbreaks of J3DV can occu::-
when the virus is in troduced into im1nunologically naive
flocks. 'frunsmission of BDV is probably most co·m.moo b~­
the oral and intranasal routes, from infected ani.Jnals or in -
fected fetuses.
Pathogenesis and Pathology
BDV infection of immunologically naivc sheep results ir:
vire1nia, with subscqucnt sprcad of thc virus to tl1e fetus ir:
pregnant animals. <Jeneralized infection of the fetus can
lcad to fetal dcath, with or without expulsion, or teratoge
nesis. The consequences of fetal infection are inversely re-
lated to fetal age, thus infection during the first trin1ester is
relatlvely more severe ttJan infectio11 later iu ge~tatiou_
Tnfection after 80 days' gf>st;ition often resul ts in vira
clec1rance without disease. Tl1e teratogenic effect of BD\-
ü1fection also is dependent on fetal age. fnfection of the
developing ce11tral nervous systcm lcads to rcduccd or al-
tered myelination and demyelinization, resuJting in the
cl1aracteristic congenital tremors of the affected newborn
1an1bs. Necrosis of the developing brain can cause more se-
rious developmental injury, resulting in hyd ranencephaly,
porencephaly, and/or cerebellar dysplasia.
Host Response to lnfection
BDV infeclion of ad ull sl1eep usually results in a pron1pl
hu1noral im1nune response characterized by the appear-
uncc of long-livcd ncutralizing antibody in scrum. T'hc
fetal immune response ret1ects 1ts gestat1onal age at the
ti me of in fection . Infection during the first half of gesta-
Liuu usually lead:s lo pt::rsisle11t posl-nalal infeclion anda
rninimal, inadequate irnmune response. Such animals
may rcmaln persistently lnfected and seronegative to BDV
for the remainder of their lives. Infection during the sec-
ond half of gestation, at a time when the fetus is gaining
immunologic competence, usually results in im mune re-
sponses t hat resolve the infection.
Laboratory Diagnosis
Tl1e clinical signs of BDV in affectecl lambs are variable, but
detailed necropsy and histologic evaluation is often diag-
nostic, espeeially when immunol1istochemical staining is
used to identify viral a11tigens in the t issues of ;ifff>ctf>cl
lan1bs.

Treatment and Control
Control of BDV is difficult. Losses can be high during ini-
tial infcction of nnivc nnimnls, whereas losses are relatively
minimal when the virus is cndemic on a tarro. BVDV vac-
cines are sometimes used.
HoG C HOL ERA V1Rus
Di sea se
Hug <.:hulera (I-TC, a l:.u kuvwu as clussicul switte feve1) is an
importilnt cliSPilSP of swinP worlclwiclP; ;ilthough it has
bee11 eliminatcd in many inten:;ive :;wine-producing
countries but otten reemerges to cause serious, economi-
cally devastating out breaks. HC is characterized by fever
(104ºF or hlghcr), leukopcnla, and loss of appetite.
Affectcd anilnals may appear dull and drowsy and crowd
togcL11cr as if ch illcd. Von1 i ting and diarrhca are con1111011,
as is conjunctivitis, erythema of the skin, and neurologic
signs such as paralysis and locomotory disturbanccs.
!nfectio n of pregnant sows can result in small litters, fetal
death, premature births, stillbirths, and the birth of
piglets wlth cerebellar ataxia or congenltal tremors.
!viorbidity and mortality are both high during epide-
nlics caused by virulent strains of the virus in fully suscep-
tible swine, whereas disease is less apparent when the
virus is endemic, making detection and eradication more
difficult.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
Hug chulera virus (HCV) is a n1en1ber of the genus Pesti-
virus , and there is considerable genetic and antigenic vari-
ation among strains of thc vi rus.
Resistance to Physical and Chemical Agents
HCV is chloroform- and cthcr-labile and relatively pH sta-
ble. Virions are quickly inacllvated by drying but can per-
sist for long periods in uncooked pork or garbage. The
virus is complctcly inactivatcd in canncd hams whcn an
interna! temperature ot 65"C is maintained for 90 min-
utes. The virus survives for 3 days at SOºC in defibrinated
blood.
lnfectivity for Other Species and Culture Systems
Domcstic swinc and wild hogs are the only naturally sus-
ceptible species.
HCV replicates in cultures of porcine cells such as
spleen, kidncy, tcstlclc, and perlphcral blood lcukocytes.
Vfost strains of the virus are noncytopathogenic and
n1ay persisl in cullure for n1any cell passages. Tl1e pres-
ence of HCV in infcctcd cell cultures is rcadily demon-
stratcd by immunofluorcsccnt or imn1unohistochcmical
Chapter 58 Togaviridae and Flaviviridac 359
stainlng rechnlques. Sorne cytopathogenic strains have
been repnrtPcl .
Host-Virus Relationship
Distribution, Reservoir, and Transmission
HCV occurs worldw1de, but it has been eradicated from
Nnrth AmPrir;:i, l.ri>;:it Rrit;:iin ancl lrPlancl, Scanclinavia,
Australia and Nelv Zealand, and portio ns of Europe. It is cn-
demic in extensive areas of Asia, Africa, Europe, and South
and Central Amcrica.
uomestic swine and wild hogs serve as reservoir hosts,
often as inapparent carriers. Pigs infected in utero may bc-
come persistently tnfcctcd carriers of the virus, analogous
to th P rP,Prvnir~ nf nthPr p1>,tivir11'P' li k P RVnV ;inrl RnV
·rransmission is by droplet, fomites, and ingestior1 of in -
fccted matcrials, particularly uncooked garbage.
Pathogenesis and Pathology
Hog cholera Is an acure, hlghly contaglous disease that is
ch;:ir;irtPri7Pci by cli~~Pmin:itPcl intr;:iv;isr11l;:ir co;:ig11l;:ition
leading to hemorrhage and infarction in many tissues. The
incubation period is short (3 to 8 days), and the virus first
replicates in the lymphoid tissues of the upper respiratory
tractor tons1ls. The virus thcn sprcads \Vtdely and repli-
cates in endothelial cells and mononuclear inflammatory
cells throughout the body. The characteristic lesions are
petechial hemorrhages on a 11 serous surfaces, lyn1ph nodes
(hen1orrhagic lymphadenitis), and kidney, and the pres-
ence of infarcts in the spleen.
More chronic forms of HC occur in somc cndcmic arcas.
Affected pigs may exhibit growth retardat1on (stuntrng),
chronic diarrhea, and secondary bacteria! pneumonia.
Host Response to lnfection
Animals that recover from hog cholera have a long-lasting
immunity. Ncutralizing antibody titcrs corrclatc with rc-
sistance to HCV intectio11. ~uck.ling pigs acquire colostral
antibodi es from the immune dan1. The half-life of this
colostral antibody Is 13 days. Pigs that have maternal anti-
body titers of 1: 1000 or above still have son1e antibody at 4
IJIUll tllS.
Pigs infected in utero with the virus are often per-
sistently infected carriers, \>Vhether or not they are healthy
at birth.
Laboratory Diagnosis
Diagnosis of hog cholera can be suspected in free areas by
cxplosive outbreaks ot severe disease in pigs, but the dla;-
nosis always requires laboratory confirmation to distm
guish it from other septicemias. Thc virus is identified in
the tiSS"1.1es of affected pigs by immunohistochemical min-
i11g Uf IJy V ÍfU:> Í:>VlcllÍUfl ÍCUlll tlH.: S)JlCCU, tun~ib. }vmph
nodes, and hlood. Sinc:P- many strains arP nonry1<''12-'-
genic in cell culture, the fluorcscent antibody :I!et=:oc ~
360 PART 111 Viruscs
1equired for tl1e deleclion of HCV. Polyn1era~e chain reac-
tion detection 11ow also is available.
The diagnosis of chronic forms of HC is more difficult
and requires caretul laboratory investigation.
Treatment and Control
Control of HC depends on whether the virus is endemic
in a particular countr y or region. In free areas, exclusion
of ll1e virus is accon1plished by regulati11g tl1e n1ovem~
(importation) of swine from endemic areas and by prc-
hibiting the fecding of garbagc and/or food scraps COG-
taining pork products to swine. In endemic areas, vacn-
nation and/or eradication are used. Vacch1es for HC'\. <:..:-:e
attenuated and, although effective in preventlng disea..~
they complicate efforts to eradicate HCV from a region -
counlry.

ORTHOMYXOVIRIDA E
:nfluenza viruses are included in the family Orthornyxo-
'iridae, and are divided into thrcc typcs (A, B, and C)
based on the antigcnic diffcrcnces beti,,veen their nucleo-
JJlU Léi11 (NP) d!IU llld l 1 Í)I (lVf) !J I Ult:i 11:..
-Uuses naturally occur in humans, horses, S\.vine, and
oirds. Typcs B and C infcct humans, and ·rypc C also in-
:ects pigs. lnttuenza A viruses are further divided into sub-
rypes. The nomenclaturc systcm includes the host of ori-
~in, geographic or!gln, straln number, and year of
solation . A description of thc two majar surfar.e antigens,
_he he111aggluLinin and ll1e 11euran1inidase, is given in
]arentheses-for example, A/swine/New Jersey/8/76 (Hl
"I ). Gy convention, the host of origin for human strains
s now omitted.
~orphology
•:1f1uen2a virus particles a rP i rrPg11 l;:i rly sh;:i perl spherical
? ar licles 80-120 111n in diameter (Figs 59.1, 59.2). The
~irion envclope is derived frorn the membranes of host
cells. Thccc are two distinct typcs of surface spikes (pe
µto n1ers); one is rod-shapcd and corresponds to the
nemagglutinin (HA), and thc other is mushroom-shaped
anct possesses ncuramlnlclase (NA) activity. Butl1 the HA.
and the NA are v iral glycoprotPins th ;:it ;:itt;:ic.h to the viral
envclopc by short sequences of hydrophobic amil10
3cids. The viral envelope surrounds a matrix protein (M)
shcll, which in turn Sl1rrounds thc gcnome of eight
seven in ·rype C influenza viruses) individual molecules
of single-stranded RNA, along with the nuc!eoprotein
:-.IP) and thrcc large protelns (PB 1, PB2, ancl PA) that are
;esponsible for RNA replication and transcription. Each
Jf Llte eigl1t ge11v111ic RNA species encades fo1 one, 01
someti1nes two, polypeptides. l'his independent nature
of the individual viral gene segments results in the phe-
:iomena of high trequency rccombination (rcassortment
of gene scgmcnts from two parental viruses to generate
progcny wlth new genotypes) during mixed infections,
'lnd explain~ thP origin of <:omp nPw p;:in<1Pmir ~trains of
!n fl u enza vi rus.
Orthomyxoviridae and
Bunyaviridae
ALEX A. ARDANS N. }AMES MA<..:LACHLAN
Viral Proteins
Hemagglutinin
All strains of influenza are capable of agglutinating ery-
Ty jJt: A i11 fl Ut:l IL<l
throcytes from humans. guinea pigs. and chickens as well
as many other species. Antibodies to the HA prevent infcc-
tion of host cclls. The HA and NA are the twO ma¡or strain-
specific surface antigens and are important to host immu-
nily. In facl, il is Lhe va1iation of lhese 1uolecules Lhal is
primarily responsiblc for cmcrgcncc of new strains of the
virus leading to ncw outbreaks of influenza, and the fail-
ure to control thcm by vaccination. ·rhe HA tunctions in
inítial virus attachment to its cellular reception, and sub-
sequent HA clcavagc allO\VS fusion of the viral envelope
with an intcrccllular mcmbrane allowing transfer of the
nuclcuca11:.i<.l i11lv ll11:: ct::ll cytuvla:.u1. The1e a1e 110\"\T 15
rc.cognized influenza HA proteins.
Neuraminidase
·rhe NA is responsiblc for the cleavage of the sialic acid-
containing rece11tur ari<.l thc elutitJ11 of lhe viral parlicle
from th<' host c.c~l l. This phenomenon prevents self-
aggrcgation and promotes release of the virus from thc in-
fected cell. Antibody against the NA does not protect
against infection but does confcr protcction against dis-
ease and reduces transmissibility. There are nine recog-
nized NA subtypes.
Nucleoprotein
'íhe nucleoprotein (NP) was originally designated the sol-
uble, or S, antlgcu <111tl i:> thc iJ111cr111v:;t cuu111011e11t uf t lte
influenza virion. lt is coiled into a double helix SO nm to
60 11111 in dia111eler and is il1timately associated wit11 each
RNA segment and the three different polymerases.
·rhe NI' is one of the type-specific antigens uscd to dis-
tinguish genera ot intlucnza virus, and can be identitied bv
enzyme-linked immunosorbent assay (ELJSA), double im-
munodiffus!on, complement fixation, single radial diffu-
~ion, ::ig;:ir-gPI prPripit;:ition, ;in<l tht> hem;:iggl11tin::ition in-
hibition tests.
361
362 PART 111 Viruses

F1G U RE S 9. 1 . Negatively stained preparation of influenza A virus. 150,000X.

(Reproduced with permission from Air GU, Laver WG. The molecular bas1s of antigenic
v;iri;itinn in influP.nz;i virus. Adv Virus Res 1986;31 :53.)

F 1G U R f ~ q. 2. Diagr;im of infl11P.nza virus. The diagram illustrates the main

structural features of the virion. The surface of thc p;:irticlc cont;:iins thrcc kinds of spike
proteins- the hemagglutinin (HA), the neuraminidase (NA), and matrlx (M2) protein-
embedded in a lipid bilayer derived from the host ce// and covers the matrix (M1 J protein
th;:it surrounds thc viral corc. The ribonucleoprotein complex making up the core
consists of at least one of each of the eight single-stranded RNA segments associateú
with the nucleoprotein (NP) and the three po/ymerase proteins (P82, P81, PA). RNA
segments have base pairing between their 3' and 5' ends forming a panhandle. Their
u1ydt1iLdliu11 dllÚ lile role of NS2 in tfle virion 1·e1n<1in unresolved. (Reproduced with
permission from Murphy 8, Weneter RG. Orthomyxoviruses. In: Fields 8, Knipe DM,
Howley PM, et al., eds Fields virology 3rd Prl NPw York· I irrinrntt-RavPn, 1996:1401.)

ANA ., ..- PB2

u--PB1
lk"--PA

-NP

.
RNP

Matrix Protein
Thc nonglycosylated matrix protein is also a type-specific
antigen of influenza viruses. I-Iowever, antibodies against
M provi.de little, if any, protection against infection. This
structural proteln surrounds the nucleoprotein to form
the inner part of thf' vir;i 1 f'nvf'lOpf'.
Nonstructural Proteins
There are at least two nonstructural proteins, NSl and NS2.
Thcir function is at this time tu1known.
Polymerase Proteins
Thc polymcrase protcins (PB1 , PB 2 , PA) localize with the
NP and viral RNA. They are Lhe largesl o( Lite viral pruteiu:;
and are responsible for RNA polymerization of the viral
gcnome. PB 1 and PB 2 are necessary for complementary
RNA synthesis, while PA and NP are required for viral RNA
synthesis.
Viral Genome
Th e influenza A genome contains at least 10 different ope.n
reading fra1nes (genes) it1 ils 8 segn1e11Ls of negative-seuse
RNA. The segme11ted genome of influenza viruses facili-
tates reassortment, resulti11g in the production of new
strains. Influenza viral variation is frequen t and occurs in
two ways, drift and shift. Antigenic drift is due to point
rnutations resulting in amino acid substitutions mainly iJ1
t he HA protein. Antigenic shift occurs with reassortment
of individual RNA segments when a cell is infected with
n vo different influen7.a vin1ses, genPr:iting nf'w vin1Sf'S
that cause pa11den1ics of J1un1an influenza.
tQUINE INFLUENZA
Disease
Influenza is an acute respiratory disease of horses. While
h orse.s of all ages are affected, thu:;e 1Jetwee11 2 a11d 6
months of age are at greatest risk. The mortal it.y rate of
equine influenza is lowwhereas the morbidity rate can ap-
proach 100%. The incubation period lasts from l to 3 days.
!)isease is manifested by a high fever, up to 106ºF, which
t:ists about .3 days. lJther clinical signs include a frequent,
&trong, dry cough lasting 1 to 3 weeks, a nasal discharge
tl1at is iuitially serou:; ir1 character tJut that later tJecorr1e.s
m ucoid, anorexia, depression, photophohia, l:icrim:ition
'ith a mucopurulent ocular dischargc, and corneal opac-
~ty (somet iroes with Joss of sigl1t). LiJnb edema and 1nuscle
soreness can occur and, in sorne severe outbreaks, acutc
,eaths due to fulminant pneurnonia have occurred.
Enteritis was described in a 1989 outbreak in northern
Cl1iua due tu a new ey_uine influenza virus, A/equine/2/
~~lin ; this virus is considered to havP PmPrgPcl from birrls
~:ltO b.orscs.
Chapter 59 Orthomyxoviridae and Bunyaviridae 363
F.q11inf' infh1Pn.za virnsf's arf' TypP A virnses. They have
been divided into two subtypes, A/equine/1 (I-I7N7) and
A/equine/2 (H3N8), based on antigenic differences in their
HA. A/cquinc/l has onc prototypc, A/cquinc/l/Praguc.
Antigenic drift has occurred among A/equine/l viruses
with the subsequent designation of two subgroups that do
not appear to differ significantly in terms of their immu-
nity after vacci.nat.io.n . Significant drift has occurred
among A/equine/2 viruses fron1 the original p rototype
A/equine/2/Miami/63 first isolated from a severe 1963
outbreak in Plorida horses. Additional prototypes
A/eq uine/Z/ J!ountainbleu, A/equine/2/Kentucky, along
wit h other variants including the 1989 Chinese virus, have
shown that considerable drift occurs in the A/equine/2
viruses, \\•hich m ay have implications for efficacious vacci-
nation. Influenza occurs in horses worldwide, wilh the no-
table exceptions of countries such as New Zealand and
Australia, and is a common and troublcsome problem
whe11 horses are congregated at shows, sales, stal)les, and
- -
r.acetracks.
Etiologic Agent
Resistance to Physical and Chemical Agents
Equine iníluen:.:a vir us is usually iuactivated at 56ºC ir1 30
minutes. Like Type A tnfluenza viruses, the virus is inac.ti-
vated by phenol, lipid solvents, detergent, formalin, and
oxiclizing agents such as ozone.
lnfectivity for Other Species and Culture Systems
Equlne Jr1fluenza virus Jufects hurses, asses, and 1nules. lt
can he experimentally adapted to infer.t m ice when intro-
duced intranasally. Ali orthon1yxoviruses, including
equine influenza virus, can be propagated in the amniotic
cavity of embryonated chicken eggs. Equine influenza
virus can also be grown in chick embryo kidney, bovine
kidney, rhesus monkey kidney, and human embryo kid
ncy cells.
Host-Virus Relationship
Pathogenesis and Pathology
Equine influenza virus infects both the upper and lower
respiratory tracts. Thcrc is an carly lymphopcnia with ac-
companying e11largement of the lymph nades of the head.
Initially there may only be a slight serous nasal discharge,
which later becomes mucold. Fatal p11eurnonia can occur
in foals ancl on occasion in older animals. Occasionally
there is ventral edema of the tru11k and lower lin1bs.
A/equine/2 has been known to cause a postinfection en-
cephalopathy in foals. Catarrhal and cvcn h en1orrbagic;;
ent eritis 1nay occur. Necrotizing bronchiolitis in which the
bronchioles are progressively obstructed is characteristic of
equine lnfluen.za. Severe necrotiZing myositis with ele-
vated serum enzyme levels has been observed wit h A2 in-
fections. Mosl a11in1als recover wilhin 2 Lo 3 weeks; Ll1ose
364 J'ART IJl Viruses
that do not may develop chronic obstructive pulmonary
disease (COPO). Prolonged recovery and severity ofdisease
appear to be related to the level of stress an affected horse
undergoes, thus adequate rest is important for recovery.
Equinc influenza virus is most commonly transmitted
vía aerosol and can spread cxtrcmely rapidly dueto the frc-
quent, viulcnt cuugh uf iiúc1.:tcu hurses. 111fecteu a1li111als
contin11c> to c;hc>cl virus for ahout .'i days after thc first signs
appcar. ]'he virus also can be sprcad by fomites.
Laboratory Diagnosis
/\. t entativc clln ical diagnosis of equine influenza can be
madc fron1 thc charactcristic r<1pirl sprt>;icl of thc> <lisp;ise,
especially an1ong stabled hor:;es, and the frequent dry
cough of affcctccl horscs. A d efinitive diagnosis requires
isolation of thc vi rus, dcmonstrntion of virnl nntigcn, or
demonst ration of risin¡; antibody titers between acute and
convalescent sera by complcmcnt fixation (CF) and
hen1agglutination inhibitlon (HJ) tests. During outbreaks
it is in1portant for the success of futurc vaccination pro-
gra111~ Lu i~lJialt: auu typt: Lhe causalive slrains of inJluenza
virus.
Treatment and Control
Vaccination is somc"vhat cffcctiYe in preventing influenza
ir111ursl':>. Prutcltiuu, huwcvcr, is ucpc11ue11L u11 Liie 111ar1-
nf'r of vaccination anrl th<' quality of the vaccine, \Vith par-
ticular cmphasis placed on proper selection of vaccine
strains. Available vaccines contain inactivated virus of
both A/equine/1 :ind A/ equine/ 2 subtypes. Classically,
A/equine/Prague/56 (H7N7) and A/equlne/l\.1iarnl/63
(H3N8) havc bccn 11scrl as thc prototype A/equine/1 and
A/equinc/2 strains, rcspectively. There is increasing evi-
dence of antigcnic diversity among conten1porary equine
influenzas that circulate in nature, suggesting that the ef-
fectiveness of the conventional vaccine strains in provid-
ing protection will become limited with time. for this rea
son, current vaccincs include sorne of the newer variant
A/equine/2 viruscs. Thc various inactivated vaccines avail-
¡itJle ilre i11curpurc1 leLI wit l1 aLljuva11l;:, l11al have p1oved lo
significan tly augment their im111unogenic potential.
lnitial im1nun1zation requires two doses of vaccine 2 to 4
\.vecks apart. Thcsc should be followed by a single booster
whcn thc horsc is 1 year of age and then repeated every 6
n1onths unt1l the horse is about 3 years of age, at which
time the booster interval may be increased to not more
than 1 ycar.
In horscs, a relationship has been shown between titers
of nasal antibody to influenza Yirus and resistance to infec-
tion. The prcscncc of prechallenge serum antibody has
also bccn sho\.vn to shorten the <luration of viral excretion
and fEbnlc response.
ln addition to vaccination, isolation and quarantinc
111ea:.u1e:. a1e advised ducing oulbrcaks to reduce the
sprt>;i<l of clisPa'\P, anci rlisinft>ction of affected premises,
equipment and clothing is csscntial to prevent n1echanical
transmission of thc virus.
SW!NE INFLUENZA
Di sea se
Swine influenza is an acutc upper rcspiratory disease of
pigs. Thc natural disease comn1only affects large numbers
of animals in thc hcrd almost simultaneously. Swine in
fiuenza occurs more frequently during colder months.
Disease caused by swin e influenza alone is usually mild;
ho,.vevt!r, disease may !Je severe wheu secu11J¡iry iilfccliuus
OCC'llT.
·rhe incubation period is short, requ.i ring at most se,·-
eral days. Discase sympton1s include fever, anorexia, leuko-
pcnia, cxtrcn1c wcakncss, and prostration. Respiratory
signs also occur, 1ncludu1g hyperpnea, dyspnea, sneezing,
paJnful coughing, anda nasal discharge. Some animals de-
velop con 1unctlvltls, pulmunary euc111a1 ur tJrur 1cltuµr 1eu-
mnnia . Most ilnimills rPcover uneventfully in 2 to 6 days
but with considerable wclght loss.
Swine influenza was first recognized during the mas-
sivc human influcnzn pnndemic in 1918, and subsequen:
studies confirm that a s1m1lar H1N1 virus caused disease
in both humans and swine. Swine influenza virus can
spread from ptgs to humans causl.J.1g illn.ess, and there Is
concern that swine may serve as a reservoir from which
i11ílue11a vi1 us can en1ergc to precipitate human epi-
demics.
Swine influenza occurs in many parts of thc world, in-
cluciing Europe, Asia, and North America. 'lhe Hl Nl virus
is most commonly responsible for disease outbreaks ~
sw1ne worldvvide, although H3NZ viruses also circulate ir.
son1e regions.
Antigenic tlifferences arnur1g straiI1:; uf :;~vine ir1flueu.a
virus have not been great enough to justify the designa-
tion of subtypcs.
Host-Virus Relationship
Pathogenesis and Pathology
()n postn1ortem examtnatlon of s"vlnc wlth Influenza, the
inucosa of thc u pper rcspi ratory tract is congestt>cl ;in el t r ...
cervical and n1ediastinal lymph nodes are edcmatous
Affected lung lobes may be atelectatic and emphysema-
tous. Thcrc 1nay be pncumonia with consolidation. Th~
spleen is often 1noderately enlarged, and the gastric mi...-
cosa is often hyperemic.
Transmission of the diseasc Is by droplet infection. Vil::.
maintenance and S'l<vinc exposurc> '"'PrP prt>viou_c;ly thougr
to depcnd on earthworms containing viral-infected Jar-r....e
of the pig lungworm (Metastrongylus spp.). Pigs fed suc-
carthworms and simultancously cxposed to Haemophí/:...,
(para) s111s often developed typ1cal s"vine influenza. l\ le.
elaborate explanation assumed that recovered anima_
actetl as vtral carrlers. Prcvluu:. evitle11Lt: uf uau:.plrtLe1 ........
infPrtion has hf'<'n rlisputed hy recent stuciies. ·1ranslll!r
sion of inílucnza viruses between pigs and birds has bccr
suggested by the isolation of an influenza from si~
turkcys of an l-llNl virus strain antigenically and gene::
cally iclentical to viruses isolated from pigs.

Laboratory Diagnosis
Swine influenza is suspected wh.enever there is an "explo-
s1ve" appearance uf upper re~µiratury ubea::.1:: i11vulvi11g
m.any pigs, p;:irtirul;:irly <luring the fall or winter months. A
definitive d iagnosis requires either viral isolation from
nasal secretions or the lung or demonstratior1 of a rising
titer between acute ancl convalescent sera. Swine influenza
virus can be cultivated in 10- to 12-day-old embryonatecl
chicken eggs and various tissue culture monolayer systems
involvir1g prirnary ur ~ta!Jle cell cullures.
Treatment and Control
Swine tllat have recovered fron1 influenza cteve!op Hl,
serum neutralizing (SN), complement fixing (CF), precipi-
la ling, and neuran1i11idase-il1l1ibiting an.tibodies. There is
a controversy as to whether recovered animals are im-
mune. Sorne researchcrs bclicvc thcy are and that subsc-
quent respiratory outbreaks in a herd in a given season
must be caused by another agent. Others have demon
srrated that pígs \.Yith neurralízír1g antíboay rnay stíll suc-
cumb to challenge with swine influenza virus and H .
(para) suis cornbined. Pigs born to i111n1u11e sows are pro-
tected for as long as 13 to 18 weeks by passive transfer of
maternal antibodies.
lmmunization using various viral preparations has
been attempted wíth little success. In the United States,
there are no commercial vaccines for swine influenza.
Treatment entails supportive measures including a
draft-free environment with clear1, <.lry, <.lu::;l-fn-:e 1Jeddi11g;
fresh clean w<'ltf>r; ;:incl a goo<l source of feed. Anti biotics
given on a herd basis n1ay help prevent secondary bacter-
ia! infection. Recovery often occurs as suddenly as the
onset of discasc.
AVIAN INFLUENZA
Disease
Avian influenza affects the respiratory, entcric, or ncrv-
ous systems of many species oí birds. Viruses of relatively
lovv virulence may cause few signs wl1ereas others cause
high rnortality. Most outbreaks produce respiratory signs
sucl1 as sneezing, coughing, rales, sinusitis, and lacrima-
tiu11. Otlit:r s.ig11::; iuclude depression, diarrhea, anda de-
cline Íl1 egg proch1ction or fprtili ty. 'fhP. oisP.aSP. ca11SP.O hy
a highly pathogenic virus was once called fowl plague.
Viruses t hat cause virulent disease should be classified as
highly pathogenic avían influenza (HPAI) viruses.
Domestic turkeys, in particular, have often been infected
b y influenza, vvhicl1 has caused substantial losses over the
years. There is n1arked genelic d ive1sily an1ong avian
viruses and at least 15 HA and 9 NA proteins have beer1
identified among avian influenza viruses. Thus, a large
num ber of su btypes of avian intluenza viruses are possi-
b le, and birds serve as the natural reservoir of influenza.
Evidence exists tllat all HA subtypes are maintained in
C'hapter 59 Orthomyxoviridae and Bunyaviridae 365
aquatic bird popu lations, including ducks, gulls, and
shorcbirds. Most infcctions in aquatic birds produce no
clinical signs. ln wild d ucks, irlíluenza virus replicates in
intestinal mucosa! cells and is excretcd in high concen-
trations. Virus has lJeen isolilte<.l frurn lakt: aJJ<.l p uu<.l
\.v;:itf>r, ;:ino survP.ys havP oP.1Tionstrated that as many as
60% of juvenile birds may be infected as they congregate
prior to migratio.n.
Host-Virus Relationship
Geographic Distribution, Reservoir, and Mode of Transmission
Highly virulent avian influenza is a major t hreat to the
world's poultry industry. To dat e, it h as been found in Asia
and South and North America, and historically has beer1
associated with HA subtypes HS and H7. In both the
Pe 1 ut~ylva11..ia uut!Jrt::ak uf 1983- 1984 a11<.l tlJe rect:r1t uut-
hreak in Mexico involv ing HSN2 virus, early isolations
yielded virus o f low pat hogenicity. It appears that the HA
gene is important in pathogenicity in that highly patho-
gcni c virut.ct. p055C55 HA5 thut are rc.-:tdily clcuvcd. Enrly
l'ennsy!vania isolates hada glycosylation si te in the cleav-
age rcgion that may l1ave blocked clcavage. It appears tl1at
a ~i11gle u1 utaliuJJ rernoved tl 1at gl ycosylalio11 sile, wl 1ich
resulted in a h ighly pathogenic virus. Similarly, in the
Mexican outbreaks differences were observed in low- and
high-pathogenicity H5N2 viruses involving the cleavage
site. As a result, it is recommended that the HA cleavage
site sequence also be determined in evaluating patho-
genicity of isolates.
The c.lisease is trar1srnit tec.I thruugh poultry fluck~
1TI<'l inly thro11gh ingPstion of virus, hut it may also he
transmitted by inhalation and by mechanical means in-
volving movement of personnel throughout flocks or be-
tween premises. f.urthermore, sorne believe that for wa-
terfowl, a fecal -water-cloacal route ot transmission may be
important in addition to the fecal-,-vater-oral route.
Outbreaks of highly virulent avian influenza roay be self-
liiniting because fe'"' birds survive the disease to servf> as
carriers.
Waterfowl have been implicated as the major natural
reservoir for ilúlucnza. Infcctcd ducks can shcd virus for
prolonged periods without sho>ving clinical signs or pro-
duci11g a detectable anti.body response. There is evidence
that influenza can persist in sorne b irds for severa!
months after infection . In the past, although avian in-
fiueuza has freq ueully beeu isola Led fron1 in1porled exotic
hirrls, prompting strirt q11;:ir;:intinf> mp;:is11rf>-~ on nPwly im-
ported birds, there is no evidence for spread in this n1an-
ner as has been demonstrated with Newcastle disease
virus. Furthermore, avían influenza may be transmitted
or1 such objects as shoes, clothing, and crates that come
in contact with infected birds or _premises. While the pos-
silJili ty uf v1:: rtical tra11~ r 11 i :s:sio11 uf aviar1 iufluellza
th rough infected eggs P.xists, no evidence exists for its
spread by this means.
.l\vian influenza viruses 11ave been found to be capable
of in fecting a wide ra11ge of mammalian spccics in addi-
tion to birds. In fact, avían strains have been ímplícated ín
366 PART III Viruses
the deaths ot mink during an epidemic in Sweden and in
the disease of seals as well as the emergence of new strains
in northern China affecting horses. Avian influenza virus
ca11 b e propagated h1 cell cult ures of hu1nan, bovine, ca-
11il1c, <.JJilkl:!lL, anll ralJlJit urig.i11.
Pathogenesis and Pathology
Tl1e patl1ogenesis of avian influenza vttries widely depend
íng on strain of virus, age and species infected, concurrent
infections, an d husbandry. At necropsy, lesions include
foci of necrosis of various sites including tl1e ski11, luu11J,
wattles, spleen, liver, lung, kicinry, intrstine, ilncl pilncreas.
Tl1ere n1ay be fibrinous exudates in the air sacs, oviduct,
pericardial sac, or peritoneum. Other lesions include pe-
tcchiation of t he heart muscle, abdominal fat, and tl1e 1nu-
cosa of the proventriculus, a nonsuppurative encephalitis,
an d a serofibrinous pericarditis.
Laboratory Diagnosis
A definitive diagnosis requi res viral isolation and identifi-
cation or th e de1non stration of rising antibody titer by Hl,
viral ncutraUzation tests, soluble antigen fluorescent anti-
body test, agar gel precipit ation, or ELISA rechnique.
Avian influenza can be cultivated in chick embryos¡ duck-
ling, calf, and rnonkey kiduey l ell lullurcs; a11d fro rn sa111 -
plrs of trar.h ea, h1ng, air sar., sinus exudate, or cloaca!
swabs. Antigen capture ELISA tests have been used for
rapid viral detection.
Treatment and Control
Recovered hirds remain imrnune to suhsequent challenge
by a 1101nologo·us strain for at least severa! mon ths. Dirds
irnn1ur1ized parenterally wi.th inactivated vaccines are
con1pletely protected agai11st challenge by a heterologous
straln wit l1 the same HA and are partially protected
agai.nst heterologous st rains possessing the same neu-
raminidase. Following immunizatiur1, l llilk1::11s ren1aiIJ
imm11nr for at least 84 clays, approximately n-vice as long
as turkcys ren1ain immune. Tt has been demonstrated that
anti-HA a11tibody is important for protection against in-
fectio11 while antlneuram.inidase antibody protects
against disease and reduces virus shedding but does not
prcvcnt infection.
In practice, however, appropriate vaccines are rarely
available to t hc poultry industry owing to great genetlc
and antigenic diversity among avían viruses. In several
states in North America (such as Mil1nesota and Califor-
nia), the rapicl isolatlo11 of virus durir1g ouLbreaks 11as per-
mittecl th e devPloprnent of inactivated vaccines felt to be
effective in lirniting t he severity of an outbrcak. There is
hope that a polyvalent vaccin e vvith a broader protective
rangc may be developed.
For the most par t, control of avian influer1za i:s
through the prevention of exposure. Carrf11l hushandry
to preve11t t lte i11lroduclio11 o f the virus into the flock is
•
important. New birds should not be introduced into c.
started flock, and careful precautions should be taken re
prevent either dir.ect or indirect contact with wil<.1, m1gra-
tory, or exoti.c birds. Since turkeys ha ve been fou11d also t-o
be susceplible Lo a sLrain of influenza Lypically associa tec.
with pigs, it is a good management practice not to h a"'e
pigs on the same farm as turkeys. Eggs for hatchin;
should come from flocks demonstrated to be free ot thé
virus. Virus l1as bee11 demonstrated to persist for 105 days
in liqu.id manure followi11g depopulation. Strict measu ro
should be employed to eliminate movement of personnt.
ar1ll c4uip1ue11l, poLeuLially co11Lan1ii1ated by nlan ure..
hen-veen flocks and premises. During an outbreak, isola-
tion of a flock along with orderly marketing of thc fl oc~·
should be considered.
Drugs such as amantadine, used for human influe=
p revention, have not been cleared for use in tJin.l:; for l un-
sumption and to date there is no satisfactory treatment fo;-
avla11 influe11za. Treatment of infected flocks with broac-
spectrum antibiotics is useful in controlling seconda0-
bacterial infcctions, and propcr nutrition and husbandry
inay help reduce mortality.
Zoo notic Significance of Animal lnfluenzas
A growing body of evidence s11ggf>sts that pandemic
sLrains of hun1an i11fluenza ar ise as a result of recombina-
tion between human and animal strains of influenza virus.
Waterfowl appear to be of particular significancc in thc
o rigin of new human isolates. Ducks appear to act as a
"melting pot" where various strains of influenza can come
together ancl undergo genetic reassor tment, resulting i1:
t he generation of new strains of influenza. $\>Vine have also
lJccu lruµlilatcll as iu tcr111.ecliale ho:>Ls of "avian-like" and
"human-like" influenza viruses. Since the interna! genes
are critica! for host range, and the HA and NA are impor-
tan t to host immunit y, recombination events can result in
t he formation of new virus strains that contain the same
or similar interna! genes but possess very dlfferent HA and
NA proteins. Tl1e novel viruses generatecl in this manne'
111ight :still lJe iufective Lo l1un1a11s bul possess su1face anli-
gens that are very different than those to which the
human population prcviously was cxposcd (and im -
mune). ·rhe result could be a substantial influenza pan -
demic as the new strain rapidly spreads through a suscep -
tible population.
In the past, major antigenic shifts 11ave takrn plar.e
among human Influenza viruses lealli11g Lo ,pande111ics ü1
the human population. Ea ch of thr lilst three shifts !1ad its
origiI1 tralcll Lo Chii1a. The role of China in the emergen ce
of these pandemic strains has i1ot been compJetely re-
solved; however, China is known to contain a Jarge reser-
voir of different influenza A viruses among wild ancl do-
mestic species, partic'Ularly ducks. Furthermore, the mass
production of ducks and the proxiulity uf hun1an 11abita-
tions to the farms, also in close association witl1 swine,
ha ve bee11 in1plicated by sorne asan ideal situation for cs-
t ablishing new antigenic strains and int roducing tl1ese
viruses to t hc human population.

3UNYAVIRIDAE
~e fa1nily 81111yaviridae in.eludes the genera Bunyavirus,
.'iantavirus. Nairovirus. Phlebovirus. and Tospovirus. There
are several hundred bunyaviruses, and v iruses of veted-
;iary significance are cJassified within the genera
Bt1nyavir11s-California encephalitis serogroup viruses and
'.kabaue virus (as well a:> Ai11u ilr1u Cacl1t Viilley viruses);
."'hlebovirus-Rift Valley fever virus; and Nairovirus-
~airobi shccp disease virus. Many of the bunyaviruses are
..:boviruses that are transmitted by arthropods such as
::nosquitocs, ticks, biting flies, etc., and thus have the
capacity to alternately replicare in vertebrate animaJs and
.nsects.
Scve1al bunyavi1uses a1e i1uvur lilrrl zuu11utic µathu-
::ens, including hantaviruses (t he cause of I Iantavirus pul-
-.:ionary syndromc and h cmorrhugic fcver with renal syn-
..:1rome in people), which are carricd by rodents and
.;ifection of humans occurs by inhalation of v irus
-..ontaminatcd rodent excreta and detrlrus; Rift Vallcy fever
·-in1c;; Crimcan-Congo hemorrhagic fever virus; and
'airobi sheep discasc virus.
Gen eral Family Properties
Bunyaviruses are enveloped, pleonltHphic viruses, 80- 120
~m in diameter, with surface projections (spikes) e manat-
ng from the envelope surface of mature v irio ns. The viri-
vflS COOSiSt Of fou r StructuraJ protei OS, including two exter-
na] glycoproteins in the envelope, a nuclcocapsid protcin
·hat encaps1dates the genome, anda transcnptase protein
:. . ·rhe envelope glycoproteins are responsible for neutral-
.l.dtiun and hemagglutination. A var1ety of nonstructural
~·otPinc; 11 l<:O i!TP PD\OOPci hy thl:' vira 1 eenome
The nuclcic acid is helical and includcd in three dislincl
.egments (large ILl, medium fMl, and small [S]), each com-
?riscd of singlc-stranded, ocgativc-scnse RNA.
rhere is consic.terable genetic diversity and serologically
~:oss-reactivity among viruses within the various genera
~r rhe Bunyaviridae.
GENUS BUNYAVIRUS- AKABANE , AINO, ANO
CACHE VALLEY VtRUSES
~1.;abancvirus is the cause of periodic outbreaks of fetal
~alfo rmation in ruminants, cspccially cattle, in Asia,
ustralia, the Middle East, and Afric;:i. Thc v iru5 Í5 tran5-
"Il.ltted by mosquitoes and Culicoides midges and causes
istinctive teratogenic defects of the musculoskeletal and
-.:rvous syslerns (ar Lhrogryposi~-li yJn111euceµha ly) in fe-
~ses that are born to dams that become infccted du ri ng
:7:cgnancy. Thc gestational age of the fetus at infection de-
ermines the type ot lesions that will be present at birth; fe-
-uses infected prior to mid-gcstation are typically born
1th thclr l!mbs held in rigid fleXion (arthrogrypos1s),
ith variable deviation of their vertebral columns. The
rains of affecLed feLuses ruay Jack ccrc!Jral hernispheres, or
Chapter 59 Orthoniyxoviridae and Bunyaviridae 367
these may be represented only as fluid filled sacs as a con
sequence of virus-mediated destruction of the developing
cerebrum during gestation (hydranenccphaly). An inacti-
vated vaccine is used to protectlvcly 11nmunize ruminants
p rior to conception.
Aino viru s causes a similar discasc as Akabane vi rus
and has much the same global distribution. Cache Valley
virus has bccn documented as the cause of outbreaks of
arthrogryposis-hydranencephaly of sheep in the western
and south\vestern United Sta tes.
CA LIFORNIA ENCEPHALITIS SE ROGROUP VIRUSES
California encephalitis scrogrollp viruscs are rclatcd (scro-
logically cross-reactive) 1nosquito-transmitted vi ruses ot
the genus Bunyavirus and include La Crossc, snowshoe
ha1e, a11u Ja111estuw11 Canyun viruses. Thcsc vlruses occur
in endemic cycles of inft>ction in rlifferent regions of North
America and, although infection occurs in a variely of
mammalian species, encephalitis is rarely documcnted
even in humans.
Othcr members of the genus Bu11yav1n1s have been spo-
radically incriminated as the cause of cncephalitis in ani-
1nals-Mair1 DraiI1 virus in 11urse~, for example.
GENUS PHLEBOV/RUS-R IFT VALLEY FE VER
Rjfl Valley fe ve1 virus (RVFV) i:> a :t.l>o11utic, n1u::>l{Uitu-
transm itted virus that causes epidemics of severe, fre-
qucntly fatal systemic disease of ruminants, especially
shecp and goats. Mortality is highest in young animals,
and pregnant ruminants often abort following infection.
RVFV causes extensive liver necrosis in affected sheep and
goats, and widespread hemorrhages are common. Enceph-
aliti:> with neurur1al necrosis also occurs in sorne affected
animals.
Rift Vallcy fcver (RVF) is endemic in sub-Sah¿tran Africa
and periodically incurs into ad jacent northern regions
such as Egypt. Climatic conditions influcncc thc occu r-
rence of epidemics, particularly h1gh rainfall that results in
rapid expansion of the populations of Aedes mosquitoes
that harl.Jor the virus. RVF is a zoonoses, and human mor-
tality can ht> vi>ry high during epidcmics.
RVFV is '"idely feared 1.Jecau::.c uf its virulence tu hu-
mans and animals, as well as its capacity for rapid spri>ad
by a varicty of species of mosquito during cpidemics. Thus,
rap1a 01agnosis is imperative, usually by virus isolation or
serology. An inactivated vaccine is availablc, and unvacci
nated veterlnarlans and laboratory staff working '.vith
RVFV-infected 1naterial are at risk of infecl'ion.
GENUS NA/ROV/RUS- NAIROBI S HEEP D ISEASE
Nairobi sheep disease (NSD) and rclatcd viruscs cause sc-
vere dlseasc of ruminants, pnnc1pally sheep and goats, in
368 P..\RT 111 Viruses
Africa and Asia. 'fhe virus is transmitted by the brov.rn ear
tick. NSD i:s chi:tri:lcterized by 11igl1 fever, ir1testinal hernor-
rhage, and death of susceptible ruminants. Pregnant ani-
.m als typically abort. NSD is a 11ighly virulent zoonotic
pathogen.
Other mernbers of tl1e genus Nairovirus that are of po-
tential veterinary slgnificance include Crimean-Congo
hemorrhagic fever, a zoonotic disease that occurs through-
011t Africa, eastf'rn F.nrnpe, thf> Midclle F.ast ancl Asia. Thf'
virus is transmitted by ticks, and both wild and domestic
animals can serve as reservoirs of the virus, although dís-
ease apparently is rare in species other th.an hu1nans.
 Paramyxoviridae,
Filoviridae, and
Bornaviridae
YUAN CHUNG Z EE
The order Mononegavlral es !ncludes vlruses withln the fam-
ilies Paramy.xoviridae, Filovirdae, l{ornaviridae, and Rhabdo-
viriúae (Chaple1 61). "fhese viruscs are a11cestrally related as
reflected by their common fcatures. includi.J1g genome of a
single-strand of ncgativc-scnsc TlNA, sin1ilar rcplication
strategy and gene arder, and virion morphology that in -
cludes an envelope.
PARAMYXOVIRIDAE
Thc Para111yxoviridac family is subdivided into two subfam-
iliPs, the. Paramyxovirinae and thc Pneu1novirinae that are
furthcr subdivided into three (R.espirovin1s, Rubulavirus,
1\!Jorbillivirus) and two genera (lJneurnovirus, Metapneumo-
virus), respectively (Table 60.1) . Viruses in this family cause
a number or senous resplratory ancl/or sysrem1c a1seases of
humans, animals, and birds.
Paramyxoviruscs art: <.:haraclerized by virio11s th.at are
Pnvt=>lopt=>cl, plc•omorphic (filamentous or spherical; ap-
proximatcly 150 nm or more in diameter), and contain a
genom e of linear, negativc-scnse, sin g le-stranded iZNA.
The viral nucleocapsid has helical symmetry and is ap-
proximately 13- 18 nm in diameter (Fig 60.1). At least threc
p roteins are associated with the nucleocapsid, including
ar1 RNA-l.Jindi11g prvlt::i11 (N 01 NI'), a phospl1oproteil1 (P),
anrl the viral polyrnerase (L) . ·rhe nucleocapsid is eo.closed
by a lipoprotein envelope derivcd from thc host ccll
plasma membrane, and contains two or three transmem-
brane viral glycoprotcins that form spikes (8- 12 nm) that
pro¡ect from the surface. The spikes are formed by the at-
tachment protcin (hemagglutinin [H] or hemagglutinin-
11eura111i 11 iua~t: [HN] in Lile Pu1u111yxovirir1ae; G pr0Leit1 it1
the P11eu1novirinae) and the fusion protein (F) that is essen-
tial for virus infectivity and for cell-to-cell spread. Another
protein, the matrix (M) protcin, lines the inner surtace ot
the envelopc. Various nonstructural viral proteins are also
formcd in infected cells.
N. ]AMES MACLACHLAN
PARAMYXOV/RINAE
MORBILLIVIRUSES
Although the various morbillivirttses are ali closely related
as evidenced by cxlensive serological cross-reactivity be-
tween them, thcy are distinguishcd on the basis of their in-
dividual host rangc, gcnocnc scqucnccs, and antigcnjc dif-
terences. ·rhese viruses al1 have a hemagglutin (H) surface
protein, but only rinderpest virus has a neuraminidase(HN).
(AN IN E D ISTEMPER
Disease
Canine tlistemper (CD) is au iluµortaul viral disease of
dogs. Ac:ntP r.n i~ rh::ir::irtt=>ri7.P.rl hy any comhination of
diphasic fever, ocular and nasal discharges, anorexia, de-
pression, vomiting, diarrhca, dehydration, leukopenia,
pncumonin, nnd ncurologic signs. The severity of clinical
signs exhibltcd by 1ndlv1dua1 an1mals can vary rnarkedly.
Thc dísease has an incubation period of 3 to 5 days. 'fhe
mortallty rate uepends largely on the irnrnune status of
thc i.nfected dog, ¡¡nd i<> hight=>st ;imong p11ppit=>s. Anim;ils
th.at survive the acute disease may develop other signs, in-
cluding hyperkeratosis of the footpads (hard pad dlsease)
and ncuro logic discasc that is charactcrizcd by any combi-
nation of convulsions, tremor, myoclonus, locomotory
disturbances, paralysis, and blindness. The onset of neuro-
logic signs frequently Is delayed until several weeks atter
the systemic signs of acute CD are manifest. Old dog en-
cephalilis is a tate forn1 of cl1ro11ic neurologic disease
caused by defective CD virus.
Etiologic Agent
Physical. Chemical, and Antigenic Properties
Caninc distcmpcr virus (CDV) cxl1ibits thc propcrtics dc-
scribed above for paramyxoviruses. '!he surface H protein
369
370 PAHT III Viruses

Ta b 1e 6 o. 1 . Major Subgroups of the F;imily ParamyxoviridaP.

Subfamily G~n1.1s Yemacular Name Natural Host

Paramyxovfrínae
R111pirovirus Bovine parainfluenza virus 3 Cattle
Human Rarainfluenz¡¡ virus 1,3 Humans
Sendai virus Mice
Rubu/avirus A"<oin poromyxovirus 2··9 Birds
Human paraintluenza virus 2, 4 Huma ns
Mumps virus Huma ns
Newcastle disease virus Birds
Pordne Rr1huf;wir11s Pigs
Sirnidn virus 5 (similar to canine
parainfluenza virus 2)
Mo(billivirus Measles virus Humans
Canine d1stemper Canioes
Phocine disternper virus
Ceta,cean Morbillivirus
Rinderpest virus Rurninants
Pe~le des pelils rurninants virus Gócts and sheep
Equine (Hendra) Morbillivirus fruit bats
Pneumovirinae
Pneumovirus Bovine respiratory syncytial cattle
virus
Human respiratory syncitical virus Humans
Murine pneurnonia virus Mice
Metapneumovirus Tu11\ey rhirrolracheitis virus Birds-

lacks 11euraminidasc activity, and is rcsponsible for attach-

F 1G U RE 6 O. 1 . Negatively stained preparations of bovine
1ne11t ot CIJV to ceus. ·rhere is only one antigenic type of
parainfluenza Type 3 virus. 120,000X. (Courtesy of A. Castro.)
CDV, although d istinct strains of the virus wit h different
biologic propertíes clearly occur.

Resistance to Physical and Chemical Agents

Canine distemper virus is labile to er1viror1rneutal fa<.:tor:,
sucl1 as extren1es oftem pi>n=i t11rP, pH, and to severa! disin-
fectants. It is inactivated by visible and ultraviolet light,
and heating at 60ºC for 30 minu tes. The virus can survive
in tissucs for 48 hours at 25°C and for 14 days at SºC. Viral
1nfectivity is Jost ahove ptt 10.4 or belov• pH 4.4, and the
virus is most stable at pH 7. CaniI1e distemper virus is read-
lly lnacrivated by disinfectar1t!;, lJ ualerua1 y an1111011iun1
compounds (surh as 0 .2% Roccal), and O. 75o/o pher1ol solu-
tion. Labile viruses like CDV persist longer in cool, sl1ady
environments or in seru.oJ or tissue debris.

lnfectivity for Other Species and Culture Systen1s

Ca11ine distemper virus intects a •vide range of an.i.mals. In
addition to dogs, other membcrs of the C~anidae (e.g ., fox,
coyote, wolf, wild dogs), Ailuridae (red parida), Hyaenidae
(byena), Must elidae (e.g., ferret, mink, skunk, b;idgi>r),
'fyassuidae (javelina:> (collared pecca1ies]), Ursidae (bears),
Viverridae (civet, mongoose), a11d Procyonidae (c.g., rac-
coon, panda) are ali susceptible. Sorne n1cmbcrs o f the Feli-
dae Oio11, tiger, and leopard) also are susceptible, a11d dev-
 Chapter 60
:ostati.ng outbreak~ of ciistemper recently have occurred
~:nong lions in Africa.
Canine distemper virus can be isolated and propagated
: :1 p rimary canine and ferret kidney cell cultures.
The v irus has been successfully adapted to embry-
onated chicken eggs and various cell cultures including
bov iIH:! kidney, Veru 111u11kt:y kid11ey, and hu111a11 an1nion
or fibroblast continuous cultures. The virus can also be
adapted to newborn Swiss micc and wcanling hamstcrs.
Host-Virus Relationship
:>istribution, Reservoir, Transmission
C.anine distemper occurs world;vide, and remains en-
demic in n1any areas despite the widespread use of vac-
cines that are highly effective i11 preventing the disease.
Dogs are the prirtcipal reservoir of CDV. Infected dogs se-
crete virus i.n their nasal and ocular secretions, and CDV is
? rcscnt in thc urinc of cxpcrimcntally infcctcd dogs 6 to
1.2 days atter infection. reces of infected dogs may also
contain CDV. Infection usually is transmitted by aerosol
or by direct contact, leading to resplratory 1nfectlon of
~u sceptible animals.
Pathogenesis and Pathology
"!'he CDV is pantropic and has an especially strong tro-
p ism for epithelium and lymphoid tissucs. Thc lungs
are also central to the pathogenesis ot CVV intection.
illitial replication of CI)V occurs in 1n.acrophages in the
b ronchial lyrnph nodes and ronsils tmmed.i ately follow-
:ng respiratory infection. The virus then is spread to other
.ymphoi.d tissues (lyrnpl1 r1ude:>, s¡..¡lee11, lhyn1us, and
bone marrow) whi>rf' f11rther n~plic:ation occurs prior to
·1iJen1ia that dissen1inates CDV to virtually all organs of
the body, including the central nervous system (CNS) and
eyes. Thts gcncralizcd disscmination of CDV to the ep-
1theli11m ot the alin1entary, respiratory, and urogenital
rracts; the skin and mucous membranes; and endocrine
glands and the CNS occurs at approxirnately 8 ur 9 <lay:>
after infection and only in rlogs that fail to rlevelop suffi-
cien.t titers of neutralizing antibodies to CDV by that
time.
Thc Jcsions of CD occur in thcorgans in whicl1 the virus
replica tes, and the respiratory and al üne11tary tracts are es-
pecially affected. Tl1e lungs of dogs with acute CD have dif-
fuse lnterstltial pn.eun1onta characterized by necrotizing
broncl1iolitis and a lveolitis with thicki>ning of thf' a lvf'olar
septae. Secondary bacteria! pneumonias are common in
dogs with prin1ary CDV-induced interstitial pneumonia.
Charactcristic cosinophilic intranuclear and intracyto-
olasmic inclusion bodies are often present within respira-
tory epithelium and in the epithelium lining tb.e stomach,
u rlnary bladder, anc.\ renal pelvis. Extensive necro:>is of
tymphocytes is characti>ri~tic of ac:11te l.11, li>a<ling to lym-
p hoid depletiOil and immune suppression. In1mune sup-
pression predisposes affected dogs to secondary bacteria!
pncumonia and may assist thc virus in gaining acccss to
the nervous system.
Param.vxoviridae, Filoviridae, and Bornaviridae 371
Dogs that survive acute CD may subsequently develop
neurologic signs that rcsult from virus-induccd dcmycli-
nation. ·111e lesions with1n the central nervous system of
tl1ese dogs begin as focal areas of demyelination that in-
creasingly are acco1npanied by Jymphocytic inflarnrn<1tiun
and accumulati.on of m.acrophages. T.i>sions occnr "\Tithin
tl1e cerebellun1, brain sten1, and cerebrum, and CDV inclu-
sion bodies may occt1r in astrocytes, ependymal cells, and
ncurons. Although CDV prcdominately replicates in astro
cytes and microgJ1al cells within the brains of infected
dogs, direct virus-1nediated injury to oligodendrocytes is
proposed to be responsible for mucr1 of tl1e initial virus-
induced demyelination that occ11rs in rlf'myi>linating
forn15 of CD.
Old dog encephalitis is a rare d isease of mature dogs
that rcsults from vcry long-tcrm pcrsistcnt CDV infcct.ion
of the central nervous system, leading to dementia. The
disease is characterized by severe lymphocytic e11cephalitis
with neuronal degeneration an<.I, often, the presence of
(~JJV inclnsion horlies. Demyf'lination is lf'ss promini>nt
than in typical neurologic CD. It is proposed that a replica-
tion defective form of c:Dv is responsible for old dog en-
ccphalitis, analogous to thc role of dcfcctivc mcaslcs virus
in subacute sclerosing pa11enecephalitis (Dawson's dis-
ease) of humans.
Host Response to lnfection
Canine distemper virus infection induces lor1g-lastü1g
in1rr1unity in dogs that rccovcr fron1 natural infcctio11.
Neutralizing antibodies first appear in seru1n of infected
dogs 8 to 9 days after infection and persist for years there-
after. Cell-mediated immune responses are also gener-
ated by dogs infected w.itb cnv and arf' likf'ly important
to recovery fron1 tl1e disease. Virus-induced immune
suppression contributes to the pathogenesis of COY in-
fection by predisposing affected dogs to sccondary bacte-
ria! infections and by facilítating spread ot the virus to
the CNS.
Laboratory Diagnosis
Since the clinicaJ signs of CD are variable and so1netimes
nonspecific, definitive diagnosis requires identiticatíon ot
C.DV by virus isolation or the staining of CDV antigen in
the censor tissues of affected dogs using cr>V-specific an-
tibodies and the immunofluorescent or immunoperoxi-
tl<1:>e technique:> . .1-\ltern<1ti vely, :>erolugical üiagr1osi:> cau
he rloni> hy rlemonstration of rising TgG titers in pairerl sera
by enzyme-linked immunosorbent assay (E.LISA).
Treatment and Control
Treatment for CD is supportive, such as the use of anti-
hiotics to pri>vi>nt si>conrlary hacterial infecti ons anrl
electrolyte solutions to restore fluids and electrolytes.
Vaccination against CD is the best means of preventíng
thc discasc. Both modificd livc and inactivatcd CD \;ral
vaccines are available, and vaccination has greatly re -
372 l'ART 111 Viruses
duced the incidence of this disease in dogs. New gene-
ration recombinant distemper vaccines are increasingly
being developed, including canary pox virus-based
vectors.
Matcrnally derivcd antibody to CDV can interfere with
the SllCCessfu} vacr..ination of puppiP.S <igainSt CDV
Puppies sl1ould be vaccinatcd for CD at a11 age when ma-
ternally dcrivcd antibody has declined to a leve! that does
not intcrfcrc with immunization. Although both inacti-
vated and modified live virus CDV vaccines safely have
been u sed to immunize a variety of ,.,.¡Jd animal species, in-
cluding foxes, ferrets, mink, bush dogs, and maned wolves,
there have been reports of vaccine-induced CD in a variety
of spccics, lncludlng mlnk, kinkajous, anc.J lesser pa11c.Jas
alter immunlzation with moc1ifiPc1 livP r.nv vac:c:ine.
S.intilarly, lnstances of vaccine-induced CD have been dc-
scribed in dogs.
PHOCINE DISTEMPE R
Phocine distemper is an infect.ious discase of seals that re-
sembles canine distemper (CD). The disease first was rec-
ognizcd in thc late 1980s whcn massive die-offs of seals oc
curred in the Baltic and North seas. ·rhe tesions and
clinical s.igns in a(fcctcd scals were remarkably similar to
those ot CD, and thc <.llsease uf seals sulJseyue11tly was
shown to be caused by phocinP distPmpPr virus (POV), a
Morbillivirus that is closcly related to CD virus. A similar
disea<;e caused hy dolphin distemper virus caused exten-
sive mortality among dolphins on the Atlantic coast of
North America in the early 1990s. The dolphin and pho-
cine distemper viruses are closely related, and other genet-
ically distinct morbllllviruses have been shown to cause
similar diseases in other pinnipeds and cet aceans. Like CD,
1.1J1euJnonia don1inales the clinical and pathological 111an-
ifestations of morbillivirus-induced distemper in marine
mammals.
EQU INE MORBILLIVIRUS (HENDRA) DISEASE
Equine 1\lforbillivirus disease is unique to Australia, where it
'"'ªs fir:;t recognized in 1994 during an outbrcak of scvcrc
rcspiratory disease that killed a number ot horses and their
trainer. The disease also is kno"vn as Hendra (and the
causatlve agent Hendra virus) after the town in which the
disease \.Yas first described. Affccted horses develop severe
iI1t<.:rstitial v11eu111u11ia a11tl JJUII11onary edema U1at rapidly
is fatal. The disease is caused by a unique paramyxovirus
that tentatively is classified as a Morbillivirus . Thc virus is
harbored asymptomatically by severa! species of fruit bat
(flying foxes). /\lthough fruit bats are thc reservoir of
cquinc Morbillivirus, the virus is contagious between
horses and to humans by direct contact with nasal secre-
tions or furr1it~:; tllat c.:011tai11 lhe viru:.. A sintila1 virus
(Nipah) is <>nrtemic in fruit hats in Malaysia and adjacent
regions of Southcast Asia, and periodically emerges to
cause fatal discase in humans and swine.
RINDERPEST
Di sea se
Rinderpest (also known as cattle plague) has long bcen rec-
ognizeel as a Clevastating pandemic viral disease of domes-
tic cattle, buffalo, and sorne wild ruminant species.
Rinderpest Is characterized by fever, lymphopenia, nasal
and lacrimal discharges, diarrhea, and erosions and ulcers
tl1roughuut tl1e oral c.:avily (ulcerative ston1aUlis). ·r11e dis-
Pase has an incuhation period of 3 to 8 days and the virus
is cxtremely contagious, so morbidity rates are vcry high.
The mortality rate also can be high (up to lOOo/o) in cattte
that have no immunity, but varíes depending on the viru-
lence of the infecting virus strain. The dlsease has been
eradicated from many parts of the world, but it persists in
parts uf Asia, th~ Mic.lt.!le Easl., a11tl Africa and ll1us contln-
11es to pose a serious threat to cattle production worldwide.
The international community has e1nbarkcd on un ambi-
tious program to completely eradicate r1ncterpest by 2010.
Etiologic Agent
Physic:il, Chcm ical, and Antigenic Properties
Tl1t:n: is u11ly 011e anligenic type of rinderpest virus, but at
Jeast three distinct genetic lineages of the virus currently
cxist. Onc lincagc is prcscnt in Asia and two different line-
ages occur in eastern Africa. Strains of rinderpest vary in
their virulence to cattle.
Resistance to Physical and Chemical Agents
Rinc1PrpPst virus is inac:tivated hy sunlight within 2 hours
and is rapidly inactívated by ultraviolet light. The virus is in-
activated by heat exponentially, but small amounts ot virus
may survive heating at 56ºC for SO to 60 minutes or at 60ºC
for 30 minutes. Rinderpest virus is relatively stable between
pH 4.0 to 10.2. It is inactivated by lipid solvents or disinfec-
tants, such as phenul ur stroug alkalir1t c.:ur11pou11ds.
lnfectivíty for Other Specíes and Culture Systems
Rinderpest virus infects ali species of the order Artioda-
ctyla, which includes cattle, water buffalo, pigs, warthogs,
sheep, and many species of African antelope. The virus can
!Je ac.Japte<l to gruw íu lal.Joratory ani1uals, such as rabblts,
mir.e, and guinea pigs, and in embryonated chicken eggs.
Rinderpest virus can be cultivated in pdmary or continu-
ous cell cultures ot bovine, ovine, and porcine ort.~in.
Host-Virus Re lationship
Distribution, Reservoir, and Transmíssion
Rinderpest historically occurred t hroughout Europc,
Africa, and Asia but never became established in either the
Amcricas or in Australasia. "I'he virus currently is enzootic
only lil certaln regions of Asia, the Middle East, and Africa.
 Chapter 60
Domestic and wild animals infcctcd with rinderpest
vi1 us :-.erve as re:;t:rvuir:; fur tlie Llisea:>e, esµecially thuse
species that have a high innate resistance to thP clisef!sP.
These species, includin g certain breeds of indigcnous cat-
tle, 'l'hompson's gazelle. and others. do not show clínica!
signs of the d isease but can shed virus fo r long pcriods of
time. Similarly, s~vine, goats and sheep can serve as impor-
tant rescrvoir hosts that disscminatc virus to susceptible
callle. Ri11Lle1pesl virus b pn:::;t:11L i11 l1igli Liter:; í11 tl1e r1asal
discharges, saliva, ocular secretions, and excretions of in-
fcctcd animals, and transmission requires direct contact of
susceptible animals with secretions and excretions of in-
fectcd animals.
Pathogenesis and Pathology
Rinderpest vi rL1s infection normally occurs th rough thc
uppcr rcspiratory t ract, ulthough cattlc can be experimen -
tally infectecl by any parenteral routc ol inoculation. 1'he
virus first rcplicates in the tonsils and regional lymph nodes,
anda prcdominantly lyn1phocyte-associatcd viremia then
cli~e1ninates the virus throughout the body. The virus repli-
ca tes in lyn1phoid Ussues (splet:u, 1Ju11e 111arruw, lymph
nades), and the mucosa of thP alimPntary and 11pper respi-
ratory tracts. Nasal and ocular secrclions contain high titers
ot virus at this time. rever (up to l07uF) and leukopenia
occur prior to the appearance of oral ulccrs and onset of di
arrhea that may con ta in b lood (dyscn tcry). Titers of virus in
the tissues and secretion s of infcctcd cattle decline after th.e
apvt:an:11 1cc uf ocutr<tliz.ing antilJouies In serun1. Th e co11va-
lescent phas<' hegins \.Vith thP hPfll ing of mo11th Jpo;ions, an rJ
complete rccovcry from the diseasc may take 4 to 5 weeks.
Lcsions of rinderpest occur in the tissues in ;vhich the
virus replicates. Oral ulccrs are charactcristic of scvcrc
cases or rtnderpest in cattle, and these begin as areas of
necrosis with the basal cell layer of the epithelium.
"ecrosls also occurs in the mucosa of the abomasum and
small intestine. ·rhe presence of syncitial cells wi.thin thc
affcctcd cµitl1elíurn is lligt1ly chélracteristlc of rinderpest,
hut nol other ulcerative disef!SPS li kP 111alignant rat :irrh;:il
fever or bovine viral d ia rrhcu. Thcrc also is extensive
necrosis of gu t-associated lymphoid tissues as a result of
virus mediatecl clestruction of lymphocytes, and siinilar
lympholysis occurs in other lymphoid t1ssues (lympl1
nodes, splccn, bone marrow).
Hosl Response lo lnfection
cattle recovered trom rinderpest dcvclop a Jong-lasting
immunity that is 1ikely rnediated by lhc prcsence of neu
trallzl ng a ntlbody in theír serum. In cattle vacciI1ated with
modified Uve rinderpest viral vaccine, both IgM and lgG 2
serun1 antibodies ap pear 2 ·weeks afler vacci 1.1C1tiou .
laboratory Diagnosis
lnitial diagnosis of rinderpest is based on the clinical signs
ancl "1>xplosive" nature of the disease in naive cattle, char-
acteristic lcsions, and herd hislu1 y vf rcccut introduction
of animals from a rinderpest-infectcd ar<'a. l.ivPn current
Para111yxoviridae, Filoviridae, and Romaviridae 373
efforts to eradicate the virus and the very serious consc
quences of incursion of rlnderpest into free areas of the
worlcl, lahoratory diagnosis is required to confirm the di-
agnosis and to differe11liale rinde!pesl f101n oLIIt:r i11fec-
tious d iseases such as bovine viral diarrhea, infectious
bovinc rhinotrachcitis, inalignant catarrhal fever, a11d
foot-and-mouth d isease. ·rhe rinderpest viru s can be iso-
lated in appropriate cell cultures. Thc prcsence of viral
anttgens or viral nucleic acíd in tissues or ocular secretions
of infPrtPcl ;inimals can rapidly be confirmed usi.ng agar gel
immunodiffusion or PCR assay, lespeclively. Tlie cu111µt:ti-
tive ELISA and virus neutralization tests are used for sero-
logical diagnosis ofrindcrpest virus infection .
Treatment and Control
'fhere is no effective treatn1enl fo1 1iuderpesl. Cu11trul is
accomplished by vaccination in endemic areas ancl rigor-
ous quarantinc of run1inants from infected arcas as out-
brcaks of rinderpest in arcas trcc of disease are always
associated with the introduction of infected animals. In-
a<.-rlvated rlnderpest virtts vaccinc was preViously used to
immunize cattle, but it has been replaccd by modified live
vaccines LhaL induce a lvugcr-lastlng immunity. Recom-
binant rinderpest vaccincs also have been developed that
induce strong protcctivc immunity.
PESTE DES PET ITS RUMINAN TS
Peste des petits ruminants (PPR) virus is a Morbillivirus that
produces a rinderpest-hke disease in goats and sheep. ·rhe
virus is closely related to rinderpcst virus, thus distinction
betv.reen the two is very lmportant g1ven the current effort
to eradicate rinderpest from the world. Of particular im-
portancc is thc facl Lhal PPR vil us infectio11s of s111all ru111i-
nants frequently are suhclinica l, th11~ sm;ill n1minants are
the major reservo ir of this virus.
RUBULAVIRUSES
·rhe rubulavi.ruses are included in the subfan1ily Para1nyxo-
vírínae, family Paramyxovíridae. Ali viruses within this
genus have both hcmagglutinin and ncurnmini.dase (HN)
proteins. Included in the rubulaviruses are nine ditterent
species of avian paramyxoviruses (avian paramyxoviruscs
1- 9) th<1t are c.listinguished on thc basls of their lacl< of sig-
nific:ant cross-nPutrali7.: ition and cross-protection. Avian
paramyxovirus 1 is the cause of Ncwcaslle disease (ND).
N EWCASTLE D ISEASE
Disease
Ncwcastle disease (l'-'D) is a highly contagious disease of
chickens that is characterized by respiratory distress, diar-
374 PART ITJ Vir11ses
rhea, and neurological signs. The severity of the disease is
dependent upon the age and lmmune status of the birds,
and on the virulence of thc strain of ND vi.rus th;it is rP-
S_1Ju11sible for tl1e ir1feclio11. The n1ost virulent strains are
desigr1atecl as velogeníc and produce mortality rates in af-
fected birds as high as 90% or more. Thc discasc causcd by
mesogenic strains is less severe and the mortality rate is
often less than 25%. The lentogenic strains are relatively
avirulent and are often used as vacclnes.
Etiologic Agent
Physical. Chemical, and Antigenic Properties
Thc properties of ND virus are sim ilar tn thosr o f other
rut:111ut:r~ of genus and subfan1ily.
Resistance to Physical and Chemical Agents
Newcastle disease virus (NDV) can be inactivated by heat,
ultraviolet light, pH, oxidation, and chemicals (lysol,
phenol, detergents, and butylated hydroxytoluene). The
rate uf viral iI1a<.:tivatio11 va1 ies will1 ll1e strain of NDV,
q11;intity of virus initially exposed, time of exposure, and
prcscncc of organic matter in thc cnvironmcnt. Infc.c-
tious virus has been recovered .trom contaminated areas
and eggs h~lls severa! weeks following an outbreak of ND,
and can survive for long pcrlods tn eggs and in frozen
carcasses.
lnfectivity far Other Species and Culture Syslerns
Newcastle disease virus infects chickens, guinea towls,
turkeys, and a large number of species of domestic and
wild birds. Sea birds are less susceptible but may actas car-
riers. Humans accideotally infected with ND\l v;hen ex-
poseu tu iI1feLteu 1.Jiru~ or livt; viral vaccines 111ay develop a
self-limiting conjunctivitis.
Newcastle disease virus can be propagated in the
chorloallantoic cavities of 10- to 12-day-old embryonated
chicken eggs. The most commonly used cell cultures for
the cultivation of NDV are prlmary chick embryo kidney,
primary chick fibroblast, and baby hamster kidney (BHK)
t:ells.
Host-Virus Relationship
Distribution, Reservoir, and Transrnission
Newcastle disease occurs worldWide and domestic birds are
the major reservoir for NDV. Although NDV has been iso-
lated from a targe n umber of wlld blrds such as sparruws,
crows, d ucks, and geese, they a ppear to pl;iy a minim;i l role
iu trau:.uülli11g Lhis disease. 1"he epizootic outb reak of the
exotic. velogenic strain of NDV in North America (Califor-
nia) in 1971 has been attributcd to thc introduction of
caged birds.
Aerosol respiratory infection is the most common
route for transmission of NDV. Infected birds begin to
shed virus 2 to 3 days after exposure from their respira-
tory tracts and continut! to ~ln.:u viru:. for severa! weeks
'íhe virus also is readily spread hy fomites because of its
stabi!ity.
Pathogenesis and Pathology
Tnitial replication of NDV occurs in the mucosa of th~
upper respiratory tract fullowi11g aero:.ol i1úeclion. Vire-
mia then clisseminates the virus throughout the body, anc
widespread multiplication of virus in cells of parcnchyma!
organs leads to a secondary viremia, which in sorne in-
stanccs leads to the infection of the cells of the CNS.
lJiseasc tal<es severa! different forros in chíckens, depend-
ing on the virulence of the strain involved. l'he very viru-
lc11t velogenic strains caust: vcry raviuly fatal il1feclions in-
volving the visc.er;il organs or the CNS. Mesogenic strains
of NDV cause respira tory and, occasionally, ncurologic dis-
ease in infected chickens with low mortality, while lento-
gcnic strains produce a mild or often inapparent disease.
lJifferences in the virulence of individual strains of ND\-
are correlated with differences in the cleavage and activa-
tlon of theír HN protein, anu thu~ tl1e:.e :.tsai11s rapidly can
be differentiated by sequence analysis of the gene encod-
i11g HN.
Lesions in ND vary greatly. lnapparent infections cause
few, if any, lcsions whcrcas hc1norrhagic necrosis that af
tects tl1e intestinal tract, resp1ratory tract, and visceral or-
gans is characteristic of more severe forros. In chickens
with CNS involvement, necrosis uf tl1e glial cells, neuronal
degeneration, perivasculilr c11ffing, and hypertrophy of
e11doll1ellal cells are often present.
Host Response to lnfection
Chickens infected with NDV produce antibodies 6 to 10
days after infection. AntibOdles to the envelope glycopro-
tein, HN, exhibit viral neutralizing and hemagglutination
inl1iuitio11 aLtivities a11d are responsible for 11ost imn1unity
to thr. disease. Humoral antibodies (lgM and IgG), secre-
tory a ntibody (IgA), and cell-mediatcd immunity ali ap-
pear to play a role in im.m unity to NlJ.
Laboratory Diagnosis
As the clinical signs and pathologic lesions of ND are vari-
able and nonspecific, definitivc diagnosis of the disease
must depend on laboratory methods to ident1fy NDV. This
now is best accomplished using PCR, and sequence and/or
nucleic acid hybridization analysts to tlistinguisll wlit;llit::r
the virus is a velogenic fielc.1 strilin or ;i live vaccine strain.
Al Le cualively, NDV ca11 be isolated by inoculating embry-
onated eggs or cell cultures with respiratory exudate or tis-
sue suspensions (splcc11, lung, or brain), and NDV antigen
in affected tissues or cell cultures can be identified by iln-
munofluorescence or immunohistochemical staining.
Serological diagnosis requlres demonstratiou uf ri~iu~
NDV antibody titers by the hemaggl11tin;ition inhibition,
i1t:utralizalio11, 01 ELISA n1cthods.
 Chapter 60
lreatment and Control
Sanitary managcmcnt to prcvcnt cxposure of susceptible
-:-hickens to NIJV is an important aspect ot control against
die disease. Since therc is only onc scrotype of NDV, vacci
C!ation with either lnacttvared or live Virus vacctnes is also
1sed to prevent ND. The majority of Iive vaccincs incorpo-
:ate lentogcnic strains of NDV adn1il1islered iu drillkiug
"l·ater or applied as aerosols.
RESPIROVIRUSES
The respiroviruses are includcd in the su bfamily
Paran1yxoviri11ae, fa111ily Pu1ur11yxovi1 iclue. All viruses within
this genus have both hcmagglutinin and neuraminidase
HN) proteins. Included in the respiroviruses are parain-
:Iuenza viruses from cattle, primates, and mice (Sendai
tj rus) . The parainfluenza virut.e$ :\re distin.guishcd bythcir
host range and scrological reactivity. They are frequently
a~~oriated wi.th upper respiratory infections in humans
and anin1als.
(ANINE PARAINFLUENZA TYPE 2 1nyxovirinae.
Canine parainfluenza virus 2 has bcen implicated as a
cause of illfecliuu:> Lraclicul.lru11clúti:s (kennel cough) in
dogs, a.long with canine acif'novirus anci HnrdP.tP.fla hron-
chiseptica. 1'he disease is characterized by sudden onset,
mild fever, slight to copious nasal discharge, and a harsh,
nonproductive cough. Parainflueoza Typc 2 virus is anti-
genically related to the SV-5 vírus of monkeys, and it ap-
pears that the same or very silnilar viruses infecta wide va-
riety of animal species. Live modified parainfluenza Type 2
viral vaccines are availablc, L1sually combined with other
canine viral vaccincs such as canh1e dislen1per and ca11iI1e
hepatitis or with a Bordetella bronchiseptica vaccine.
80VINE PARA INFLUEN ZA 3 of nasophuryngcal smears with immunofluorescent anti-
Parainfluenza Typc 3 virus (Pl-3) 11as been ilnplicaleu i11
so-called "shipping fever" of cattle, a clinical disease syn-
dromc in thc bovinc rcspiratory discase complex. ·rhe dis-
ease is charactcrizcd by high fever ( 107UF or above), con-
junctivitis, rcspiratory distress, mucopurulent rhinitis,
anú pneumonla, and occurs after cattle are congregated on
fee<llots. ThP ciic:e;ise is widcspread in the United States and
remains one of the major causes of econon1ic lo:.:ses il1 tl1e
cattle industry. A number of viruses including PT-3 anci
bacteria, cspccially Mannhcitnia hae1nolytica, have been
implicated 1n thc pathogenesis ot "shipping fever," but the
precise role of PT-3 virus in the disease, iJ any, is poorly
defineú. Similar vlruses havc been isotated from upper res-
pi ratory infPttions of humans, sheep, horses, and water
buffaloes.
Param)1xoviridae1 Filoviridae, and Bomaviridae 375
Inactivated and modified live PI 3 viral vaccines are
available, usually comblned with otl1cr viral vaccines or
\'\/ith bacterms.
PNEUMOVIRINAE
P NEUMOVIRUSES
'fhe genus Pncurnovirus is includcd in the subfamily
Pneu111ovirinae in the family J1aramyxoviridae (see 1'able
60.1). Viruscs w ithin the genus include respiratory synci
tia! viruses that lnfect humans, cattle, and mice, as well
as thosr. that infPrt <;h<'r.p and goats, which are closely re-
lated to bovinc respiratory syncitial v irus. The viruse~
within th·is gcnus are distinguished on the hasis of thPir
host rangc and lack of cross neutralization. The members
of this group lack neuraminidase and the nucleocapsid
has a diameter of 13-14 nm, which is smaller than the
nucleocapsld of the paramyxoviruses (18 nm). Ali mem-
hers of thf> gPnn<: Pnr 11n1ovir11s are antigenically related
1
but are antigcnically distinct fro111 olhe1 _l)arau1yx-
oviruses. Their hcmagglutin protein is designated as G, as
compared to H or HN in mcmbcrs of thc subfamilv , Para-
'
l
-
J -
B OVINE RESPIRATORY SYNCYTIAL VIRUS
-
e
BoviI1e respiratory syncyttal virus (BRSV) causes an acute
pneumonia in r;ilvP<;, which show such clinical signs as
coughing, fever, anorexia, nasal discharge, and respiralory
distress. The pneumonia is characterized by b ronchitis
and alvcolitis with multinucleated syncytia and alveolar
epithelial hyperplasia. ·rhc disease has been reported in
North America, Japan, Europc, and Australia. Not ali cattle
infected with BRSV show clinical signs of the disease.
Tnfected animals serve as the reservoir of the disease and
the virus is shed in the nasal secreliuu~ uf iufectetl ani-
mals. BRSV infection can be diagnosed hy direct staining
body to HKSY.
Cattle infectcd with BRSV develop serum-ncutralizing
antibody 3 days after viral exposure, reaching a peak after
3 to 4 weeks. There is also evidence of a cell-mediated im-
111une 1espo11:.e i11 cattlc to BRSV. An attenuated live
bovine respiratory sync:ytial viral varrinP is available.
METAMOVIRUSES
Metapneumoviruses are disti ng11i<;hPd from pneumo-
viruscs on the basis of their gene order and virion proleil1
composition. ·rurkey rhinotracheitis virus (aVian pneu-
movirus) is assigned to this group. This virus causes upper
respirarory tract disease ín young turkeys and chickens.
376 PART TII Viruses
FILOVIRIDAE
Viru~es wilhin lhis fan1ily are characterized by envelopcd,
p leomorphic (filan1entous) virions and a gcnome of
singlc-strandcd n1olecule of negative sense RNA. There are
two major groups of filoviruses: Ebola and related viruses,
and Marburg and related viruses. Viruses within these
groups cause severe, fatal diseases of humans that are char-
acteri:zed ;is "hemorrh::ieir fPvPrc:" hpc;i11c:P of thf' spi>ctacu-
lar diseascs they cause in affectcd hurnans. These viruscs
infect primates and can be adaptcd to laboratory animals.
BORNAVIRIDAE
Viruscs within this family are characterized by enveloped
spherlcal virions of approximately 90 r1u1 ill Llia1ueler, a11d
a gcnon1e o[ single-stranded, ncgative-sense RNA. Barna
dlscase virus (BDV) is tl1t: prululy_¡Je virus of lhis fan1ily.
ShPep ¡¡ncl hnrses are considered to be the natural reservoir
host of BDV, but infcction occurs in a varicty of othcr ani-
mal species including humans, cattle, rabb1ts, goats, dogs,
cats, and selected species of birds. The BDV is neurotropic
and it has been incriminated as a cause of a variety uf ue-
havioral disorders in humans and encephalitis in animal~ .
.13orna dlsease is descrilJetl a:> :>t:vt:1 e 11eurologic disorder of
horscs that largely is confincd to regions of central Europe,
whereas apparently asyrnptomatic BDV infection of hu-
mans and horses apparently occurs worldwide. The precise
significancc ofBDV as a veterinary pathogen remains to be
adequately defilled.
 Rhabdoviridae
YUAN CHUNG ZEE
- -abdoviruscs (the Greek word rhabdo means rod) are
...:iet-snaped, envetopea, s1ng1e-strandcd KNA viruses
-at have a broad host range. Members of the family Rhab-
- iridu.e are iru.:luded ili. fuur geuera (Lyssavirus, Vesicu-
irus, Ephern1erovirus, Novirhabdo viru.~) a nrl infPrt verte-
;atcs, invcrtebrates (arthropods), and plants. Rhabdo-
~ses cause a variety of important diseascs: rabies (and
~,ies-related virus diseases), vesicular stomatitis, bovinc
::"lemeral fever, and diseases of fish that 1nclude spring
:.:emia of carp, infectious hematopoetic necrosis, and
--11 l 11::murrhagic septicemia of salmon (fa ble 61. 1). Rhab-
:. •iruscs sharP a rommon morphology, reguire a viral
- ~-.\-depcndcnt polymerase for replication, and llave 1na-
-.;:e virions that bud from plasma mem branc or into intra-
-.oplasmic vacuolcs (figs 61.1, 61.2).
-• ABI ES
-- .sease
...
--bies virus infects ali \'varrn-blooded ani1nals, includu1g
-mans, causing a severe and invariably fatal disease of
- e central ncrvous systcm (CNS). Thc incubation period
prolonged, varying from 2 weeks to 6 months and even
!1ger in cxceptional circumstances. Clínica! signs h1volve
~e CNS but vary depending on the spectes of animal that
affpctPcl . 'íhP t>arly signs ilnd symptoms of human rabies
_;:e headache, exl1erne lhirsl, vu111illug, <1ud anorexia.
·ore advanced signs and symptoms inrl11clP painful
pa:;m:; of thc pharyngcal mu:;clcs "vhc:n drinking (hy-
_::ophobia), exciternent to sensory stimuli, and general-
-2d paralysis. Oeath is the inevitable outcome once clini-
~ signs d evelop. ·rhe course of rabies In dogs usually lasts
1!y ~ to R cli'iys. Rehavioral changes are common during
·-e early phases of disease, and Ieve1, dilaliu11 uí tl1e
~.ipils, and photophobia are sometimes present. The furi-
- llS form of rabies occurs latcr, when the affected dog be-
_on1es nervous and progressively irritable. Affected dogs
::iay bite without provocation. Clinical signs include pro-
-Se sa!ivation a nd frothing at the mouth, difficulty in
· ~ llnwing an<I rlrinking, convulsions, and muscular inco-
:dination. Sorne affected dogs exlubil 011ly a ¡.iaralytic ur
~umb" stage without going through a furiou.-; phasP.
21aracteristic clin ical signs of this form of rabies are paral-
'""SlS of thc muscles of the pharynx and lower jaw, and in-
.:oordination that progresses to coma and death. Thc clin-
N. JAMES MAcLACHLAN
ical signs of rabies in cals a1e siuülar lt1 l11u:.e i.I1 dugs, but
rabid cats have an even greater tendency to hide in se-
cluded places and are often more vicious t han affected
ctogs. Clln lcal. signs of rabies 1n horscs often include pro-
gressive ataxia and eventual paralysis, and difficu lty in
swallowing. Calllc affected with rabies are usually excit-
able and restless. Typical signs inrl11clP salivation, choking,
abscnce of rumination , rectal straining, and paralysis of
the hindquarters.
Several viruscs that are closely related to rabies virus can
also cause a rabies-like disease in intectcd animals and hu-
mans (see Table 61.1). Bat s are an important reservoir host
uf severa! of rhese viruses.
Etiologic Agenl
Physical, Chemical, and Antigenic Propertles
·rne virion ot rabies virus is shaped like a cone ora bullet,
approximately 180 nm in length and 80 nrn in width, and
contalns an envelope with short spikes (peplomers 6 nm
to 7 nm in length). A central cylindrical core of ribonucle-
op1olei11 ru11s tluuugl1out tl1e longitudinal axis. The virus
has a huoyant rlPnsity of 1.?.ílg/rm3 in cesium chloride.
The viral RNA is single-stranded, negativc-sense, a11d ei1-
codes ftve subgenomic mRNAs t hat are translated into five
n1ajor prot eins designatcd as L (thc RNA-dcpcndcnt RNA
polymerase), G (the glycoprotein that for ms the envelope
spikcs, N (the nucleoprotein), P (a part of the viral poly-
merasc) and M (or M 2 for rabies virus) . The major surface
glycoprotein G contains the neutralizing epitopes of the
virus. The nucleoca¡.>sid Lu11sisls uf v!Jal RNA a11u ll1e N,
NS, and L proteins, and tl1e M protein links the nucleoc.ap-
sid to thc lipid cnvclop that contains the G glycoptotein.
5tralns of rabies virus isolated trom naturally occurring
cases are referred to as "street virus," and attenuated labo-
ra tury stralns are referred to as "fixed virus." These strains
cliffpr in their bi.ologic properties in laboratory anirnals-
for exan1ple, virulence, lengll 1 oí thc iuculJation period,
and distribution and nature of h istologic IPsions in target
tissucs. Thc antigcnic variation betwccn street and fixed
strains of rabies virus can be distinguishcd by their reac-
tion with monoclonal antibodies. It has bcen shown that
vacclnatcd mice were protcctcd against challenge 'vith the
field strain that was more closely related to the vaccine
strail1 lhan 11101ea11Uge1úcally di:>tant stralns. Thus, the se-
Iection of appropriate strains nf thP virus for vaccine pro-
duction can be critical.
377
378 Pl\ICT JIJ Viruses

Table 61.1. Mdjur Ger n:~r d of Rhabdoviruses

uenus Virus Natural Host Dlsease

Vesiculovirus
Vesicular stomatitis Horses, cattle, swine Vesicules ~nd ulccrs
Alagoas virus
lndiona virus
New Jersey virus
Spring viremia of carp Carp Abdominal distention
Lyssavirus
R~hi~ Ali warm·blooded animals Rabies
Au)lfalian bat lyssavirus Bat Rabies·like
European lyssavirus (1 & 2) Bat Rabies·like
lagos bat B~t Rabies-like
Mokola Shrews Rabies·like
Ephcmcrovirus
Bovine ephemeral fever canle Systernil i11le1.liu11, rt!Vt!I
Novirhabdovirus
lnlt!<.liuu) 1i~111i1lopoietic necrosis Salmonids Systemk; nervous system
Ungrouped
Viral hemorrhagic septicemia ~lmonids Tissue necrosis and
hemorrhage

Resistance to Physical and Chemic:al Agents

F1G U RE 6 1. 1. Vesicular stomatitis virus budding trom plasma
membrane of an infected L ce//. 40,000X. (Reproduced with
permission frou1 lee YC, Hatkell AJ, Tdlen L. Vesicular ston1atitis virus
maturation sites in six different host ce/Is. J Gen Viro/ 1970;7:95.)
Rabies vir us is i11activated t)y heatlng at 56ºC for 30 rnin-
utes and by cl1emical agents such as formalin (1 ºA>), creso!
(3%), and beta-proplolactone (0.1 %). ·r11e virus u1ay JJt:rsi::.l
in infected brain tissue for up to líl <iays at room te1npera-
Lu1e a11d íor severa! weeks at 4°C, but is relatively suscepti-
ble to disinfection.

l lnleclivity for Other Species or Culture Systems

Rabies virus infects and repllcates in aH warm-bloodcd an-
imals. The virus can be rea<lily propagated in chicken em-
bryo or duck embryo as well as in a 11u111uer o[ ct:ll cul lure~.
especlally in b<iby hamstPr ki<lncy cells (BHK21) and
hun1an diploid cells (Wl-26).

1•
b snvír11s .is closely rPlatPc1 to r;ihics virus and causes aniden-
Rabies virus is included in the genus Lyssavirus, along
with rabies-related viruses like Australian bat lyssavirus,
European bat Iyssaviruses l and 2, Lagos bat viru::., a11d
Mokola virus, all of which can in<1111P a rabiPs-like disease
i11 l 1u111a11s (see Table 61.1).
Host-Virus Relationship
Distribution, Reservoir. and Transmission
Rabies is distributed throughout the world, with the no.
table exception of Australia (altl1ough AusL1alia11 bal Lys-
tical disease in humans), thc United l<i.ngdom and Ircland,
Cyprus, Ja pan, New Zealand, and Scandinavia. · rhe di sease
is cndcmic in many countries in Africa, Asia, North and
South America, and Western Europe. Extensive epizootlcs
of rabies have occurred in Asia and Africa in particular.
Rabies recently has been eratli<..:ate::u fn.>111 exle11sivc por-
tions of EuropP hy vacci nation of wildlife reservo ir hosts of
ll1e virus, especially foxes.
Of the many animais that are susceptible to rabies,
dogs, foxcs, skunks, wolvcs, raccoons, mongooses, coyotes,

_\.u1lks, and bats ali can serve as rcscrvotrs of rabies Virus.
~e <;ignificanci> of inclivi<h1al ri>~i>rvoir host species differs
bt:twccn gcographic regions. For example, foxcs are an in1-
70rtant reservoir host of rabies virus in Western Europe.
Similarly, foxcs are the most important reservoir hosts of
:abics virus in portions ot Canada, Alaska, and thc dcscrt
southweste rn regions of the Vnited Sta tes, where:is skunks
and raccoons are the critica! reservoi r of rabies virus in
Dther regions of North An1erica. 'fhe mongoosc is a11 in1-
po1La11L1es1::1 vui1 of rabie:> viru::; iu Afric.:a a11J A::;ia. ·rhe tlu-
mestic dog, however, remains the most important source
of rabies for humans, especially in the developing coun-
tries of Africa, Asia, and Latin America.
Most rabies cases stem from thc bite of a rabíd anímal-
the saliva of most infected animals contains infectious
virus. Howcvcr, transmission from bats is an increasing
problcm, and rabies virus maybe transrnlttcd from bats to
h uman~ \'\lithnnt any histnry of" hitP Thibies rnay be ac-
quired by inhaling aerosol containing rabies viru~ in in-
fectcd ba t caves or by virus passing through intact n1ucous
me111brancs.
Pathogenesis and Pathology
Thi> prim;iry ro11ti> of infection in rabies is through the bite
of a rabid animal that contains infectious virus iI1 ils
saliva. Severa! factors, including the virulence of the iI1-
fecting virus strain, thc quantíty of infcctious virus in thc
saliva, and the susceptibility of the spccies, are important
in determining whether or not infcction is established in
the rec.:lplent animal. Foxes, cattle, hamsters, and coyotes
arp pnrticnl;:irly <;11scPptible to rabies, <ind dogs and cats are
more susceptible than either hu111ans or rode11ts. "!'he
length of lhc incubation period after viral exposure varíes
greatly and dcpcnds on thc anatomic distancc between
the hite s1te and the CNS, the sevcrity of the bite, and thc
amount of infcctious virus in the saliva.
Follow1ng viral cxposure, the rabies virus persists in the
loral m11scle tissues for hours or days, and viral replication
Chapter 61 Rhabdoviridae 379
F 1G U RE 6 1 . 2. Vesicular stomatitis virus budding
into the lumen of cytop/asmic vacuo/e in an infected chick
fibroblast. 17,000X.
rakes place in str1ated muscle cclls near the inoculation
si te. Thcrc is no evidence that a viremia is necessary for dis-
se111i11aliu11 uf ral>ies virus to the CNS; rather, rabies virus
is taken up at motor and sensory ni>rvi> i>ndings and the
virus then ascends vvithin Lhe axons oí 11e1 vt:: c.:e::_ll::; lu Ll1l!
ventra l horn cells of the spinal cordata rate of 12 mm to
21 mm pcr duy. Antibodics do not inhihit viral transpoct
once virus is present within thc axon. Viral replication oc-
curs in the spinal cord cells and subsequently spreads to
the IJrain. '!'he virus then dissernlnatcs wlthin the brain
li>acling to thi> rlinical <;ign<; and symptoms of rabies. lt
should be pointed out, however, that thc virus ca11 persisl
in the brain of infected skunks, rats, raccoons, bats, and
foxcs for many months without producing overt clínica!
signs. 1t has been sho\vn experimenta Uy in rodents that ra-
bies virus after peripheral inoculatíon can be found in the
spinal cord 24 to 72 hours after iooculation and in the
brain 96 to 192 hours after inoculation. Rabies virus ulti-
n1alcly spreads fron1 tl1e CNS Lo 0Lhe1 oi¡~a11:s, :sucli as ::;ali-
vary glands, cornea, and tonsils, via the peripheral nerves.
Viral rcplication in salivary glands occurs rapidly, and in-
fected saliva is the major source ot infection.
Lymphocytic polioencephalomyelitis with perivascular
cufflng is thc most prominent histologic lesron in thc
brains of animals with rabíes, although the severity of this
chaugt: i:> liiglily varial>le. The pre:;ence of acidophilic ln-
tracytoplasmic inclusion hocties (Ni>gri hoclii>s) in the in-
fcctcd neuron s of the hippocampus or cerebellum is char-
actcristic ot rabies.
Host Response to lnfection
A11i111als aud l1umans vaccinatcd wlth rabies virus vaccine
develop c:irculating ni>11trali7ing antibodics to the vir11s
within 2 to 3 weeks of vaccination. High tilcrs of neultali:t.-
ing antibody may occur in aoimals with rabies; thus it is
Jikely that antíbodics must be prcscnt at thc time of expo-
sure to rabies virus if they are to be protective. Both cellular
and humoral immune responses are important in prevent
380 PART III Viruses
ing rabies in vaccinatcd animals, although ncithcr mecha-
nism is eítective in preventing disease in naive animals.
Laboratory Diagnosis
·rhP rliag nosis of human rabies centers on a history of an
animal bite and, logicully, cvcry cffort :;hould be takcn to
locate and quarantine the suspected animal. Clinical diag-
nosis of rabies in animals requires differentiation from
other infectious or noninfectious diseases of the CNS.
Rabies should be suspected in endemic or epizootic are;is
wl1c11 a11 a11i111dl exl.ül.Jil:. al.J11u1111al behavior and/or paral-
yc;is. The definitive laboratory diagnosis is typically done
by direct immunofluoresccnt antibody staining of sec
tions of brain, which is a vcry rapid and accurate ctiagnos-
tic test. Dircct immunofluorescent staining of the skin can
be used asan antemortem diagnostlc test al so. Highly se11-
sitive PCR assays have heen developed for laboratory con-
firmation. The diagnosis cau l.H.: cu11fi11ned by intracere-
bral inoculation of yo11ng mice with brain suspension
frun1 suspected animals. lnoculated mice usually develop
clinical signs within 17 days of inoculation, and Negri
bodies are usually prcscnt in thcir ncurons at the time of
death. Monoclonal antibodies directed aga1nst the glyco-
protein of rabies virus can be uscd to distinguish rabies
virus from rabies-related vlruses, and to confir111 ca:.e:. uf
vaccinc-induced rabies in dogs, c<1ts, ;inri foxe.s.
Treatment and Control
Rabies in animals can be controlled by eliminating or im-
munlzlng wlld animal reservoir ho~t~, <u11.l 1.Jy v<11.:1.:i11ali11g
susceptíblP ciomf'sti< anímale;. Strict quarantine of im-
ported wild and domestic animals for up to 6 months from
rabies-endemic regions has kept island nations such as
New Zealand, Australia, the Unitcd Kingdom and Hawaii,
free of rabies. 'fhe elimination of wild animal reservoir
hosts is difficult and increasingly unacceptable in many
countrles, tl1us vacc1nanon of i:tu:!se hust:. l~ a u1ure accevt-
a ble and successfu 1 stratPgy.
Va.rious types of inactivatcd and live attenuated rabies
vaccines are commercially available for vaccinating dogs
and cats, including ncw gcncration rccombinant vaccines
that do not include intectious rabies virus. New generatjon
vaccines do not suffer from the instances of vaccine-
tnduced rabies that sporadically occurred With tbe original
live-attenuated low-egg passage vaccine Caution shonlc1
be exer<.:iseu wlte11 live vacci11es are used, especially in a dif-
fPrPnt species.
ihere is no ef-fective treatment for rabie:; in animals.
VESICULAR 5 TOMATITIS
Disease
Vesicular stomatitis (VS) virus causes an acute febrile dis-
casc of horscs, cattle, and swine in the western hemisphere.
The disease in cattle is characterized by the formation of
vesicles in the mucosa] lining of the mouth and tongue.
Lcsions ra11ge from mild punctatc rrosi.ons on the. rle.ntal
µau lo :.t:vere ulcers on lhe lo¡1gue. Forn1ation of vesiclcs is
also ohserved on the teats, on the skin of the coronary
band, and in t he interdigita l spaccs of thc foot. The lesions
of VS in cattle and p igs mimic those of foot-and-mouth dis-
case, and in swine they also mimic those of swine vesicular
disease and vesicular exanthema; thu~ thc 111ajor in1pacl of
VS is the restrictions impo~Prl on animal movement that
adver:.ely iluµa<.:L lrade and co1nn1erce. The important dis-
tinrtion, however, is that only VS occurs in horses, and
hor:;e:; frcqucntly are thc scntincl spccics affccted during
outbreaks ot v::;. Jndeed, spectacular epidemics of VS oc-
curred in the past when horses were congregated in the
United States during times of military confllct.
Vesicular stomatitis is a relatively benign ;ind self-
limltíug <.li~t:a~t:, a11u infecled an irnals usually recover un-
PvPntfully. Hovvever, milk production can be markedly
impacted in dairy cattle, and affcctcd animals and the oral
lesions can be painful so that affected animals cease eating
and lose condition. The VS virus produces an acute, in-
fluenza-like illness in humans, and severe con¡uncti viti~
when virus directly is introduced to the eye. Experiment;il
lnfec..lion of mice wlth VSV can pruuuce fa tal encepl1ali tis,
and the susceptibility of VSV in mice is age-dependent.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
1'he VS virus ger1erally resembles rabies virus in its mor-
phology, genome, and protein structure. Three diíterent
spccics of VS virus occur in the Americas; New Jersey,
Indiana, and Alagoas v1ruses.
Resistance to Physical and Chemical Agents
V1:::.h.. ula1 :.luJ11dlili:> vi1 u s is inactivatcd by high tempcra-
tures (50ºC to 60ºC for 30 1ninutes) and by light, ultravio-
lct light, and lipid solvents. lt is also scnsitive to common
disintectants such as sodium hypocl1lorite and quaternary
ammonium compounds (Clorox, Roccal, respectively)
phenol, and formalin. The virus Is more re~i:.taul Lu l)e
(NaOH); 2% to 3% !ye fails to inactivate VS vi rus after an
t:xpu:.ure uf 2 hours. 'I11e virus can be preserved for years at
- 70ºC and by freeze-drying under vacuum.
lntectivity tor Other Speties or Culture Systems
Vesicular stomatitis virus 111fects anll causes ui~eases :.
horses, mu les, cattle, swine, dcPr, ;incl humans. Mo
mamrnalia11 svecies can be infected witl1 VSV, and t h.
viruc; can he propagated in laboratory animals such as mic.
and guinea pigs and in cmbryonatcd chick eggs. Vesicula-
stomatitis virus readily is propagated in a wide variety ....
cell cultures and produces a cytopathic effect and visib. ~
plaques. 1'he virus can also replicate ir1 u1u~4uiloes a1.~
certain other insects.

Host-Virus Relat ionship
Distribution, Reservoir, and Transmission
vesicular sromatitis is a disease of North, Central, and South
.l\merica. Vesicular stomatitis virus is endemic in regions
closesLLO ll 1e equalur, auu t:¡Ji<leuli<.: Íll<.:UISÍUIIS uf the virus
into ad jacent temperate regions (such as thc {Jnitecl StatPs)
occur at regular intervals (3- 10 years or so). Doth Ncw Jersey
anct Indiana viruses cause epizootics of VS in North
An1erica, although New Jersey now is more common .
Yl.olecular epldemiologic studies indicate that each VS epi-
demic in North America is caused by a single genetic variant
o f VS vi1 us, suggesli11g Lhal Ll 1e virus er ncrgcs frurn an en-
demic focus further to the south. Multiplc gcnctic linP.agPs
of VS viruscs oc<.ur in tropical regions of the Americas.
The definitive reservoir host ot VS virus is uncertain, and
insects havc bccn implicated as biological vectors. Direct
<.:ontact Is an lmportant mode of transm1ss1on of VSV as
la rgP q11ilntities of virus are present in the vesicles in t he
mouth, thus contan1j11ation wilh itúected saliva <.:ar1 trans-
mit the infection to healthy animals. Cattle can also he ex-
perirncntally infcctcd by thc aerosol route. Severa! insects
estable fly, tabanids, mosquitoes) have been demonstrated
to be mechanical carriers ofVSV and presumably can trans
mit the dlsease. Sandflies perpetuate the vlrus in tropical
anrl ~11htropical regions of the Americas, including Ossa-
baw Tsland off the coasl of Geo1gia i11 ll 1t Uuitetl States.
Epizootics of VS typically end after the fi rst killing frost in
tcmperate regions of North Am erica, and the seasonal oc-
currence of outbreaks of VS in the warmcr months furt her
implicatcs insccts in thc disscmination of the virus.
Pathogenesis and Pathology
Vesicular stomatitis has a short incubation period in ani-
mals lnfccted through an abrasion, after which the virus
rapidly disseminates to a wide variety of tissues. Cattle with
VS are febrile (103ºF to 104ºF) and develop oral lesious Lhat
begin as papules that progress to vesicles which rapidly ul-
ccratc, lcaving punctate erosions on the dental pad and ul-
cers that can be cxtensive on the tangue and oral mucosa.
In severe cases, sloughing of the mucous membrane on t he
~urfac.:e of the tangue causes extenstve salivation and
anorPxiil. lJ lcerated vesicles can be extensive in the mouths
of affected horses. High titers of virus a1e p1esenl i11 Lite v1::si-
clc fluid and saliva but not in the blood. 'fhese lesions usu-
ally hcal rapidly, although rccurrcnt and/o r chronic ulcers
can develop. Similar vesicular lesions that rapidly ulcerate
occur on the feet and teats of affected cattle and swine, and
tl!csc <.:an IJec.:ome secondarlly lnfected wlth bacteria.
Histologic changes in affectcd arcas of oral mucosa and
skin adjacenL to lhe hoof ü1<.:lutlc c¡.¡ltl1elial hyperplasla
with marked intracellular and interccllular edem a. Neut ro-
phils infiltratc affcctcd rcgions, whic h rapidly undergo ne-
crosis leading to eplthelial ulceration.
Host Response to lnfection
lufcLtc<.J or vaccinated antmals develop high neutraliZing
antihorly titl'r<: (~3?,,000; 80o/o plaque reduction assay) 10
Chapter 61 Rhabdoviridac 381
days aftcr cxposurc. Scrologic response can also be meas-
urcd by the complement tixation test and enzyme-linked
immunosorbent assay (ELISA). Tmm.unity, however is tran-
slent, and animals wit h high titers of neutrali2ing anti-
body can be susceptible to reinfection with t l1e homolo-
gous virus. ·r11ere is liLLle crv:.s-µru lt:<.:lio11 IJet vvcen the
Indiana and New Jerseyviruses.
Laboratory Diagnosis
Diagnosis of VSV is important because its lesions resemble
thosc caused by foot-and-mouth discase; horses are not af-
fPctPrl by foot-and-mouth disease, whereas they are com-
monly affected it1 oulbreaks uf VS. Lauoratury diagno::;is is
required for the definitive identification ofVSV infertion .
The ani1nal inoculation test was once the only one avail-
able for the differential diagnosis ot vesicular diseases, but
now the following laboratory tests are used to detect VSV:
1) electron mícroscopy or immunoclectron microscopy on
vl'<:irle fluid , 2) immunofluoresccnce antibody staining of
vesicle tissues, 3) isolaliou uf VSV usi11g susceptible cell
cultures, and 4) demonstration of a rise in antiho<iy titl'r
by thc ELISA mcthod.
Treatment and Control
A varicty of VSV vaccines are available to prevent eco-
nomic losses from interruption of milk production in
dairy cattlc. There is no effect1ve treatmcnt for the disease
nthPr th;in providing good nursing care. Sick anirnals
should be separated fron1 clinically lieallhy 011es.
BOVINE EPHEMERAL fE VER
Bovme ephemeral fever (ilEr), also known as 3-day sickness,
is a noncontagious disease of cattle and water buffalo that
ls characterlzed by fever, la1neness, anct nasal discharge.
The morbidity of the disease is high but the mortality is
vcry lo"Vv. Econon1ic l os:> is il:>:>v1..ialcu will1 lv:>:> vf u1ilk ¡Jrv-
ductíon in infected cows. Bovinc ephemeral fever virus
(BEFV) can infcct othcr spccics of ruminants. The virus can
be propagated in suckling micc, mammalian cell cultures
such as BHK-21, Vero cells, and insect cell cultures such as
Aedes albopicrus cells.
l .ikP rahies and VS viruses, BEF viruses are bullet-shaped
or cone-shaped particles wilh a le11gllt uf 140 1101 to 200
nm anda diameter of 60 nm to RO nm . The genome and
virion organization also are similar. The virus is inacli-
vated by heat (10 minutes at 56ºC) and by high pH (12.0)
orlo~"' pH (2.5).
Bovlne ephen1eral fevcr is enzootic in tropical and sub-
tropiral regions of Africa, Australia, the Middle East, and
Asia. It has ncve1 been repurtt:<l ir1 tl1e An1erlcas. The dis-
ease is not transmitted by direct contart, and BEFV has
bccn isolated from mosquitocs and bitiJ1g n1idges
(c;ulicoides) that serve as vectors.
Lesions described in fatal cases of BEF include serofibri-
382 PART III Viruscs
nous pulyserusiti:. wilh accw11ulation of fluid in thc joints
and pericardial, peritoneal, and pleural cavities, and con-
gestion of lymph nodcs. Histologic lesions include necro-
sis oi 1nuscte, generali.zed J1yperemia and edem a, and vas-
culitis that apparently is mcdiated by neutrophils.
lJiagnosis is accomplished by the dcmonstratiur1 uf
BEfV by isolation in cell culture or intracerebral inoc11lri-
tlon of suckling mice witl1 ulvvd of infccted aI1imals 1 or
by the dPtPction of a rise in neutralizing antibody titer in
paircd serum samplcs. Animals infcctcd with BEFV are
immune for severa! years. Experimental attenuated live,
rccombinant, and inactivated vaccines have been de-
veloped.
•
D ISEAS ES OF f lSH ( AUSED BY RHABDOVIRU SES
lnfeclious 11e111atopoietic necrosis, Spring vircmia of carp
;inct viral hemorrhagic septicemia ot sailnonids are diseases
of fish that are all caused by rhabdoviruses, and tl1ere ar"'
several others. Infectious hematopoietic n ecrosis is a dis-
cnsc of salmon and trout that cause substantial losses ir
hatcl1er1es in North America, Asia, and Eurupe. Di:>ea~e :
characterized by anemia and hPmorrhrige. Viral hemor-
rhagtc septiceuua is the cause of a similar disease of salmo-
nict~. Spring viremia of carp virus causes a fatal discase Oi
carp that also is chnrnctcr izcd by hcmorrhages and edema
ot abdominal viscera leading to abdo1ninal distentiun.
 Coronaviridae and
Arteriviridae
UDENI B. R.
The vi rus families Coronnviridae and Arteriviridae are in
cluded i11 tire orut:r Niduvirales (Table 62.1), alo ng with
the newly ident ified fa m ily Rnniviridae. All members of
the order Nidovirales are enveloped viruscs will1 lir1ear,
positive-sense, single-stranded RNA (ssRNA) genomes.
Thcy share a strikingly similar gcnomc organization and
replication strategy, but d1ffer considerably in their genetic
complexity and virion architecturc. "Nido" derives from
l11e Lali 11 wurd nidus fur nest, which rcfers to the 3' co-
tcrminal n ested suhgr.nomic v ir;i l m RNA that is p roduced
during replicati.on of thcsc v iruses.
1'he family Coronaviridae is further dividr.<l into two
genera, Coronavirus and Torovirus. The Coro11avin1s genus
conta1ns numerous pathogens ot veterinary significance,
and coronaviruses commonly are associated with respira-
tory and enteric infections of a variety of animal species.
·rhe toroviruses infect humans and ani mals (horses, cattle,
and pigs) a11d are predo1ninantly assu <.:lalt:d with enteric
disease. The family Arteriviridae (gen us Arterivirus) con-
tains four species- specific viruses that infcct horses and
donkeys (equine arteritis virus), pigs (porcine reproductive
and respiratory syndromc virus), monkeys (simian hcmor-
rhagic fever virus), and mice Oactate dehydrogenase ele-
vating viru s) . l'he outcome of Arterivirus infection s is
Jiigllly varialilt: dud iu<.:ludes p ersistent asymptomatic in-
fections, respirato ry dis~ase, reproch rctivP f;.iilure (abor-
tion), and lethal hemorrhagic fever. 'fhe family Roniviridae
(gcnus Ronivirus) contains a number of closely related
viruses that infect farmed prawns in Australia and Asia, but
wilJ not be further discusscd here because these viruses are
not yct wcll characterized.
CORONAV/RUS
Coronaviruscs have a unique morphologic appearance.
l hey are spherical, enveloped vi rions with large club-
shaped surface projections (peplomers) cxtending from
the viral envelope. Thc envelope en closes an icosah edral
int1•rn::il con> <;tr11rh1rP within which is a helical nucleo -
capsid (f.ig 62.1). Virion size ra nges fron18011111 tu 220 r1m
in diameter. Coronavirus genomic ssRNA (linear, positivP-
sense) is the largcst viral fl.NA gcnome and rangcs in size
from 27.6 to 31 kb.
B AL/\SURIYA ] EFFREY L. s-rorr
Two viral-spccificd structural glycoprotcin s (spike [S]
and M) are present in t h e envelope. c;1ycoprotein S is
largely external to the m embrane perimeter and forros
tite club-shapcd pro¡ections (approximately 20 nm in
lr.ngth) from the virion membrane. Neutralizing anti-
bodies and cell-1nediaLed cytutoxlclty are directed
against epitopes in the S glycoprotein, which ;.il.;o is rc-
sponsible for binding of virions to host ccll membranes.
G lycoprotein Mis a trans1nembrane protein that is more
deeply embedded in t he envelopc. Antibodies d ircctcd
against lvl may ncutralize t he virus in the presen cc of
complement. The small envelope protein (E), together
wilh the M prolt:in, µlays an essential role In Coronavírus
particle assembly. Sorne of the coronavin1sPs contain a
hcmagglutinin-csterase (HI:,) protcin that forms short
surface projections. ·rhe n ucleocapsid protcin (N) is a
basic phosp h oprotein tl1at forms a long, flexible, hclical
nucleocapsld enc1os1ng the gen omic RNA. The M and N
p rotcins fo rm an interna) core structure in at least two
coronavir uses (Lra11:>111i:>:>ilJle gdstroenter1tls virus and
mouse hepatitis virus).
Coronaviruses infcct a '-vide range of nian1n1als (includ-
ing humans) and birds. They exhibit a marked tropism for
cpithelial cells of the respiratory nnd enteric tracts, as well
as macrophages for sorne viruses. Coronaviruses cause a rc-
markably diverse spectrum of d iffcrcnt diseases in di ffer-
enLhosLs (st:t: Tal.lle 62.1). They typically have a restricted
host range, in fec:ti ng on ly t heir nat1.1ral host and closely re-
lated animal species. Based on tl1ei1 se1ological crus:>-
reactivity, coronaviruses are divided into three an tigenic
groups, including tv.•o groups of mammalian coron-
aviruses an da single group of avia11 coronaviruses.
T RANSMISS IBLE G ASTROENTE RITIS V IRUS ANO
Po RCI NE RES PI RATORY (ORONAVI RUS
Disease
Transmissible gastroenteritis (TGE) is a hlghly con tagious
enteric disease of swine caused by TGE virus (TGEV). The
discase is characlt:rit:e<l liy :;evere diarrhea, vomiting, dehy-
dration, and high mortality in young piglets (less than 2
383
384 PART 111 Viruscs

·1ab1 e 6 2. 1 . lmponant Animal Vlruses in the Order Nidovirales, Which lntlude) Tluee Vi1 us Fan1ilies. Coronaviridae (Genera Corona virus and
Torovirus), Arteriviridae {Genus Arterivirus), and Roniviridae (Genus Ronivirus)

Family Genus Species Primary host Diseasemssue affected

CurufldVÍI iúae Coronavirus Transmissible gastroenteritis virus (TGEV)• Swine Enteric infection
Porcine respiratory coronavirus (PRCoV)ª Swine Respiratory infection
Fclinc coronüvirus (FECoV)ª Feline Enteric infection
Feline infect1ous pentonrtis virus (flf'V)• Peritonitis, respiratu1y, e11le1it, and
neurofogical infection
Canine coronavirus (CCoV)º Ca ni ne Enteric infection
Rabbit coronavirus (RbCoV)ª Rabbit Enteric infection
Humen coronoviru> (l ICoV 229[)• Humon Re<piratory infoction
Porcine epidemic diarrhea virus {PEDV)• ~wine Enteric infeaion
Porcinc hcmagglutinating encephalomyelitis (HEV)b S\vine Enteric. rPspiratory, and neurological
i11feLtiu11
Canine respiratory coronavirus (CRCoV)b Canine Respiratory infection
Bovine coronavirus (BCoV)b Bovine Enteric and respiratory infection
FntPric hovinPcoronavirus (EBCoV)
Respiratory bovine COfonavirus (RBCoV)
Mouse hepatitis virus (MHV)b Mouse Hepatitis, enteric, and neurologlcal infection
llat coronavirus-syn. Sialodacryüdcnitis virus (SADV)b Rat Sialodacryadenitis
Human coronavirus (HcoV·OC43Jº Human Respiratory infection
Scver acute respiratory syndrome coronavirus (SARS-CoV)b Human Re1piratory infection
Equine coronavirus {ECoV)b Equine E11teric infection
lnfectious bronchitis virus {IBV)< Avían (chicken) Respiratory, reproductive, and kidney
infection
Turkey coronavirus (TCoV)' Avian (turkey) Respiratory and entenc mfection
Torovirus Equine torovirus (ETV) Equine Enteric infection
Bovine torovirus (BoTV) Bov1ne Enteric infection
Porcinc torovirus (PoTV) Porcine Enteric infection
Human torov1rus (Hul V) Human Enteric infection
Arteriviridae Arterivirus Equine arteritis virus (EAV) Fquine Respiratory and reproductive infection
Porcine reproductive and respiratory synú1 orne vir u~ (PRRSV) Po reine Respiratory and reproductive infection
Simian hemorrhagic fever virus (SHFV) Simian Hemorrhagic fever
lactate dehydrogenese elevating virus (lDV) Murinc Neurological infection
Roniviridae Ronivirus Gill-associated virus (GAV) Prawns Lyrnphoid organ
lymphoid org~n viru~ (LOV) Prawru lymphoid nr!J•n
Yellow head virus (YHV) Prawns lymphoid organ

• An.t1genK group 1
• Antigenic group 11
<Antigenic group 111

weeks of agc). Murlality in oldcr pigs (greater than 5
wPPks) is 11s ual ly low.
Porcinc rcspiratory coronavirus (PRCoV) is a deletion
mutant ofTGEV (delction ot nucleotidcs 621 to 681 in the
S gene) and has a tropism for respiratory epithelium and
alveolar m acrophages. PRCoV is nonenteropatl1ogcuic
and can. i.nfcct pigs of all agcs by aerosol or direct rontar.t
transmission. PRCoV infectiu11~ art: generally subclinical,
but strains of the virus cliffPr in the severity of thc clinical
~ig11~ t111::y induce. Clinical signs include moderatc to sc-
VPTP rPspiratory di.scase with i.11terstitla\ pneumonia. Ln
addition, PRCoV infection can be concurrently associ-
ated with other respiratory virus infcctions, such as wlth
porc\nc rcproduct\ve and resp\ratoty syndrome virus,
which ca11 alter thc scvcrity of diseasc and associatcd clio-
ical signs.
Etiologic Agent
Physical, Chernica\, and Antigenic Properties
TC;Ev is antigenicauy related to coronavtruses of human~­
dogs, and cats. Only one serotype of TGEV is recogni1E>d
and TGf.V and PRCoV antigenically c1oss-react, although
sorne antigenic sites of TGF.V are ahsent from PRCoV be-
cause uf tl1e delelion in the amin.o terminus of thc S pro-
tein \n ~R\,oV .

F 1G U RE 6 2 . 1 . Negatively stained preparation of bovine
coronavirus. 204,000X. (Courtesy of R. Nordhausen.)
Resistance to Physical and Chemical Agents
TGEV and PRCoV are inactivated by lipid solvents (ether
;u1ll chloroforrn), ~oúiurn hypochlorite, c.¡uaternary arn-
monium c.ompouno, iooinf's, hP;:iting ;:it S6°(~ for 4.5 min.-
utes, and exposure to sunlight. Both viruses are stable
when frozen but somewhat labile at roorn ternperrih1re.
1·GEV resists inactivation by trypsin and acidic pl1 (p11 of
:-{) and is relatively stable in pig bi .le.
lnfectivity for Other Species and Culture Systems
TGE has only been described in swine; however, ·rc.JEV has
been isolated from the feces of cxperimentally infected
cats, dogs, foxcs, and starlings (Sturnus vulgaris) for up to
lO days. Serologic stuclies have also suggested natural in-
fection of skunks, opossums, 1nuskrats, and hurnans.
Virus has also been demonstrated in house flies (Musca
domestica Linneaus) following experimenta] a11d natural
infection.
'f'GEV has been propagated in various cell cuJture sys-
tcms, ir1cluding pig kidncy, tcstis, salivary gland, and thy-
roid; organ cultures of esophagus, ü1testine, and nasal ep-
itl1elium; canine kidney cell cultures; and embryonated
chicken eggs (arnniotic cavity). Pig kidney and swine testi-
cle (S1') cell lines have been the choice of cells for virus iso-
laliou fror11 tl 1<.: feces ur gut contents of infected pigs.
Development of cytopath·ic effec.t rn;:iy rPcprire multiple
passages in cell culture.
l'KCoV replicates in pig kicl11ey a11cl s·r cells, as well as a
cat fetus cell line.
Chapter 62 Coronaviridae and Arterivirid.ae 385
Host-Virus Relationship
Distribution, Reservoir, and Transmission
'fGEV infection of swinc occurs worldwidc and has been
documented in North, Central, and ~outh America; .Eur-
ope; and Asia. Epizootics of 'l'GE are often seasonal in the
Uniteú States, occurring ir1 the winter months. The pri-
mary modf' of 'T'GF.V tr;insmissi.011 appears to be ingestion
of feed contaminated with infected feces (fecal-oral).
Persistencc of 'fGEV in nature is likely vía the fecal
carrier/shedding statc in rccovcrcd swinc; thus the virus is
maintained 1n e11demically infected herds through ongo-
ing fecal-oral i11fection of susceptible pigs. Infection of
swine in endemlcally infected herds is often subclinical or
rnild, whf'rf'a<; thf' virus spreads rapidly in nonimmune
herds and can cause devastating outbreaks of disease. I11
aclclition to movement by infected swine, 1'GEV is poten-
tially transmitted bctwccn hcrds by fomites and other
animal s.
PRC~oV virus has been isolated in North America and
Europe. The vlrus is not shed in the feces, and therefore
there. is no f'viclf'nr.e for the fecal -oral transmission.
Tnfectcd pigs shed virus in tl1eir respiralory secreliou:;, arH.l
PRCoV may persist in herds by continuous infection of
newly weaned pigs.
Pathogenesis and Pathology
TGEV survives in tl1e gastrointestinal tract after ingestion
because of its resistance to low pli and trypsin, thus it
passPs through t1.1e stomacl1 without inactivation. Six to
twelve hours following il1L1agaslric inoculaLio11, viral
replication occurs in villus epitl1elial cells of the small
intcstinc with highcst titers of virus in t he jejunum.
·rGEV infects and destroys tl1e columnar epithelial cells
lin.i ng the intestinal v illi, rcsulting in atrophy of thc villi.
Villous blunt1ng and 1ncreased crypt <1eptJ1 (as a con-
sequence of replication of the progenitor cells in the
cry pls i11 au effurl Lu repopulate tl1e Jenudeú villi) occurs
24 to 40 hours postinfection and coinc.ioes with thf' oc-
curcc11ce of severe diarrhea. The loss of entecocytes lining
the villi results iI1 malabsorption and maldigestion,
which in turn results in diarrhea and dchydration.
Uodigested lactose in the intestinal contents passes to
the large bowel, where it exerts an osmotic effect that fur-
Llrer ex<:1<.:erbiltes the diarrhea.
Affected piglets ;:irp clPhydr.ated with fecal staining
around the pcrineum. 'l'he typical lesions i11clude Lhin-
ning of the interstitial wall, villous atrophy, and gastroin-
testinal distension with ycllow fluid containing curds of
undigestec1 milk.
·rhe characteristic microscopic lesion of PRCoV infec
tior1 is c.liffuse intestinal pneumonia, whereas the intestine
does not exhibit atrophy of the ~rilli .
Host Response to lnfection
Neutralizing antibodies develop within approximately 7
days of'I'GEV infcction of swine. 'fhe presence of secretory
386 PART 111 Viruses
IgA plays a major rolf' in protPctivP im rnunity and viral
cleara11ce. Tntra1nuscular imrnunization of pigs with TGEV
rcsults in development of a humoral IgG response but not
protcctivc immunity. Conversely, pigs immunized orally
with ·rc;Ev develop protecttve vtrus-specific IgA in thelr
intestinal mucosa! secretions. Infection of sows with TGEV
results in the secretlon of protecttve IgA in culustruru (su-
ca!led lactogenic immunity), whirh i.~ protPrtive in suck-
llug JJigs. Cell-1nedialed in1niunity also is likely ilnportant
in the im mune response to Tc;Ev infection, as passive
transfer of mononuclcar Jcukocytcs from immu ne donor
pigs to susceptible histocompatible p1glets results in re-
duced disease expression. High levels of Type 1 interferon
are produced by infected intestinal cells, \>Vhich alsu ruay
play a role in controlling viral replication.
Plgs infecte<.l with PRCuV develop ncutraliz.ing antibod-
ies to t hP virus. Antihodies against PRCoV p rovide partial
protection against TGEV, and t hcrcforc thc incidcnce and
severity of TGE may decline in swine herds with endemic
PRCoV infection.
•
La borato ry Diagnos is
TGEV ii1fection of young pigs is usually diagnoscd
through the demonstration of viral antigen in mucosa!
scrapings or frozcn scctions of jejunum and ileum by i1n-
munohistochemical or lilUTiunofluorescent staining with
virus-specific antibodies. Definitive diagnosis can be done
by viral isolation through inoculation uf arúruals (µigs 2 Lo
7 days old) or cell Cl1ltures (pig kiclnPy, tP~tis, or thyroid).
Elcclron rnicroscopy (EM) or in1n1uno-EM can also be used
on fecal cont ents or intestine.
Serologic diagnosis is appropriatc when acute and con
valescent sera are available. Hovvcver, serologic diagnosis is
complicated by the finding that both PRCoV and TGEV in-
duce neutrallzlng antlbodies that cross-ucutralU.e:: e::ach
other. Therefore, l'GEV strains arP not c1istinguished from
ils nonenleropatliogenic variant PRCoV by the virus neu-
tralization t est but can be distinguished by a b locking
E.LISA test.
Treatment and Control
Tr1:::at111enl of 1"GEV-ilúected pigs is usually unrewarding.
Rcplacement of fluids and antihacterial drugs to reduce
complications associated with enteropathogenic Esche-
ric/7ia coli may be of benefit.
Inactivated and modified live attenuated virus vaccines
are avallable for vacc!n<:1tiu11 uf µigs Lu _preve11l 'fGE. These
can be used for vaccination of newborn piglets or jmmu-
ni¿atiu11 uf sows, or bolli. Vaccination of prcgnant sows
provides lactogenic immunity that is passively transferred
to piglets vta colostrum. Vaccincs havc bccn variably suc
cesstul in preventing rGt... ural 1mmu111zat1on p rovides
optima! stimulation of local immunity (secreto ry IgA) in
the intestine. The practice uf i:ufccti11g :-.uws will1 virulcnt
TGEV at least 3 weeks prior to farrowing to induce an im-
mune rcspur1st uy _provid.il1g colostral immunity to piglets
i~ romplic.ated hy the fact that this practice contaminates
the environ1ncnt with virulent TGEV that subsequently
can sp rcad to susceptible pigs.
HE MAGGLUTINATING ENCE PHALOMYELITI S V IRUS
Disease
HemaggJutinating encephalo myelitis virus (HEV) is tlle
cause of vomiting a11d wasting disease (VWD) of young
swine, a disease that is charactcritcLI l.Jy e11cepl1alon1ye-
litis, vomiting, and wasting. VWO occurs in pigs less than
3 weeks of agc, although older Sl>vine may exhibit mildcr
signs of the disease. VWD in young piglets is characterized
by anorexia, lcthargy, vomiting, constipation, and signs of
central nervous system disturbance (hyperestllesia, muscle
trem ors, paddling of the legs). Mortality is h igh, up to
100º/Ó; pigs also may develop chruuic i11feclions a11d even-
tually die from starvation or sPcondary infections.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
1-IEV is antigcnically rclatcd to bovine coronavirus. 'fhe
virus hemagglutinates chicken, rat, mouse, hamster, and
turkey erythrocytes.
Resist ance t o Physical and Chemical Agen ts
HF.V is sPnsitive to lipid solvents, including socliu m deoxy-
cholat e; it is also heat labile and rclativcly stablc when
frozen.
lnfectivity for Other Species and Culture Systems
HEV grows in priruary µig kidney (PK) or pig thyroid (P'f)
cells \vith formation of characteristic syncytia.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
HEV was first isolated and associated with VWD in
canad1an sw1ne 1n 1958. sunsequenrly, the virus has l.Jce::u
identified in swinc in many areas of the wnrlc1. Pigs are the
only kn u w11 110::.L of HEV, and subclinical or inapparent
r;:i rriPr states likely exist. Nasal secretions contain virus
and horizontal aerosol an d dircct anirnaI contact are
mechanisms ot transmission.
Pathogenesis and Pathology
The patllugeuc:.ls uf JiEV i11f1::1..tiv1¡ has been. ch.'1racteri:z:ed
hy Pxperimental inoculation of colostrum-deprived day-
old pigs. Following oroJ1asal inoculation, primary viral re-
plication occurs in the epithelial cells ot the nasal mucosa.
tonsils, lungs, and small intestine. Virus subsequen tl~
spreads atong per1phcral nerves to the central nervous sy!)-

tf'm (\.NS). Prior to ctisease expression, viral antigen is
present in the trigen1inal, inferior vagal, a11d superior cer-
vical ganglla. solar and dorsal root ganglia of the lower
thoracic region, and the int estinal nerve plexuses.
lnfecti.on in the brain stem is initiated in the trigeminal
and vaga! sensory nuclei and subsequently spreads to
0 L11e1 11uclei a11d Lile roslral porLion of lile brain Slt:111.
Later stages of the infection may be charact erized by viral
rcplication in the cerebrum, cercbellum, and spinal cord;
virus is typically tound in nervous plexuses ot the stomach
late in infection.
There are fevv characteristic gross Jesions in natural HEV
infections; a mild catarrhal rhin itis is sometimes evident
in ei1c.epl1alon1yelitis cases, and gaslroenLerilis is so1ue-
ti1nes observed i.J.1 VWD.
Lcsions in thc CNS are o f a nonsuppurativc cnccpha-
lomyelitis characterized by perivascular cuffs of mononu-
clear cells, for1nation of glial nodes, neuronal degenera-
tion, and meningit is. Respiratory tract lesions consist of
foc;i 1 or diffuse interstiti.a 1 peribronchiolar pneumonia
with cellular infiltrates con1posed of n1onocytcs, lyn1pho-
cytes, and neutrophils.
Host Response to lnfection
Humoral immune responses may be quantitated by viral
neutralization, hemagglutination inhibition (HI), and
agar gel inu11unodiffusio11 (AGID). Tl1e clinic.al disease is
self-limiting in pig populations and is dueto the rapid de-
velopment of maternal antibodies and their transfer by
colostrum.
Laboratory Diagnosis
Diagnosis of HEV e11cephalomyelitis or VWD in piglets re-
quires immunohistochemical staining of viral antigens in
the tissues of affected pigs, virus isolation on primary PK
or PT cells, or demonstration of a rising antibody titer by
nP11tr;:i liz;ition or hf'm;igglutin;it ion in hi hition ;iss;iys.
Treatment and Control
No effective treatment has bee11 described fo r HEV-
induced encephalomyelitis or VWD. Clinical outbreaks
are self-limiting. No vaccines are available and good ani-
rnal l1usbandry practices are essential for the preventlon
anrl control of the rlisPase.
PORCINE EPIDEMIC DIARRHEA VIRUS
Coronavirus-like viruses have been isolated from swine
w!th diarrhea, thus their dcsignation as porcine epidem ic
ctiarrhf'il vin1Sf'S (PF.DV). These viruses are antigenically
distinct fron1 'fGEV and HEV, and cause diarrl1ea, von1it-
iI1g, and dehydration in. inoculated swine. Pathogenesis
studics indicatc that PEDV rcplicatcs in both the small and
large intestines, but lesions are contined to smalJ int es-
Chapter 62 Coronaviridae and Arteriviridae 387
tinf'S. Afff'cteci pigs have sm.all lntesti.nes that are distended
with yellow fluid; the lesions are sil11ilar to those of TGE.
PEDV particles can be demonstrated in the feces of in-
fected pigs by direct EM, and the virus can be propagated
in so1ne African gree11 monkey (Vero) cell lin.es and not t.n
others. Viral growt h depends on the presence of trypsin in
Ll1e Lt'.11 t:ulture 11u::diu111. Generally, fielu strains of PEDV
need to be adapted to grow in cell culture heforf' t hey c;in
be used as a routin.e diagnostic assay. A direct lf test and
immunohistochemical technique applied oo a section of
small intestine are the most sensitive, rapid, and reliable
inethods of diagnosis of PEDV. A serologic diagnosis ca11 be
made by demonstration of PEDV antibodies by IF and
ELISA.
Currently there is no vac.c.inf' for prf'Vf'ntion of PF.OV in-
fection in pigs, thus control is dependent on managen1ent
and husbandry practices.
80VINE (ORONAVIRUS
Disease
Bovine coronavirus (BC:oV) is an e11teric pathogen that is
responsible tor both neonatal diarrhea in nevvborn calves
(1 to 3 weeks) and winter dysentery in adult cattle. The
clinícal disease in newborn calves is characterized by
anorexia anda liquid, yellow diarrhea that pers;ists for 4 to
5 <.lay::>. WiI1t<::r <.lysentery is a sporadic acute disease in
adult cattle that is c.harac.terize<l hy f'xplosivf' hloo<iy cti<Jr-
rhea accompanied by decreased milk production, depres-
sion, and anorexia. The BCoV strains that are isolated frorn
d iarrhea fluid or intestinal f.lui.d are now identified as cn-
teropathogenic bovine coronaviruses (EBCoV) .
Other strains of bovi11e coronavirus more recently have
been identified as respiratory pathogens in cattle; these
strains of coronavirus have been isolated from the nasal
secretio11s artd lu11gs of cattlc witl1 severe sl1ipping íever
pneumonia, and are designated as respiratory bovine
coronaviruscs (RBCoV) . Rcspiratory discasc causcd by
RBCoV typically occurs in calves aged 6 to 9 months, and
is characterized by fever, nasal discharge, and r.espiratory
distress.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
BCoV particles hemagglutinate erythrocytcs from ham-
sters, mice, and rats. BCoV is antígenically related to coro-
naviruses of other species. Although there are significant
pl1er1otypic, antigenlc and genetic differences between
ER\.oV anrl RRC:oV strain.s, the precise relationship be-
tween enteric and respiratory strains ofBCoV is uncerlain.
Resistance to Physical and Chemical Agents
BCoV is acid stable (pll of 3.0), but is inactivated by lipid
solvents, detergents, and high temperatures.
388 PART 111 Viruses
lnfectivity for Other Species and Culture Systems
lnfcction with BCoV is limited to cattlc (bovids), but the
virus has been propagated in suckling mice, and following
such passage, will infect suckling rats and hamsters by
both lntracerebral ano SUULUtallCUU:> ruule:.. EBCoV has
been propagatecl in M;iclin-l)arhy hovine kidney (MDBK),
Africcu1 green n1onkey kidney (Vero), bovine fetal thyroid
(13fTy) cells, and bovine fetal brain (BfB) cells. ·rrypsin
trcatmcnt of thc lattcr two fetal cell cultures enhanccs
plaque forn1ation and cell fusion. Certain isolates are diffl-
cult to propagate in vitro and may require passage in the
natural host. in contrast, only a hurnau rt::ctal lu111u1 Lell
Une (f-TRT-18) is permissive for initial i~olation of RBCoV.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
'fhc distribution of DCoV is \vorldwide, and transmission
of EBCoV is likely fecal-oral by ingestion ot virus from con-
taminatcd fccd, tcnts, and fomites. RBCoV is shed in respi-
ratory tract secretions of mfected animals, and thus is
spread horizontally by aerosol.
Pathogenesis and Pathology
Diarrhea develops within 24 to 30 hours tollowing oral in-
fcction of calves with EBCoV. Four hours after onset of diar-
rhea, viral antigen is dete(table ln the epithelium of the
small intestinc and colonic crypts. lnitiation of infection is
facilitated by proteolytic enzymes iI1 tl1t! i11tt!slü1al lracl
since trypsin trf'atrnPn t of coronaviruses in cell culture re-
sulls in enhanced viral growth. Thc virus also infects the ad-
jacent n1esenteric lymph nodes. Destruction of the mature
cntcrocytcs that Une the intestinal villi leads to atrophy and
fus1on of affecte<l vi.JI!, Witn suosequenr J.ntesnnal maltllges-
tion and inalabsorption, rapid loss of fluids and electrolytf'~,
and, il1 :>t::vt::rc ca:>e:>, dehydraU011, acidosis, shock, and death.
HKC:oV infection in calves causes interstitial pneumo-
nia with conge:;tion, t1emorrhagc, and edema ofthe inter-
lobular septa of the lung. Histologica lly there is interstitiaJ
pncurnonia with infiltration of mononuclear infla111ma-
tory cells and th1cken1ngs of alveolar septa.
Host Response to lnfection
Boll1 EDCoV and RBCoV infections in calves result in a hu-
moral in1mune response that readily can be quantitatcd by
viral ncutralizntion, hcmagglutination inhibition (Hl),
hemadsorption inhib1t1on (HAI), and ELISA tests. Local im-
mune responses play an important role because drculating
antiboclies do not prote<-t calves fruu1 iJú1::cliv11. Neonatal
ingestion of colostral TgA protPrt.<: th<' intestinal lumen
agai11sl EBCoV infcction for a limited pcriod of time.
Laboratory Diagnosis
Oiag1tu:>is of EBC0V-i11duced neonatal diarrhea requires
irtPntification of the virus in fecal samples or intestinal sec-
tions. This can be achieved by viral isolation, electron mi-
croscopy, fluorcsccnt antibody or immunohistochcmical
staining. Nasal swabs collected <.Iuring the acute stage of
upper respiratory tract disease are the spccilnens of choice
for the diagnosis of RBCoV. Rt:s¡Jiralury epil11elial cells
present in the nasal swabs arP .c;pottPrt onto slides for exam-
inalio11 by dirccl fluorescent antibody test.
Treatment and Control
Treatment is dlctated by the sevt:rity a11d lype of disease.
Electrolyte solutions can hP artministered for dchydratíon
ü1 calves wilh diarrhea caused by EilCoV infections, and
antibiotic therapy may be used to control secondary intec-
tions. AH BCoV infcctions are best controlled by good
management practices to minimize exposure to these
viruses, such as by avoiding introducing new (infected) a n-
imals into an lntensive calviug uµ1:::1alio11 . Il is difficult to
control enteric disPasf' hy vacci nation because very young
calves are 111ost affected, beforc they have the opportunity
to respond to vaccination. The altemative is to immunize
the dam to incrcasc antibody lcvcls in colostrum.
(ANINE (ORON AVIRUS
Disease
Canine coronavirus (CCoV) infection ot dogs is highJy
contagious nnd gcncrally causes inapparent or mild gas-
troenteritis. ·rhe virus occasionally is associated with more
severe enteric disease, and puppies are most susceptible to
CCoV-induced enteritis. Signs iru..:ludc a11ur1::xia, lelhatg) ,
vomiting, and rliarrhf'a. Morf' rf'cently, CCoV also has
been proposcd to be an importnnt cnusc of trnchcobron-
chitis ("Kennel-cough") in dogs as well.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
canine coronavlrus (CCoV) i:> au1ige11ically rclalcd 10
other coronaviruses, inclu<ling transmissible gastroenteri-
lis virus, feli11e enteric coronavirus, and feline inicctious
peritonitis virus. Multiple antigenically and geneticall}
distinct strains of CCoV have been recognized, including a
nove.I respiratory coronavirus.
Resistance to Physical and Chemic;:il Agcnts
Canine coronavlruses are iI1aclivalt!d by lipid solvents and
are heat-labile. Tht> vin1se..c; are acid-stable (pH of 3.0) and
relain i11fcctivity under cool conditions.
lnfectivity for Other Species and Culture Systems
Caninc coronnvirus infects domestic and wild canini:
spet.ies; severa! prunary and continuous canine cell cu:-

tures, including primary kidney and thymus; and contin-
uuus liut:s uf thyu1us, erul>ryu, syuuviun1, and kidney (line
A-72). The v irus al.so·infec:ts feline kic1ney anc1 emhryo fi-
broblast cell lincs.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Ca11i11t: curuuavirus was first isulated in 1971 from an epi-
demic of c1iarrhca in dogs in Germany. ThP virus .sincP has
been recognized virtually worldwidc, including North
America, Europe, Australia, and Asia.
lnfected dogs excrete virus in their fcccs for 2 wccks or
longer, and fecal conta1nination of the env1ronment is the
primary source fo r it s tra.nsmission via fecal-ora l infection .
Pathogenesis and Pathology
rollowing an incubation period of 1 to 4 days, the virus
causes infection of intestinal epithelial cells that progrcs-
sively passes through the gastrointestinal tract. Diarrhea
occurs 1 to 7 days postinfection, virus being present in
íeces wiú1iJ1 1 Lo 2 days folluwi11g Lile aµµcarance of clini-
cal signs. Viral replicatíon in the intestinal epithelium
rcsults in desquamation and shortening of the villi. The
diarrhca associated with CCoV infection occurs as a conse-
qucnce of intestinal maldigcstion and malabsorption.
While Cl~ov infection is widespread , mortality is typi-
cally very l o~v. Thus, necropsy of CCoV infected dogs is
un usual.
Host Response to lnfection
Mucosa! immunity appears to be protective because dogs
orauy infectcd with CCoVbecome immune and those im-
munizcd parcntcrally do not.
Laboratory Diagnosis
Vin1<; or vir;il ;intigens can be visualized by electron mi-
croscopy (EM) or fluorescent antibody (fA) stai11i11g of feces
or necropsy tissues. Antiserum, spccific for CCoV, is com-
monly used to aggregate virus prior to negativc staining for
EM. Virus can be isolated from teces or intestinal tissue on
cell culture. A reverse-transcriptase-polymerase chain reac
tion (RT-PCR) assay has been developed to detect CCoV in
feces. Serum virus neutralization anci F.l .ISA tPsts for detec-
tion of CCoV antibodies have been dcvcloped.
Treatment and Control
Treatment of CCoV-associatcd gastroenteritis is limited to
relief of dehydratíon and electrolyte loss in severe cases.
Inactivated and modified live virus vaccines are avail-
al>lc for µarcntal administration for protection against
C.C.oV infPction. HowPvPr, thPir use is questionable dueto
the apparcnt importance of local in1n1unily al Lhe leve! of
thc intestinal mucosa.
C hapter 62 Coronaviridae and Arteriviridae 389
fELINE INFECTIOU S PERITON ITIS ANO fELINE ENTERIC
(ORONAVIRUS
Disease
Feline infectious perito11ilis (Fil') is a co11Lagiou:;, ¡Jrogrc:.-
sive and highly fatal disease of domestic and sorne wild
fcline species. The signs are highly variable and rcflcct
the tlssues affected by the disease, but persistent fever,
weight loss, lethargy, dyspnea, and abdominal distension
ali are:: 1.:0111111011 diilical signs. 'fhe dlsease can occur In
cats of ali ages hut is i>spPcially common in young and
very old cats.
'l"wo distinct forms of f IP are recognized: 1) an effusive
(wet) and 2) a noneffusive (dry) form. "fhc cffusivc form,
which Is two to three times as common as the dry form, is
ch aracterized by accumulation of protein-rich fluid (exu-
da Le) i11 Ll 1e µ eri tuneal cetvity. "l'he noneffuslve form is
characterized hy formation of granulomas in interna! or-
gans, CNS, and eyes. Mortality rates are high.
FIP has an unusual and highly complex pathogenesis
that involves the mutation of relativcly nonpathogcnic fc-
line enteric coronavirus (FECoV) 1nto FIP virus (rll'V) that
replicates in macrophages to produce an immune-medi-
aled disease i11 a.ffectt:d cat~.
Etiologic Agent
Physical, Chemical, and Antigenic Properties
FIPV is antígenícally related to other coronaviruses anci is
vcry closcly rclated to feline enteric coronaviruses (ffiCoV)
that do not produce FIP; it now is evident that FIPV arises
by mutation of FECoV. The virulence to cats of strains of
FIPV is directly correlated to the1r abihty to gro"v in cul-
tured feline macrophages. The virus is rcsistant to add and
tryµsin L>ut is readily inactivated by most disinfectants, in-
cludíng lipid solvents.
lnfectivity for Olher Species and Cul ture Systems
In addition to infecting domestic cats, FIPV has been asso-
ciated with disease in wild Felidae such as lions, rnountain
liuns, leopards, ¡aguar, lynx, caracal, sand cats, and pallas's
cats. Young pigs can be experimentally infected with FIPV,
resulling in devetov111e::11t uf lt!sions similar to those in-
duccd by"fGEV. Suckling niic:e are suscPptihlP to infPction,
with vi rus rcplicating in thc brain. f.IPV primarily repli-
cates in macrophages, but virus can be propagated ín vitro
in feline organ cultures, cell lines, and mononuclear
phagocytes.
Host-Virus Relationship
Distribution. Reservoir, and Transmission
FECoV and FIPV occur worldwidc. Persistently infected
cab :.t:rvc as tl1e virus reservolr, and the virus ts readily
spread horizontally hetween cats.
390 PART 111 Viruses
Pathogenesis and Pathology
Tlie pall1oge11esis of FJP is complex, and much remains to
he resolved. Initial FECoV infectio11 rarcly is characterized
by obvious discasc, but rcsults in persistent, low level in-
fection of macrophages in many tissues. 1he developmcnt
of clinical FIP is associated \.vith incrcased viral replication,
usually secondary to sorne immunosuppressive event that
suppresses cellular imn1unity. h1crcascd virus replication
results ill the cn1ergence of vlr<1l varia11L:-. (FIPV) ll1at repli-
cate with increasing pfficiency in 1nacrophages where they
persisl, safe fron1 immunc clearance. Antibody cxaccrbatcs
tl1c disease, which suggests that FIP is at least in part an
immunc-mcdiatcd discasc. Virus specific antibody actu-
ally tacilitates uptakc of FIPV by phagocytes in which
pathogenlc strains of the virus replicate.
Botl1 "wet" and "dry" forms of FIP <lf\.'. cl1araclerized by
thc appearance of p, r<1 ni Jlnn1;is (or pyogran ulon1as) around
1.Jluuu vessels. ll is pro posed that dcposition of complcxes
of FTPV and spccific antibody (ilnm une complexes) in the
walls of blood vcsscls is rcsponsible fo r the charact eristic
perivascular location of these lesions. l 'hese perivascular
granulomas can occur in the bo'l>vel, kidneys, liver, lungs,
C NS, eyes, and lymph nades of affccted cat$, anu are c:.µe-
cially common on thc serosa of the abdomi n;il visce.r;i in
cats with the wt:l fvri 11 of FIP.
Host Response to lnfcction
·rhe IJasi:. vf i111111u11ily lo FIPV is poorly understood. Cats
di>ve.lop humoral and cellular immune responses soon
after FECoV infection, and tl1csc responses hold the in
fcction in check until so1ne stress or concurre11t infection
causes imn1une suppression. Antibodies to FECoV cross
react witll flPV, and these cause diseasc expression rather
than protcction; this antibody facilitatP<; vir;il 11pt;ikp
IJy ¡Jl1agucy lic 1..ells vv hece ll1e virus effectively replicatcs,
anrl viral antigens complexed 'l.vith specific antibodies
(and complement) contributc to immunc-mcdiated vas
cu litis.
Laboratory Diagnosis
FIP is a common disease of cats that often can be diag-
nosed based upon the characteristic cllnlcal signs coupled
with serology and he matology. Fluid accumulation in thP
peritoneal o r pleural cavity, (1$ uel1::n11ined by paracente-
sis, in assoc:i;:itinn vvith a positive serum or fluid antibody
li lcr i:; indicative of c ffusive PIP. Noneffusivc FIP is more
difficu lt to diagnose and must be ditterentiated from
othcr infcctious, granulomatous, and neoplastic condi-
tions. Histolog1c examination for pyogranulomatous or
fibronecrotic inflammatory lesions and vasculitis, in asso-
clation ivlth scrology, faLilitate~ uiC1g11t1sis. Reverse tran-
scri p tion-polymer;isi> rh;iin reaction (RT-PCR) techniques
have been developed for identification of FIPV sequcnces
in clinical material, as has immunobistochemical sta.in-
ing fo r FIPV.
:>erologic diagnosis may be determined by viral neutral-
ization, ELISA, o r indirect fluorescent antibody tech-
niques. A serum titer of greater than 1:37.00 s11pports th
lli<tg11v~i:. uf FIP al L11ough cals -¡,vilh f-TP can have low titcr
of virus-specific antibody and, conversely, unattected cat:.
may havc high titcrs of antibody to the virus.
Treatment and Control
No treatment fur FIP lias been dcscribed that consistcnt!:
rPVf'TSPS the discase pIOCeSS.
A temperature-sensitivc mutant FIPV is available fe,:
vaccination of cats. lts use is n ot recom rnended in seropos-
itive cats. Control of FIP is best realized by decontaminat-
mg (with quaternary ammonium compound$) i11fe1..ll:\.l
premises, isolating serologically positive c;its from tho<.e
with no titer, <IIIU :>cree11iug i1ewly acquired cats for ~erurr
a n tibo<ly.
MousE HEPATITIS VIRU S
Mouse hepatitis viru:. (MHV) is highly contagious anc.
causes "explosive" onthreaks of disease in mouse coloni~
lhrougl1ou l Lbe world. The scverity of thc clinical discos..
depends on severa! factors. These tactors can be broadh
distinguisl1cd into viral (strain, dose, and route of infec-
tion) and host factors (strain of mi ce, agc, and im1nune sta-
tus). There are many different strains of MHV, each with
characteristic tissue tropism and assuciatt:<.l cliuical 1nan1-
festatio11s. Infection of mice \<ITith c1iffPrPnt virus strain
can c<1ust: e11tt.:rili:., hepatitis, ncphritis, and dcmyelinat-
ing Pncephalomyelitis. For example, the A59 strain or
MlIV in.duces moderate to scvcrc hepatitis, whereas the
MHV-4 strain does not.
The enteropathogenic strains of MHV cause severe di-
arrhea in suckllng mice, with nearly 100% rnortality. Tht.
intestines are distended and filled with yPllowish fluid
01<.ler ruice llt.:velov jau11dice, lose weigl1t, and cease
hrPerling. C:haracteristic histological lesions include
blunted (cl ub-~haped) intestinal villi with cxtensive syn
cytiun1 formation. AH 01ice develop acute hepatitis witl1
focal hepatocellular necrosis and inflammatory cell infil -
tration. Othcr strains of MHV are the cau$e uf respi1aL01~
and central nervous system (l.NS) disease in mouse
colu1úe:.. CNS infeclio11 with neurotroplc ::;trains of MH\
causes paralysis dueto den1yelination (used as a model 01
chronic demyelinating dlscascs of human s, such as multi-
ple sclerosis}.
MHV infection in mouse colonies is diagnosed with the
detection of characteristic gross and hí:>tulugical lesions in
the intestines and liver, altho11gh sorne strains of MHV are
lligltly alle11ualed a11d cause littlc pathology or discasc.
The diagnosis is confirmed by immunohistochemistr\
and serology using an cnzyrne immunoassay. Virus can be
isolated u sing any ot severa! mousc cell lines.
The v irus pcrsists following epidemics in persistently
infected mi ce that contin ually inft:ct ::;u:;ccplible n1icc l hat
are introduced into the colony. \.ontrol is achicved b)
brt:al<iug thi:. cycle of transmission through thc use of
~trirt quarantine measures.

SIALODACRYADENITIS VIRUS (RAT (ORONAV IRUS)
Sialodacryadcnitis virus is the etiologic agcnt of sialo-
dacryad(~n i tis in laboratory rats. Tt is a scvcrc, sclf-limiting
1nflammatory d1sease of the salivary and nasolacrin1al
glands of rats. The v irus is highly contagious and causes
higl1 morbltllty anti lo\v mortality In lnfcctcd colonies.
\.linical signs of infPction incl11clr ~wPlling of the face and
ncck, excessive Jacrimation and blinking, squinting, and
exophthalmia. Lcsions in the lacrimal duct may lead to
cornea! drying. Lesions typically resolvc within 2 weeks.
lhe virus is transmitted by aerosol, direct contact, and by
fomites.
EQUIN E (ORONAVIRUS
Coronavirus-likc virions have been isolated fron1 both n1a-
ture horses and foals expericncing enteritis. The viruses
found in horses were antigenically distinct from group l
1c.g., T(1EV and HCV 229E) a11d group :5 (e.g., lBV and
TCoV) coronaviruses, but hada close antigcnic and/ or ge-
111::Lil.: rclatiouship witl1 group 2 coronavlruses (e.g., BCoV).
Howcver, thC' pathogenic.ity of thPsP P<J.11inf' coronaviruscs,
as well as thcir role in entcric discasc has nol bee11 exa.ru-
ined in dctail.
AVIAN INFECTIOUS BRONCH ITIS VIRUS
Disease
..\vian infectious bronchitis virus (!BV) causes rcspiratory
disease in chicks 10 days to 4 weeks of age; however, all
dge:., sexes, an<l lJree<ls are susceµtilJle to ir1fection, al-
~hough mort·ality is low in hirds grC'atC'r than 6 WPPks of
age. Thc virus also causes discasc of thc rcproductive tract
and kidneys. The respiratory disease is characterized by
rcspiratory distress, rales, coughing, nasal discharge, and
deprcssion. Thc clinical course lasts 6 to 18 days. Morbidity
is lOO<Yo and mortaJity may cxcccd 25ºA>. Chicks with no
1nal1::n 1al a111ilJu<ly 111ay <.:XJJL'.rie11cc pl'rn1aueut ovitluct
damage and fail to lay eggs when maturC'. lnfPction of lay-
ing flocks results in a drop of cgg production and hatcha-
bility. Pullets in good condition return to normal produc-
tion within a few weeks.
!BV-associatcd renal discase is ctependent upon Vlral
strain. Many viral strains with an affinity for the kidneys
ca.use OJJly 11til<l ur i11apvar1;;11L r<.:sµiratory signs, but can
cause substantial mortality in susc:cptihlC' hi rds.
Etiologic Agent
lhysical, Chemical, and Antigenic Properties
:BV is autigl'uically tlistinct from other coronaviruses, and
;nultiplP di~tinrt vin1s strain~ h;ivp bccn identified and
.::roupcd by serologic techniques, polypcplide palle1n5 i11
ChaplL'r 62 Coronaviridae and Arterlviridae 391
polyacrylamide gel electrophores1s (PAGE), oligonu-
cleotidc fingerprinting, and nucleotide sequencing.
Strai r1-sµ<.:cifíc <:!pitopes for viral neutralization and hemag-
glutination have he.en idPntifiPd on thP Sl protein with
monoclonal antibodies.
Resistance to Physical and Chemical Agents
Most strains of lBV are inacti.vatcd within 15 minutes at
56ºC . ·rhe virus is quite stable at cold temperatures.
Stability at acidic pH is strain-variable; with sorne stralns
survlVlng at pH 3 for 3 hours at 4ºC. Lipid solvents inacti-
vate the virus.
lnfectivity for Other Species and Culture Systems
Thc chicken is the only k'Tiown natural host of 1HV.
1-Towcvcr, quail and gulls have becn experimentally in-
ft:Clt:u. Suckl iug ulice car1 ue infected by l ntracerebral inoc-
ulation. The virus c.an hP cultivated in developin g avían
embryos, ccll cultures, and organ cultures. Turkey en1b1yos
have also been succcssfully infected with IBV, but less
efficiently.
lBV can be gro..vn in ch1ck cmbryo ceLls (kidney, lung,
and livcr), cmbryonic turkey kidney cells, and monkey kid-
ney (Vero) cells. Organ cuJtures have been used to propa-
g;itp TRV including tracheal and oviduct cultures.
Host-Virus Relationship
Dislribution, Reservoir, and Transmission
llJV has a \>'lOrldwide distribution. ·rhe virus Jíkely persists
in persistently infected birds and/or continuous cycles of
transmtssion. Virus has bccn recovered for up to 49 days
from infected chickens held in isolation and for even
lo11ger pcriu<ls i11 tl1ose l1el<l uu<ler natural conditions.
Viral transmission oc.c:urs hy inhalation, thi> rPspiratory
tract being the primary site of infcction. Virus is shed in
rcspiratory and fecal materials, with subsequcnt spread by
contaminatcd fomites and aerosol.
Pathogenesis and Pathology
Thc incubation period of lBV infection is 18 to 36 hours.
Viru:- gains entry via the respiratory tract and the respira-
tory form of fRV rf's11lts in trac-heitis and bronchitis.
Mortality may be as high as 25% in young chicks. Strai11s
exhibiting an affinity for thc kidneys damage the renal
tubules, resulting in renal failurc.
lnfection produces pr1marily a serous, catarrhal, or
cascous exudate in the trachea, nasal passages, and si
nuses. 'f'he ai.r sacs may contain a caseous exudate, and
small foc i of hronchopneumonia may be apparent. Young
chi<.:ks may experience a more scvere ii1fecU011, will 1 le-
sions <leveloping in the oviduct. Chickcns infected with
nephrotropic viraJ strains dcvclop renal tubular necrosis
charactenzed by swollen and pale kidncys with accumula-
tion of uric acid crystals causing distension of the tubules
anú ureters.
392 PAitT 111 Viruses
Microscopic lesions of the respiratory tract include cel-
lular infiltration, mucosal edema, vascular congestion,
and hemorrhage.
Host Response to lnfection
TBV elicits humoral and cellula r immune responses.
Humoral responses can be measured by viral neutraliza-
tion and hemagglutination inhibition (HI) tests. Titers of
passively transferred maternal antlbody decrease to negll-
gible levels within 4 weeks of hatching.
Cllicke11:. rt::<.:uvt::rt::LI fro1n natural ilúecU011 are resistant
to homologous viral challPngP. ·rhP ciuration of immunity
is variable and difficult to determine dueto the multiplic-
ity of lBV strains. Passive transfer of maternal antibody
does not confer to tal protcction to thc chick but reduces
disease severity and mortality.
Local tracheal immunity appears to play a rnajor role
in resistance to IBV. 'Ihe relatlvc lmportance of !1urr1ural
versus cellular immunity is 11nclPar. 1-he ohservation
tl1al chickens u1ay be protccted in the absence of demon-
strahle antibody would suggest an important role for cell-
mediated immunity.
Laboratory Diagnosis
Dlagno:1i:. of il1fectiuu:. 1.Jru11cl1ilis 1nay be based upon di-
rPct visualization of viral antigen in tracheal smears using
immunohistochemical or fluorescent antibody staming,
viral isolation, or serology. Intectious bronchitis must be
differentiated from other acute respiratory diseases of
birds, such as Newcastle disease, Jaryngotracheitis, and in-
fectious coryza.
Vi1al i5olaüo11 i5 conducled by inoculation of tracheal
or rPspiratory exudates in to the chorioallantoic sac of 10-
to 11-day-old embryonated chicken eggs (ECE). Serial pas-
sage in ECEs may be required betore embryo dwarting or
mortality occurs.
Scrologic diagnosis of IBV infection requires paired
serum samples and the use of 113V-specific viral neutraliza-
tlon, he1nagglutination inhiuitiu11 (Hl), agar gel ünn1un-
oc1iff11sion (AG 11)), or F.T.TSA assays.
Treatment and Control
No specific treatment for infectious bronchitis is availahlP.
Propcr husbandry practices that reuuct:: e11virorune11Lal
stress are logical. Control of infectious bronchitis may be
approached through managcment procedures and vacci-
nation. Spread of the virus may be reduced by strict isota-
tion of an affccted flock, and restocking >vit h day-olcl
c hicks reared in isolation. Attenuated anct inactivated vac-
cines have been developed for the control of IB.
Inacrtvated vaccmes induce neutralil.il1g a11Ul.Jodies, but
their efficacy has been <}11PstionPci. Live viral vaccines at-
lenualed by serial passage in cmbryonated chicken eggs
have reducect pathogenicity but also decreased immuno-
genicity. Vaccincs may be administered via aerosol or in
drinking water. High passage vaccine viruses apparently
have a reduced invasiveness and gencrally require aerosol
administration.
The multiplicity of IBV stralns an<.l serotypes 11a:. HléH.le
it difficult to develop efficacio11<; varrines. No single strain
11as bee11 ideJ1tified as capable of inducing more than lim-
itcd protection to heterologous viruses. Multivalent vac-
cines are availablc, butin ccrtain instances prolonged reac
tio11s to vaccination and sorne interference between
vaccine strains have been reported.
T URKEY (ORONAVIRUS
Di sea se
Turkey coronavirus (TCoV) Is the causative age11t of curu11-
avi rus enteritis (CE) of t urkPys. lt is an acute and highly
cuulagious disease of turkeys of all ages. Synonyms of the
disease include bluecomb disease, mud fever, transrnissi-
ble enteritis, and infcctious enteritis. It is of major eco-
nomic importance to the turkey industry a11d affects
turkeys of all ages. The disease affects primarily the ali-
mentary tract and is characterized by depression, sul.Juor-
mal body temperature, anorexia, inappetence, loss of hoc1y
welght, a1H.l wt::t Llruvviugs. Darken.lng of the head and
<;kin anci h1cking of the skin over the crop are characteris-
tics of infectcd growing turkeys. A rapid drop in cgg pro-
duction with formation of chalky eggshells occurs in pro-
ducing brceder hens. Morbidity is essentially lOOo/o, and
mortahty var1es w1t!1 agc and envtronmental condttlons.
Etiologic Agent
Physical. Chemical. and Antigenic Properties
·rcov is mactivated by lipid solvents and detergents, bnt is
re:>i:.ta11t tu acidic vH (JJH of 3 al 20ºC for 30 n1inutes). The
virus is resistantto SOºC for 1 hr. TCoV is apparently unre-
lated antigenically to other coronaviruscs. TCoV aggluti-
nates rabbit and guinea pig erythrocytes, but not thosc f1om
cattle, horse, sheep, mouse, goose, monkey, or chi<.:kens.
lnfectivity for Other Species and Culture systems
TCoV infection is confincd to turkeys. Laboratory propa-
gation of thc virus has been limited to turkey and chicken
embryos, and attempts to grow ·rcov m cell cultures have
been unsuccessful.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
CE has been described in North America and Australia.
The virus persists in turkeys for lite tollov.ring recoverv
from thc discase. Tt is stable in frozen feces and survives
throughout the winter months in infected droppings.
thus transmission of TCoV principally is fecal-oral from
the lnfected teces of carrier 1.Jin.l:.. T11lroduclio11 of virus

onto a premise may occur via carrier turkeys, teces-
contaminated fomites such as personnel and equipment,
and possibly mechanical transmission by free-flying birds.
Pathogenesis and Pathology
The incubation period of CE varies from 1 to 5 days. Gross
lesions are largely confined to the intestinal tract, with pe-
techial hemorrhages sometimes apparent on the serosa!
surf<H.:c. l.c:;iu11:; drc ruu:,t di:;t.inct i11 the jcjur1u1n but mé:!y
;:ilso occur in the duodenum, ileum, and cecum. Gas and
fluid typically distend the sn1all intestine and ceca. Tl1e
breast muscles are typically dehydrated and the carcass
generally appears emaciated.
Microscopic lesions include destruction of enterocytes
lining the intestinal villi, leading to theír shortening, loss
of n1icrovilli, and inononuclear cell i11fillraLiuJJ oí tl1c lau1-
ina propria of the affected bowel.
Host Response to lnfection
Turkeys respond Lo 1'CoV iufecliu11 witlt 11u1uural arH.l ccl-
lular imn1une responses. Serum antihodies (TgM, TgA, an<i
IgG) develop follo\"7ing infection but by 21 day:; only IgG is
present. Local lgA antlbody appears in intestinal secretions
and bile for at least 6 months. Passívc transfcr of maternal
immunity has not been observed and administration of an-
tiserum to young poults does not afford protection.
Laboratory Diagnosis
Definitive diagnosis of CE requires identification of viral
a11tige11 in i11testil1al tissue sections by i111111unohisLo-
chemical staining, viral isolation, or demonstration of
serum antibody. Virus may be isolated by inoculation of
embryonated turkey eggs or young poults and its presence
confirmed by fluorescen t antibody staining of infected tis-
sues. Serologic diagnosis can be conducted on paired
serun1 sarnples by viral neutralization or indirect FA tests.
Treatment and Control
No spec.ífic. treatment is effec.tive in re<iuc.ing morhirlity of
CE, although various treatments may be used to prevent
other intestinal infections. TCoV vaccines are not available,
butprevention ofinfection ispossible. Thcdiscaschasbccn
eliminatecl in sorne areas by depopulation and decontami-
nation of affected premises. An alternative to viral elimi na
t1011 ls exposlng poults (5 to 6 weeks of age) to recovered
c'arriPr hircl-; 11ncl0r icl0;il environmi>nt;:il ronrlition~ for the
purpose of inducing protective immunity. Such a progran1
is reco·m mended only on farms with continued problems
and when all other methods of control havc failcd.
T0Rov1Rus
·roroviruses resemble coronayjruses in their genome or-
ganizalion and replicaLl011 SLraLegy lJut u iffer in virion
Chapter 62 Coronaviridae and Arteriviridae 393
morphology. ·roroviruses are p leomorphic and n1easure
120- 140 nm in diameter. Spherical, oval elongated, and
kidney-shaped particles have all been observed. Torovi-
ruses are enveloped with a tubular nucleocapsid of helical
:;y n11neL1 y. The 11uck:v1..ap:,iu fv1111:, a uvugl111uL-:>haped
structure, and the envelope contains a large number of
spikcs that rcscmblc thc pcplomeres of coronaviruses.
Torovirus particles consist of at least tour structural pro-
teins: nucleocapsid protein (N), u nglycosylated membrane
protein (M), spike glycoprotein (S), and hemeagglutinin-
esterase protein (1-IE). The Toro11irus genome consists of a
polyade11ylaled, vosi l.ive-:;e1.1se, li11ear rnulecule of ssRNA,
which is estimated to he 20 to ~O kh in lengt h . ·rhe mem-
bcrs of this gcnus Torovirus include equine torovirus (E1'V;
formally named Heren virus), bovine torovirus (Borv; orig-
inally narned Breda virus), porcine torovirus (PoTV) and
human torovirus (HuTV). Toroviruses have also been de-
tec.tecl hy f>lf>c.tron m ic:roscopy in the feces of dogs and cats.
Toroviruses infect the epitl1elial cells of l11e sn1all a11d
large intestine extendiI1g from 1nid-jejunum to colon. ETV
was originally isolatcd from thc rectal swab of a horse with
d.i arrhea. BoTV has been idenütied as a pathogen causing
gastroenteritis an d diarrhea in calves and possibly pneu
rnonia in o lder cattle. Antlbodies to BoTV have been
demonstrated in severa] countries around the 1Norld. PoTV
was origi11ally isolaled fror11 Lhe íeces uf piglets tt1at did
not show any signs of enteritis or diarrhea, hut it sinc.e has
been isolated from pigs with diarrhea at the time of wean-
ing. Much remains to be determined regarding the patl10-
genic irnportance of Torovirus infections of animals.
ARTERIVJRUS
Thf> mf>mhf>rs of f;imi ly Arteriviridae, genus Arterivirus, in-
clude equine art.eritis virus (EAV), porcine reproduclive
and respiratory syndrome virus (PRRSV), lactate dehydro-
gcnasc-clcvating virus (LDV), and simian hemorrhagic
fever v irus (SHrV). rhree ot th.ese viruses >vere first discov-
ered and characterized long ago (EAV-1953, LDV-1960, a11d
SHFV-1964), whereas PRRSV was first isolated tn North
America in 1987 and in Europe in 1990. The arteriviruses
are each. antigenically dislincl, bul LDV and PRRSV are
most closely related to each other. There are two geneti-
cally distinct subspecies of PRRSV (European, Type I;
American, 'lype 11). '!'he arteriviruses are hjghJy species-
specific, but sl1are many biological and molecular propcr-
ties, including their virlon morphologies, unique proper-
ties of t heir structural proteins, and the ability to establish
pcrsistent infection. EAV and PRRSV are of veterinary sig-
nific.anc.e an<i V\rill hf> rlisc11S'(f'd in cli>t;iil
Etiologic Agent (EAV, PRRSV, LDV, and SHFV)
Physical, Chemical, and Antigenic Propertíes
Jlrtcrivirus virions are spherical, 45 to 60 nm in diameter
and consist of an isometric (probably icosahedral) nucleo-
capsid of 25 to 35 nm il1 diameter, surrounded by a lipid
envelope. ·rhe envelope includes 12-15 nm diameter ring-
394 PART 111 Viruses
like structures. ·rhe large spikes that are typical of coron-
aviruses are absent from the !lrtcrivir11s envelope. The
Arterivírus genome is a linear, posítive-sense, ssRNA mole-
cule oí 12. 7 to 15. 7 kb. The buoyant density of arteriviruses
rangcs from 1.13- 1.17 g/cm 3 ir1 sul'rost!, wl1ert!as tllt: scJi-
mentalio11 coefficíent is ?.14S to z:~OS.
Arterivirus particles consist of a nucleocapsid protcin
(N) and six envelope proteins (E, GP2, GP3, GP4, GPS, M).
However, thrcc additional cnvclopc protcins (GP2, GP4b,
and <.11'7) have been reported for SHFV. The GP5 (glycosy-
latcd) and M (unglycosylated) are thc major envelope pro-
tci ns of the arteriviruses and thcy form a disulfide-linked
heterodimer in the maturc virus pa cticles. Tn acldition, the
Arleriviru:> e11velope conlai11s a hcterotrin1er of three
minor memhrane glycoproteins (GP2, GP3, and GP4) and
an unglycosylated envelope protein (E). Thc GPS protcin
contains the known neutralization determinants of EAV,
PllilSV, and LDV, and monoclonal antibodies (MAbs) spe-
cif1c to the GP4 of PRRSV also neutrallze the virus indicat-
ing that sorne of the neutralization epitopes are ;ilso Jocal-
lzed in tllis prott::i1i.
Resistance to Physical and Chemical Agents
Both EAV and PRRSV are rcadily inactivated by lipid sol-
vents (ether and chloroform) and by com1no11 disinfec-
tants and detergents. EAV survives 75 days at 4ºC, between
2-:~ ctays at 37ºC, and 20 to 30 rriinute::. dt 56nC. Ti::.sue cul-
ture fluid or o.rgan s;implPs ront;iining f./\V can he stored at
- 70ºC Ior years without significant loss of the infectivity.
PRRSV inícctivlty is lost within 1 week at 4ºC, but is stable
for a long time (months to ycars) whcn frozen -70ºC or
-zuvc). PIU{.'.)V is rapidly inactivated by lo>v and high pli
(below 6 and above 7.5). LDV is not stable at -20ºC: and
raptdly loses its infcctlvlty.
lnfectivity for Other Species and Culture Systems
EAV can infcct horscs, donkcys, mulcs, a11d zebras, and
rcplicates in pcimary cultures of equine pu!Jnonary artery
cndothelial, horse kidney, rabbit kldney, and hamster kid-
ney cells. It also replicates In ccll llnes such as Bli K-21, RK-
1 .~. African green monkey kidney cells (Vero), rhesus mon-
kcy kidncy (LLC-MK2), hamster lung (HmLu), SV-40
transformcd equine ovary, and canine hepatitis virus-
transformed hamster tumor cells (HS and HT-7). PRRSV
primarily intects pigs, but recently, chlckens and ducks
that wcre exposed to PRRSV in drink.ing "''ªter shed the
virus in their feces, suggesting that they are susceptil>le tu
infec.."tion witl1 the virus. North AmPrir;in PRRSV (Type TI)
i:.ulaLes repllcale i11 porcine alveolar macrophages (PAM),
C:llL-11171, and African green monkey cell line (MA-104),
and derivativcs thcrcof (CL2621 or MJ\.RC- 145). Most, if
not ali, Europcan 1'!\KSV ( l'ype l) isolates repllcate best or
exclusivcly in PAtvfs. However, Europcan PRRSV isolates
have becn adapted to grow in CL2621 cell lines. Vaccine
strains of PRRSV replicate much morP i>ffiriPntly (100 to
1000 luues) i11 derlvalivcs of nionkey kidney cell lines than
in PAMs. LDV primarily replicates in primary cultures ot
mouse macrophages from 1- to 2-wcck-old mice and other
mouse macrophage cell lines, but not other cell lines.
SHfV intects and rep!icates in primary cultures of per1-
toneal macrophages from rhesus and patas monkeys and
also replicares in the MA-104 cell llne.
EQUINE ARTERITIS VIRUS
Disease
EAV is thc causativc agcnt of cquine viral arteritis (EVA) ir.
horscs, a respiratory and reproductive disease that occurs
throughout the world. ºfhcre is o nly one serotype of EA\·-
howcver, field strains differ In their viruler1l'e aBd 11t:ulrdl-
ization phenotype. 'l"he clinical signs displ;iyed hy EA\--
iufc1.:tt:J ltorses depend 011 a variety of factors, including
thr. age and physical condition of the horse(s). ch allenge
<lose and route of infcc..i:ion, strain of virus, and environ
mental cond itions. 'J'he vast ma1ority ofEAV infections ar(
inapparent (or subclinical), but acutely infected animals
may develop a Wide range of cllnical sig11s, il1cTudi ng P> •
rexia, depression, anorexia, dependPnt t><lema of (scrotum
veulldl Llu1lk, a11d li111bs), slÜÚlC):S of gait, conjunctiviti'
lacrimation and swelling around the eyes (periorbital and
supraorbital edema), rcspiratory distress, urticaria, and
leukopenia. Tl1e incubation period ot J to 14 ctays (usuaUr
6 to 8 days following venereal exposure) is followed b~
pyrexia of up to 41 ºC that may persist for 2 to 9 days. The
virus causes abortion in pregnant mares, and abortion rates
during natural outbreaks of EVA can vdry Iro1u 10 to 60%v.
infected mares. EAV-induced ahortions c.an occur at an:
Li111e belwee11 3 a11d 10 n1onths of gestation. EAV infectior
of neonatal foals can cause a severe fulminating interstitia
pneumonia, and of oldcr foals a progrcssive pneumoen
teric syndrome. A high proportion of acutely infected stal-
1ions (30 to 60%) become persistently infected and shed thc
vlrus in semen; however, thcrc ls no evidence uf ar1y aualv-
gous persistent infection of marPs, gt>ldings, or fo;ils . 11'
víius persisls iI1 Ll1e an1pula ofthe male reproductivc traC"
and the establishment and maintenance of the carricr stat.c
in stallions ls tcstostcronc-dcpcndent.
Host-Virus Relationship
Distribut ion. Reservoir, and Transmission
Thc EAV is distribl.1ted throughout thi> worl<l, although tr
::.c::r~iprevalence of EAV infcction varíes between countrit
and horses of dilierent breeds and agc in the same countr
in the Unitcd Statcs, sorne 70 to 90% of adult Standard bree
horses are seropositive to EAV, as compared to only 1-3
of thc Thoroughbred population. Similarly, a high pe:-
centage of European warmblooc.l horses are scropu::.i li v1:: •
EAV. Seroprevalence to E.AV incrPilSt>S with age, inc.licati r.~
LI 1al ho1ses 111ay be rcpeatedly exposcd to the virus as the'
age. Persistently infected carrier stallions function as thr
natural rcscrvoir of EAV and disseminate the virus to sus
ccptible mares at breed1ng. The two principal modes
EA.V transmission are horizontal transmission by aeroso -
zation of infectious res pi catory tract se1.:rc::Liu11::. fru1

acutely infected horses and venereal transmission during
natural or artificial inscmination with infcctivc semen
from pcrsistcntly infectect stallions. EAV also can be trans-
mitted through indirect contact with fomites or person-
nel. Congcnlral lnfection results from transplacental
tr;in~mis~ion (vertical transm.ission) of virus when preg-
nant n1ares are infected late in gcslaUon.
Pathogenesis and Pathology
Most information on the pathogenesis of EAV infection is
derived from experimental studles ln horses inoculated in-
tranasally with a highly vir.ulent strain of EAV. lnitial mul-
tiplication of virus takes place in the alveolar n1acrophages
in thc lung. and the virus soon appears in the regional
lymph nodes, especially thc bronchial nodcs. Within 3
days the virus is present in virtually all orp;ans anct tissues
(viremia), where it replicates in 1nacrophages and en-
dolhelial <.:t:lls.
EAV infec.tion of aclult horsf'.~ is almost never fatal. EAV
infection of pregnant mares can result in the abortio11 of fe-
tuses, which are usually partially autolyzed at the time of
expulsion. Aborted fetus may cxhibit intcrlobular pul-
monary edema, pleural and pencard1al effusion, anct pe-
techial and ccchymotic hemorrhages on the serosa! and
mucosa! surfaces of the small intestine. Neonatal foals occa-
~ion;illy rlPVPlop ;i VPry <:PVPrP, :ir11tP intPrstitial pneumonia.
The characteristic histologic feature of EVA is a severe
necrotizing panvasculitis of small vessels. Affected muscu-
lar artcrics show foci of intima!, subintimal, and medial
necrosis, with ecterna and intiltration ot lymphocytes and
neutrophils. Prominent vascular lesions are also seen in
the placenta, brain, liver, and spleen of the aborted fetuses.
Lungs of affected neonatal foals have severe interstitial
pneu111onia.
Host Response to lnfection
Animals that recover from EAV infcction or thosc that are
vaccinated with either inactivated or attenuated strains of
EAV dcvclop neutralizing antibodies and are resistant to
suuse4uc111 1.:halle11ge with EAV. Neutrallzing antibodies
are detected within 1 to 2 weeks following t>xpo~11rf' to the
virus, reach maximum titers bctwccn 2 to 4 months, and
persist for 3 years or more. With the exception of persist-
ently infected stallions, EAV is eliminatcd from thc tissucs
of infected horses by 28 days after the exposure. foals born
to imn1une mares are protected against clinical EVA by pas-
sive Lra11:.fer uf ncutralizing antlbodles In colostrum.
?-leutralizing antihodies appear a few ho11rc: ;:iftf'r rolostrum
feeding, peak at 1 week of age, and gradually decline to ex-
tinction between 2 to 6 .m onths of age.
Laboratory Diagnosis
EVA is uncommon in horses, ancl othf'r, more important
viral respiratory infections clinically resen1ble EVA.
Contormation of a diagnosis of EVA currently is based on
virus isolation and/or serological dcmonstration of raising
neutrali2ing antihody titers (fourfold or greater) in paired
Chapter 62 Coronaviridae and Arteriviridae 395
serum samples taken ata 21 - to 28-<lay intPrv;i 1. F.AV can be
isolated from nasal swabs or anticoagulated blood col-
lcctcd from adult horses with signs of EVA, or the tissues
(including placenta) of aborted cquinc fctuscs. Carricr
stallions are first ictentified by serology because they al-
ways are seropositive, and persist.cnt infection is con-
firr11 t:u uy virus isulation from semen In ce!l culture, by
test-hreeding using st>ront>g::itivP m;ires (and monitoring
these for seroconversion to EAV after breeding), or by RT-
PCR to identify viral nucleic acid in semen. Histopath-
ologic examination coupled with immunohistochcmical
staining also is useful for diagnosis of abortion in particu-
lar, as are RT-PCR assays.
Treatment and Control
There is no specific treatment for horscs infcctcd with EAV.
Currently there are no 111eans available of eliminating the
carrier state in stallions persistently infected with EAV
olher Llia11 <.:astration. Modified llve attenuated and killed
virus vaccines are av;:ii l;:i hit> for prevention of EAV infection
in horses. Colts should be vaccinated wilh a11e11ualeu
vaccines after they lose colostra1 immunity but befare
puberty.
Outbreaks of EVA can be prevcnted by the identifica-
tion of perslstently infected stallions and the institution of
1na11age1 ut:nt µractices to prevent the lntroduction of EAV-
infected horses. C:a rrit>r st;:illions should be kept physically
isolatcd and bred only to mares that are seroposilive fron1
previous natural exposure or vaccination. Mares should be
kept isolated from othcr scroncgativc horscs after being
bred by carrier stallions.
PORCINE REPROD UCTIVE ANO RESPIRATORY
SYNDROME VIRUS
Di sea se
The European ('Iype I; prototype Leylystacl virus [LV]) and
American (Type 11; prototype VR-2332 virus) isolates of
PRRSV represenl genelically a11u autigeuically distinct
groups of the same virus. Both virus0.s ::irf' ;:issoci::itt>d with
outbreaks of similar reproductive and respiratory disease
in pigs, dcspite the fact that there is only 55 to 70o/o nu-
cleotide identity in the various genes of thc two typcs.
Cllnlcal slgns of porcine reproduct1ve and respiratory syn-
drome (PRRS) are extremely variable and influenced by
sLrain of vi1 u:., i11lllluuc status uf the herd, and manage-
ment practices. Low-virult>nr.f' str;:iins of PRRSV may result
in widespread infection of SVl'ine with n1ini1nal occu1rence
of disease, whereas highly virulent strains can cause severe
clinical disease in susceptible hcrds. Ali agcs of pigs are sus-
ceptible to infection with PRRSV in immunologically
naive herds. Acute PRRSV infection of susceptible pigs is
cha1a1.:lerizttl uy ilnorexia, fever (39º to 41 ºC), dyspnea,
and Jethargy. Afft>rtt>cl swinf' are lymphopenic, and exhibit
transient cutaneous hyperemia or cyanosis of exLreutltics
that is most visible on the ears, snouts, mammary glanrls,
396 PAR1· 111 Viruses
and Vl1lvas. Transplacental transmission of PRRSV occurs
n1osl efficie11lly in lhe Llúrd trin1cster of pregnancy (usu-
ally aftcr 100 days of gestation), and the abortion rate in
uffcctcd :;ow:; con rangc from 10 to 50%. Sow mortality is
cons1derably lower. Affected liters contain a variable miX-
ture of normal pigs, weak small pigs, stillborn pigs, and
partially or complctely mummified fetuses (so-called
SMEDI: stillbirth, mummification, embryonic death, and
iI1fcrtility). J11fe1..tel.l :.uw:. ctl:.u cdu exl1ibil nervous signs
such as ataxia and circling. PRRSV infected boars may con-
tinue to shed virus in their semen for prolonged periods of
time.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Despite lts very rccenL rccognition, PRRSV appear~ tu \Je;;
endemic in virtually ;ill ~winP-pro<iurin g c:ountries of the
world. Th c original source of the virus a11d the circum -
stances undcr which it was introduced into the domestic
swine population a re unknown. ·rransmission of PRRSV
usually occurs by close contact benveen infected and unin-
fected animals. s~vine are susceptible to PRRSV by a num-
l)er of routcs of exposure, lncluding oral, intranasal, vagi-
nal, intrarnuscular, an<l intraperitoneal. PRRSV is sl1ed in
re:.piralury l1c11..l :.eL1eliun:s, saliva, :sen1en, 111arnn1ary :se-
cretions, urine, and feces of infected animals. Susceptible
pigs are naturally infected by inhalation of infectious
aerosols or ingestion of PRRSV-contaminated tood.
Congenital infection results from transplacental transrnis-
s1on (vertical transnússion) of virus. ·rransmission of PRRSV
to females has been demonstratcd during breeding 11vith
~c1ucu fru111 pcr:.ble11Lly i11 fecleu cctrrier IJoars. PRRSV aJso
can he transn1itted through indirect contact with fomites or
personnel, and sorne pigs harbar the virus in their tonsils
long atter the virus is cleared trom other tissues.
Pathogenesis and Pathology
PRRSV rcplicatcs Jn macropl1ages and dendritic cells, espe-
cially thosc in thc lungs and lymphoid tissues. Viremia oc-
curs :.uu!I afl1::1 i11fe1..:liu11, a11d ca11 lasl for 1 Lo 2 wecks in
n1at ure animals and 8 weeks in young pigs. Gross lesions
are usually observed in only a few organ systems (e.g., rcs-
pirato ry and lymphoid). Microscopic lesions ot PKK.S in-
clude diffuse interstitial pneumonia, myocarditis, vascu-
ht1s, and encephalitis. Lymphoid tissues exhibit lymphoid
hyperplasia and follicular necrosis with mixed inflam-
matory t:cll i11fillratiu11. Cliilical oulb1eaks of PRRSV can
hi> compliratt'd hy hacterial pneumonia, septicemia, or
enteritis.
Host Response to lnfection
Pigs infected with PRRSV mount a11 immune response and
virus-specific antibodics have been identified using a varl-
ety of assays. 'l'he antibody response is detected by th e ap-
pearance of viru:.-s¡.11~t:iflt: IgM \Jy 7 U.ay:; and specific IgG by
14 rlays afti>r infPrtion. An libody titers peak by 5 to 6 weeks
postinfi>ction an<i pPrsist for thP lifP of commerc:ial feeder
pigs. Ilowever, neutralizing antibodies appear slowly, usu-
ally between 4-5 weeks postinfection, and peak at about 10
,..,ccks postinfcction. Piglets born to immune sows acquire
anti-PRRSV antlbodies by ingestion of colostrum. Thc ceU-
mediated immune response to PRR.SV has not been well
characterlzcd; however, T lymphocyte proliferatiou re;;-
sponses in pigs infected with PRRSV havi> heen demon-
slratcd bctween 4 and 12 weeks after infection.
Laboratory Diagnosis
Diagnosis of PRRSV may be complit:atec.I lJy tl1e;; f<tt:l lhctl
many infections are in<ipp;irPnt, hut PRRS should he con-
:sidered when Lherc are clinical signs of respiratory disease
associated with reproductive failure in a 11erd. PRRSV anti-
bodies are dctccted by usir1g n vnriety of serological assays,
including I::LJSA, immunofluorescence assay (lfA), in1-
munoperoxidase rnonolayer assay (IPMA), and serun1
virus neutralizatlon assay (SVN). Serolugit:al exa1ninalion
of acute and convalescent pig ser;i may provide evidence of
:;e;;ru1..:u11ve1 sion. Virus lsolation from clinical specimens
such as hronchioalveolar lavage fluid, lung, lymph node,
buffy coat, and serum can be done in PAMs, MA104 and its
derivatives. and CRL-11171 cell lines, although different
strains or isolates of PRRSV vary in their ability to replicate
in different cell types. Various PCR assays have been devel-
oped to detect viral nuclcic acid in blood, semen, and
other clinic.:al spet:irr1en~.
Treatment and Control
There is n o effective treatment for PRRSV infection in pigs.
Both rr10<.1ified live attcuuate;;l.l (MLV) ct11d killed vaccines
arE' ;ivailahlf' for prevcntion of PRRSV infection. They can
be used to immunizc sows o r wcanling piglcts; howcvcr,
there is considerable variation in t he relative safety and ef-
ficacy of MLV vaccines. Ml,V vaccines induce long-Jasting
protectlon as c.:ompared to l<illed vacc111es, but do not t:om-
pletely prevent reinfection with wild -type virus and subse-
quent viru:. tra11:.111i:.:.iu11. Furlhe11nore, ll1erc have been
reports of under-attenuation and reversion of MLV vaccine
viruses to a more virulent type th.at can be spread from
vaccinated to unvaccinated swinc. Control programs have
been developed to eradicatc the virus from infected herds.
LACTATE DE HYDROGENASE -ELEVATING VIRUS
Lactate dehydrogenase-elevating virus (LDV) can cause a
lifelong persistent infcctíon in inbred laboratory strains of
mice, nlthough infection of laboratory mice now is very
uncommon. The vi rus has also been isolated from wlld
house mice (Mus rnusculus domestia1s) in several countries,
although the wurl<.1wiú1: i11t:ille11ce is uol known. Atten1pts
to infE>rt PP.rnmysru.~ mi ce, rats, guinea pigs, and rabbits
with LDV have been unsuccessful. LDV replicates in per-
missive macrophagcs ln the spleen, lymph nodes, thymus,

a1H.I liver of infectcd mtce. ·rhe subpopulation of macro-
phagcs thrit riri> pP.rmis~ive to LL)V infection also are re-
sponsible for the i1orn1al clcarance of lhe laclale uel1yuro-
genase (LDH) enzyme from circulation. '!'he continuous
dcstruction of these macrophages by LDV leads to elevated
levets ot LIJH in blood. thus the name of the virus. The
persistent infection that characteri~es LJ)V infcction of
1uice is maintained by replication of LDV in new pern11s-
sivc mac.roph;igps th;it ;irc continuously regenerated from
apparcntly nonpern1issive precursor cells. ()l11er thar1 the
elevated LDH leve! and subtle cha11ges in host immunity,
persistent LDV infection of n1icc gcncrally is asympto-
matic. Ho~vever, micction of rnice ot certain strains (C58,
AKR, PL/J, and C3H/f.gl3oy) by neurovirulent strains of
LDV can leatl to fatal age-depcndent poliomyclitis.
Anti-LDV antihocliP~ hPein to appear 4 to 5 days postin-
fcction and peak at J to 4 weeks. Antibodies ll 1al rieu tral-
ize LIJV appear in n1icc only after 4 wecks postinfection,
and these antibodies are tlirccted against thc GPS protein.
of the vi rus. The rep1icat1on of LDV in macrophages allovvs
it to avoid host defense mechanisms, although the precise
n1echa11i:.111 uf iJJ1rr1une evaslon and persistent infection 1s
still unclear.
I.DV transmission betwcen mice is relatively i11efficie11t,
despitc the lifelong vircmja that occurs in infected mic.P.
and the secretion of the virus in urinc, fcccs, and saliva.
LDV transmission from mother to offspring through the
p lacenta or vía breast milk is highly efficient if the mot her
.s i111111unologically naivc. However, transmission via these
; o ut<'<; from persistently infected mothers is rare because
anti-LOV antibodics block t1ansplace11tal transmission of
the virus and its release into milk. Generally hori7.ontal
transmission of LDV bctween m ice is restrictcd by n1uco.sal
barriers, although horizontal transmission of LDV does
occur among laboratory m ice that fight and bite onc an-
'Jther. -rhe sexual transmission of LDV has not been
demonstrated.
LDV can only ve quantltated by an end-point dilution
assay in mic.e, whirh i<; h;ispcl on the incrcase in plasma
:..OH activity that accompanies LDV infection in n1ice. 1'11e
;>resencc of LDV in mice or in other materials is readily de-
•ected by injecting p lasma, tissue homogcnatcs, or other
:nater1a1s (e.g., transplantable mouse tumors) into groups
·f two to three mi ce and assaying their plasma LDH activ
.ty 4 Lo 5 uays later. ·rransplantable mouse tumors can be
·eadily freed of LDV hy ;¡ ~-wPPk in vitro propagation or
7assage in a host animal other than micc.
StMI AN HEMORRHAGIC FEVER VIRUS
5.n1ia11 hc1nurrhagic fever (SHF) virus (SHFV) was first iso-
:ated from rhi>s11s m;.ir¡¡qucs suffering frorn hemorrhagic
:cver, in primate research ccntcrs iI1bolhll1e foru1er SoViet
_ nion and the Unitcd States. African monkeys of thrPe
~enera (/\frican green monkcy [Ceropithecus aethiops1,
Chapter 62 Coronaviridae and Artertv1ndae 397
patas ft:1ythrocebus patas¡, and baboon 1.Papio anuibus]) are
persistently infected in the w ild with the SHFV, and thcy
clo not exhibit any cll nical signs of d 1seasc. Accidental
tr;insmission of SHFV from African monkeys to any of
Lliri::t:: species of Aslan macaque monkeys (Macaca mulalla,
Macaca arctnides, and Mnrnra fasciculnris) results in a gener-
nlly fotol hcmorrhagic fcvcr. Clinical :.i~u:> uf StiP i11
macaques include anorexia, fever, cyanosis, skin petechia,
and hemorrhages, nosebleeds, facial edema, bloody diar-
rhca, dehydration, adipsia, and protc1nuria. Veath gencr-
rilly occurs Sto 25 days after the onset of clinical signs, and
mortality approachi::::. lOOo/o . The high lethality observed
in macaque monkeys may hP cl11P to ;in extreme sensitivity
of thcir macrophages to cytocidal infeclion by SHFV.
Jnfection ot captive patas monkeys with SHFV from per-
sistently infected patas monkeys results in pcrsistent infec-
tlon without any clinical signs. However, infection ot cap-
tivP patas m onkeys with SHF\1 from diseased macaques
results in transienl 111ild disease, indlcatlng selection of
more virulent variants during i>pi7ootics in macaque mon-
keys. 'fhc humoral immune response againsl SHFV varíe;)
with the specics of monkey and the infecting virus strain.
SHFV induces neutralizing antibodics in putas monkeys 7
days after experimental infect1on. However, onty lo'l-v lev-
Pls nf anti-SHfV antibodies are found in rnany persistently
infected patas 111onki::y::..
'l'he prevalence and incidenre of SHFV in fcction among
/\frican monkeys in endemic areas of Africa is not know11,
but the incidence of persistent subclinical infections in
wild patas monkeys appears to be high. The method of
rransmission of SHFV among African monkeys in the wild
is unclear. Jnfcction rnost likely occurs through wounds
and biling, 1.JuL sexual transmission has not been ruled out.
SHFV is not transmlttP<l trflnsplricentally from persisteotly
infected mothcrs to their offspring. Severa! epi;:oolics uf
SHF in captive macaque colonies originated from acciden-
tal mechanical transmission of SHfV from asymptomatic,
persistently iiúected African 1no11keys. Once illness bc-
comes apparent in the macaque colony, the SHFV spreads
rapidly throughout thc colony, most likcly by direct con-
tact and aerosol<\.
Pcrsistently infected n1onkeys cau 1.Je ideutified tJy the
presence of SHPV that replicates in primary c.ulturi>s of
peritoneal rnacrophagcs from rhesus and patas monkeys.
The most sensitive mcthod for detection of persistent in-
fection is experimental inoculation of macaque monkcys,
tintl currently there are no molecular d1agnostic assays tor
clPti>rtion of SHFV. fndirect immunofluorescent assay
(TFA), ELISA, and neuL1aliz.aliu11 asst1ys are available for
serological diagnosis of SHFV infertion . Howi>ver, these as-
s<iys are not rcliablc because of thc low lcvel of ai1li-SHfV
antibodies found in many persistently infected monkeys.
Accidental transmission of SHFV from pcrsistently in-
fectcd African monkeys to macaque monkeys in primate
C'Pnters can be minirnized by strict adherence to proper
sanitary condilious a11<.l anirntil care practlces.
 Reoviridae
N . J AMES MACLACHLAN ] EFFREY L. STOTI

Viruses in the Reoviridae fan1 ily infect a wide ran ge of

spccics, including mammals, fish and shcllfish, in sccts, F 1G U RE 6 3 . 1 . Negatively stained preparations of avían
and plants. Ali of the vi ruses in t his family have a seg- orthoreoviruses. 204, 000X. (Courtesy o f R. Nordhausen.)
men te<l genome of doublc-stran<led RNA (dsRNA), ai1d t he
number of tndtvtdual genom e segments varies 1)etween
genera. ·rhe family is grouped into nin e genera: ()rthoreo-
virus, Orbivirus, J<otavirus, Aquareovirus, Coltivirus, 01yzavi-
rus, Cypovirus, J>flytoreovirus, and Fijivirus . The latter four
genera are conflncd to insccts, plants, or both . The Colti
virus genus 1ncludcs Colorado tick fevcr virus, which is an
insect transmitted human pathogen. Orthoreoviruses (Fig
63.l) and rotaviruses (fig 63.2) infecta wide variety ofver-
tebrate species. ·rhe host range of orbivíruses includes both
vcrtcl.Jratc:> <:111<..l iuvcrtcl.Jratc:>, with 1.Jluctuuguc viru:; :;crv-
ing a~ the prototypc. The aquarcovirus host range includcs
fish and shellfish, \-vith goldcn shiner virus serving as the
prototype. Table 63.1 includcs a list of genera and species
that are important in veterinary medicine.

ORTHOREOVIRUSES

MAMM ALIAN ÜRTHORE OV IRUSES

Di sease
Rcovi rusc:; (gcnus Orthoreovirus) have been isolated from
t he respiratory anc.1/or gastroi ntestin al tract oí m any ani-
mal species, including non h uman p rimates, roden ts, F1G U R E 6 3 . 2 . Negatively stained preparations of bovine
l1orses, cattle, sheep, sw1ne, cats, and d ogs. Reoviruses are rotaviruses. 204,000X. (Courtesy of R. Nordhausen.)
usually isolated from healthy animals, thus their designa-
tion as "respiralory enleric orpha11" viruses because Lhey
typically are not associated with any disease. However, re-
oviruscs are somctimcs isolated from animals with mild
respiratory and/or enteric disease, and reovirus in!ectio11
of infant (neonatal) mice can cause severe system ic dis-
ease. Experimental infection of kittens with reovirus
sProtypP ~ h;i~ r<111~P<l ronj11nrtivitis, photophohi;i, gin-
givitis, serous lacrimation, and nasal discharge. Ali three
serotypes of reovirus have been isolated from sheep, and
experimental infcctions with scrotypc 1 havc bccn rc-
ported to cause enteritis and pneumonia.

398
 Chapter 63 Reoviridae 399

Ta b 1e 6 3. 1 . Genera With in the Revviridae Thol l nliuue Vir u~e~

Etiologic Agent
Relevant to Veterinary Medicine

Physical. Chemical. and Antigenic Properties

Genus Serogroup Mioimum Number
There ate three serotypes (1, 2, and 3) and many stratns of o.f Serotypes
111au1wallar1 reoviruses. SLrai11s of reovirus that vary in
vi rulen ce have been identified by sequence analysis of in- Orthoreovirus Mammalian
, 3
dividual viral genes and protcins. Mammalian rcoviruscs Avían 11
all possess a genome ot ten distinct segments of dsl<.NA. Orbivirus Bluetongue 24
The genomt~ segments are of different sizes (grouped as Epizooti< hemorrb:agk di;ease 8 (pt:>~5ihly 9)
large, medium, and small). Each encodes a single protein African hor.sesickness 9
except the Sl gene, which includes two distinct open Equine ente¡:itialosis 1
reac.ling frames . Tl1e cou1plete reovi rus parlicle has 110 e11- Palyam 1'f
velope and exhibits icosahedral inorphology with a diam- Rota virus 5 majorgroops Uncertain
eter of approximatcly 85 on1.. Thc rcovirus particlc con- Aquareovirus Not designated Uncertain
sists ot eight structural p roteins arranged into inner and
outer protein capsids (coats) . ·rhe inner protein core con-
tains the viral RNA-dependent RNA polymerase (tran-
scriptase), as well os otber enzymes that mPciiatP mRNA
synthesis and capping, helicase activity, and other func-
tions that are necessary for virus replication. 'l'he p redom-
inant outer coat protein sigma 1 is the primary determi-
nant of virus serotype and hemagglutiI1atio11 and also is Pathogenesis and Pathology
the cell attachrnent protein. Enzymatic digestion of the Studies in mice have shown that reoviruses can intect ei-
outer capsid protel.11 sigma 3 from lntact reov1rus partlcles ther intestinal or respiratory epithelial cells after, respec-
genera tes infectious subviral particlf'S, <in<I rf'mova 1 of the tively, orofecal enteric or aerosol respiratory infection. Ini-
outer ca.psid proteins si.g ma l, si.gma 3, and mu 1 gener- tial virus replication ocet1rs in regional lymphoid tissues
atP.s c.ore partic.les. All three particles are important in the afler reovirus infeclion of eilher Lhe gaslroil1testinal (Peyers
lifecycle of reovirus replication. Genetic diversity of patches) or respiratory (bronchus-associated lymphoid tis-
strains of reovirus occurs through accumulation ot muta- sucs) tracts. 1'hc virus somctimcs gains cntry to thc sys-
tions within individual viral genes (genetic drift) and hy temic circulatio11 in infected neonatal mi ce, leading to pan-
the exct1ange of entire ge11es (reassortment) between creatitis, myocarditis, m.y ositis, encephalom.y letitis, or
viruses during mixed infections with more than one re- hepatitis, with the specific disease process and pathogene-
ovirus strain or serotype. sis reflecting the properties of the indivici11al infPcting
slrain of reovirus as well as the age and resistance of the in.-
fected mouse. Reoviruses can cause encephalitis and hepa-
Resistance to Physical and Chemical Agents
titis in primates.
Reoviruses are stable at low temperatures (4 "C to room
tcmpcraturc) and are resistant to high temperature (SSºC)
for short periods of time. Reoviruses are also resistant to Host Response to lnfection
detergents and many disinfectants and stable over a wide Botl1 respiratory and enteric infection of n1ice with mam-
pH range (pH Z to 9). T11ey are inacLivaLed by exposure Lo malian reoviruses results in induction of humoral and cel-
9SºAl ethanol and sodium hypochlorite. lular immunc responses, natural killcr cclls, as wcll as in-
terterons and other cytokines, ali of which are potentially
important in terminating infection.
lnfectivity for Other Species and Culture Systems
Reoviruses infect most mammals and they replicate in a
variely ()f cell cuILures.
Laboratory Diagnosis
Reovirus infections can be diagnosed by virus isolation or
Host-Virus Relationship
detection, and by serolob'Y· Virus can be isolated from tis-
sues ¡¡nd from rectal, nasal, and t hroat swabs by c:ell culture
Dlstribution, Reservoir, and Transmission
techniques, although blind passage n1ay be required before
Mammalian reoviruses l1ave a wide geographic distribu- cytopathic effect (CPE) becomes visible. Virus isolates can
tion and are commonly present in river water, untreated be scrotypcd by cithcr hcmagglutiI1ation inhibition (HI) or
sewage, and stagnant water, likely reflecting fecal contam- virus ncutralization (VN) testing with serotype-specific
ination hy infected animals and/or humans. The mode of antisera. Reoviruses can be identified in tissues or cell cul-
transrnission is apparently l.Jy tlirect co11tact or exposure to ture by lmmunofluorescent antibody staining (FA) or im-
matPrials contaminatr.cl hy vir11s-infr.c.tr.cl fr.c.r.s (orofPcal) m un ohistochemistry. Serologic testing is done using paired
and/or respiratory discharge. sera a11d VN or ELISA.
400 PAJlT 111 Viruses
Treatment and Control
Since reovirus iJ1fectior1 in marnmals is usually mild, treat-
ment is i1ot requircd. No vaccincs or control mcasurcs
have been described and are unlikely to be developed in
the future unlcss rcoviruses are determined to be of greater
in1portance as anlrnal patl1ogens.
AVIAN ÜRTHOREOVIRUSES
Disease
.! \vian reoviruses (ge11us Orthoreovirus) are of economic sig-
11ificance to tl1e poultry industry. Syste.m ic reovirus infec-
tio11s of poultry can cause a variety of clínica! syndromes,
including gastroenteritis, hepatitis, myocarditis and peri-
carditis, pneumonia, and ill-thrift. Acute reovirus infec-
Lions also lead Lo inc1eased n101Lalily a11d ca1cass co11den1-
nations in affected ±1ocks, as well as poor growth and food
conversion efficiency (stunting syndro1ne). Arthritis a11d
tenosynovitis are common in birds that survive acute re-
ovirus infection; reoviruses are a major cause of avían
arthritis, and this disease occurs principally ill broiler
chickens and, less often, in !ayer birds and turkeys.
Etiologic Agent
Physical, Chen1ical, and Antigenic Properties
'l·he avian reoviruses Jargely resemble their mamn1alian
counterparts but differ in t hat t hey produce cell fusion in
ce11 cultures (syncltia), lackl1emagglutinating activity, and
arf' typic;:¡ lly 11nt1 hli> to grow in man1mal ian cell 1.i11es.
Avían and mamn1alian reoviruses exhibit varying degrees
of antigenic relatedness. At least 11 serotypes of avian re-
ovirus havc bccn dcscribcd, and strains of avian rcoviruscs
vary significantly in their virulence. AH share common
antigens as determined by agar gel immunodiffusion
(AGID) and complement fiXation (Cf).
lnfectivity for Other Species and Culture Systems
Avian rcoviruscs rcplicatc in cmbryonatcd chic.kcn cggs,
primary avían cell cultures, and, once adapted, certain es-
tablished mammalian cell lines.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Avian reoviruses are prevalent worldwide in chickens,
turkeys, and other avían species. Avían reoviruses persist
in nature tl1rougn environmental contamination and by
continued transmissio11 of tl1e virus from infected birds,
including those persistently infect ed with t he virus, to sus-
ceptible birds. Transmission oC(urs both horizonta.11y and
vertici:llly. Huril:o1rtal tra11::.uli:.::.iu11 b predo111i11a11lly lJy
the o rofecal route and occurs through both direct and
indirect contact. Vertical transmission has been demon-
:;tratell fulluvvi11g urdl, trdclit:~dl, dnd Ildsdl inuculdtiur1 uf
hreeder chickens.
Pathogenesis and Pathology
Infection with avían reoviruses is usually inapparent,
and the occurrence of disease reflects the age of the bird at
infection (your1g bin.ls dre predisposed), tl1e virule11ce of
the infecting virus strain, and the route of exposure.
Reovirus-induced arthritis is initially characterized by
acute inflammation within affected joints that progresses
to pannus formation \vith erosion of articular cartilage;
thus, reovi rus-induced arthritis in chickens somewhat
mimics h uman rheumatoid arthritis. Cross lesions iI1 af-
fected chickens often also include extensive :;wellir1g of thtt
digital flf'xor and metatar.~al extensortendons that can lead
to chronic hardening a11d fusion of the tendon sheaths.
Host Response to lnfection
Antibody responses to avían reoviruses have been demon-
strated by AGID, CF, and VN tests. l'he mechdnism or rne-
chanisms responsible far protective imn11.1nity t1rf' poorly
defl11ed, a11d variable degrees of cross-strain protection
have been reported.
Laboratory Diagnosis
Reovirus-induced avían arthritis must be differentiated
frou1 artl1riti:; d11d syr1ovitis c<Luseu lJy otl1er viru:;e:; auu
hacteria. A defi nitive d iagnosis requi res demonstration of
reoviru::; infection by dircct f.A staining of tissues (tendo11
sheaths), virus isolation, or serology.
Treatment and Control
There is no treatment for avían viral arth ri tis, and the in-
fection is be::;t controlled by proper management pro-
cedures and vaccination with either attenuated or killed
vaccines.
ORBIVIRUSES
ürbiviruses are iinportant pathogens ot Uvestock. four-
teen serogrou ps ha ve been described and five are of real or
potential veterinary sig11ificance: 1) bluetongue virus
(BTV), 2) epi.zootic hem orrhagic disease virtts (EHDV), 3)
Palya111 viru:;, 4) A(ricaJJ ho.rsesickness v irus (AHSV), and
.S) equine encephalosis virus (EEV). Like mammalian re-
oviruses (orthoreoviruses), orbiviruses have a doublc cap-
sid structure, possess a segmented dsKNA genome ( LU seg-
ments), a11d replicate in tl1e cytoplasm of infected cells.
OrbivJruses are dlstinguished from ortl1oreoviru:;e:; dILU
rotaviruses because they repllcate in both insects a11d
1uaII11ual:;, a11u lJy tl1e fact Ll1al Lhey are nol e11Le.ric pall10-

gens of vertebrares. Orb1v1ruses replicare wtthln the diges-
:i\·e tract of the hematophagous (blood sucking) inst>cts
.hal l1ansn1il Lhcsc viruses between susceptible mam-
:nalian hosts.
BLUETONGUE VIRUS
Di sea se
Sluetongue (BT) Is an artltropotl-trar1:s111ittctl viru:s <.li:sca:se
nf clomt><;tic ;incl wi lcl r11min;int spec.ies c.a11sed hy RT virus
BTV). BT occurs most commonly in shecp (soremuzzle,
catarrhal fever) and ccrtain species of wildlife, particularly
1vhite-tailed deer. BT in sheep and deer is characterized by
congestion, hemorrhage, and ulceration of the mucous
membranes of thc mouth, nose, and upper gastrointesti-
:1al tract. Otl 1cr ella racLcri::.lic 11:::.i vns i ncl ude hyvere111 ia of
-he coronary btind :-ind nPcro~is of hnth c;ircliac ancl skt>lt>-
tal muscles. B'rV infection of sheep and deer is sometimes
~atal. with terminal occurrence of disserninated intravas-
cular coagttlation. Sheep that survive severe bouts of B1" are
:requently emaciated, wcak, and lame, and have a pro-
rracted convalescence duringwhich they are susceptible to
~couc..lary iltft!ctio11::.. Brt!ak:; 111ay vccur i11 ll tt:: wool íiber of
convalesc.ent shcep. C.attle are commonly infected with
BTV in endemic arcas, but clinical disease is extremely un-
common. Vaccinc strains of BTV and those propagated in
cell culture can cross the placenta of prcgnant shccp and
cattle to infect the developin_g letus, leading to tetal death,
abortion , stillbirths, or teratogenic defects in progeny.
The ma¡or economlc lmpacr of BT is that it is included
in List A of the Organization Internationa le des F.pizootit>s,
aloug wilh fooL-and-n1ouLh disease and son1e fourteen
othcr diseases t h at are considered to have major adverse
economic and societal ramifications. As a consequencc,
the intcrnational movement of rumiI1ants and their
gcrmplasm from BTV endemic countri.es is frequently re-
strícted by non-tariff trade barriers pertaining to BT. The
validity of thesc tradc barriers is highly conjectural given
that c..li.stiucl :>lrcaiu:> a11d se10Lypes of B1'V, alo11g 1-vith
unic¡ue specics of insect vectors, occur in different regio11s
of the world, implying that the global spread of BTV was
nota recent event and certainly not associated with recent
trade in animals or their germplasm.
BTV recently was recognizcd as a potential pathogen of
carnivores. lnadvertent infection of pregnant dogs with a
BTV-cunta111i11alt:u vacciut:: 1:aus1:d abo1lion a11d deall1.
Serologic evidence of Bl'V infection has also been demon-
strated in African carnivores.
Etiologic Agent
Physical, Chemical. and Antigenic Properties
The genome of BTV and other orbiviruses includes 10 seg-
ments of dsRNA, cach of whlch cncodes at least one pro-
tf>\n (l':\h\f> f\'\ ?) 11-\f> \\i\1 l':\Tt\r\f> \<. C'C"lffi\'C"l<.f>c\
structural proteins, nvo of which form the outer coat and
Cllapter 63 Reoviridae 401
Ta b 1e 6 3. 2. Molt:!tular Con~lilut:!rtb uf Oruiviru~~~
Gene Encoded Role
protein
1 VPl RNA polymera5e; minor component of viral tore
partid e
2 VP2 Re<eptor binding; serotype determination; romponent
of outer capsid
3 VP3 lnteracts with gonomic RNA; rlructural componont of
viral core partlcle
4 VP4 RNA rapping Pnzymes; minor component viral core
particle
5 VP5 Structural interactions with VP2; component of outer
capsid
6 NS1 Virus tubules; not a virion cornponent; nonstructural
7 VP7 Group antigen; structural component of viral core
partlcle
8 NS2 Binds RNA; viru1 indusion bodies; honstructurnl
9 VP6 Helka1e; bind1 RNA; rni1101 co1nponent of viral tore
particle
10 NS3/3A Virus egress frorn infected cells; nonstructural
five the inner core. The outer capsid protein, YP 2, contains
the serotype-specific epitopes recognized by neutralizing
anc..l l1ernagglutinatlun-ir1hiuitirtg (HI) a11tiuutlit!:>. Tl1t!
other outer coat protein, VPS, contributes to the confor-
111alio11 of Lhe neul1alizil1g epilopes on VP2. Four i1or1-
structural (NS) protcins also occur in BTV-infected cells,
with NS-1 forming cytoplasmic macrotubular structures
that are charactcr1st1c of orb1v1rus-1nfected cells. The core
protein VP7 contains epitopes that are common to all
serotypes and stralns of BTV, 1'Vhich is the basis of group-
specific serological assays such as agar-gel immmunodiffu-
sion (AG ID) and con1pelilive enzyn1c-linked in1n1unosor-
bent assay (cELlSA).
There is considerable hctc:rogcncity within thc B'[V
serogroup, with l4 distinct virus seroty pes and many
strains, each with potentially distinct biological propcr-
ties. This gen etlc dtverslty has arlsen as a consequence of
both genetic drift of individual gene segments as well as
Ll 11:: rea~sv1l11 1 1::11 l of geue seg111enls d u1ing n1ixed infec-
tions of either insect o r ruminant hosts vvith more than
one 13'l'V serotype or strain . lnterestingly, recent sequence
and phylogenetic analyses have shown that prolonged co-
evolution of BTV with the different species of vector in-
sects that occur In various regions ofthe world has resulted
in strains of BTV that are uniquc to each rcgion, so-called
viru.s "Loµvtyµc!) ."
lnfectivity for Other Species and Culture Systems
B'l'V commonly infects domestic (sheep, cattle, and goats)
and wild (deer, antelope, wild sheep species, etc.) rumi-
nants. The viruses can be adapted to growth in suckling
mice, cmbryonated chlck eggs (ECEs), and a variety of
C"I~ <.f>Vf>n m:\mm:\\\:\n :\ne\ \n~f>rt C'f>\\ rn\hlTf><. \{f>I'\\r:\nC"ln of R\\l \n
ECEs is facilitatcd by an incubation temperature of 33.SºC .
402 PART 111 Viruses
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Bluetongue virus has heen isolated from ru minants in ali
continents exccpt A.ntarctica. 1I1fectio11 occurs ll1rougl1oul
tropical, subtropical, and temperate regions of the '.vorld,
coincide11t with tl1e distribution of susceptible rurninants
a11c1 co1npetent vector insects (Culicoides spp.). ·rhe virus is
not contagious betwee11 rumir1ants; rather, infection oc-
curs onJy following the bites of BTV-infected Cu/icoides in-
sects. Subclinic:al and/or asymptom.atic RTV infection of
r u1nina11ls occurs lhroughoul endernic regions of lhe
world. Outhreaks of BT occur only sporadically, most often
at the northern and southern extremities of the virus'
range following incursions of BTV into immunologically
n.aive populations of ruminants.
Vector Culicoides insects become persistently infected
with BTV after feeding on a viremic rumir1ant. 'fhese
hematophagous insects are tr ue !Jiulugic vecturs uf BTV
ht>causf> they only trans1nit virus to other ruminants after
an extrinsic incubation period of approximately 10 days.
During this time the virus disserninates from tl1e n1idgut
of thc infcctcd inscct to its salivary glands. Rcplication of
H·rv within the insect vector is dependent on ambient
temperature; thus increased replication of BTV in vector
insects occurs at higher temperatures, but the lifespan of
these insects is inversely propo.rtion.al to temperature.
Tra11sn1issio11 of BTV ca11 occur year-rou11d il1 clin1ates tl1at
permit insect (Culicoides) activity in ali seasons, with the
virus pcrsisting il1 a pcrpctual vcctor-rumina11t cycle of in-
tection . In contrast, transrnission oí H'l"V is highly seasonal
in regions of the world at the northern and southern ex-
tremities of the virus' range (approximately 35 ctegrees lat-
itude south and 45 degrees latitude north). In these areas
BTV t ransn1ission typically occurs 011ly il1 ll1e la le sun1n1er
and fall '"'hen vector populations peak a11d wl1en ambient
tcmpcraturcs are highest (likcly reflecting thc influe11ce of
temperature-dependent virogenesis), 'fhe traditional
global range of BTV has recently expanded into Mediter-
ranean Europe after the northern spread of competent
vectors, perl1aps as a consequence of global warming.
Pathogenesis and Pathology
Sheep and sorne species of dee.r are most susceptible to RT,
whereas cattle are resistant. The patbogenesis of BTV in-
fection appears to be identical in ali ruminant species. The
virus n1ultipliC$ initially in thc lymph nodc(s) draining thc
si te of infectior1, and viremia occurs as early as 3 days later
with a subseque11t febrile response. Upon systemic distri-
bution, virus repllcates in mononuclear phagocyti.c cells
and the endothelium of small blood vessels, resultine in
vascular injury, thro.n1bosis, and il1farctio11 of tl1e affected
tissues; these include the upper gastrointestinal and respi-
ratory tracts, thc coronary bands, thc hcart, and skclctal
muscle. Visseminated intravascular coagulation likely
contributes to tl1e vascular injury, hemorrhage, a11d tissue
l1Jfarctiu11 tl1ar are cltaracteri:sLic uf :severe ca::;e:s uf BT i11
sh<-~ep and deer.
Viremia ir1 l3TV-infcctcd ruminants is higbly cell-
associated. Virus initlally is associated with ali blood cell
typP.s, ;inri titP.rs of vi rus in P.<1í.'h rPll fr::ic'tion rPflPrt thP príl-
portion of each cell type in blood; thus s1·v initially is most
associated with platelets and erythrocytes, and less so
leukocytes. Late in the course of infection, bowever, virus
appears to be exclusively associated with erythrocytes. lt is
this association of BTV with erythrocytes that facilitates
both prolonged infection of ruminants as well as infection
of the hematophagous Culicoides i nsect vectors that feed on
lheuL Il i:> Lv lJe :>Li.e:>:>ed Lhal although BTV in!eclion oí ru-
minants can be prolonged (up to approximately 60 days),
pcrsistcnt B'fV infcction of ruminants <loes not occur.
'fhe gross lesions oí 1rr include tacial edema and hyper-
emia, with or without hemorrhage (petechial and ecchy-
motic) of the oral and nasal mucosa, skin, and coronary
band. Ulcerations and erosions also may occur in and
C1ruunu tl1e ruuutlr, e:sµecially 011 lile hard palale. He11101-
rhages at the hase of the pulmonary artery are very charac-
teristic of severe cases of B1'. Petecl1ial hcmorrhagcs may
also occur in the myocardium, pericardium, sl<eletal mus-
culature, a11d the tissues of the upper gastrointestinal tract.
Host Response to lnfection
RTV-infected r11min;:ints dt>velop hoth humoral and cellu-
lar in1n1une responses. Virus-neutralizing (serotype-
specific) and non-neutralizing (group-specific) antibodies
dcvclop 7 to 14 days aftcr infcction. However, virus canco-
exist in blood •vith high titers of neutralizing antibody for
several weeks because of the intimate association of BTV
with the cell membrane of infected erythrocytes. Varying
degrees of cross-serotype viral neutralization may occur
following infeclion of an anin1al will1 a sil1gle n·rv sero-
type and subsequent exposures to additional serotypes can
ge11erate production of broadly cross-reactive neutralizing
antibody, including antibodies that neutralize serotypes ot
BTV other than those witb wbich the animal was infected.
Laboratory Diagnosis
Initial diagnosis of BT in sheep aod <leer is bascd on thc
characteristic clinical signs of affected animals in l<nown
B'fV-endemic areas. BT typically occurs in the late sum mer
and fall. Conflrmatíon of the diagnosis requires virologtc
testing, usually by eitl1er polymerase chain .reaction (PCR)
assay or by virus isolatio11 usil1g i11oculation of susceptible
sheep, ECEs, sucklir1g mice, or cell cultures. The cell cul-
tures most often used include Vero and BHK; multiplc
blind passages are otten required before cytopathic effect is
observed. Upon adaptation to cell culture, virus can be
identified by fluorescent antibody stalnlng or virus neu-
tralization assay. BTV is very commonly detected in the
blood of 11ealll1y run1i11anls il1 ei1dcn1ic areas, especially if
sensitive nested PCR assays are used because these can de-
tcct BTV 11ucleic acid for up to 200 or 111orc days aftcr infcc-
tion. 'l'hus, the mere demonstration of J:5 rv or .B1' V nucleic
acid in the blood of ruminants certainly is not proof of dis-
ease causallty.
Serologic diagnosis can he performed using tests for
group-specific (AGID, CF, ELISA, IFA) or type-specific (viral

neutralization, HI) antibodies. Paired serum sam ples are re-
quired to dcmonstrntc scroconversion oran increase in titer.
A single serological test ts often meaningless beca use a high
proportion of ruminant s are seropositive in BTV-endemic
areas, and the vast majority of these animals never experi-
ence obvious clinical disease following BTV infection .
Treatment and Control
There is no specific treatment for BT, although stress ap-
pears to exacerbate expression of disease. Furthermore,
trans1nission of B1'V to unaffected ru1ninants can be pre-
vt::JJ Lt::LI l;y 111oving anin1als ii"ldoors (if fca .5i])le) 'l'vher e in -
sect vectors are 11ot present . Vaccination of sheep with at-
t.cnuatcd strains of BTV has been practiced for mallY years
1n South Africa and North America; the South African vac-
cine incorporates a large number of different BTV sero-
types, antl requires va<.:<.:i11alio11 on Lhree d iffere11L occa-
sions. Potential disadvantages of live attenuated vaccines
include rcversion to virulence and their ability to be t.rans-
m itted il1 n.ature. Furthermore, live attenuated vaccines
potentially can reassort t heir genome segments witl1 field
strains of BTV to create novel variant viruses. Finally, live
attenuated vaccine strains of BTV repeatedly have been
shown to !Je a u le tu <.:rus::. ti 1e place11La Lo cause felal dealh
o r inj11ry, \~>hereas fíeld strai ns of the virus apparently can-
not. Recon1binant baculovirus-expressed virus-like parti-
cles recently have been used as a BTV vaccine, avoiding the
proble1n s inhcrcnt to livc attcnuat ed vaccines.
EPIZOOTIC H EMORRHAGIC 0 JSEAS E ANO IBARAKI
VIRUSES
Epizootic hernorrhagic disease (EHD) is an arthropod-
t ransmitted virus disease of wild ru1ninants caused by
EHD virus (EHDV). EHDV is an important cause of disease
and n1ortaJity in white-tailed deer in North America and,
to a lesser extent, prongl1orn antelope and bighorn sh.eep.
Altt1ougl1 Elil)V luíecliur 1. uf Lluu 1e~ liL 1u1uiua11l:. i:. 1..v1u-
mon in endemic a reas, EHDVis not regarded as a pathogen
of don1estic livestock, and authentic cases of EHD in do-
mestic livestock are lacking. A notable exception is lbara.l<i
discasc of cattle in Ja pan and Korea; t he causative agent,
Ibaraki virus, is closely related to EHDV serot.ype 2. EHDV
shares many features "'' ith BTV, and EHD of white-tailed
deer closely resern!Jles fulu1l11a11l '[Jlue tougue will1 hype1-
emia, hPmorrhage and ulceration of the upper gastroin-
Leslinal Lracl, 11ec1osis of cardiac and skeletal muscle, and
terminal disseminated intravascular coagulation with
\.videspread blecding. Addition.al fcaturcs of lbaraki discasc
ln cattle in.elude rnarKeC1 dyspnag1a as a consequ ence of
necrosis of muscles of the larynx, pharynx, esophagus,
anc! tongue.
l.ikP 1rrv. F.HDV infection occurs throughout tropical
and temperate regi.ons of the world, and EI-IDV in fection of
ruminants has been described in Africa, Asia, and the
Amcricas. EHDV closely reseo1bles BTV in terms of its epi
demiology, including dissemination by Culicoides insect
Chapter 63 Reoviridae 403
vectors, pathogenesis of infection of ru1ninants, repHca-
tion strategy, molecular structure, and 1nethods of diagno-
sis. There currently are eight recognized serotypes of
EHDV- nine if Ibaraki virus is classified as EHDV serotype
7, as recently has uee11 µruµused . Wilh Lhe exceplion of
lh::ir::iki virus, vaccines are not widely available for EHDV.
PALYAM VIRUS
Palyam viruses a re insect-transmitted orbivruses that
cause abortlon and teratogenesis a111ong cattle in Afri<.:a,
A ~ i;i , ;inri A11~tr;1l i::i . FPfl 1sPs t h::it s11rvivf> infPction w ith
Palyam viruses prior to m id -gestation may develop brain
malformations, including hydranencephaly and/or talse
porencephaly. Like the ot her orbiviruses of vet erinary iln-
portance, Palyam vi..ruses are disseminatecl by Culicoides
insect vectors. There are ll serotypes of Palyam viruses,
a11tl tlie µa tlioge11e:si:s a11d Leralogeuic effecls of ChuL:an
(Kasaba) virus infection of fet al cattle have been especially
well déscribed.
AFRICAN HORSESICKNE SS VIRUS
Di sease
African horsesickness (Al IS) is an arthropod-transn1itted
Orbivirus disease of equids, includi11g horses, mules, and
donkcys. 'l'hc cnusnt ivc ngcnt, A.HS viru~; (J\HSV), is zoo
notic, alt hough fatal infection of humans only rarely has
been described. Fatal AHSV infection of dogs also has been
described. AHS of horses varíes greatly in severity, tlepentl-
il<g on the infecting strain of virus anrl thf> _.;uscPptihility of
Lile infected horse. Severa! distinct forms of Al-IS have been
descrihed, including 1) a peripheral form characterized by
edema of the head; 2) a central forro chnractcrizcd by pul-
monary edema, high fcver, severe depression, coughing,
dischnrgc of fluid from the nostrils, and rapid death of
many affected horses; 3) an intermediate for1n tl1at is char-
acterized by fever, eden1a of the head and subcutis (supra-
oruital e<.1e111a is highly characlerislic), and sig11ificant
n1ortal ity of affected h orses; and 4) horse sickness fever,
wl1ich is a febrile di.sease with a more benign course.
Et iolog ic Agent
Physical, Chernical, and Antigenic Properties
The causative agent of AHS belongs to a distinct serogroup
within the genus Orbivirus. Ni.ne serotypes of J\HSV have
been identif1ed by cross-neutralization <.n1aies in mice. All
types share co1nmon group-specific antigens.
lnfectivity for Other Species and Culture Systems
AHSV infections have been documented in horses, don-
keys, rnules, zebras, goats, dogs, and large African carni-
vores such as lions. Disease has been described in horses
404 PART 111 Viruses
and dogs. ·r11e virus can be propagated in suckling mice and
adapted to grow in ECEs a11d cell cultures (Vero and BHK).
Host-Virus Relationship
Distribution, Reservoir, and Transmission
African horscsickness occurs tl1roughout southern Africa,
with periodic epizootics in northern Africa, the Middle
East, Asia (the h1dian subcontincnt), and, on occasion in
the past, Mediterranean Europe (tbe Iberian Península).
AHS is not contagious; rather, the virus is transmitted only
by Culicoides i11secls Lhal serve as lrue bivlvg.ic vt:ctvr::.. Like
bluetongue, AHS occurs n1ost commonly in the late sum-
mer and faH in endemic areas, an.d the distribution of AIISV
is dependent on the presence of compete11t insect vectors
and ambient temperatures that facilitate temperature-
dependent virogenesis within these vectors. Zebras have
been in1plicated as potential n1ammalian reservoirs of
AHSV ir1 :;vutl1err1 Afric«, !JecC1u:;e AHSV iufectiur1 i:; C1:;yrup-
tomatic in zehras and AHSV viremia is more prolonged in
zebras tha11 iI1 horses. Dogs may become infected with
AHSV by eating infected horse 1neat, and serological sur-
vcys l1avc sl1own that ar1tibodies to AHSV are common
among large wild carnivores in soutl1ern Africa.
Pathogenesis and Pathology
The incubation pcriod of AHS is gcncrally less than 7 days
following the bite of an AH.SV-in.tected <.;u/icoiaes insect.
The incubation period is shortest il1 11orses infected with
virulent strains of AiiSV, and mortali ty in susceptible
horses iI1fected with highly vir1.tlent AHSV can reach 9SºA>.
AHSV replicales i.J.1 n1011onucleai cells of Lite ly111vl1 llVUt:::i,
spleen, thymus, and pl1aryngeal inucosa, and in vascular
endotheli1un. Thc lcsions of AHS rcsult from vascular in-
jury to small blood vessels, although it is uncertain whether
the vascular injury that characterizes AHS is a result only of
direct Virus-mediated endothelial injury or if vasoactive
mediators released from AHSV-infected mono11uclear
pl1agocylic cells also co11Lribule. 1'he severe c1::11lral fun 11 uf
AHS i.s cl1aracterized by pulmonary edema, hydrothorax,
and hydropcricardium, with epicardial and endocardial
hemorrhages. Subcutaneous edema can be extensive iI1
horses that suffer a more protracted form of tl1e disease.
Host Response to lnfection
Ali serotypes of AHSV share con1mon group antigens and
induce devclopment of antibodies that may be recognized
by CF, AGID, and in.direct FA. Viral neutralizing antibodies
:ilso rlPvPJl)p fl)Jlowi ng infPction; the~P. arP. pn~r.lomin<1n.tly
serotype-specific, but so111e cro.ss-neutraliz;atio11 activity
has been observed.
Laboratory Diagnosis
ri.eld diagnosis of AH.S, especially in n.o nendemic ar.eas,
should be supported by viral isolation or serolob'Y to distin-
guish other infections that ca11 produce similar clin l.cal
signs. Polymerase chain reaction tests are available, wl1ich
provide a rapid and accurate resul t if done propcrly. 1'11e
presence of virus also can be rapidly identified in tissue
specimens using an AHSV-specific capture ELlSA. Virus is0-
latiun i:; a slower but accurate method of virus detectlon .
lnt r;:irerebr;:i l inocu l;:ition of suc:kling .tnice with blood or
tissue suspension is a very sensitive n1etl1od of isolating
AHSV, although time-consuming serial passage may be re-
quircd for viral adáptation. Cell cultures are notas cfficicnt
in isolating virus as mouse or horse inoculation. Virus can
be identified by virus 11eutralizatio11, Hl, or PA.
Serologic diagnosis requires paired serum sa1nples for
demonstrating seroconversion or an increase in ;:intibody
ti ter. Assays routinely used for such ptirposes include con1-
petitive ELISA and CF, which are group-specific tests and
detect antibodies to AHSV regardless of the infecting virus
serotype, and vi.ral neutralization assay, which is very sen-
sitive but serotype-specific.
Treatment and Control
No specific treatment of AHS is available. Hyperimmune
horse serum confers transient .rrotection. Stabling of
horses in insect-secure facilities is assumed to reduce expo-
sure of animals to the Culícoídes vector. Annual vaccina-
lion of l1orses wilh allenualed virus vaccü1es tl1at i11clude
the nine recognized serotypes of AHSV is widely practiced
in southern Africa, and to control incursions of AHSV into
Europe. "l'here are severa! potential problems inherent to
use of multivalent modified live /\HSV vaccines, including
lack of protection against ali serotypes of the virus, rever-
sion to virulence of vaccine strains of virus, acquisition
C1r1u ui:s:;1::1uini:ltiu11 uf Vi:lccine viruses by vector insects, and
reassortment of gene segn1 ents between different v irus
strains/serotypes during n1ixed infections.
EQUINE ENCEPHALOSIS VIRUS
Equine encephalosis virus (EEV) has been isolated from
horses with hep;:itir lipidosis ;:ind v;ig11P ni>11rologic signs,
but its iinportance as a primary pathogen is uncertain. ·rhe
virus has also been isolated from aborted fetuses. Serologic
studies have shown that EEV infection is very v1idespread
and com1non among 11orses in southern Africa. The seven
recognized serotypes of equine encephalosis virus (EE\7)
sl1C1re cummon group antigens anu closely rest:!rnble
African horse sickness vinises (AHSV). 'fhe epiderniology
of LEV infcction is also like that of ATISV, with transmis-
sion by Culicoides insects.
ROTAVIRUSES
Disease
Rotaviruses cause enteritis and diarrhea in many mam
rnalian species (lncluding huma ns) and birds. Rotavirus in-

fections are an important cause ot diarrhea in young farm
animals, including calves, lambs, foals, and p.iglets. Th.e
Virus infects mature adsorptive cells at th.e tips of the intes-
tinal villi with resulting malabsorption, maldigestion, ;:incl
<.liarrl1ea. The clinical severily of tl1e infection depends on
factors such as t he ageand susceptibility of the affected an-
imal, the viruler1ce of the i.nfecting strain of Rotavirus, and
the presence of other enteropathogenic organisms.
Etiologic Agent
Physical. Chemical. and Antigenic Properties
Rotaviruses represent a distinct genus within the family
Reoviridae. Tl1e viru.se.s are J1011e11veloped, conlain a seg-
mented (11 segments) dsRNA genome, and exhibit a dou-
ble-.shelled capsid morphology 1.vith an overa U d iamctcr of
the mature virion of approxjin.ately 100 nm (see rig 63.2.).
At least 13 differe11t rotavirus proteins bave been identi-
fied, with 2 of the 11 gene segments encoding 2 distinct
proteins. Of these 13 proteins, 7 are stn1ctural virion com-
po11e11t.s (i11cludi11g e11:¿y1nes) and 6 are i1011slruclural pro-
teins that are produced in Rotavin1s-infected cells but that
are not incorporated into virions.
1'he rotaviruses currently are organized into five major
groups (A E), with two possible additional species (F,G).
Many distinct strains and/or serotypes of Rotavirus occur
within each group. Rotaviruses within each group share
common antigens, can rea.s.surt tl1eir genon1e seg1ue11Ls
during mixed infertions, h;:ivp ronsidc~rahle sequence hn-
n1ology of conserved viral genes, a.nd tend to infect the
same species of animals. Neutralizil1g antibodies are in-
duced by thc outcr capsid protcins VP7 and VP1, whereas
VP6 expresses determinants common to each rotaVirus
group and subgroup.
lnfectivity for Other Species and Culture Systems
Although rotaviruses obtained from one species can some-
times infect other specics, strains of Rotavirus are largely
species-specific in their tropism. Rotaviruses are difficult
to propagate in cell culture. A major advance in the propa-
gation of rotaviruses was the discovery that low concentra-
tions of trypsin are required to initiate virus infection and
replicatiu11 iu cell cul Lure. 'frypsin cleaves ll1e viral outer
coat protein, VP3, to facilitate infection of cell cultures,
and once adapted rotaviruses grow well i.n ccll culture. The
most common cell lines used are kidney epittlelial cells, es-
pecially the rhesus monkey kidney cell line, MA104.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
Rotaviruses are considered to be distributed worldwide, and
occur in many different animal species. High titers of virus
are cxcretcd in the feccs of infccted animals, and the virus
is very stable in the environment if associated with feces.
Transmissior1 to other animals occurs by ingestion of virus,
following elther director lndirect orofecal transmission.
Chapler 63 Reovirhlue 405
Pathogenesis and Pathology
The pathogenesis of Rotavirus infection is similar regard-
less of the animal species affected. After oral infection, thc
virus infects the mature adsorptive epithelial cells that line
the apical (lumenal) aspects of the intestinal villi. The in-
fection progresses from t tle upper to the lower portions of
the small intestine, and in some species, to the colon.
Destructiu11 uf the.se 1uature vUlus e!l Le roe y tes leads to vil-
lus atrophy, and the mature absorptive cells t hat line the
vflli are replaced by more immature cells from the intes-
tinal crypts, leading to maldigestion, small intestinal mal-
absorption, and diarrhea. Interestingly, the NSP4 protein
of rotavirus alone can induce intestinal hypersecretlon
from crypt cells, su.ggesting that both malabsorption and
hypersecretiu11 uf fluid au<.l t~lectrulyle.s cuulril!ule Lo Lile
diarrhea of rotaviral enteritis. The disease is worstin young
animal.s and can rapidly lead to fatal acidosis, dehydration,
and hypovolemic shock.
/\nimals t hat die of Rotavirus enteritis are dehydrated
and have very liquid intestinal contents. Diarrhea is fluid
and yellow/white (the so-called white scours), which fre-
tiuently .stair1.s tlu; µeriueu 1n of affecte<.l aniHtal~. Hi~to­
logic lesions in elude Villus atrophy with loss of mature ad-
sorptive cells covering the villi, and hyperplasia of the
immature cells within t he intestinal crypts.
Host Response to lnfection
Animals infected With J.<otavirus develop local and sys-
temic humoral immune responses that can be identified
by various serologic technigues. Vira.1-neutral!zlng antl-
body is serotype-specific and directed at VP4 and VP7. Tht>
Rota.virus ELISA detects antibodies to group and subgroup
determinants. Local immu11ity within the bowel is very
important in preventing scverc rotaviral enteritis in young
animals; thus ingestion ot colostrum with high titers of
Rotavirus neutralizing antibody provides temporary im-
munity against disease in neonates.
Laboratory Diagnosis
Diagnosis of Rotavirus-induced diarrhea requires identifi-
cation of t he virus in teces or in tissues obtained at
necropsy. Electron microscopy (EM), immune EM, and flu-
OI(;!.St:eut a.ntil!o<.ly (JFA) $Lai11i11g ofreces and/or inlesli11al
tissue sections ali facilita te direct visualization of virus or
viral antigens, but Rotavirus in feces is most easily detected
by antigen-capture ELISA. ·rhe ELISA is very sensitive and,
depending on the capture antibody used, can distinguish
different types. Direct examination of the dsRNA genome
of rotaviruses in the feces of animals can be done by poly-
acrylamicle gel electrophoresis, which also identifies t!1e
spec:ific: gro11p of Rntavirus that is present. Polymerase
chain reaction assays also are available.
Virus isolation is usually done on MA 104 cells in the
presence of low concentrations of trypsin. /\vian ro-
taviruses are isolated on primary chicken embryo hver and
kidney cells.
Serologic diagnosis can be done by ELISA or virus neu-
406 l'A1u 111 Vlruses
tralization assays; however, the utility ot data obtained
usually is uncertain beca use of the widespread distribution
of Rutavirus lnfectlon, whlch means that a high proportioo
of animal' arP 'Propositive rcgardless of disease st atus.
Treatment and Control
'freatment of clinically ill animals is guided by disease
severity, and treatment of scvcrc cases involves replace-
ment fluid therapy to treat dehydration and acidosis, min-
i111iza lion of environ1nenlal sl1ess, a11d lrt:al1111::ut uf :ieL-
ondary infcctions.
Control is often difficult because of the stability of the
virus in teces lead ing to long-term environmental contam-
ination. Although often challenging to implement, strin -
gen t sanltation practices can rninimize exposure. vaccine
stratcgics are bcst directed at the dams of suckling neo -
nates, to cnsure that high titers of antibody a re present in
the colostrum and mili< of thcsc animals.
AQUAREOVIRUSES
Aquareoviruscs are 111orpl1ologically a11d pl1ysicocheuú-
cally similar to orthoreoviruses, but have 11 segments of
dsRNA. Scvcn of thc 12 protcins (gcn o m c scgmcnt 11 en-
codes two proteins) are structural, wit h V F7 rep resen ting
the major capsid protein. Six genotypes (A- F) have been
proposed fo r Aquareovlrus. Viruses in thls genus infect
bot h fish ;ind shellfish, causing 11ccrosis in th e pare11chy-
n1al organs of infected fisb a nd h.igl1 i11ort ality i11 fish
hatcheries. Aquareoviruses replicatc in fish or shellfish cell
lincs at 16ºC and can in d uce syncytia formation .
 Birnaviridae
N. JA1vfES MACLACI ILAN
The fam1ly Birnaviridae includes three genera: Avibirnavirus
(infects poultry), Aquabimavin1s (infects fish), and F.ntnmn-
birnaviru~ (i1 Lft:.:1.:L:> i11:>ecls). lnfectious bursal disease virus
(genus Avibirnavirus) is the best studied. lnfectious pancre-
atic necrosis virus (gcnus Aquabirna~·irus) is a significant
pathogen o t salmonid fish.
INFE CTIOUS BURSAL DISEASE
Disease
lnfectious bursal disease (IBL)), also known as r.11mhnrn di.~­
ease aft4::!r tite Luwu in Dela,'\Tare (North Ani.erica) where it
was first identified, is an economir.ally important virus dis-
ease of young cl1ickcii.s. The IBD virus replicates in imma-
ture B lymphocytes iI1 the Bursa ot Fabricius, leading to re-
duccd immunologic responsiveness. The virus can cause
relatively high mortality in chickens 3 to 6 weeks of age,
and profound iminunosuppression in birds infected earlier
in life. The clinlcal disease in lJirtls uvcr 3 wt:.:eks of age is
characterized by soiled vent fPathPr~ (hirdc; often peck at
th4::!ir uw11 vt:.:uls), diarrhea, depression, anorexia, trem-
hling, s<'vert• prostration, dehydration, and eventual death.
In the United States, economic losscs are typically due not
so much to bird death but rather to reduced weight gain
and carcass condemnation due to hemorrhages in skeletal
muscle, whereas highly virulcnt stralns uf IBD virus tl1al
cause high mortality in affected floi'k~ arP inr.reasingly im-
portaut lu Eurupe and olher rcgions of the world. Infection
of hircis less than 3 weeks of age results in economically
devastating inapparent infection. Such birds are exten-
sively i1nmunocompromised and exhibit increased suscep-
tibility to a variety of other infectious diseases. Further-
more, affected birds respond poorly to vacclnation.
Etiologic Agent
Physical. Chemical, and Antigenic Properties
lnfectious hursa 1 c1isPase vi rus (TBDV) particles are nonen-
veloped with icosahedral symmetry (60 nm in diameter;
Fig 64.1). The genome includes two segments ot double-
strandcd RNA (dsRNA), designated A and B, that encode
five protems. Segment A has two open reading trames and
encodes a nonstructural protein (VPS) as \'\Tell as a polypro-
teln that Is cleaved into two structural µrutci11s (VP2, VP3)
anti tht: viral prulea:.e (VP-l). Gene seg111c11l B encocles the
viral RNA-dependent RNA polymerase (VPl). There are
two serotypes of IBDV and markcd antigcnic and gcnctic
variation within each typc. Strains ot LBIJV serotype 1
cause disease in chickens throughout the world.
Resistance to Physical and Chemical Agents
Tnf<>c:tious bursal disease virus is extremely stable and re-
sists i nactivation following a cid trcatmcnt (stablc at pH 3),
lipid solvents, various disintectants, and heat (survives
60ºC for 30 minutes).
lnfectivity for Other Species and Culture Systems
lnfectious bursa 1 c1isPase virus pri nci pally affects chickens,
although turkeys, ducks, and some other species of domes-
tic and wild birds also can be intccted with the virus. ·rne
virus can be p ropagated in cmbryonating chicken eggs,
wtth subsequent embryo morrallry, as well as in a varlety
of avian cell cultures.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
lnfectious bursal disease occurs worldwidc in intcnsivc
poultry-raising areas. 'l'he virus persists in nature because
of its stability.1.he v irus has reportedly persisted in poultry
houses, following depopulation, for over 100 days. There is
no evidence of a true carricr state in birds.
Transmissiun uf IBDV uccur~ IJy i11geslion ofvirus fron1
feces or fpcpc;-contaminateci fomites, feed, and \~>ater.
Pathogenesis and Pathology
lnitial repllcatlon of IBDV occurs in the intestinal traer
within hours of ingestion of the virus, from wbere it dis-
semlnatcs to numerous tissu4::!s, lnclutliI1g the IJursa o f
fabricius (BF). Other lymph()icl ()rgans surh as thymu\,
spleen, and toi1sils also niay be affected, and disseminated
Jymphoid atrophy is especially characteristic of birds in-
fcctcd with vcry virulcnt strains of IBDV.
·rhe striking age-dependent nature of the response of
chickens to IBDV likely reflects the maturation of the BF at
the time of infection. Speclftcally, blrds are most suscepti-
ble to 1BL1 from 3 to 6 weeks of ag<.>, wherea5 hur~Prtomi7.Prl
bi1ds of lhe san1e age do i1ot develop disease. Birds older
407
408 PART TII Viruses
F1G U RE 6 4. 1 . Negatívely staíned preparations of ínfectious
bursal dísease virus. 250,000X. (Courtesy of R. Nordhausen.)
than 6 weeks also do not dcvclop scvcrc discasc, nor do
those infected prior to 3 weeks of age. ·rhe impaired iln-
munologic responsiveness that occurs in chicks infected
early in life has bee11 attributed to bursal injury, which re-
s11lts in failure to seed B lyn1phocytes to peripheral lym-
phoid organs and d i111i.nisl1ed11un1oral in1n1 uui Ly. Cell u lar
irnmunity also is reduced in IBDV-infected young chicks.
Gross lcsions may includc dcl1ydration, darkened pec-
toral m.uscles, and hemorrhages in the pectoral and leg
muscles. The appearance of the BF is dependent on the
state of disease. The BF illitially becomes enlarged d ueto
P.dc>ma <inci hyperemia, but this rapidly is followed by pro-
gressive atrophy th.at is n1arked by app1oxi111alely 8 <.lay:;
after infection . Bursal necrosis and he1norrhage are char-
actcristic of advanccd discasc.
H1stolog1cany, the ep1t he1Ja1 surtaces ot t11e HF nave
multiplc erosions, and there is extensive necrosis of lym-
phocyles wiLhil1 ly1uµl1uiL1 fullicles so that these become
ciPpleted of lymphocytes. Edema and infiltration of het-
erophtls occurs il1itially il1 ll1e aifec Led BF, followed by for-
mation of cystlc cavities bound by columnar epithelial
cclls and lymphoid dcplction. Pathology in other lym-
pl1oid organs is less severe and recovery more rapid, except
in birds infected with very virulent strains of IBDV.
Host Response to lnfection
following infection, birds develop a humoral immune re-
sponse that is 111casured by viral neutralization, agar gel
imn1unodiftusion, and enzyme-linked immunosorbent
assay (ELISA) tests. Adult birds transfer maternal antibody
to developing en1bryos, and if present in sufficient titer,
the antibody lends protection to the hatched cbick for
variable periods of li1ne.
Laboratory Diagnosis
Field diagnosis can usually be made based upon the ch<ir-
actcristic clinical signs and high morbidity and rapid re-
covery of most attected birds. Atrophy of the cloaca! bu rsa
is characteristic of inapparent IBDV infection in young
chicks. Definitive diagnosis of IBD can be carried out by
direct fluorescent antibody (FA) stainit1g of sectioned tis-
sues 01 viral iSulallu11 fru1u tlrc u ursa c111<.l spleeu. Isola-
tion can be made by inoculation of emhryonated chicken
cggs or cell cultures. Serology is also useful for diagnostic
purposes.
Treatment and Control
No treatment has heen descrihed for affected hirds.
Control of the disease may be facilitated by proper sanita-
tion practices. Vaccination programs are widely used to
control IBD. Immu11ization of breeder flocks is done to fa
cUitate passive transfer of immunity to chicks. Vaccination
of chicks is also practiced, but to be effective, levels of ma-
tt::r11al dlltiuu<.ly lllUSt ue luw. Butl1 atte11uateL1 a11L1 killed
vir11s vaccinc>s <ir<> av<iil<ihlc>.
INFECTIOU S PANCREATIC NECROSIS
Infectious pancreatic 11ecrosis virus (IPNV) is the cause of a
highly contagious and fatal disease of salmonid fish (trout
and salmon) that is increasingly important to the aquacul-
Lure i11<.lu:;try wurl<.lwi<.le. Tl1e <.lisease is especially severe in
fish that are less than 6 months old, hut <ilso occi1rs in
older fish following strcsses such as that associated with
relocat ion from fresh to salt water. Affected fingerlings ap-
pear dark in color and swim with a rotating action (whirl-
ing). Petechial hemorrhages may be present in the abdom-
inal víscera of affected fish, along wit h necrosis of the
va1 H.: reas. Tl1e virus also affects commercially raised ycl-
lowtails with high mortality and is classic<illy rc>fP.rrc>ci to <is
viral ascites due to accumulation of fluid in the abdomen
tascites) and associated abdominal ctistension. Eels can be
infected with resulting clinical disease. Fish that survive
infection with IPNV become carrlers and serve as a source
of infection to other fish, especially u11der 11atcl1ery condi-
lions. The virus ls very slaule iI1 tl1t:: t::11viru111ue11t. The
virus can be identified in the tissues of affected or carrier
fish by virus isolation in fish cell cultures or reverse-
transcriptase-polymerase chain reaction (Rf-PCR) tech-
niques. Up to 10 serotypes of IPNV have been described,
with the neutralizing epitopes being mapped to VP2.
P.fforts to clc>v<>lop efff'ctive vaccines are complicated by the
multiple serotypes of IPNV and the d.ifficulty in in1111uni.z-
ing very you ng fish that are especiaily susceptible to the
virus. Co11trol is based on husbandry efforts that ma,'<i-
mize hygiene and sanitation and minimize crowding,
stress, and introductlon of infected replacernent stock
and/or eggs. Vigorous identification and culling of carrier
fish is advocated as a method of controllin.g the disease.
 Retroviridae
RlcHARD M. DoNOVAN
Rctroviruscs (family Retroviridru~) are enveloped, single-
stranded RNA virusc:> lhal replicate th rougl1 a DNA inter
n1ediate u~ing an RNA-dependcnt DNA polymerase (re-
\ t:1se uanscriptase). ·rhis largc and diverse family includes
membcrs that are oncogenic, are associated with a variety
of in1munc system disorders, and cause degencr;itivf' and
neurologic syndromes.
Classification
1"hc fa111ily Retroviridae is classificd in to scven genera (Table
ó.S. 1). Classification is based on genomc structure and nu-
cleic acid scqucncc, in addition to older classification cri-
teria based 011 1norphology, serology, 1Jiuclle1nical fea-
tures, and the species of animal from which the retrovirus
>vas isolated .
The genus Lentiviru.s (latín lenti, mcaning slow) in-
cludes tll<.: ltun1an i1nmunodeficiency viruses (HIV) as well
as many important animal retroviruses. Lentiviruses are
rnu:.t. ofLen associatcd ~vith chronic immune dysfunction
and neurologic diseases. ·rne genus spurnavtru:; (Laliu
spu1na, n1caning foan1) are nononcogenic virusPs fou nd in
spontaneously degenerating cell cullu1 es, causing the for-
mation of multinucleated vacuolatPd (foamy) giant cells.
No d iseascs have beeu (1:.socialed with spumaviruscs in hu
mans or animals. ThP rPmaining retrovirus genera are
termed tl!c oucoviruses (Greek 011kos, meaning tumor) or
thc RNA tumor viruses becausc ot their ability to induce
neoplasia, althou gh t hey are now known to cai.1se othPr
kinds of discases as weJJ. ·r11ese genera are Llie Alpharetro-
virus (e.g., Avian Ieukosis virus), RetaretrovinJS (Mouse
mammary rumor virus), Garnrnaretrovirus (e.g., Murine
lcukemia virus), n1~ltaretrovirus (e.g., Bovine Ieukem ia
virus) <Htu Epsilonretrovirus (e.g., Wallcyc der1nal sarcoma
virus).
Severa! additional features need to be considered in thP
classificatio n and description of the Retruviriúue. Exogen-
ous retroviruses spread horizontally (or vertically but non-
genetically) from animal Lo anin1al, similar to thc mccha-
nism of transroission of other kinds of viruses. ln contrast,
endogt:Jl OLl.S retroviru.ses are transmittcd genet.ically. 'fhcse
rf'tro,riruses persist as integratcd UNA proviruses that are
passed from gcncration to generation through the DNA in
the gamctes of thc host animal species. 'I"hus, Lhe et1doge-
no us proviral genomc occurs in earh C0. ll of thc animal.
Man y vertebrates pu~:;e:;s su ch endogenous retrovi ral DNA
sequences. Thf'~P Pndogenous retroviruses are usually not
FREDERICK J. F ULLER
pathogenic for their host anlmals auu are oflen not ex-
pressed. When replication of Pn<logenous viruses docs
occur ln the host cell of origin, it is usually restrictcd. Cclls
from anim:il sp<>c:ies other than thc host specics are some-
Lin1cs unrestricted, howevcr, and can support the replica-
tion of the retrovirus in an exogcnous manner. Thc cn-
dogeno us modc of transmission occurs in many of the
oncoviruses, but is oot known to occur In the lentiviruses
or thc spumaviruses.
'
~! ' 1
'
Some membcrs of tite u11coviruscs are also classified by 1
their intpr;irtion with cells of diffc rent specics. Ecotropic
strains replicate only in cclls from animal spccies of origin dt 1
and xenotropic strains replicate only in cells of other
spccics. Amphotropic strains replicate in both. Most of thf' 1
endogcnous retroviruses are also xt:nutrupic.
·rhe morphology of rctrovin 1sPs in transmission elec- '1
tron microgra¡.¡h is also useful in classification (Fig 65.1).
1'hc si1f' rangc of retrovirus particles is from 80 nm to 130
11111. Type f\ particles occur only i.nside cells and consist of
a ring-shapcd nucleoid surrounded by a membranc. B-
typc virions havc an cccentric core, and C-type virions a 11'
central core. D-type virions have a morphology inter111eui- 1
ate between B ande virions with an Plongated, dense core.
The core of lentlviruse:. has a shape that resemblcs an icc-
cream cone.
GENERA L fEATURES OF RETROVIRUSES
Many o f the fcatures ot retroviruses are kno\-vn in great uc-
tail because of the exten sive '""ork done on thc onc'oviruses
in cancer research and thc lentiviru:.es i11 acquired im-
munodeficiency syndrome (ATD~) r<'search. The members
of the family Re/ruviriúae share many common fcatures in
their composi tion, organization, and lite cycle, although
thc delails of individual retroviruscs vary.
Components of Retroviruses
A typlcal retrov1rus viriu11 is con1posed of 2% nuclcic acid
(RNA), 6QOJ, protPin, ::\.Sº/o lipid, and 3% (or more) carbohy-
urale. 1Ls buoyant density is 1.16 to 1.18 g/rn L.
Retroviral Lipids
Retroviral Iipids are mainly phospholípic1 anci occur in the
viiion envelope. They fun11 a bilayered structure similar to
409
410 PART 111 VirnSf>S
Table 65.1 . Genera and Selected Species of Retroviridae
Genus . Spedes
Alpharetrovlrus Avfan Leukosis Virus
Avian Erythroblastosis Virus
Avían MyeloblastosisVirus
Avian Myelocytomatosis ViFus
Rous Sar.coma Virus
Betaretrovícvs Mouse Mamma¡:y Tumor Virus
Símian Type O Rctrovirus•
Ovine P.ulmonary Adenocarcínoma v¡rus
Squirrel Monke¡r Retrovirus
Gd111111d1e!1 uvi1 u; Mufirn~ Leuk~rnid 'l'ifu~
Feline leukemfa Virus
Porcine Type C Oncovirus
FeUne Sarcoma Viruses
Mur.ine Sarcoma Virus
\1Voolly MonkeY. Sarcoma Virus
Avían Reticulendotheliosis Virus
Delraretrovírus Bovine Leukemla Virus
Human Í'lymphotrork Vin1~ i
Hunran T·lympholropic Virus 2
Simian T-lymphotropic Virus
Epsilonretrovítus Walleye Dermal Sarcoma Virus
Lentivirus VisnaiMaedi Virusb
Caprine Arthritis Encephalitts Viws
Equtne inrecuous Anemia virus
.Boyine lmmunodefiáency Vir11>
Feline lmmunodeficiency Virus
Primate Lentiviruses (HIV-1, HIV-2, SIV)
Spumavirus fluman Spumavirus
Simian Foamy Virus
-Bovine Sy.ncytial Vírus
feline-Synq'tial Virus
ªSimian AIDS·related virus (SRV).
~Ovine progressive pneumonia virns is.synonomous with maedi virus.
the outcr ccll mcmbranc from \vhich thc rctrovlrus enve-
lope is derived.
Retroviral Nucleic Acid
Retroviral RNA. Retroviral parlicles co11lain RNA as Lheir ge-
netic material. This genomic RNA is present in each viral
particlc as a dimcr of two linear, singlc-strandcd, positive-
scnse copies that are noncovalently joined near t heir 5'
ends (Fig 65.2A). Hence, the virion is d iploid. 1·he genomic
R1'1A has a se<.lluit::11tatlun stze of 60 to 705 In neutral su-
crose gradients. lJpon dt=>nati1ration, t=>arh RNA copy h.as a
sedimentation coefficient of 38S. The molecular we.i ght of
n1onomcr RNA-determined by electrophoresis in polyacry-
lamidc gcls is approximately 2 to 5 x 106 daltons, or about
! to 11 x 10:~ bases. Host cell transter !:{NA (tl{NA) is associ-
ated vvith genomic RNA near the 3' terminus and serves as
a primer for the synthesis of DNA by the reverse poly-
mt=>rasf>. "fhf> typt=> of tRNA parkagt=>ci in the vi.rion. is useful
in the classificatio11 of rctroviruscs. ·rhc 3' tcrminu:; oí
each RNA monomer has a poly (A) tract. The 5' terminus
b.as a n1ethylated nucleotide cap .
Pro virul DNA. Witlli11 <1 cell, tl1e retroviral RNA geno1ne
is reverse tran.scrihed into a DNA copy, and it is t hf> provi-
ral DNA fonn that serves as the intracellular ret roviral
genome. The retroviral DNA is several hu11dred bases
longer than the retroviral RNI\ genome duc to duplication
of repeat ed and unique terminal sequences prese11t in the
RNA genome during the reverse transcription process.
Tl1ese se4ueuces furrn the long t errninal repeats (LTR) thac
flank the genes in the retroviral ONA (see Fig 6S.2B). The
p roviral DNA is covalently intcgrated in thc DNA of the in-
tected host ce.LL ·l"his integration is lacilitated by a viral en-
zyme, appropriately termed integrase, which is encoded in
the polymerase open reading trame.
Retroviral Nucleic Acid Structure and Sequence. The se-
tiuence of structural genes of retroviruses, frurn the 5' ene
to the ~ ' end of genomic RNA, is Gag-Pol-Fnv. Sorne retro-
viruses, sucl1 as the lentiviruses, spumaviruses, and delta-
retroviruses have additional genes (tax ancl rex) that regu-
late expression of the retroviral geno1ne and othe
accessory functions. Highly oncoge11ic retroviruses ofter:
have an oncogene in place of a portio11 of the Pol and/o:
E11v gene.
R~troviral Prot~ins
Rctroviral Structural Protcins. Rctroviral structural protcins
are e11coded by tl1e Gag gene and the Env gene (Fig 65.3).
Gag (group specific antigen) proteins form the core of the
virus and consist of three major proteins. The nucleocap-
sid (NC) is a srnall protein (about S kd to 10 kd) t hat inter-
acL:; witl! rt:trvvir<1l RNA. 1'he capsi<.l (CA) protein (about 25
kd) forms t he major structural element of the rf>troviral
core. ·rhe matrix (MA) protein (about 15 kd) serves to join
the retroviral core •vith the retroviral envelope. In sorne
retroviruses, there are additional SJ.llal! core proteins.
The Env (envelope) gene is responsible for th.e sy11thesi.S
of two glycoproteins that are non-covalently linked. T.hese
lwo glycupruteius fvr1n tri1neric II1ultiiners in sorne retro-
v iruses. The glycoprotein outsideofthe retrovirus (SlJ, sur-
face) is a knob-like glycoprotein (about 100 kd) that is re-
sponsible tor binding the retrovirus to its cellular receptor
during 111fection. The other glycoprot ein (TM, transmem
bra11e) is a spike-like structure (about 50 kd) that attaches
the SU protein to tl1e retroviral envelope.
Relrovirul Enzyrne~. TI tt: Poi ge u e t:r1c0Jt:s sevt:ral IJrott::iI1s
with enzymatic activities t hat are important for the replica-
tion of retroviruses. 1'l1ese enzyrnatic proteins are found
wit hin tl1e retroviral particle, but in a n1uch lower molar
concentration than the retroviral structural proteins.
The reverse transcnprase (RT) enzyme Ls responslble for
t hf> procluct ion of tl1e retroviral DNA genon1e from the
retroviral RNA ge11on1e. To accon1plish this, reverse tran-
scriptase possesses severa! catalytic functions, inclucling
an RNA-dependent DNA polymerase and an RNase H ac-
tivity. 1rr req uires the preseoce ot a d.ivalent cation to tunc-
tion, and the type of divalent cation (magnesiurn or man-
ganese) that a particular retrovir us requires is useful in
retrov:ira l class.ification. Tl1e measureme11t of reverse tran-
 Chapter 65 Retroviridae 411

F 1G U RE 6 5 . 1 . Transmission electron photomicrographs of budding and mature virions of

feline immunodeficiency virus (A,B); feline leuki:111Íd viru) (C,D), feline syncytium-forming virus
(E,F); human immunodeficiency virus (G,H); simian immunodeticiency virus (l,J); and v1sna-maed1
virus (K,L). Uranyl acetate and lead citrate stain. (Reprodvced with permi~~inn frnm Y;imamoto JK,
Spa1ye1 E, Ho EW, et al. Pathogenesis of experimental/y induced feline immunodeficiency virus
infection in cats. Am J Vet Res 1988;49:1 246.)

FIV FeSF V

~~' ;:oq: "),~ ;

. -
. . ,
,.,

-~ ~ ~~ ~
,
HIV

Gag Poi Env

A. 5'1 "-':..._illl________/// AAAAAAA 3'
cap R U5 U3 R

Gag Poi Env

B.
U3 RUS U3 RUS
F 1G U RE 6 5 . 2. The nucleic acid of a retrovirus.
LTR LTR Rctrovirul RNA (A); rctrovira/ DNA (B).

scriptase activity is one of the principal laboratory meth-
ods for thc detection and assay of retrovlruses.
'fhe Poi gene also encodes other enzymes. The retrovir;i 1
prutcasc (PR) 1uediates cleavagc of Gag and Poi polypro-
tPi ns d11ring retroviral assembly and maturation. The
retroviral integrase (IN) functions to covalcntly link thc
retroviral DNA into the host cell's lJNA as an integrated
provirus. Sorne retroviruses also cncode a deoxyuridine
trlphosphatase enzyrne (uUTP) tl1at b rt:quired for virus
rPplir.:ition in nonclividing c:ells.
Otl1er Retro viral Proteins. Forman y members of the Retro-
viridae, only the proteins encoded by the Gag, Pol, and Env
genes are prcscnt. Othcr rctroviruses contain additional
genes whose products serve functions such as controlling
4 12 PART 111 Viruses

RT, Reverse Transcriptase

-..--_ IN, Integrase

RNA FIG URE 6 5. 3 . The struct ure o f a retrovirus.

FI G URE 6 5.4 . The life cycle of a retrovirus.

Cell-free
Retrovirus

~
~\Blndlng
Penetr /
and ---

/ Reverse • .A e t ro v~·-1
1ra
1/ Transcription lntegrated DNA
"""IJ lntegrat~on ·'t \ Transtation

lntegratio n w~ 1,
Complex ~\~
Rett ira l
P t ei ns

"'Budding
la\
\l!I
Maturatíon ~

the Ievel of provirus transcription, facilitating transport of
retroviral mRNA, and e11hancing retroviraJ replication u1.
specific cell types. · the nucleus of the cell, allowing such retroviruses to repll-
Retroviral Replication
¡\general scheme of retroviral replication is shown in Figure
65.4. A rctroviral particle bi11ds to a specific receptor on the
surface of a target cell via the SU protein. The retrovirus
penetra te::. tl1e cell a11Ll Ll1e It'.LrO\firal core u11dergoes specific
stn1c.tural c.hanges. The retroviral RNA within the modified
core is reverse transcribed by RT using the associated t RNA
primer, first toan RNA/ DNA hybrid form, then to a linear
double-stranded DNJ\ form with long terrninal repeats. ·rhe
newly made retroviral DNA is still associated with sorne
viral core proteins and enzyn1e <ictivitiP.s in a structi.n:e
Lern1ed Lhe irztegratiorz cornplex. 111 son1e retrovi ruses, infec-
tion must occur v\Tithin dividil1g cells so that tl1e integration
complex ca11 access the 11ost DN.I\, wh ile in other retro-
viruses tbe integration complex is actively transported lnto
1.:aLe iu uvudi vidlng Of Lern1inally differe11Lia ted cells.
The retroviral DNA is in tegrated into the h ost cell's
DNA by th e activity of the lN enzymc. Thc intcgration of
ret roviru ses is not ata specitic si te within the cellular DNA,
rath.er integration can occur at many sites. "fl1e integrated
DNA provirus behaves very n1uch as a eul<aryotic gene. lt
may be transcribed into inRNA and genomic RNA i1sing
l1osl cell enzyn1es to produce n1ore virus, or it may remain
latent for long periods of time a11d replicate when t he cel-
lular DNA is replicated by thc cclL
New retroviral particles are produced by budding from
cellular membranes. lmmature retroviral Gag polyprotein
and genomic RNA assemble and acqulre envelopes as they
P)(it in fPrtPrl rPll <; h y hnrlrli ng thro11gh thf> pl;:isma m~n1-
branes into '"'hich retroviral SU and TM envelope proteins
have been inserted.

In the final step, the retroviral protP.asP. (PR) cleaves the
Gag polyprotein in to the mature structural proteins of ma-
::ix, capsid, and nucleocapsid.
. !"t1 MUNOLOGIC (HARÁCTERISTICS OF RETROVIRUSES
:1etroviral protcins posscss various types of antigenic sites.
. ype-specific antigens that defin.e the serologic subgroups
are associated with the envelope glycoproteins. Group-
specific antigens are shared by relatetl viruses arH.l, in ge11-
eral, are associated with the virion corP. protP:ins. There are
al:su iI1terspecie:s a11ligeus Lhal are shared by otherwise un-
related viruses derived from differe11t host species. Reverse
unnscriptnsc (KI") is nlso nntigcnic and contain$ typc ,
group-, and interspecies-specific determinants.
ÜNCOGENIC VIRUSES ANO ÜNCOGENES
Oncogenic viruses can produce inappropriate cell growths
in the tissues of susceptitJle l1o:st:s. Cancer:s are 111aliguauL
tum.ors th;it ;irp charac.tP.rizP.d hy loss of normal cellular
controls resulting in unregulated growth and ability to in-
vade adjacent tissues and to metastasize to other parts of
thc body. Canccrs are classified by their tissue of origin:
sarcomas are malignant tumors of connective tissue (mes-
.enchy1ne); carcinomas are maligna11t tumors of epithelial
origin.
The ability of oncogenic viruses to cause canci>rs 11nclPr
either nat urdl or experin1enlal conditions has been the
suhjP.ct of intense study for almost a century and this work
h as made significant contributions to thc un.derstanding
of viruses, neoplasia, and cell biology. ·rhe fundamental
discovcry in the field is that oncogenic viruses cause can-
ter via genes they carry or activate. These genes are terrned
oncogenes.
Oncogenesis by Retroviruses
There are sever<ll ruecha1ü:s1us by vvhich relrovir uses are as-
sociatPcl with cancer. Highly oncogenic or acutely tra11s-
forming retroviruses cause cancer rapidly and efficiently,
often within days or weeks of infection. Such retroviruses
are rarc in natural animal populations, but are used exten
sively in the laboratory for the study of cancer. Rous sar-
coma v irus (RSV) o f chickens, IAlhich was discovered in
1910, is the prututype llighly oncogenic relrovir us.
In 11ighly oncogenic retroviruses, all or part of an onco-
gene exists in the viral genome, usually in place of viral
genes. Th is retroviral oncogene is responsible for the ability
of a highly oncogenic retrovirus to cause oncogenic trans-
forrnation of a cell. There are more than 20 different retro-
viral oncogenes known. Each of these has a corresponding
gene tl1dt cau l.Je found iI1 Lhe genon1e of norn1al cells. The
normal gene that corresponds to a viral oncogene is termed
a c-oncogene or proto-oncogene, and the viral version is called
a v-oncogene. In a normal cellular environment, the gene
Chapter 65 Retroviridae 413
products of proto -oncoge11es usually have sorne function in
growth regulatory pathways, such as protcin k.inases,
growth tactors or their receptors, G rP binding proteins, or
transcriptional activation factors. When they are part of a
retroviral genome, these proto-oncoge11es are under the
control of the retroviral LTR rather than being regulated by
nurutdl cellu1ar 1uechanisn1s, a11d a.re oftc11 expressed at
high levels. The v-oncogene mayal so b e truncated, contain
point mutations, orbe fuscd with anothcr rctroviral gene.
·rhis aberrant expression of n1utant p rotein can lead to ab-
normal growth of the infected cell and the b eginning of
progression to neoplasia.
For example, in RSV the src oncogene is responsible for
sarcomatous transformatiun. 'l'he v -src; ger1e wa~ uriginally
acq11irPcl from thP normal c.-src cellular proto-oncogene
°l'vhen illegitimatc rccombjnt1ti on occurrcd bctwccn i:ctro-
viral a11d tbe cellular genomic sequences for c-src. ·rhe c-src
gene product is a 60 kd protein that has p rotein kinase ac-
tivity and is Jocated near the inner surface of the plasma
membrane. The kinase activity is part of a11 .intrica te cellu-
lar signa! Lransduclion pa lh>-vay that n1ediatcs ccll growtl1.
Other examples of v-oncogenes with c-oncogene counter-
parts in normal cells are found in o tl1cr acutc transforming
retroviruses, for example, myb (avían myeloblastosis virus),
erb (avían erythroblastosis virus), myc (avian myelocyto-
matosis virus), and ras (mouse sarcoma virus).
Highly oncogenic retroviruses are usually defective. The
reason they are defecrive is that tl1ey lack their full con1ple-
ment of Gag-Pol-Env genes because the v-oncogene takes
the p lace of a portion of the retrovi·r.al geno1n c. In ordcr to
replicate, these detective retroviruses require a replication-
competent helper virus t o supply the missing gene prod-
ucts. 'fhe helper retr ovirus ts usually a closely related retro-
virus that is not defective and contains the usual Gag-
Pul-Env cu111pleu1e11l of geues. Since Lhe defective, higl1ly
oncogP.nic virus is packaged into a virion composed of the
envelope proteins of the helper virus, the host range of the
highly oncogenic virus is dependent u pon the helper virus.
'fhc prescncc of the genome of one retrovirus with the pro-
tein components of another virus is termed a pseudotype.
Weakly oncogenic (nonacutely transformi.ng) retro-
viruses cause neoplasia less rapiuly a11u u1ucl1 less effi-
cien t ly t han clo highly oncogenic retroviruses. Such
viruses exist in don1estic animals. Weakly oncogenic retro-
viruses do not carry a v-oncogene and do not requiré a
hclpcr virus. HO'-'<Cvcr, examination of the tumors pro
duced by the '"'eakly oncogenic viruses usually shows a
clona! proliferation of c<o~lls with a retroviral genorne near a
cellular oncogene. For exan1p1e, avian leukosis virus is
o ften intr~gr<i ted n e;i r or in t hP 1'-myr gPnP ' l'hp rnPrh;i -
nisn1 by wl1ich weakly oncogenic retroviruses cause cancer
is known as insertional or cis-activational oncogenesis. Dur-
ing retroviral rcplication, proviral DNA is inscrtcd into
man.y random locations in the host genorne. Occasionally
the integration of the provirus occurs close to a cellular
proto-oncogene. ·rhis can sometimes produce inappropri-
ate transcription of the oncogene, either by read-through
íron1 tl1e rclrovlral pron1oter, or by c11l1ancer activity by
the retroviral LTR. Integration of the provirus near a proto-
oncogene tends to be a very rare event and therefore oc-
curs m uch less frequently and at much lower efticiency
414 l'ART 111 Viruses
than when the retrovirus carries its own oncogi>nP. The tu-
mors are clo11al in origin because, although many cells are
infected, only one rare cell has undcrgone insertional
oncogenesis and progresses to a tumor. In addition, thc in-
appropriate activation of a proto-oncogene is ¡ust one
event in a multifactorial process that leads to cancer.
A lhird n1echa1 ú~1u uf retruviral onc:ogenesis occurs in
the bovine leukemia virus-h11m::in ·r lymphotropic virus
genus of Rctroviridae. These viruses have a regulatory ge11e
called lax in addition to Gag, Poi, and Env. The protein
product of Tax functions as a transactivator to uprcgu.Iatc
rcrrov1ra1 rranscription by t>1n<11ng to spec1fic U NA se-
qucnccs in the L1'R of the retrovirus. Under sorne circum-
stances, lhe 'fax p1olei11 ca11 ~u 111eli111c~ al~u !Ji1H.l tu tran-
sc:ription;il ;ictiv;itor si>qnences in cellular genes and may
disrupt regulatory pathway:; of the infeclcd cell. U11like i11-
sertional oncogenesis, the integrated provirus is not neces-
sarily adjacent to a proto oncogene, since it is tl1e Tax pro-
tcin that produces the oncogene activation (in trans),
rather than tl1c retroviral DN1\ itself. Like insertional onco-
genesis, the transacrivation of a proto-oncogene is just one
f'Vf'nt in;¡ m11ltif;irtori;il 11rocP<><> th::it fp;¡<i<; to r::inrer
. and more invasive than non-FeSV-induced tumors .
Oncogenesis by DNA Víruses
1vfany of the DNA vi ruses, including the adenoviruses, pa-
povavlruses (polyoma, papillorna), herpesviruses, hepad-
naviruses, and poxviruses, havc oncogenic potential. In
conLi'asL lo highly 011c.:ogeulc.: n.:lruviru:;es, the v-oncugenes
of DNA viruses are not ceJlular dcrlvatives but are true viral
genes. 1'hc normal functio n of these viral genes is to acti-
vate ccllular path,-vays for UNA replication. ·rhis activation
is requircd for DNA viruses to multiply in resting cclls that
lack the enzymes and materials the virus needs for its own
D A replication. The mcchanism of neoplastic transfor-
mation by oncoge11ic DNA viru:. i:. tl1at the vi ral genes that
actívate cellular DNA replication ::trf' f11nction;:il. hut thP
genes for viral production for sorne reason are not. This
causes the infected cell to get inappropriate activation sig-
na ls without the subseguent viral production that dcstroys
the cell. The result is inappropriate cell ac:t1vation and di-
vision, and is one of the initial steps that can lead to the
devclop1nenl of a ca11cer. Like ll1e wcakly uncogenic retro-
viruses, insertion and transactivation mi>ch;:inisms arP ::i lso
known for D~A viruses.
FELINE LEUKEM JA/SARCOMA VIRUS
Disease
feline le ukemia virus (FeLVJ causes a variety of important
diseases of cats. The most significant conseguence of per
sistent feLV infection is severe immunosuppression that re-
sults in the development of opportunistic secondary infec-
lio11s. Cli11ical ~y1u.lruu1es pruuuceu l>y FeLV infection in
cats also include tumors of th<' h<'molymph::itic systi>m
(lymphoma, leukemia), refractory anemia, ulceration of the
oral cavity, a feline panleukopcnia-like syndrome, FeLV-
induced neurologic syndrome, and immune-complex
glon1erulonephri üs.
Lymphoma (lymphosarcoma) is the most c:ommon
ncoplasm in cats, although only about 70% of all lym-
phomas in cats are caused by feLV infection. Multicentric
lymphosarcoma that affects a variety of tissues (including
!!ve r, gastrointestinal tract, kidneys, spleen, bone marrow,
and central nervous system) is the most common tumor in
FeLV-i11fecleu ca~, wl1ereas ·thyulic <HH.I alimentary (gas-
trointestinal) forms predomina te in u ni nfec:ti>cl c;:its.
Young cats with lymphoma tend to be infected with feLV,
w hereas oloer cats with lymphoma ten<! not to be. cats
with Iymphoma typically present with weight Joss, often
ac:companied by any comblnatlon of respiratory difficult y,
diarrhea, vomiting, and constipation. FeLV can also cause
ab1101111al prul iferatiu1L uf erytl1ruiu auu 1uyeluid cells, rt!-
sulting in a variety of myeloprollfcrative disorders includ-
ing lcukcmias.
'rransmissable fibrosarcoma in cats is associated with
infection by feliI1e sarcoma viru s (FeSV) and typically oc
curs in young cats. The more common fibrosarcomas that
occur in older cats are not associated with FeSV. The PeSV-
induccd fibrosarcon1as le11d lo be poorly dilfere11lia led
Etiologic Agent
Classi fication
·rhrec subgroups ot exogenous reLV (A, B, and CJ are dis-
tinguished by viral interference tests and antibody neu
trallzatton tests. These two properties are associated with
the envelope glycoprotein.
Feli11e sarcuu1a viruse~ (FeSV) are replication defectlve,
highly oncogenic (acute transforming) vin1sP.<> th;:it h;:ivp
acquired an oncogene through rccombination of the FeLV
genome with one of severa! cellular oncogenes. FeSVs are
thought to arise de novo in FeLV-infected cats and not to
be naturally transmitted from cat to cat.
r.:ats also have endogenous feline retroviruses such as
RD-114 lhaL are lra11:.111lttcu gcrn.:tically. Multiple copies of
thc RD-114 provirus are found in ali cat cells. T hese P.n-
dogcnous viruses are not associated with any known felin c
disease.
Physical, Chemical, and Antigenic Properties
Morphologically, the feline rerroviruses are typlcal mam-
malian Type <~ retroviruses of the Carnmaretrovirus genus.
FeLV is con1posed of two ei.1velope proleins, gp70 (SU) and
p15E (TM) . and three Gag proteins, plO (NC), p15 (MA),
and p27 (CA). The Gag proteins are produced in grcat cx-
cess in infected ceus and are usetul m laboratory diagnosis
of FeLV infection of cats.
Resistance to Physical and Chemical Agents
T.ikemost enveloped viruses, feLV is sensitive to inactiva-
tion by lipid solvents a11d detergents. FeLV is rapidly il1ac-
tivated at 56ºC, but only minimal inactivation occurs at

~ for up to 48 hours in culture m edium. The virus is
""!!>1dly inactivated by drying.
_;;ctivity for Other Species and Culture Systems
-=.::..V-A replicates exclusively in cat cells, whereas FeLV-B
~d FeLV-C replicate in a variety of c:ell types, inrh1rling
~<nan e.e\\:,. "\'\11: \10:;\ nYngc :;pcc.\'iic.\t.y <:>'i 1'tLV i:1 a:1:1oc.i-
:::ed w ith t he envelope glycoprotein, gp70. No relation-
-ip has been shovvn hetween FeLV and human disease,
-=id there is no evidence that FeLV disease is trans1nissihle
-~ humans.
Since the much rarer .sdrcuu1a viru~, FeSV, i:s defeclive,
""'1E' host r::ingP. of this virus is dependent upon the helper
~ukemia virus that supplies the protein for its envelope.
!ost experimental studies have been conducted wit.h the
eSV (FeLV B) pseudotype. FeSV ca11 transform fibroblasts
>:om nonfeline species, including dog, mouse, guinea pig,
~r, mink, sheep, monkey, rabbit, and httman. FeSV has
.:ieen fou11d Lo be 011cogenic in inany of Ll1e anin1al species
~ested, although inoculation of fetal or newborn animals is
;enerally required to show oncogenesis of PeSV in species
.:;ller tha11 cats.
-fost-Virus Relationship
Jistribution, Reservoir, and Transmission
~LV lnfectlon of cats occurs t hroughout tl1e wurl<.l, ar1<.l
m e cat is the only knO\Nn reservoir of thP. v irus. Ahout 2%
of the cats in the United States are seropositive, indicating
eit11er past or current infection, and sorne 50°16 of these
seropositive cats are positivc for l'cLV antigens by im-
:nunofluorescent antibody test (!FA) of their peripheral
nlood leukocytes, h1dicating current infection. FeLV-A
occurs in infected cats eithcr alone (50o/o) or in combina-
-áon with feLV-B or FeLV-C.
FeLV is excreteu i11 ::;aliva and tears a11d possibly tlle
urine. Transmission appears to occur during close contact
':ia biting or licking (grooming). Tt is possiblc that infcc-
tion may occur v ia contaminated feeding dishes. l'ro-
longed, extensive cat to-cat con taet is required for efficient
spread. In environments \.Vith multiple cats, the presence
of oneinfected cat greatly increases the risk of infection for
other cats. FeLV i.s al~u tra11s111illed congenilally, a11d n1osl
k ittens exposed in utero or befare 8 weeks of age become
persistently viremic.
Pathogenesis and Pathology
Upon penetrating the oral, ocular, or nasal me1nbranes,
the FeLV replicates in lymphocytes in the local Iymph
n odes of the head and neck. Acute FeLV disease, mani-
fested by fever, lyn1phadenopall1y, and n1alaise, develops 2
to 4 weeks after infectio11; however, these signs are seldom
conspicuous. Tn about onc·half of infcctcd cats, thc ani-
mals recover quickly and becorne feLV antibody positive,
FeLV antigen negative. Some of these cats have probably
cleared the virus and the virus remains latent in others.
The long-term significance of latent feLV infection has
Chapter65 Retroviridae 415
not been determined, and FeLV-viremia 1nay be reacti-
vated under conditions of stress or cortlcosteroid therapy
in sorne of these cats.
In cals lhal do not n1ou11t an adequate in1n1une re-
sponse, the FeLV replicates in the rapidly dividing cells of
the bone marrow. 'fhese cats are persistently infected with
fe LV and are positive for FeLV a11tigen by the IFA test in pe-
r~?h~ral blood leuko<:ytet. The <:y<:le of infection it com
plete after viral replication in epithelial ce lis of the salivary
glands, where infectious FeLV is shed in the saliva .
1'he lin1e fron1 ll1e onset of vire1uia to the appearance of
the later signs of FeLV infection is tern1ed the induction pe-
riod. This period ranges from months to years, with an av-
erage of about 2 years. Most persistently vire1nic cats di.e
within 3.5 years of it1fection. Persistently infected cats
often develop leukopenia, immune deficiency, and sec-
ondary opportunistic infections. FeLV-induced immune
deficiency n1usl be disliI1guisl1ed fron1 ll1al induced by fe-
line immunodeficiency virus (FIV), which is a different
retrovirus .
Host Response to lnfection
About half of FeLV-infected cats produce protective
a111ounls of neulralizü1g anlibodics to lhc n1ajor e11velope
glycoproteins while FeLV is confincd to cells of the local
lympl1 nodcs, a11d thc virus is climinatcd or rcmains la-
tent. ·rhese cats do not becon1e persistently infected wit h
FeLV, a11d usually live out a nor1nal life span. The response
to FeLV infectio11 depe11ds on the age of the cat, the dose of
virus receivecl, and probably other genetic and virologic
facturs. Kilte.1 1s Le11d lO respond poorly and, as a resull, are
predisposed to persistent FeLV infection.
Laboratory Diagnosis
Because sorne cats are able to ciear a PeLV infection, and
many cats have been vaccindted dgaiust FeLV, tt:st~ for au-
tihocly to Fel.V nrP. of li1n ited utility. The inost useful tests
for FeLV diagnosis detcct feLV antigens. An enzyme-linked
immunosorbent assay (ELISA) is availahle for FeLV anti-
gcns in scru1n or saliva and is especially useful as a rapid
screening method. An immunofluorescence antibody test
(IFA) is used to detect FeLV a11tigens inside of infected ce lis,
which is evldence that the virus is replicating in the bune
marrc.Y1.v and that the cat is persistently virem ir.
Treatment and Control
Vaccines against Fe LV are available, although th.e ir efficacy
under field conditions is controversia!. Current FeLV vac-
cines either contain the inactivated ("killed") whole virus
or a subunit protein preparatío11 of tl1e virus. Kitte11s
should be vaccinated twice starting at 9 to 10 weeks of age,
with thc sccond <lose of thc vaccinc givcn ?, to 4 wccks
tater, and with annual booster vaccinatio.n s. Eight-tive per-
cent of cats under 12 weeks of age, ·if exposed, be.come per-
sistently infected, but cats over 6 months of age have only
a lüo/o- 15% chance of becoming persistently infected if ex-
416 PART ITJ VinJSf'S
posed. Use of FeLV vaccines are thus potentially most ben-
eficia! in young cats.
FPl .V infPction in c:;itteries c;111 be co11trolled by test and
removal procedures and can be con1bined will1 FeLV vacci-
11ation, si11ce vaccination will not interfere witl1 the labo-
ratory detection of PeLV antigen in infected cats.
A diagnosis of FeLV infection does not 11ecessarily dic-
tate euthanasia, since an FeLV-positive heaJthy cat n1ay
Uve for years. Because the cat is probably shedding virus
tl1at could infect other cats, however, precautions to re-
duce the cha11ce of spreading Lhe virus and conlact wilh
opportunistic pathogens should be instituted.
SIMIAN TYPE 0 RETROVIRUS
Disease
Simian Type D retrovirus, '.\Thich is a member of tl1e Be-
taretrovirus genus (Simian AJDS-related virus, SRV), pro-
duces a falal iu 1111 UllO:>U!JIJft!SSi ve ui:;t:a~e Íll lllUllkt:y:;.
Infected animals show an initial generalized lymphadeno-
pathy and spleno1negaly acco1npanied by fever, weight
loss, diarrhea, anemia, lyn1phopenia, granulocytopenia,
and thro1nbocytopenia. Profoundly immunosuppressed
animals develop diseases caused by opportun1st1c patho-
gens, tl1e most comn1on of which is dissen1inated cytome-
galuviru:; (CMV) infection.
Etiologic Agent
Classification
Primate rctroviruscs are rcprcscnted in four distl11ct gen-
era: I) Betaretrovirus genus, which in eludes the simian type
D retrovirus (SRV); 2) the Deltaretrovirus genus, whicl1 in-
cludes the sinúan T-lymphotropic viruses (STLV) a11d
hum;in T-lymph.otropic viruses (HTLV); 3) t he prin1ate
lentiviruses, vvhich include Ll1e 11un1a11 ü11n1 Lu1odefi-
ciency viruses 'fypes 1 and 2 (HIV-1 and HIV-2) and the
simian immunodcficicncyviruscs (SIV); and 4) thc sitnian
and human spu1naviruses. Alt hough .)!{V is in a separate
genus from tl1e prhnate lentiviruses (HIV and SIV) that
cause acquired llnmunodeficiency in humans (AJDS) and
siln.ians (SAIDS), many aspects of the imrnunüdeficiency
an.d associatcd opportunislic ir1feclions are sinlilar.
The Mason-Pfizer monkey virus (MPMV) was the origi-
nal SRV to be isolated.
Physical, Chemical, and Antigenic Properties
S.RV i.s a Type D retrovirus (Betaretrovirus genus). The Type
D viruses are characlerized by lhe forn1alion oí cytoplas-
mic 1'ype A precursor core particles. Mature 'fype D viruses
are plco1norphic in shapc, spheroid, ei1veloped, and 80
nm to IUU nm in diamet er. ·rhe r1ucleocapsic.1 is isometric
to spl1erical with an asymmetric, spherical nucleoid.
111e R'f of SRV has a preference for Mg2 ... and uses tRNALys
as a prirner far negative-strand DNA reverse transcription.
SRV consists of at least five serotypes based on ncutrnl-
ization properties of the envelope.
lnfectivity for Other Species and Culture Systems
SRV infccts scvcral spccics üf monkeys. Scrologic surveys
have shown no conclusive evidence ot .'.>RV iníection in an -
imal handlers who work \.Yith rnonkeys.
SRV isolates replicate in both T and B lymphocytes as vver:
as in macrophages. Various hun1an and rnonkey cell lines
of T aud B ly111¡.¡l1ucyte, 111dcrupl1dge, dHli filJrulJlast urigi.r:
support the growth of SRV. SRV induces syncytia in Raji
cells, which can be used as a method to quanti.tate the virus.
A human counterpart of SRV- a human Type D retro-
virus- has been reported. The distribution and clinicai
significance of t his virus remains to be determined, as does
its relationship to SRV.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
SRV is indigenous and widespread in Asían macaques but
<loes not 11aturally infect African mo11key speci,~s. In one
study, about 25(Yo ot captive macaques in United .'.>tates
primate centers were seropositive; 11owever, the prevalence
varies widely based on the Iocation and the species
Stl..tdied.
SRV is transruittell priruarily iu the saliva by h iting.
Mortali ty has he.en Psti matPcl to hP :~OO/o to SOºAi, anrl of-
ten occurs at an early age. lnapparent carriers that are
vire1nic but antibody negative n1ay be an important reser-
voir for SRV.
Pathogenesis and Pathology
SRV infects hoth T ;inrl R lymphorytes in vivo and causes a
profound depletion of both of these kinds of Iymphocytes
leading to fatal immunosuppressive disease. 1'he absolute
Iymphocyte count decreascs but thc CD4/CD8 ratio rc-
mains relatively stable. In the ly.n1pl1 11odes there is a de-
pletion of lymphocytes and an absence of p1asma cells.
SRV al:>u i11fect:; IIldCIUIJhage:;, out IlUt grar1ulucyte:;.
Host Response to lnfection
Sorne infected monkeys die acutely 7 to 20 weeks after ex-
perimental inoculation, wnereas sorne remain persistently
infected, a11d sorne develop neutralizing antibody and be-
con1e nonviren1ic and ren1ain healthy.
Laboratory Diagnosis
Serologic screening methods include ELISA and Western
immunoblotting. I3ecause infected monkeys may be
seronegative, 11ov.7ever, it is necessary to incl.ude virus iso-
lation as part of the screening process. Techniques based
on a11tigen capture and polymerase chain reaction (PCR)
have also becn developed.

Treatment and Control
It is important to establish and maintain specific retro-
virus-free breedlng colonles, both for animal health as well
as improving the quality of nonhum;in prim;itp<; 11sf'<l in
bio1nedical 1esea1cl1 and, potentially in the future, trans-
plantation. A serial test and removal p rogram can elimi-
natc SRV infcction in group-houscd monkeys.
Vaccines against S!ZV have demonstrated effectiveness
under experimental conditions.
AVIAN LEUKOSIS/SARCOMA (OMPLEX
Di sea se
The avían leukosis/::;<1n.:u1na t:urn¡.¡lex uf viruses (ALSV) iI1-
rl11cf' ;i wi<lf' Vílri<>ty of <liseases in c:h ickens. These have
been of great cconomic importance to the poultry indus-
try, as wcll as being important research tools for the under-
standi ng of canccr. These diseases include lymphoid leu
kos1s, erythrob1astos1s, myeloblastosis, myelocytomatoses,
sarcomas, osteopetrosis, hemangiomas, and nephroblas-
toma. 1'he signs of tli~ease prutlut:<::tl uy tl1e ALSVs ar<:: IlUt
<>pPciñc, an<l <lifff'rf'ntial <liagnosis requires careful histo-
pathologic examination and laboratory testing.
In lymphoid leukosis, the most common and econom-
ically important discasc causcd by ALSVs, the comb may
be pale, shriveled, and occasionally cyanotic. Inappe-
tence, emaciation, and weakness occur frequently.
Enlargement of the llver, bursa of Pabricius, kidneys, and
thc nodular nature of the tumors can sometimes be de-
tect<::d 011palpaliu11.
AJ _<;y ;i lso c:auses sporadic cases of nonlymphoid tu-
1nors, such as erythroblastosis, myeloblastosis, and myelo-
cytomatoses. C linica l sig ns of these diseases include
lethargy, general wcakncss, and pallor or cyanosis of thc
comb. In more advanced disease, weakness, emaciation,
diarrhea, and occasionally profuse hemo:rrhage from
feather follic:les are ol.Jservetl.
()stPopPtrosis, in whic:h thc long bones ofthe limbs are
commonly affected, is also caused by ALSV. Thickening of
the diaphyseal o r metaphyseal region can be detected by
inspcction or palpation. Affected chickens are usually
stunted, pale, and walk with a stilted gait or limp.
Reticuloendotheliosis virus (REV) is a retrovirus found
in chic:kens and turkl!ys tl1at i:> u11reléit<::d to th<:: viru:;<:::; uf
thP Alpharetrnvirus g<'nus (lcukosis/sarcoma group). REV is
actually classified in the genus Ga111111arctrovirus based on
nucleic acid homology and biochemical properties. REV
causes n eoplastic disease and nonneoplastic runting in
several spccics of poultry.
Etiologic Agent
Classification
ALSVs urc classificd into fivc subgroups, A to E, on thc basis
of differences in their viral envelope glycoprotein antigens
Chapter 65 J<etroviridae 417
that determine v irus-serum neutralization propertíes and
viral intcrfcrcncc pattcrns with membcrs of the same or
different subgroups. Subgroup E viruses include ubiqui-
tous endogenous leukemia viruses of low pathogenicity.
Addinonal subgroups (P, G, H, 1) comprlse retrovlruses
from pheasants, quail, and partridges and have antigenic
p1ope1 Lies and hosl 1ange disliucl frou1 U1at of thc viruscs
in subgroups A to E.
It is important to note that many of thc highly onco-
genic avían alpharetroviruses (type C) that are used in re-
search studies are defective and require a helper virus to
replicate. 'fhese v1ruses are packaged as pseudotypes usíng
the envelope proteins of a helper virus. They therefore take
on the inle1fe1ence and 11eul1alizalio11 properlies of 1l1cir
helper virus.
Physical, Chemical, and Antigenic Properties
In size, shape, and ultrastructural characteristics, viruses of
thc avían leukosis/sarcoma complcx are alpharetroviruses
(Typc C) and are indislli1guishable fro111 011e a11ot11er.
ALSVs within a subgroup cross-neutralize to varying
cxtcn ts. Vi ruscs of diffcrcnt subgroups do not cross-
neutralize except tor partial cross-neutralization between
subgroups ll and D.
Resistance to Physical and Chemical Agents
1'he infectivity of ALSVs is abolished by treatment with
lipid solvents such as ether or detergents (sodium dodecyl
sulfate). ·rhesc viruscs are rapidly inactivated at higher
tcmperatures, whcreas viruses of this group can be pre
9
served for long pcnods at temperatures below 26 C . The
stability of viruses of this group changes little between pH
5 anti pH 9 . Ou l~itlc lhi:> raoge, howeve1, inaclivaLion rales
are markedly incrcascd .
lnfectivity for Other Species and Culture Systems
AL:SVs occur 1n cn1c1<ens ana nave a1so been isolated trom
pheasants, quail, and partridges. More distantly related re-
troviruses ot:t:ur In turkeys. Exp<::riiuent<1lly, :surue uf Lli<::
A1.SV<:. havi> a wirlP host rangi>, rspP.ciíl lly RSV. Sorne strains
of RSV induce neoplasms in other species of birds and
even mammals, including monkeys, although only very
young or immunologically tolcrant animals generally are
susceptible.
The avian oncovi ruses, likc many retroviruses, are not
cytocldal for the cells In \-Vhlch they replicare. In chic:ken
cmbryo fibroblast ccll culture, RSV and other highly onro-
ge11ic men1bers of the ALSV group induce rapid transfor-
mation of cells characterized by alterations in cell growth
propcrtics and cell morphology. These cells proliferate to
produce discrete colonies or foci o f transformed cells
within a few days. The number of transformed foci is in
versely proportional to the viral dilution and can be used
as a gauge of viral concentration. Various strains of sar-
co111a virus can il1ducc l1ansfo1n1aU011h1111ouse, ral, a11d
hamster embryo fibroblasts, as well as in chickens.
Although members of the weakly oncogenic ALSV
group induce neoplastic disease, they produce no obvious
418 PAllT Ill Viruses
cytopathic effects or detectable levels of transformation in
chickcn fibroblast cult ure. 'f heir presence is as::;essed by an
immunofiuorescence tocus assay with type-specific
chicke11 antisera or by their ability to induce resistance to
tra11::;furu1atiur1 by RSV. This resistance occurs when the
glycoprotr.ins (attarhmPnt sites) of <ln identical or related
virus block ll1e cell recepLors for lht: super-iI1fe<.:tiI1g virus.
Stocks of leukosis virus originally detectcd hy intr.rfr.rr.nrP
with RSV are referred to as resistance-inducing factor (Rlf)
strains.
Host-Virus Relationship
Distribution, Reservoir, ¡¡nd Tr¡¡nsmission
ALSVs occur nat urally in chlckens and most flocks of
chic:kens worldwide 11arbor various strains of ALSV, except
for those derived fron1 specific pathogen-free (SPF) birds.
Even in infected flocks, the frequency of lymphoid tu1nors
is typically low and mortality is usually 2<Yo or lcss, al-
though sometimes losses can be much higher. l"he reser-
voir host for ALSV is the infected chicken.
·rransmission can be either vertical (fro1n hen through
egg) or horizontal. Vertically infected chicks are immuno-
logically lolera11l lo lhe virus and fail lo p roduce 11eulral-
izing antibodies, and remain viremic for life. Horizontal
infcction is t hrough infected saliva and feces and is charac-
terized by t rm1sitory viremia tollowed by the development
of a11tibodies. Tumors are 1nore frequent in vertical than
horizontal infections.
Endogenous leukosis viruses, such as those of sub-
group E, are usually Lra11:;r11itt1::u gE:!llE:!ti<.:cilly in the germ
cells in the form of a DNA. provirus. Many o f thr.sP r.n -
dogcnous ALSVs are defective, but sorne (RAV-0) are re-
Jeased in an intectious .torrn and can be transmitted hori-
zontally, although n1ost cl1ickc11s are genetically resistant
to infection.
Pathogenesis and Pathology
ALSV.s tnduce a wlúe varlery of neoplas1ns. The patho-
gr.nPsis of infPrtion of hircts with AT.SVs depends on
"l>vhcther the particular ALSV in question carries an onco-
gene or not. ALSVs containing a v-oncogene are highly
oncogenic retroviruses, transform cells in culture, are
usually defective, and are 1nost often products of the
research laboratory and occur only sporadically in na
ture, if at ali. ALSV strains that contain a particular
v-011cogene usually cause a rapid and relatively repro-
ducil.Jle tyµe uf 11eupla$lic tlisea:;e i11 a liigh µer<.:e11Lage uf
infected chickens.
In contrast, naturally occurring ALSVs are •veakly onco-
genic, cause disease by insertional oncogenesis, do not
tra11sfor1n cells at detectable levels in culture, are usually
not defective, and áre naturally transmitted. The onco-
genic spectrum of non-oncogene-containing strains of
ALSVs terH.ls tu uvE:!rl<1p, .su t11at a giver1 str<1in uf A LSV <.:an
induce many kinds of t111nors clepending on other factors,
such as thc arnount of vi.rus, age, and gcnotypc of chicken,
and rou te of infection. This is consiste11t with the concept
of insPrtional oncogenesis .for these viruses in which the
ALSV infects and replicates i11 a variety of cell types bul, L .
order to produce neoplastic transformation, must int.e-
grate near an appropriatc ccllulnr proto oncogcnc.
Under natural conditions, the most common disease
caused by ALSV is lymphoid leukosis. Transformation o:-
lymphocytes occurs in the bursa of Fabriclus, usually at a
fpw months aftPr infection . These ear.ly ALSV-induced le~
sions son1etimes reg.ress, w.h ile oth.ers e11large an.d evenlu-
ally spread to other visceral organs. Grossly visible n eo-
plasms are of variable sizc and organ distribtition, almost
always involve tl1e liver (a synonym for lyrnphoid leukosiS
is hig Ji ver dísease), spleen, and bw·sa of Fabrich.1s. Indi-
vidual 11eopla$111s are sufl, s111uuLh, a11d glisle11i11g aut:
are usually miliary or diffuse, hut may he nodular, or a
combination of these forms. These neoplastic masses are
composed of large B lyn1phocytes that express surface im-
munoglobulin. 'rhcrc are oftcn no consistcnt or sig11ifi -
cant hematologic changes il1 circulating blood, and fra11k
ly111phoblastic leuken1ia is rare. Fully developed ly1nphoid
leukosis occurs in birds at about 4 months of age and
olde.r.
Erythroblastosis occurs sporadically in ALSV-infected
cl1icken flocks. The liver and spleen are enlarged by a dif-
fuse Wiltration of proliferating erythroblasts, and the
bone marrow is ettaced by the same cells. Attected chick-
ens become al1emic and t.hrombocytopenic. Blood smears
show an erythroblasrtc leukemia. Indu.ction of erythrol>-
lastosis by naturally occurring, slo,.,ly transfor1ning ALS\.
ü1vulve:; iJLtiviJtiu11 uf the <.:ellular uucugeue c-erbB by in-
sertional oncogr.nesis. High ly onc.ogr.nic., lahoratory
strains of ALSV carry tl1e v-form of this oncogene and are
termed avian erythroblastosis virus (AEV). Sorne strains of
AEV can kili chickens by erythroblastosis withir1 a wcck
after experimental infection.
Myeloblastosis is relatively uncon1mon under natural
<.:outlitiuu:; arH.l tenlls to occur in atlult chickens. The target
organ in this ctisf'aSP is honP marrow, and the first neoplas-
tic alteration is in the form of inultiple foci of proliferating
myeloblasts, •vhicl1 is followed by leukernia and invasion
of other organs, especially liver, kidney, and splcen.
Microscopic examination reveals massive intravascular
and extravascular accumulations of inyeloblasts vvitl1 vari-
able proportions of pron1yelocytes. The v-rnyb gene is car-
ried hy the highly oncogenic avian myeloblastosis virus
(AMV) strams. These laboratory strains produce mortality
a few '"'eeks after experimental infection.
Myelocytomatosis is another forxn of leukosis that oc-
curs sporadically in chickens. In this disease, tumors char-
acteristically occur on t he surface of bones in association
wi lh Lhe periusteu111a11tl11ear carlilage, and al ll1e costo-
chondral junctions, posterior stemum, and cartilaginous
bones of the mandible and nares. 'fhey consist of compact
masses of uniform myelocytes. Earliest changes occur in
bone marrow in which there is crowding of intersinu-
soidal spaces by myelocytes, destruction of sinusoid walls,
and eventual overgrowth of the borre marrow. Tumors 1nay
<.:ruwtl througl! the lJ011e a11d exleud Lhrough lhe perios-
teum. The v -rnyc oncogene is carried by the higl1ly 011co-
genic avían myelocytomatosis virus.
Various benign and malignant connective tissue tu-

Drs occur sporadically in AT.SV-infP.c.tt>d c.h ickens. There
.::e a nun1ber of laboratory strains of avían sarcoma virus
..5\'), the most famous of which is Rous sarcoma virus
"1.SV) . AS\ls induce sarcomas (tumors of connective t is
s::e), induding fibrosarcoma and fibroma; myxosarcoma
-:Jd Inyxoma; h istiocytic sarcoma, osteoma, and o s-
3lgenic sarcoma; and ch.ondrosarcorna. 1'hese higl1ly
=:icogenic ASVs carry an oncogt>nf' suc.h as src (in RSV), fps,
'"'lS, or yes.
Infection with ALSV is important in its own right. As
rompared with specific pathogcn-frcc chickcns, ALSV-
-=úected chickens exhibit poor grovvtl1 and egg produc-
.:-0n even in the absence of tumor formation. The pat.ho-
_enesis of subcllnical ALSV infectíon of liin.ls .i s µoorly
-nder.stood .
- ~s.t Response to lnfection
~hickens exposed to ALSV virus fall in to four classes: 1) no
~rem ia, no ant ibodies; 2) no virem ia, antib ody; 3)
'1.remia, antíliolly; e:u1u 4) virt:iuia, 110 anlibody. Calego1y
· inc.h1clt>s h irds that are genetically resistant. Most ex-
;x>sed chickens are included in category 2, and antibody
.;iersists throughout the life of these birds and is passed via
-~e yolk t o progeny chicks. ·rhe passive immunity pro-
4 ded by such antibodies generally lasts for 3 to 4 1veeks. In
addition to t he neutralizing antibodies clirected against
:he envelope proteins, a11tiliodies are procluced lo Ll1e in-
-ernal group-speciñr antigens (Gag proteins), which are
::101u1eutralizing and not protective. Althou gh virus-
:ieutralizing antibodies restrict the amount of virus. they
h ave little dircct cffcct on growth of the virus induced
neoplasms. J<ew chickens occur in the third category,
·,·hich may represent chickens that are in t he process of
clearing an acute infection wit h ALSV. Most chickens in
category 4 acquire ALSV vertically when in the egg ;in<l ;irP.
immunologically tolerant to lhe virus. He11s i.n category 4
-r;insmit vir us to a high proportion of their progeny
through the egg.
Since there are mult iple subgroups (A to V) that co1n-
monly occur in chicken flocks and are not cross-neutralized
by antibody, the status of a chicken for one subgroup of
..\LSV is independent of ot her viral subgrou ps.
Laborat ory Diagnosis
.l\LSV can usually be isolated from plasma, serum, t umor
tissue, and albumin, or from the embryo of infected eggs.
Since ALSV is generally not cytopathogenic, complement
fixatio11, fluorescent antibody, o r rauioi11u11u11oassay
( RrA) tP~ts m11st hP 11st>ci to cit>tt>c.t and idt>ntify the viruses
in cell culture. A.11 ELISA test is used for direct detcction of
virus in egg albu1nen or vaginal swabs. ·rhese tests have
bccn uscd d ircctly on test materia.! (egg albumin) or indi
rectly on the cell cultures used for viral isolation. All tests
require a source of chicken embryos free from e11doge-
nous ALSV.
Another means of icft>ntifying virus is hast>d on ph~no­
typic n1ixin.g of viruses. Chicken fibroblasts that are trans-
formed with envelope-defective strains oí RSV are nonpro-
Chapter65 N.etroviridae 419
ducers (NP) of infectious RSV. Superinfection of NP cul-
tures by anot hcr ALSV act ing as a helper virus results in
production of infectious RSV, vvl1ich produces trans-
forn1ed foci on susceptible chicken embryo fibroblasts.
Treatment and Control
Allen1pts to produce effective vaccines have been largely
unsuccessful. Moreover, congenitally infected chicks,
vvhich constitute the majar source of virus and are most
likely to develop neoplasms, are immunologically tolerant
to AL.<;V and cannot be immunized.
It is possible to eradícate ALSV Cro1.n chickens t>y estatJ-
lishing breeder flocks that ;ire frt>f' of t>xogt>no11s AT.SVs.
He11s are selected that are negat ive for ALSV antigens in
their eggs. The fertile eggs laid by t he selected hens a:re
hatchcd and thc chicks rcarcd in isolation in small groups.
·rhe birds without leukosis virus antigen or antibody are
used as the breeders for a leukosis virus-free flock . The
flock 1nust t11en be n1ai.n tained in isolation fro1n llntested
chickens.
BOVIN E LEUKEMIA VIRUS
Disea se
llovine leukemia virus (BLV) is a cause of lyrnphon1a (lym-
phosarcoma; lymphoreticular neoplasia) in older cattle-
so-called enzootic bovine leukosis (EBL), whicli occurs
very sporadically in BLV-infected cattle. Cattle with F.RI.
are u:;ually older lhan :) years, ><Vilh tl1e peak of tun1or 1n -
c.idt>nc.e hetween .S and 8 years. Affected cattle typically
are afebrilc with nonpainful cnlargcrnc11t of peripheral
lymph nodes (ly1nphadenopathy). Depending on t he in -
volvement of d ifferent organs, affected cattle can exhi-
bit signs of gastrointestinal dysfunctJon, paralysis, ex-
opthalmu s, and cardiac dysfunction . Neopl;istic lympho-
cyLes can i11vade lhe blood to cause lyn1phoid leukemia
in sorne affected cattle. The forms of lymphomas tl1at
occur in calves (less than 6 mont hs of agc) and juvcnilc
cattle (6- 18 months ot age) are not associated w ith BLV
infection.
Etiologic Agent
Classification
Because of its genome structure, nucleotide sequence. a11d
sizc and amino acid scqucncc of thc structural and non-
structural Viral proteins, BLV has recently been grouped in
a genus (I>eltaretrovirus) with the human T-lymphotropic
viruses (HTLV-J and HTLV-TI), and the closely related
simian T-lympbotropic viruses (S1'LV). "l'hese viruses can
inLluct: Lliseases wi lh si111ilar pall1ologies, cl1aracLerized by
low viremia, long latency period, and a Iack of preferred
proviral intcgration :;itcs in thc tum<>rs (i.c., thc provirus is
11ot necessarily found near an oncogene).
•
420 PART 111 Viruses
Physical. Chemical. and Antigenic Properties
Morphologically, BLV resen1bles other·rype C retroviruses.
AntilJody to gp51 is neutralizing.
Resistance to Physical and Chemical Agents
'fhe infectivity of BLV is abolished by lipid solvents, perio-
date, phenol, trypsin, and formaldehyde. Infectivity is rap-
idly destroyed at 56ºC but can be retained for prolonged
pcriods at less tha11 SOºC. Pasteurizalion deslroys Lhe infec-
tivity of: this virus, which is of interest because infected
lyrnphocytcs ar<.: found in thc rnilk of lnfccted dalry cattle.
lnfectivity for Other Species and Culture Systems
BLV h.as been shov.rn to be in fectious for severa! animal
spec.ies other tha11 catt le, i11cludi11g sl1eep, goats, a11d plgs.
Under natural conditions, the oncogenic potential of BLV
appcars to be cxprcsscd only in cattlc and shccp. Sincc
tt1ere are no significant antigenic or genetic differences be-
tween bovine and ovine isolates, the agent designated
ovtne leukemia virus is regarded as BLV infecting a heterolo-
gous host.
BLV 1eplicates i11 cell cullure fr(!lll a wide V<triely or ~¡;e­
cies, including bovine, human, simian, canine, caprine,
and equine cells. Alt hough 13LV replicates in human cells,
humans are not known to be intected. Seroepictemic stud-
ies among high-risk humans (veterinarians, farmers, ani
mal keepers, and slaughterhouse personnel) revealed no
infections, and BLV 11as not been associated ~.vith human
11ev¡;las111s.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
·rhe geographic distribution of BLV is worldwide. 1'he
rcscrvoir is infcctcd cattlc. Discasc is dircctly rclatcd to BLV
prevalence, which can vary widely but is highest in intc11-
sive dairy areas (up to 50% or even higher). In addition to
productlo.n losses associated with BLV infection of cattle,
;i<iclition:il losses result fH)m exportation restrictions by
foreign countries t hat halt export of BLV-positlve cattle 01
semen from infected bulls.
BLV ir. trun5rnittcd horizontully undcr condi tion~ of
close contact, and rnost con1monly occurs when heifers
are introduced into the milking herd. The virus is highly
cell-associated, ar1d transmission is by blood o r tissu_e con-
taining lymphocytes between a11in1als, by trauma, co11-
tan1inated veterinary equipn1ent, or other lcss defi11cd
routes. Transmissíon of BLV can occur t hrough t he skin
and thc rcproductivc, alin1cntary und rcproductivc tracts,
and is easily transmitted to susceptible calves or sheep by
as few as 2500 lyn1phocytes from infected animals.
Experimental transmission has also been accom plished
with milk and colostrum, both of which contain lympho-
cyles, bul Lhis route is likely uJ1in1porlanL In ule.ro tra11s-
.1uission of BLV has been documented, but occurs ilúre-
qucntly. Thc transmission of BLV by hcmatophugous flics
and ticks has been demonstrated experimentally; hovv-
ever, field observations do not support a major role for
such vectors. DLV-infected cattle with virus-induced per-
sister1t lymphocytosis are major reservoirs of the virus and
pose the greatest risk for transmission.
Pathogenesis and Pathology
Most BLV infections are asymptomatic. There is a brief
viremia soon after infection of susceptible cattle, followed
by a long incubation period when the virus remains quies-
cent as provirus that is randomly integrated into the
genome of infected cells. Only a low percentage of BLV-
in fected a11irnals ever develop lyrnphoma, suggesting tbat
Lhe i11culJalio11 ¡;eriud for i11duclio11 of neoplasia is longer
than the lifespan of rnany infected animals. Sorne cattle
develop only a transient viremia \-vithout seroconversion,
and after 3 to 4 months viru s can no longer be isolated,
whereas others develop persistent lymphocytosis within
months or years after infection.
Neoplasms in cattle with enzootic bovine leukosis typi-
cally ü1volv!::! auy cou1lJiuallo11 of inLernal and superficial
lymph nodes, heart, abomasum , intestines, kidneys.
uterus, liver, spleen, epidural space of the lumbar spina!
cord, and retrobulbar fat (of the eye). Distribution of the
tu1nor is unprcdictablc, but blood is often not involved.
Botl1 ·r lymphocytes and B lympl1ocytes can be ilúected
with BLV but t he tumors are composed only of proliferat-
lng B lymphocytes.
Host Response to lnfection
Most BLV-infected cattle develop antibodies to 13LV struc-
tural proteins. A greater response is usually detected to t h e
glycosylated proteins gpSl and gp30 than to the interna!
proteins p24, pl5, pl2, and plO and to the reverse tran -
scriptase. Whereas mostiofected cattle develop high titers
of v irus-specific anUbodies, so1ue re1ual11 ¡;er::.isteutl~
seronegative.
Antibodies to 13LV are a lso detected in the milk an d
colostrum and are partially p rotective against infection of
calves. Antlbodies do not provide protection against tumor
development tn infected animals, however, and do n ot
prevent the spread of infectious BLV by carriers.
Laboratory Diagnosis
A variety of serologic tests (agar gel immunodiffusion
[AGID), in1munofluorescence, and ELISA.) that detect BL\ ·-
specific antibody can be used. 'fhe animal usually becomes
seropositive 4 to 12 weeks after viral exposure.
Bovine leukemia virus induces syncytia in target cells.
Treatment and Control
Infection, once established, appears to be lifelong in in -
fecled call.le. Tl1ere ls J10 Ueatn1ent for lyn1phon1a or BL\ -
infection in cattle. BLV can be eliminated from a herd by
rcpcatcd scrologic tcsting and immcdiatc rcmoval of p osi-
tive animals.

VISNA/MAEDl/PROGRESSIVE PNEUMONIA VIRUSES
ANO (APRINE ARTHRIT IS ENCEPHALITIS VIRUS
Disease
These viruses cause severa! different diseases that involve
the lungs, joiI1ts, n1ammary glands, and central nervous
system of affected sh.eep and goats. Inttial signs of visna are
subtle and insidious, consisting of a slight aberration of
gail, especially of Lhe hi11dquarlers; lren1bling of lhe lips,
unnatural tilting of t he head; and in rare il1stances, blind-
ness. 'fhe signs progress to parcsis or cven total paralysis.
rever is absent. Unattended a11imals die ot inanitio11,
bence the name visna, which means "wasting" in Tcelan-
dic. Signs of visna typicauy occur in sheep over 2 years of
age, and tl1e clinical course is protracted.
Maeui and progressive pueun1011ia viruse::; cau:;e a :;üui-
lar chronic pneumonia in infected sheep. Early manifesta-
tions includc progrcssivc loss of condition accompanicd
by dyspnea. Eventually, breathi11g requires the use of ac-
ct~ssory inuscles and is accompanied by rhythmic jerks of
the head. There is sometimes a dry cough, but 110 nasal dis-
charge. The clinical phase is protracted, although affected
anilnals ofLen die f1on1 seconda1 y baclerial p11eun1011ia.
Caprine arthritis-encephalitis virus (C.l\.EV) induces
severa! discasc syndromcs i11 domcstic goats, including
chronic progressive arthritis, mastitis, and occasionally in-
terstitial pneumonia in older goats, and an acute paralytic
syndrome in kids that is characterized by ·h ind limb ataxia,
weakness, and paralysis.
Etiologic Agent
Classification
The agent of visna/maedi/pr.ogressive pneumonia and
th.e closely related organism of CAEV are lentiviruses. ·r11e
n.ame designations are largely historical and refer to the
sile of vi1us isolalio11 or Ll1e predo111ina11l patl1ology in an
individual animal. OviI1e progressive pneun1onia vi.rus is
synonomous with macdi virus.
Physical, Chemical, and Antigenic Properties
The virion is cornposed of four structural proteins desig-
nated gp135, p30, p16, and p14. Minar structural proteins
include a reverse transcr iptase, integrase, and dUTPase.
Neutrallz;aLiuu Le:;t:; have :;l1ovvn ll1al variatlu11:; iu viru:;
strains occur during infection in individual anitnals. Tf an
animal is inoculated \.Yith plaque-purified virus, many
months latera virus can be isolated that is not n.eutralized
by antiserum that neutralizes the original inoculum
strain. Both the inoculum strain and the variant strains
can be isolated simultaneously, indicating that new strains
do uoL replace pare11lal viru:;. Willt tirne, rreut.raliz;ir1g au-
tibodies are produced to the ne\'\' strains.
The RT of these viruses has a pr.eference for Mg2.+ and
uses tRNALys as a primer tor negative-strand DNA reverse
transcription.
Chapter 65 Retroviridae 421
Vi~ua/11raedi/progressive p11eu111onia virus a11d CAEV
show extensive cross-reaction by immunodiffusion assays
involving the 1najor structural protein.
Resistance to Physical and Chemical Agents
Lcntiviruses are relatively resistant to ultraviolet irradiation.
Infectí.v ity is abolished by lipid solvents, periodate, phenol,
trypsin, ribonuclease, formaldehyde, and low pl-I (less than
4.2) . I11feclivily is rela lively slable al O lo 4ºC in lhe presence
of serurn, but infectivity is rapidly destroyed at 56ºC.
lnfectivity for Other Species and Culture Systems
Visna and ovine progressi.ve pneumonia have been de-
scribed only in sheep anc\ goats. Sorne breeds of sheep ap-
pear tu ve ruure su:;ceptilJle, especially Icelandic sheep,
which are highly inhred. The visna virus inferts c.ells <le-
rivc:d fron-i rnany vcrtcbrutc spccies but replicates effi-
ciently only in sheep cells. Unadapted vir us isolates repli-
cate best in macrophage cultures.
Host-Virus Relationship
Distribution, Reservoir, and Transmission
These viruses cause disease in sheep and goats throughout
much of the world. Tl1e freguency of infection varíes
widely based on control programs, but can range to greater
than 75% in sorne flocks in the United States. lnfected
sheep serve as the reservoir.
Tra11s.mission is via respiratory exudates and aerosol.
Virus i~ excreleu iu Ll1e urilk, a11d larul>:; raised ou iufected
e\'\Tes are infected at a young age. Infection rates are in-
creased by p ractices tl1at pool n1ilk. Intrauterine tra11sn1is-
sion is infrequent.
Pathogenesis and Pathology
'l he ovine lentiviruses infect cells of the monocyte-
macrophage system. Visna is a chronic and progressive en-
cephalomyelitis characterized by multifocal areas of
chronic inflammation with accompanying demyelina-
lion. 'fhe process begins in1n1edialely benealh lhe epen-
dyrna bordering the ventricles. but spreads throughout
the brain and spinal cord.
The lungs of sheep with progressive pneumonia are
markedly expanded, vvith as much as a two- or threefold
increase in lung weight. The histopathologlc changes ln-
cht<l<' thickening of the intera lveol<! r septa as a rest1lt o f in-
filtration of lyn1ph.ocytes, n1onocytes, a11d n1acrophages.
·rhe thickening may be so pronounced as to obliterate the
alvcoli. Lymphoid accumulations with the formation of
follicles and germinal centers are scattered throughout the
lung parenchyrna.
Sorne adult sheep infected with avine lentiviruses de-
velop chronic arthritis and/or mastitis.
Caprine a1tl1Iitis encepl1aliUs virus (CAEV) causes a pe-
culiar motor spinal dysfunction in goat kids at 2-4 months
of agc. The lesions in affected goats resemble those of
422 PART III Viruses
visna. Goats that survive infection with CAEV as l<ids
oftPn ciPvPlop progrPssivP, chronic arthritis, mastitis, anci,
occasionally, interstitial pneumonia that resen1bles ovine
progressive pneumonia.
Host Response to lnfection
Most lentivirus infections of ruminants are subclinical,
likely because of the prolonged incubat ion per:iod of the
diseases th.ese viruses il1duce. Lesions in all of these d is-
eases include chronic, ongoing inflan1mation; t hus t he le-
sions t hcmsclvcs likcly rcsult in part from thc host's im-
mune response.
In experimental infections, complen1ent-fixing anti-
bodies appear a few weeks after inoculatio11, rise to a max-
imum wit hin 2 months, and remain consta11t thro ughout
Lhe course of disease. Neulralizi11g a11libodies appear later,
reach a maximum at about 1 year, and then ren1ain con-
stant. Ho\ovever, thc virus pcrsists dcspi tc a vigorous hu-
moral immune response, possibly because most infected
cells are l10t producing vira] antigens and are therefore un-
detectable by the imrnune surveillance mechanisms.
Laboratory Diagnosis
In its early stages, visna is dífficult to dístinguish from other
central nervous system (CNS) diseases¡ however, th.e pro-
gressive protracted course, the absence of fever, and the
pleocytosis in the cerebrospinal fluid (CSI<) are all character-
islic of visna. ·rren1or of ll1e 11ead, grii1ding of leelh, and il1-
tPnse itc.hing arP 1nore c.ha rac.teristic of scrapiP than visna.
Virus can be isolated frorn CNS, lung, spleen, peripl1eral
blood leukocytes. and CSF, but because of the limited viral
replication that occun: in vivo, tissue explantation and
blind passage are often required. Group-specific tests t hat
detect antibodies that appear early a11d are m aintained
tl1rougl1out tl1e disease are preferred over seru111 neulral-
ization for serologic diagnosis. Neutralization tests are of
less value in diagnosis because they become positive much
later in disease and are strain-specific. Thus, serologic tests
such as agar gel immunodiffusion are now used to identify
lentivirus-infected sheep and goats.
Treatment and Control
No effective vaccine currently exists ai1d no useful thera-
peutic agents are available. C:ont rol of these viruses and
their diseases is by serologic testing and the elin1ination of
infected animals. Visna and maedj were eliminated from
l\.:t'.hu1u a:, llie rt'.::;ull uf au eradicalio11 p1ogranl.
EQUINE INFECTIOUS ANEMIA VIRUS
Disease
Equine infectious anemia virus (EIAV) can cause severe
anemia in horscs, but thc ciinicru prcscntation of EIA is
highly variable. In the acute form, slgns develop suddenly
7 to 7.1 dilys postinfection . Signs can incluclf' ff'vf'r, ano-
rexia, thron1bocytopenia, and severe anemia. 'fhere may
also be profuse sweating and a serous discharge fro1n the
nose. Such a ttacks often last for 3 to 5 days, after which
the anilllal appears to recover. Horses in the acute stage of
the disease are se.ronegative for ETA.
Subacute disease otten follovvs the acute infectior1 after
a convalescence of ?. to 4 1Nf'f'ks. Ar11tP signs arf' rPpPatP.d
along with '~reakness, eden1a, petechiae, lethargy, dcprcs-
sion, anemia, and ataxia. 'fhe animal again appears to re-
covcr and thc cyclc n1ay then recur.
Chronic EIA is the classical presentation of so-called
"swarnp fever," whlch rese1nbles the subacute form but is
mllder and seldom leads to death. 111e cycle of fev1:.'.r, welght
loss, anorexia, and clinical signs ran rPc.11r six or more ti1nes.
Each cpisode usually lasts 3 to 5 days, a11d the interval be-
tween cycles is irregular (weeks to months). The frequency
and scvcrity usually decrease after 6 to 8 episodes, usually
~.vithin tl1e first year. Most horses are tl1en without dinical
signs but carry the virus for the remainder of their lives. EIA
ca11 be induced by stress or irrnnu11osuppressl vt:: drugs.
EIAV il1fection in horses gPnPra ll y resnlts in clinical
sig11s lhal are inapparent, subclinical, or mild. These
horses remain asymptomatic but have anti body to tl1e
virus and are lifelong carricrs of thc virus. Asymptomatic
but chronically viremic animals have been observed for
periods in excess of 18 years.
Etiologic Ag ent
Classification
EIAV is a Lentivirus, and was thc first anilnal discasc to be
1aent1t1ed as caused by a tllterabie v1rus (1YU4).
Physical. Chemical, and Antigenic Properties
ElAV is composed of two er1velopc-e11coded glycoproteins
(gp 90 = SU and gp 45 = TM) and four 1na¡or nonglycosy-
lated proteins (p26 = CA, plS = MA, pll = NC, and p9). 1'he
_p26 is lhe inajor core _proleü1 a11d den1011strates group speci-
ficity, •vhile the envelope-associated glycoproteins demon-
stratc hcmagglutination activity and are typc-spccific.
'l he ElAV genorne is highly mutable. When the virus is
placed under selective pressure by the l1ost immune sys-
te1n, individual nucleotide substitutions (mutatlons) pro-
duce novel antigenic variants of the gp 45 and gp 90 f'nvf'-
lope _proleins. ll is ll1ought t hat these antigenic variants
cause EIA's characteristic episodic recurrence. In cell cul-
ture (v.rhcrc thcrc is no immune selection), antigenic types
remain stable and neutralizable by serum antibodies from
the horse from 1"7hich the virus '"'ªs isolated. Wl1en intro-
cluced into a new horse, these same strains prouu\.:e r1t'.1.,,
antigenic viral variants that no longf'r ari> nP11 tralizPci by
lhe original antibodies.
Reslstance to Physlcal and Chemlcal Agents
EI1.\V is readily inactivated by common disintectants tha t
contain dctcrgents. The virus is also inactivated by sodium

hydroxitle, so<.liurn hypuchlurite, 1uust urga1lic sulve11t:;,
and c.hlorhexidine. ETAV heated in horse serum at 58ºC for
30 minutes shows no infectivity for horses. However, at
25ºC, EIAV remains infectious on hypodermic needles for
96 hours.
lnfectivity for Other Species and Culture Systems
Horses, ponies, donkeys, and mules are susceptible to in-
fectlon by EIAV. There is only one report of human infec-
tion, ;inc1 no c;isps of EIA-likP <1ise;isp have heen i<1PntifiP<1.
Attempts to propagate the virus in lambs, mice, hamsters,
guinea pigs, and rabbits have laíled. Primary ísolates of
EIAV can be propagated only in equine leukocyte cultures,
where it grows in cells of the monocyte/macrophage line-
age. Laboratory strains of EIAV can be propagated in a va-
riely of cell llnes fro111 severa! species, including hu1nan
fetal lung fibroblasts. These laboratory strains display sig-
nificant scqucncc diffcrcnccs from primary isolates, par-
ticularly in the U3 region of the LIH..
Host-Virus Relationship
Distribution, Reservoir, and Transmission
The llistril.Jutiur1 uf EIAV is wurlllwille l.Jut is rnost prevalent
in warm climates. lnfection rates vary widely but the dis-
ease is increasingly rare in countries such as the United
States. Horses, donkeys, and mules are the only known
reservoirs and natural hosts of the virus.
Mechanical inoculation of blooa is considered t11e
major mode of ELl\V transmission. EIAV is naturally trans-
mitted by hematophagous insects, especially deer and sta-
ble flies. E.IAV does not replicate in the insect cells, but flies
cau tra11:;1ujL Lhe virus by sin1ple n1echanical lransfe1 oJ in-
fected hlood. The transmission of EIAV vía blood can also
be through contuminutcd nccdlcs; thus it is importunt not
to share needles or use unsterilized needles in veterinarv
,
procedures. Viral transmission to the nursing foal from a
carrier mare is well documented. EIAV can also be trans-
mitted in u tero but tl1is is probably rare.
Pathogenesis and Pathology
:\cute EIA is related to massive viral replication. Anemia re-
flccts rcduccd lifc span of red blood cclls (RBCs) as a consc-
q uence of hemolysis a11d erythrophagocytosis by acti-
vated macrophages. A decrease in complement levels and
the presence of complement-coate<.l erythrocytes have
hPPn ohsPrvP<1 in F.IAV-infPctrcl horsrs. DrcrP<lSPd erythro-
poiesis levels and pertubations in iron metabolism also
contribute to anemia in chronic cases.
Lcsions of EIA rcflcct thc duration and scvcrity of infcc-
tion and dísease, and can include widespread hemorrhage
and necrosis of lymphatic ti.ssues, anemia, edema, and
e1n<1ciatiun. Micru:;cupic lesions inclu<.le activation of the
mononuc:lear rhagoc.ytic systrm in ali lymphoid tissues,
activation of Kupffer cells, and hemosiderin deposition in
many organs. Immune complex-mediated glomerulon-
ephritis and hcpatic ccntrolobular necrosis are common,
Chapter 65 Retroviridae 423
tl1e latter as a cunsequence uf severe, acute-unset anernia.
Granulomatous ependymitis, meningitis, choroiditis,
subependymal encephaliti:s, and hydrocephalus are assocj-
ated with ataxia.
Host Respon~e to lnfection
Hurses infected with EIAV develop persisting antlbody
titees •-vithin 45 days. Most animals become ELISA-positive
within 12 days a11d AGID-positive will1in 24 days of ir1-
fection.
laboratory Diagnosis
Laboratory diagnosis depends 011 lhe delecliu11 uf specific
antibody using an agar gel immunodiffusion test (the
Coggins test). More sensitive ELISA tests are also n.o w
available. r
Treatment and Control
No specific Lrealrnenls are available. Sup¡;orlive Lltera¡;y i:;
the most import ant factor iI1 recovery.
Affcctcd animals should be either euthanized because
t ne virus is contag1ous or physically isolatecL ~pread ot
EIAV can be reduced by control of stable flies and mosq_ui-
w
toes. Repeated use of hypodermlc needles and transfusio11s
from untested donors must be avoided .
Infected stallions should not be bred to seronegative
mares, altho11gh the reverse need not he true. Uninfected
foals can usually be obtained from positive mares and pos-
itive stallions if they are isolated from the infected mare
and her milk.
A v accine against EIAV is used in sorne co1mtries (Cuba,
China) but probably does not provide broad protection
agai11sl ali varianls of EIAV.
80VINE IMMUNODEFICIENCY VIRUS
Disease .
Despite its provocative nan1e, the significa11ce of bovil1e
immunodeficiency virus (BIV) as a cause of immune dys-
rcgulation and chronic inflammation in cattle is uncer-
tain. Unsubstantiated reports implicate HIV as a cause ot
lethargy, mastitis, pneumonia, lymphadenopathy, and
chronlc dermatitis, but these reports are viewed \<Vith in-
creasing skepticism.
Etiologic Agent
Classification
BIV is a Lentivirus that is not closely relate:d to any othPr
known Lentivirus.
424 PART 111 Viruses
Physical, Chemical, and Antigenic Properties
Tl1e 1norphology and physical properties of BIV closely re-
sen1ble those of other lentiviruses. BIV 11as an SU glycopro-
tein of 100 kd and a -rM glycoproteirl of 45 k.d , and Gag
proteins, MA, CA, and NC, of 16, 26, and 7 kd, respectively.
BIV also produLes several 11011stru<..tural µrvtei i1s. Tite RT
of RTV has ;i prPfPrPnrP for !'vfg2+_
lnfectivity for Other Species and Culture Systems
Experimental infection of rabbits and sheep with BlV is
possible, but these ani.mals do not develop disease. BIV can
be cultur1::u ilt c~l.l:s fro111 a variety oí species, ü1cluding
boviI1e, rabbit, and canine, b11t not primates or humans.
Host-Virus Relationship
Oistribution, Reservoir, and Transmission
The distribution ot Hl V is probably worldvvide. In the
United States, the prevalence of BIV infectio11 is low but
may be mucl1 higher i.n individual herds. Herds lnfected
with BIV are often also illfected with BLV.
Pathogenesis and Pathology
Cells of the monocyte/macrophage support replication of
BIV ill infected cattle.
Host Response to lnfection
RIV infPrtion in c.attle result·s in a strong host antihody re-
spo11se. Jlowcvcr, like most other lentiviruses, DIV induces
a chronic lifelong infection. The vast majority of infec-
tions are subclinical.
Laboratory Diagnosis
lnfected cattle can be detected by serologic tests for anti-
bodies to BIV. BIV isolation from blood can also be used to
detect infected animals.
Treatment and Control
There is no vaccine or treatment for BIV infection. 1'11e im-
portance of BIV infection as a pathogen of cattle remains
most uncertaiI1.
fELINE IMMUNODEFICIENCY VIRUS
Disease
Feline imn1unodeficie11cy virus (FIV) iníeclio11 of cats pro-
duces ;icntP fpvpr and 1ymphadenopathy, followed by an
asyn1ptomatic carrier phasc. In sorne cats, FIV infection
causes µroívu11d iuuuu11udeficiency Ieading Lo secondary
chronic infections. FIV infection of cats shares cornmon
features with AIDS of huma11s, and PIV infcction of cats
has become an important animal n1octel for AllJS re-
search.
Etiologic Agent
Clossificotion
FIV is a Lenlivirus Lhal is nol closely related to any other
known Lentivirus.
Physical, Chen1ical, and Antigenic Properties
'!'he morphology a11d physical properties of FIV closely re-
semble those of other Ientiviruses. FIV l"l<ls an ST Tglyrnpr0-
teil1uf95ku a11d a TM glycop1oteil1 of 41 kd, and Gag pro-
teins, 1vfA, CA, and NC, of 16, 27, and 10 kd, respectively_
rrv also codes for several nonstructural proteiI1s. 1'he RT of
FIV has a preference for Mg 2 +.
Resistance to Physical and Chemical Agents
FIV is inactivated by appropriate concen trations of uisin-
fectants such as chlorine, quatern;iry am moni111n com-
JJOu11ds, phenolic con1pou11ds, and alcohol. It survivcs at
60ºC for only a fe'"' minutes.
lnfectivity for Other Species and Culture Systems
FIV infects dornestic cats, althougll th.ere is serologic evi-
dence that FIV-like viruses infect wild Felidae in Africa
(lions, cht:!etahs) arH.l tl1e Ai 11ericas (pun1a, bobcals,
jaguars). FTV isolates replicate in primary cultures of feline
n1ononuclear cultures stimulated to divide with m.itogcn
and supplemented wit h interleukin-2 (lL-2; ·r cell gro,-vth
factor) . Sorne isolates of FIV are also able to replicate in es-
tablished feline cell lines. FIV does not replicare in nonfe-
line cell lines. 'fl1en~ is 110 li.nk between FTV an<l any human
disease, including AIDS.
Host-Virus Relationship
Oistribution, Reservoir, and Transrnission
FJ V is enc.temic in cats throughout the world, although th e
virus is notas cont agious as FeLV. FIV is shed in saliva, anc
the most important route of transmission is pro!Jabl_
through bites. Free-roaming rnale c.ats, which are mas:
likely to fight, are most frequently infected with fIV. Tu-::
virus is not efficiently spread by casual, nonaggressive cor:-
tact an1ong cats. Sexual contact probably is al.so nota pri-
mary means of spreading FIV. lransmission from an iI:-
fected queen to her kittens can occur.
Cats rema.i n infected witl1 FIV fvr life, allhough Lhe re.c.-
jority of FTV infec.tions are c.linically silent.
Dual infection of FIV and FeLV is 11ot uncommon , a:-_
cats infected with both FlV and FeLV appear to hav~ _
more scvcrc discase course.

Pathogenesis and Pathology
Thcre are no definitive gross or histologic changes in the
tissues of FTV-infected cats, even in more advanced stages
of disease. Following initial infection, the virus replicates
in regional lymph nodes, then spreads to lymph nodes
throughout the body, sometimes resulting in a transient
geuerali:t.ed ly1npl1<1c.le11up<1tl1y. Must infectiuns are
asymptomatic, whereas lymphoid depletion and immune
suppression occur in sorne cats that are then susceptible to
secondary opportunistic infections. FIV appears to infect
both CD4 and CD8 lymphocytcs, as wcll as macrophages
1n vivo. Many cats marufest an absolute decrease in the
numbcr of CD4 lymphocytcs with an inversion of t he
CD4/CD8 ralio.
Host Response to lnfection
lnfected cats typically respond with vigorous hun1oral (an-
tibody) and cell-mediated immune responses. These re-
~ponses appear to be sufficient to limit the initial acute
phase of thc discasc. Likc n1osl lc11liviruses1 howeve1, FIV i:.
never eliminated. It probably produces various degrees of
subclinical immunc dysfunction in thc majority, and clin-
tcally significant immunodeficiency and associated sec-
ondary infections in a minority, of infected cats.
Laboratory Diagnosis
FIV infectton ts most eastly diagnosed by detecting anti-
bodies in the blood. Antibody to FIV can be detected using
ELISA te:>Ls, Weste1 n i1nn1unobloLLing, alld indi1ect nuores-
cent antibody (IFA). ELISA is often used as a first screening
test, followed by Western blot as a confirmatory test. fIV in-
fection can also be diagnosed by virus isolation and poly-
mcrasc chain rcaction (PCR) to detect FTV nucleic acid.
Young kittens may be ant1body pos1t1ve (and thus have
a positive test result) without actually being infected with
FIV due to passive transfer uf FIV antlbudies frum their
rnothPr.
Treatment and Control
Treatment of f.TV-associated disease is largely supportive.
Secondary and opportunistic infections are treated with
appropriate antimicrobial therapy. Control of FIV infec
tion is by avoiding contact with stray cats and avoiding cat
fights. Experimental vaccines appear promising.
SIMIAN IMMUNODEFICIENCY VIRUS
Di sea se
Tl1e ~Irnlan immunodefic.iency virus (SIV) comprises a
numhPr of IPntivin1sPs inrligenous in many simian species
living in the >vild in Africa. In their natural AfTica11 sin1ia11
hosts, these viruses apparently cause Jittle orno disease. ln
Chapter 65 Retroviridae 425
contrast, the Asian macaques, which are not infected with
SIV ir1 the wild, are susceptible tu a fatal immunosuppres-
siv<> .~yncl rome c:allecl simian A l/)S (SA ff)S) when inferterl
by sorne strains of SIV.
SIV-infected Asian macaq ues often develop a transient
skin rash soon after infcction. Lymph nodcs und splccn
may be inítially enlarged. r11e architecture of Iymph nades
becomes disrupted and eventually atrophies. ·rhe main
cUHical features uf SAIDS in Asian macaques are wasting
ancl persistent diarrhea. Opportuni~tic infertions orrnr
and often persist in the immunocompromised monkey.
Virtually aJI Asían macaques infected with pathogenic SIV
strains dcvclop fatal SAIDS within 2 months to 3 years.
·rhe tatal immunodeticiencv discase caused by SIV in
Asian macaques is the major animal model for AIDS in hu-
111a11s. Further, an awareness of the hlology of SIV ls Impor-
tant for thP. oc:c:upational hPa lth of animal Ci'lretakers,
technicians, a11d veterinarians who handle 1nonkeys, as
well as use of primates in biomedical research and medi-
cine. Artificially generated Simian/Hurnan Immunodcfi-
ciency virus (SHIV) strains of virus have been constructed
that contain HIV-derived envelope genes with SIV-derived
g1::r1u11res tltat llave beer1 used extenslvely In the laboratory
for studying immunity and pathogpne~i~ to HTV envPlope
proteins in a nonhuman primate modcl system.
Etiologic Agent
Classification
The primate lentiviruses existas a bread continuum. For
example, the prototype SIV Lsolate from Aslan macaques
(rlP~igna terl STVmac) is only about 50% related to HIV-1,
but is 75% related to HIV-2 based on nucleic acid seque11ce.
Other STV isolates from chimpanzees (SIVcpz) are much
more closcly rclatcd to HIV-1 than to HIV-2. Further, sorne
SI V isolates trom other Atri can primate species are even
closer to HIV-2 than is SIVmac. Many isolates of SIV and
HIV have been made and thelr nucleic actds sequenced
and classified in phylogenetic trees of sequence related-
ness in an efforl to u11dersland Lile origin of AIDS a11d Lhe
diversity and epiderniologic potentials of the primate
lcntiviruses.
Physical, Chemical, and Antigenic Properties
Thc morphology and physical properties of SIV closely re-
semble those ot other Jentiviruses. Sl V has an SU glycopro-
tein of 120kd and a TM glycoprotcin of 32 kd, and Gag
proteins, MA, CA, and NC, of 16, 28, and 8 kd, respectively.
SIV also cedes for severa) nonstructural proteins that
funclion in regulallou u[ vinil 1.:xpressiuu anll accessury
functions .
The RT of S!V has a preference for Mg'-+ and uses tRNALys
as a primer tor negative-strand DNA reverse transcription.
On the basis of seroepidemiologic data, as many as 30
d1Stlnct SIV strains may be harbored in their African mon-
key hosts. The prototype SIVmac strain is antigenically
11ro1e clu:;t:ly relatec.I tu HlV-2 than to HTV-1, in agreement
with the sequenc:e homology ovPrall. STV isolated from
 "'°"""
Translllissible Spongiforlll
Encephalopathies
BRADD C . BARR
Transmissible spongiform encephalopathy (fSE) is the col-
lecttve term given to a unique gro uµ uf µrugre:ssi vt:, u11 i-
form ly f;:it;:i 1, ctegener;:itive, central nervous system (C~NS)
diseases of humans and a11imals that share a similar pat-
tern of clinical disease, neuropatl1ology, and etiology.
While sorne human 'fSEs are inherited or sporadic, most
TSEs are 1nfectious diseases t hat can be transmitted to sus-
ceptible hosts. For many years the identification of the in-
fectious cause ren1air1ed elu:sivt:, lJul Sta11ley Prusi11e1 pos-
t11l;:itect in 1982 that an ahnormal host protein called a prion
(derived from protein.aceous ínfectious partícals; abbrcví-
ated as PrPRes) >-vas the infectious agent anci that propaga-
tior1 of this protein was a post t ranslational event, not re-
quiring nucleic acid or genetic material. Prusiner's prion
hYI)Othesis incorporated mechanisms to explaín both tl1e
heritable and infectious disease presentation:s uf the TSE:s.
Since his initíal proposal, a large bociy of t>xq11isite research
work by Prusil1er, colleat,rues, and other researcl1crs (work-
ing mostly 'With sera pie) has been amassed that supports
bis original hypothesis, and Prusincr rcccivcd the Nobel
Prize for this work in 1997. ·rhe concept of the prion as an
infectious agent is now widely accepted, and few believe
that genetic material still may be found. Counter hypothe-
ses in elude 1) the virion hypothesis, which suggests the in-
fectious org<11lisu1 l 1as a $111all ce11lral core of nucleic acid
protected and/or surrounded by protein; and 2) sorne sort
of unconventional virus. Givcn thc vast amount of rc-
search work supporting the prion hypothesis, and the ap-
parent lack of data supporting the other hypotheses, the re-
mainder of this discussion is based on the premise that the
prion is the infectious etiology for this group of díseases.
·rsEs g(;!JJl:!rally ha ve a lin1iled hosl ra11ge. With one ap-
parent exception, the human 'fSEs are limited to humans
(excluding experimental studies). Thcy includc two forms
of C~reutzfeldt~Jal<ob disease (C.JlJ), Gerstmann-Straussler-
Scheinker syndrome (GSS), fatal insomnia (FI), Kuru, and
newvariant CJD (vCJD), which is the exception because of
its relationship with bovine spongiform encephalopathy
(BSE). So1ne of these human 'rSEs (farnilial CJD, GSS, a11d
fatal familia! insomnia) are inl1erited, while sporadic C.TD
occurs spontaneously in a small pcrccntagc of humans.
·rhe a11ima1 ·r::;i::.s are ali considered to be infectious and,
with t h e notable exception of BSE, they have a narrow host
range. The animal TSEs and their respective natural hosts
íncludc scrapie in sheep and goats, BSE in cattle and other
YlTAN CHlTNG ZEE
species (exotic ungulate species, felids where it is known as
feli11e spongiforn1 encephalopalhy, and co111pelling evi-
dence to suggest it is also responsible for vCJD), transmis-
sible mil1k encephalopathy (TME) in inink, and chron.ic
wasting disease (CWIJ) in deer and e.lk. While there are
m.any similarities shared by all TSE d.i seases, there are also
salient differences in the behavior of the various TSEs,
most notably related to t heir tissue distribution and d.etec-
Lio11 wilhín hosts, ll1eír n1ea11s of transn1ission 1 a11d very
importantly the zoonotic potential of BSE. Scrapie, t he
prototypi.cal TSE has bccn studicd most cxtcnsivcly.
SCRAPIE
Disease
Scrapie is a chronic, progressive, and. uniformly fatal de-
generative CNS disease that occurs naturally in only sheep
and goats. ·rhere is no evidence to indicate that sera pie is a
zoonoses. Scrapie occurs throughout the world, \>Vith a few
exceptions (e.g., Australia, Nl:!\-V Zt:ala11d). The disease íncí-
dence, h;:ist>cl on c11rrent detection methodology, is low in
endemic countríes. Scrapie is caused by an abnormal prion
protein designa ted PrP5 c (PTion Protein. Scrapie). Within
tl1e world's sheep population, there is a varying degree of
susceptibility and resistance to scrapie following exposure
to PrPSc_ The reasons for this diversity in disease suscepti-
bility are complex and poorly understood, altl1ougl1 sl1e::ev
breed ;incl gf'notypt>, ;:is w~ll ;:is Prrsc str;:iin diversity, likely
ali play son1e role. For sera pie the genotype of individual
sheep at three specific DNA sites encoding amino acids
within thc prion protcin scquence is a major factor in de
termining the degree of d1sease susceptibility and/or re-
sistancc. Like all TSEs, scrapie has an extremely long incu-
batlon period fron1 infection to the onset of clinical signs,
rangíng roughly from 24 to 60 m ontbs. The progression of
cli11ical :sigu:s is dirt:ctly correlaLed with Lhe progressive ac-
cumulation and spread of PrP~c throughou t the CNS, and
is accompanicd by a uniquc pattcrn of dcgcnerative and
vacuo lar lesions in the CNS that is shared by alJ TSEs.
Sera pie is spread by direct contact with infected sheep,
probably through oral infectlon (Le., lngestlon) of Prrsc. It
is also possible that natural infectio11 could occur húre-
427
428 PART 111 Viruses
quently by direct il1oculation, such as through scarified
mucous membranes. Scrapie can be experimentally trans-
mitted by inocu.lation, including transn1issio11 through
large volume bloocl transfusions from infected sheep. This
suggests there is a low lr.vt>l of Prrsc in tht> hlon<l nf srr;;ipie-
infected sheep that cannot be detected by other means.
Natural co11tact transmission occurs most frequently when
naivc shccp or goats are cxposcd to infcctcd ewes shedding
PrP5<- at, or soon after, lambing, and this trans1nission likely
results from tl1e expulsior1 of PrPSc infected placenta and/or
contaminated fetal/uterine fluids. ·r11ere is as yet no clear
evidence of true vertical transmissio11 (either true genetic
transn1ission or i11 utero tra11s111ission). La111bs appear to be
more susceptible to infection than adults. The tern1 "mater-
nal tra11smission" has been adopted to define transmissi.on
trom intected ewe to ottspril1g at, or shortly alter, .Lambing
and to differentiate it from vertical transmission. Epiden1i-
ologic studies suggest that indirect transmission of scrapie
also occu rs, through exposure to contaminated environ-
n1enLs w here scrapie-infecled sheep llave previously bee11
kept. This likely occurs more often where tl1ere has been
prcvious high-density confincmcnt of scrapic-infcctcd ani-
mals and especially \.Yhere previous lambing has taken
place. One study suggests that scrapie can be transrnitted
vía hay mites in contaminated environments.
1'he clinical signs of scrapie are progressive and co11sist
of 011e ur 1uore of tl1e folluvviI1g CNS :;ig11:;: l>chaviorcll
changes, locomotor prohlems (i ncoordination, paresis,
proprioccptivc limb dcficits, l1ypcrmctriél), mild 11cud or
neck tremors, hyperesthesia, and pruritis, often resulting
in patchy wool loss from r ubbing or biting. Behavioral
changes include withdra;val fro1n tbe flock, nervousness,
or aggression. Scrap ie-infected sheep also progressively
lu:;e body conclitiun. A classic behavior often used to aid in
clinical d·i agnosis of scrapif' ronsists of opwarcl t>Xtt>nsion
of the head and neck witl1 a.11 accompanying licking, nib-
bling motion, or t eeth grinding in response to rubbing the
sheep's rum p region. However, it should be en1phasized
that the particular clinical signs seen 111 any individual in-
fected animal are highly variable.
Etiologic Agent
It is generally accepted that the etiology of scrapie is a
p rion, an abnormal isoform of a normal host protein of
sheep (PrPS<"). PrPSc is very similar in its size and shares
many similarities in its amino acic.l sequence and biochem-
ica I physicochr.m irill p ropt>rtit>s to ilhnormill prion pro-
teins responsible for other TSEs. Mu ch of the following ex-
p anded discussion ot Pr Ps~ (and scrapie) also holds tr ue tor
other abnormal prions and the respective disease t hey
cause. In the following discussion t l1e abbreviation prpRcs
(referring to "resist ant" PrP) will be used to reference all ab-
norrnal p rion p roteins as a group, and PrP5'" to reference
tht> spt>cifir ahnorm;;il prions Ci111sing scrapir..
Prion Origin, Structure, and Biochen1istry
PrrRcs is clerived from normal cellular proteins ("cellular
prion protein" or PrPC) t hat are found in multiple tissues
of ali n1ammalian species. Tl1ey are cell mcn1branc-
associatecl proteins found as a constituent part ot a larger
surface sialoglycoprotein. PrPC is a 35- 36 kDa p rotein that
is especially abundant in the CNS (approximately 50 times
mo re than other tissues) witl1il111eurons a11d glial cells. It
also occurs in cells of the 111011onuclear phagocytic syste111
(macrophages, dendritic cells, follicular dendritic cells).
Thc precise fu nction of PrPC is unccrtain. Possiblc func-
tions include aspects of copper metabolism, interactions
~·Vith the extracellular matrix, and apoptosis and signa!
transduction. Prrc ctoes not cause t1isease. rrr1tc>s are iden-
tical in ami110 acid seguence and lengtl1 to PrPC of the l1ost
species vvl1ere tl1ey 11ave replicated, and differ only in their
secondary a11d tertiary spatial co1úor.mation from their
originating Prrc. Thc tcrtiary conforn1ational changc in
J>rJ>R~s is the putative basi.s for transforn1ation into an in-
fectious pathogen t hat propaga tes and spreads within l1ost
tissues to cause disease. rrrRcs has a protease-resistan t core
(27- 30 Kl)a; 142 amino acids) desig11ated PrP?.7-30, whirh
11as bee11 fou11d il1 braiI1s fron1 hu.r nans and anin1als with
prion diseases. This sn1aller portion of the PrPRes retains
infectivity. i\n even smaller segment of the PrPRes, consist -
íng of 011ly 106 amino acids, .is sufficient for replication of
Pri>Rcs. These smaller segment s of PrPRes are termed
miniprions. 'fhe PrP27- 30 segment of PrPc is derivect from
a single copy mammalian gene in humans and animals
(PRNP a1Hl Pr11µ, re:;µectively) . Wlúle ll1e ¡;recise nalure of
these structural changes hetween PrPc and PrPKes is diffi-
cult to dctcrn1ine, thc c<::ntréll fcélturc thought t o be rc-
sponsible tor the pl1ysicochemical and in.tectious trans-
formation of PrPRes is a change in the portion of an
alpl1ahelical and coil structure of PrPC to a larger percent-
age of refolded and rigid beta-pleated sl1eets in PrPRes (the
percentage of beta-pleated shects íncreases from 3% to
43ºAI v.rith th.e transformation to PrpRcs) . Once .in a suscep-
tible host, the propagation of nevv PrPRcs occurs by a post-
translational event (not involving DNA or RNA), \-vhereby
existcnt host PrPC is transformcd to PrPRes. Experimental
evidence suggests t11at the l'rf'Res actually serves as a phys-
ical templa.te for refolding of PrPC ;vhen the latter approx-
imates the PrpRcs template. The process also appears to re-
quirt> tht> p rest>nce of a second spt>cit>s-specifi< host protein
("X") that binds to PrPc and facilitates the transformation
to ne'"' PrPRes_ Once completed, the i1ew PrPRcs releases
from the tem pla te and can then serve as a new te1nplate for
further propagation of PrPRcs. In t he case of inherited TSEs
of humans, mutations or insertions in the human PH.NP
gene are postulated to cause t he resultant alterecl f'rpC to
spont;;int>o1 1sl y convt>rt to nf'w PrrRes_
Iliocl1emically, PrPC is labile and can be destroyed rela-
tively easily !)y a variety ot insults, such as enzyme destruc-
tion a11d heat. In contrast PrPRes is very resistar1t to .insults
like enzyrne digestion, heat, ultravlolet light, iooizing ra-
diation, acids, bases, certair1 autoclavlng procedures, for-
rnalin fixation, aud disir1fecta11t:;. Becau~e uf ll1e resisla11ce
of PrrRes to dt>_~truction or inactivation, effective proce-
dures to inactivate f>rpltess are extre1n.ely li111ited in n u mber
and very severe in nature, including autoclaving at
13·1 138º(~ for 18 minutes for porous-load and gravity-
displacement autoclavir1g, autoclaving at 132ºC for 1 hour,
exposure to lN NaOH for 1 hour, or both. One authority
 Chapter 66
suggcsts that thc only means to effectively achieve com
plete decontamination involves either exposure of Prl'Res
to strong sodium hypochlorite solutions or to hot solu-
tlons of Na OH. This resista11ce to <.lestructio11 t:X(Jlaius tl1e
potential longPvity of PrrRes in thf. P.nvironment. lt also al-
Jows for transrnissio11 of the ·rsEs even tl1rough conta1ni-
nated feeds that a.re cooked or processed, as well as trans-
mission through thc use of inadequately autoclaved and
contaminated surgical instrument s, or via human/veteri-
nary b iologicals derived from tissues or fluids harvested
from infectell aniruals/hu111ans.
Evidence suggests that not all Prrsc causing scrapie is
ll1e san1e; .catl1er, the1e are n1ultiple Prrsc strains. Initially,
PrPsc strains were discovered through experimental inocu-
lation studies in laboratory animals Vlhere differences were
noted in the laboratory animal susceptibility profile, d is-
ease incubation periods, and tl1e CNS les ion p:rofile within
these laboratory animals. Similar differences have also
hPPn notP.cl following P.xpP.rimP.ntal transmission sh1cliP.s
in sheep.
Host-Prion Relationship
The ho5L-prion relalionships for scrapie and 0 Lhe1 TSEs are
complex, as expected with diseases caused by infectious
agents that actually represent modification of the host
proteins.
Host Genetic Polymorphisms
The susceptibility of sheep to contract scrapie following
p,psc exposure appears to be controlled by a poorly under-
stood co111µlex of faclors Lhal include ll1e sl1eep genolype
(referred to as its genetic polymorphism), the strain of the
scrapie PrPsr. to which it is exposed, and other poorly un-
derstood tactors such as sheep breeá. üne major tactor
that affects both the incubation period and the suscepti-
bility of sheep to scrapie is the sheep's genetic makeup at
codons 136, 154, and 171 of the PrP gene. Each possible
ge110Lyµe al Lhese siles is referred to as a gerletic polyn1or-
phism. At codor1 136, valine (V) is associated with suscep-
tibility and alanine (A) with rcsistancc. At codon 154, his-
tiáine (H) is linked to susceptibility and arginine (.!<.) with
resistance, and at codon 171, glutamine (Q) and histidine
(H) are associated with susceptibility and arginine (l~) with
resistance. Of the possible polymorphic combinatio11s at
lhese Lhree codo11s, only five have bee11 foui1d witl1 a11y
frequency in nature, including: Ai36R1s1R17i. A136R1s1Q17v
At36H154Q17v A136R154H171, and V136R154Q171· Codon 171
appears to be the n1ost significant in determining sheep
susceptibility or resistan ce, at least within North America,
while codon 136 is next in importance and codon 154
seems to be less of a factor. In particular the QJQ polymor-
phisn1 at codo11 171 ls linked witl1 a l1igl1 deg.ree of suscep-
tibi]jty for scrapie, while OJR and R/R polymorphisms at
codon 171 are associatcd with rcsistancc to scrapic. In
North America, scrapie is diagnosed most commonly in
sheep with the QJQ polymorphism at codon 171. Scrapie
has only been very rarely diagnosed in Suffolk sheep with
the QJR polymorphism at codon 171, and only one
Trans1nissible Spongiform Encepha1opathies 429
scrapie positive Suffolk sheep has ever been diagnosed
with the R/R polymorphism at codon 171. Further data
suggests that when scrapie sheep are found with the QJR
µoly1norµJ1i~111 al co<.lon 171, lliese :.lieev al:.o are 111ore
likely to have the A/V polymorphism at codon 1:~6. In
North A.tnerica, the "United States Department of Agricul-
ture sera pie flock clean up program" h.as used these ge11etic
susceptibility patterns to establish criteria for removal or
movement-restriction of scrapie-exposed sl1eep. The crite-
ria are based first on determining the polymorphism of
tlie µositively <liaguost:d scraµie -i11fecte<l sht:eµ . This l1elµs
establish information on the potential strain of S<:Tapie
preser1t . Once the scrapie-infected genotype pattern is
known, criteria are established for removal or restriction of
remaining sheep in the flock with susceptible genetic
polymorphisms, as a means for disease eradication. These
criter.ia are lengtl1y and complex, but salient fea tures in-
clude the following: Jf positive scrapie sheep are Q/Q at
coclon 171, ali rPm;;iining Q)Q sheep in the flor.k are re-
moved or under restricted moven1ent. In rare instances
where scrapie is diagnosed in a OJR sheep, it has been
found almost cntircly in sl1ccp with A/V 136 Q)Il 17t poly-
morph1sms. n 1erefore if scrap1e 1nfect1ons are áetectea in
.fl../V136 QJR171 sheep, then all A/V136 QJR 171 sheep, in addi-
Lion Lo Lhe QJQ171 sl 1eeµ, are Largele<l for re1uoval or re-
stricted movement.
PrP genetic polymorphisms are also docu.m ented in
goats, although there is much less data regarding the im-
pact of specific polymorphisms on susceptibility/resist
anee of goats to scrapie.
Prion Strains
Data f.ron1 various arlin1al studies indicates that strains of
prions can exist within a single prion disease. ()nly one
strain howeyer has bccn idcntificd for many prion dis-
eases. !Jitterent strains ot scrap1e are reportea. ·rhe aata
supporting different PrrRes strains initially carne from two
observatlons: experimental inoculation of different PrPsc
isolates into susceptible hosts resulted in variable clisPast>
incubation tin1es and t he brain lesions induced by these
isolates also differed in both distribution and nature.
Subscqucnt studics havc shown that thcsc isolatcs consist
of different isoforms of the f'rp.Res, although the specific
factors responsible for "strain" differences are unclear. The
strain of prion, which seems to be enciphered in the con-
for1nation of PrPSc, conspires with the PrP sequence,
wl1icl1 is specified by t l1e recipient, to deternú11.e the terti-
ary structure of the nascent PrP5c. This infers that each
strain has a uniquc tcrtiary structurc, but also suggcsts that
the affect each strain has on a .h ost is dependent to sorne
degree on the PrPC amino acid sequence of the host,
\<Vhich is d ictated by the host genome, and which takes
part in determining the final conformational st:ructure of
lhe Prl'Res.
Scrapie Disease Pathogenesis
The putative pathogencsis of scrapic includcs thc follow-
ing: Prrsc is first detected in follicular dendritic cells and
1nacrophages within the tonsils and gut-associated lym-
430 PART JII Viruses
phoid tissues (GAL1; especially ileal Peyer's Patches) after
oral exposure of genetica1ly susceptible sl1eep. l'rrsc multi-
plies at these sites and then spreads througl1 the lymph
vascular sysle111 Lu peri plieral 1y111 ph Lissues, i11cl ud i11g lhe
spleen, and numerous lymph nodes \Vhere it is next de-
tected. PrPs,- may be detected at this time in the retropha-
ryngeal lymph nodes and in lymph follicles of the third
eyelids of sheep. PrPSc also is found in spinal ganglia of the
autonomic nervous system, the adjace11t thoracic spi11al
cord, and in the dorsal vaga! nucleus of the vagus nerve
\-Vithin the caullal IJrain ste111. 1'llis early CNS iuvasiu11
likely occurs through invasion of nerve endings \-Vithin the
lymph follicles of the ileum or elsewhere in the gastroin-
testinal tract and travcls through these nerves of t he auto-
nomic ncrvous systc.m an<l va¡,,'US nerve to the thoracic
cord a11d vaga! 11ucleus of the brain stem, respectively.
Once withiI1 the CNS, the PrPSc 111ultiplies and spreads.
'l'l1e iIH.:reast'.U PrPsc produclio11 wiLhin lhe CNS is directly
related to the progressive development of CNS degenera-
tive lesions and the onset of clinical sig11s.
There is no humoral or cellular imn1une response to the
Prrsc bccausc thc protcin is idcnticul in its amino acid se
quence to the host l'rl'c. ·rhus, there is 110 seru1n antibody
test to allow for antemortem detection of exposure, and
cellular infiltrates are not present wlth.i.11 the CNS of af-
fected sheep. Lesions associated witl1 scrapie or an.y othPr
TSE are foL111d 011ly '"'itl1in tl1e CNS (aside froni. seconclary
lesions such as hair loss). These 1esions are bilateral in dis-
tribution and limitcd to tbc gray muttcr arcas. Thcy consist
o t three basic changes: spongiforn1 change, which refers to
vacuolation within the neuropil of gray matter that repre-
sents intracytoplasmic vacuolation of nerve processes;
neuronal degeneration/loss, t"1hich includes intracytoplas-
nlic vacuolalio11 of neuro11s, sl1ru11ken, dark, a11gula1 neu-
rons, rare necrotic neurons, and neuronal loss; and astro-
cytosis or thc hypcrtrophy and hypcrplusiu of ustrocytcs
within the gray matter (see f ig /1 .4) . !he lesions occur first
in the caudal brain stem and progress from there to other
regions of the brain stem, cerebellum, and cerebral cortex.
PrPSc c;in hf' il111str;itpcl f'Vf'n i1rior to cl f'tf'ction of r.NS l f'-
sions by the use of irnmunohistochemistry. The PrPs,. de-
posits around and within neurons and glial cells within
the neuropil.
Recent studies u1 pregnant scrapie-infected sheep indi-
cate t bat fetal genetic susceptibility plays a major role re-
gardtng both maternal transmission of infection to the
fetus and th.e potential spread of scrapie within a flock.
Th.e results i11dicate tbat in utero fetal infection does not
occur in infected ewes. I-Iowever, the fetal membranes can
bccomc infcctcd witl1 PrPSc during gcstation, scrving us a
source for shedding of Prpsc int.o the environment both at
and for sorne ti me after Jarn bi11g. Whether the placenta be-
comes infecte<.l is controlle<.l by the fetal ger1etic puly111ur-
phism. Asan examplP, Prrsc tv;is fo11ncl in pl;icenta of in-
fected ewes where the fetal genot ype was c¿Q at codon 171
but was not detected in the p lacenta if the fetus was a re-
sistant QR gcnotypc ut codon 171. At lambing, place11tal
.Pr¡>St: is a high risl<. source for infection, and very young
lambs witl1 susceptible genetic polymorphisms are at the
higl1est risl< for infection. The reason for this increasetl sus-
ceptibility of young anirnals is nnknown, h11t it is theo-
rized that the more developed GALT in. young anirnals
(which atropllies in older animals) may be responsible.
Cross Species Barriers
for most TSEs, the susceptible host spccics rangc is vcry
limited and specitic. ·r11e barrier limiting cross species
transmission is termed the cross species barrier. Experin1en-
tally, these cross species barriers can often be overcome,
albeit. with so1ne difficulty. The factors that are used to pro-
1110Lt: Lraus1Hissio11 across ll1ese barriers are direct intra-
cerebral inoculation of the candidate PrPRes in to the resist-
ant host species and rnultiplc serial passagcs of infcctcd
CNS tissue through the resistant species. T'l1e exact basis
for these species barriers is poorly understood an<l proba-
bly very complex, but contributing facturs k.r1uwu tu affect
the species barrier incl11clP:
l. 'rhe degree of homology in the ami110 acid se-
quence of the llonor anu recipie11L oI PrP. This ge-
netically deterrnined host PrPC amino ac.icl se-
quence is ilnporlanl as it is thought to have a direct
affect on the abi1ity of PrPC to refold in a manner
that rnirnics the PrPltes tertiary conformation.
2. The strain of prion. As 1nentioned alJove this is enci-
phered in the conformation ofthe PrPRes.
3. The species specificity of the helper "X" protein.
This may also be an important deterrninant in the
specie:> barrier.
As a side note, evidence now suggests that the process of
experimentally adapting the ca11didate PrPRes across the
previous cross species barrier actually creates resultant
modifications of the origin.al Pr.PRes, ;:inc1 th11s PSSf'ntially
creates a oew strain of PrPRcs. For this reason, PrPll.es isolates
are now designated not only according to their original
natural host, but ulso by any spccies tl1rough. whjch it has
been adapted experime11tally.
Subcfinical Carriers of lnfection
1'here is sorne experiment al evidence to suggcst that,
under certain circu1nstances, and >vith certain PrJ>R~•s from
certain TSEs, PrPHes in fcctions can result in subclinical car-
riers that do not develop disease. ln addition, it may be
very difficult to detect the PrPRes in these carriers except by
bioassay. As a11 exau1ple, exµeriu1e11lal i11(ecLion across lhe
spPc.ies harrif'rs in rodents rnay require multiple serial pas-
sages through tl1at rodent species. In such instances fol-
lowing initial inoculation of the resistant host, the l'rl'R~
may not be detected, and t he host may live i.ts entire life
wit hout contracting a ·rsE. e ·o;vever, when brain material
from such a host is subsequently serially passed tl1rough
tl1at sa111<:: ::.pecie:;, a TSE disease n1ay eve11tually result as
th e cross species barrier adaptation results.
Laboratory Diagnosis
Severa! diagnostic tests for prion clisease have been devel-
upeu fur speclfic deleclion of f'rpRes ü1 tissues. These tests
rely on the use of polyclonal or monoclonal antibodies to
 Ch.apter 66
thr. PrP protr.in . Since these antibodies cannot differenti-
ate between PtPr. and PrPR"\ thc test mcthodologics utilizc
th e biochemical differences between PrPC and PrPRes to
first destroy the PrPC epitopes using digestion or denatur-
il1g procedures (Le., proteinase K or formlc acld digeston,
aod/or denaturation by autoclaving), leavi.ng only PrPRcs
witlt its intact a11Ligenic epitopes. This group of tests,
which includes immunohistochemistry (lHC) and
enzymc-bnkcd immunosorbcnt assay (ELISA) nlethodolo-
gies, has been utilized successfully for detection of PrPRcs
in a variety of TSE diseases.
Scraple can be dlagnosecl anternortern by irnrnur1ohis-
tochemical (IHC) detection of Prrsc in thircl PyPlicl hiop-
sies. ·r11e l>iopsy <.:011::;ist.s of a s111all aggregate of Lhe lyn1-
phoid fo!Hcles that can be visualized and excised on the
inner surface of the tbird eyclid. A ncgativc test rcsult <loes
not completely ensure the animal is scrapie free for the fol-
lowing reasons: t he animal may be in the early incubation
stages of infe<.:tior1, or represent 011e of tl1e su1<:lll per<.:e11t-
age of clinically affected sheep where Prpsc is not present/
detectcd in lym.p h tissue p rior to CNS infection; or t here
may not be sufficient lymphoid tissue present (this seems
to occur more in very aged animals and possibly in sorne
sheep breeds more than others).
The postmortem diagnosis in clinically affected ani-
mals is often made by hlstopathologic evaluation of the
hTain (<.PP Pig 71 .4) ThP PXtPnt of thf> 1Psi<ins, anrl therPforf>
the accurac-y in diagnosis through routine histology, is di-
rectly related to the severity of the clinical disease at death.
Treatment and Control
There is no treatment available for scr.apie, so thP rlisPasP is
llaJ1dled by prevenlion, co11trol, or eradication. In North
America there is a federally regulated program t o eradicate
the disease, which includes:
l . A "S<.:rapie Flo<.:k Cerlificalion Progran1" ll1al n1011i-
tors flock disease status over an extended period
with the goal of assigning status to flocks with no
evidence of scrapie. ·rhe program has requirements
for individual animal identification, record keep-
ing, reportin.g, and restricrtons on flock addítlons to
ensure scrapie is not introduced into flocks free
froin deLeclable serapie.
2. The eradication of scrapie through disease survejl-
lance, and diagnosis, identification of infcctcd/
exposed tlocks, elimination of positive or suscepti-
ble/exposed animals, ar1d monitoring of sheep
movement to control potential spread of scrapie
and to allow for .successful trace-back from posi-
Li vel y liiaguu:,eli aui111al:. lu Lliei1 IluLk:. uf u1igi11.
Within an individual flock, determinatior1 of the flock
status and strict limitations on the addition of new ani-
111als are tl1e best 111ea11s for preventi.ou a11d con trol. Ini tial
determination of tbe flock status can best be obtained
through thc Scrapic Flock Ccrtification Program. Main-
tenance ot a disease-free fJock is achieved by maintaining
a closed flock, particularly a closed ewe flock. If n.ew intro-
ductions are to be made, care should be taken to ensure
'lransmissible Spongitorm Encephalopathies 431
that new additions come from flocks with clisease-free sta-
tus a5 can bcst be dctcrmincd (oftcn difficult). ·rhird cyclid
testing of animals over 14 months of age can be considered
in making this assessment but is not foolproof. Genotype
testing to select for new a<l<lítions with resístant ge11otypes
is ;:ilso 11sr.f11 I, although researc:h has not elim inatr.ci thr.
possibility of a resistant genotype carrier animal. ·rh.e feed-
ing of ruminant ineat and bonemeal to rumjnants is pro-
hibited in many countries.
80VINE SPONGIFORM EN CEPHALOPATHY
Disease
Hovine spongiform encephalopathy (HSE or "mad cow dis-
ease") is a chronic, progressive, and uniforrnly fatal degen-
erative CNS Jisease tltat o<.:<.:urs 11aturally iu <.:attle, exotí<.:
ungulates, and domestic and exotic felids, and is the likely
cause of vCJD, a newly recognized TSE in hun1ans. BSE
emerged as a newly recognized TSE in England in 1986.
The inciden ce of BSE in cattle rapidly increased, Jargely in
dairy cattle throughout the United Kingdo·m (U.K.) over
the next few years. Shortly thereafter, additional new TSE
diseases were then recognized in both captive exotic ungu-
liítP<; anrl ff>lirl.s from zoos in F.ngl;:inrl, ancl ;:iLso in rlomPstir
cats in the U.K. Experimental studies and characterization
of the PrPRcs from this latter group of prion diseases estab-
lished that PrPBSE was the cause, indicating that 13SE, un
like ottler T'SEs, had an unusually \-Vide susceptible host
range. Significantly, vCJD also emerged in a small number
of young hurnan patieuts íu EuglauJ at tl1e sau1e:: Lüue.
lJnlikr. sporaciic: C'JD, vC:JD occurs in humans ata much
younger age and has a diffcrent time course, EEG pattern,
and pattern of clinical CNS slgns. Sheep, pigs, primates
(macaqucs), marmoscts, lcrnurs, a11d mice are experimen-
tally su sceptible to BSE infection, altl1ough natural occur-
rence of disease has not been described in these species.
Sheep are susceptible experimentally to oral inoculation
vvith RSF., whirh raisPs suhst;:int ial conc:erns as to how i1'
would be differentiated from sera pie. Because of the asso-
ciation with vCJD, BSE is considered a major threat to pub-
lic hcalth, which is thc basis for national cradication pro-
grams in all countries where it eXists, and extensíve
national surveillance and prevention programs in coun-
t ries where the disease does not.
Following the widespread detectio11 of BSE in the TJ.K.,
cases of BSE occu1-red in other countries through the ex-
port of infected meat and honemeal, infected live anima Is,
and possibly other infected animal products. As of August
2003 a total of 23 countries had reported cases of BSE.
These include severa! cou.n tries in Europe, as well asJapan,
Israel, and Canada.
Epidemiologic analyses strongly suggest that the pri-
111ary n1ode of i1alural BSE transn1ission is Ll1rougl1 Lhe il1-
gestion of contaminated meat and bonemeal contai11ing iI1-
fcctcd offaL Horizontal transmission from animal to animal
does not occur to any signiticant extent. Young cattle ap-
pear more susceptible to infection than adults, and there is
an i.ncreased risk for BSE among calves born from BSE-
432 PART III Viruses
affected cows, suggesting a lovv leve! of 1naternally associ-
atcd transmission. Howcvcr thc rncchanisrn rcsponsiblc for
this in creased calf riskis unknown (i.e., what role does direct
cow-to-calf n1aternal il1fection vs. ge11etic predisposition vs.
feed-borne exposure play?). There is no strong evidence to
suggest tr;:insm ission from a contamil1ated environment, al-
though the occurrence of a sn1all 11un1bcr of 11ew BSE cases
in the U.K. followi11g the enforced anilnal protein feed ban
in animal$ born aftcr August 1, 1996 is disconcc.rt.ing.
The initial source oft he infected material responsible for
the emergen ce of BSE in the U.K. n1ay never be known. T'"'º
theories have been offere<.I to explain the emergence of BSE:
1) BSE arose from the feeding of meat and bonemeal con-
laining sheep offal conlanlinaled will1 scrapie lo cal lle or
2) BSE arose from a single spontaneous case of BSE in cattle
tl1at \"las thcn rcndcrcd and fcd as contaminatcd mcat and
bonemeal to other cattle. A third theory- that BSE arose
from the feeding of infected offal from sorne other man1-
malian species- has also bee11 proposed. Epidemiologic ev-
idence suggests tl1at a change in the rendering process in
E11gla11d lu Ll1.e Ja le 1970s u1ay llave allowed for incteased
survival of PrPRcss, regardless of the source (offal from in -
fected sheep, bovine, or other species), increasing exposure
of cattle to these PrPRcss. Additional epidemiologic data
support ing scrapie as the lnltiaJ sou.rce for BSE incl ude the
fol lowing: the sheep population increased significantly in
the 1980s, when BSE likely emerged, possibly increasing
the prevalence of enllern ic :;crapie in the U.K.; anll meat
ano honemeal was ;:¡ cheap feed so11rce liste<i for ct;:iiry c;:ilf
starter feeds. I Iowever, it is difficu lt to explain by these
point source theories (whether it arose initially fron1
scrapie-infcctcd sl1ccp or a BSE-lnfcctcd cow) how BSE si-
multaneous1y emerged at multiple sites in tlle U.K., unless
this single source ,,v as "''ldely distributed. Once BSE infec-
tions were established in the cattle population, tra11sm1s-
sion c.011 Id he e;:isi ly ;:implifiect hy repeated recycli ng of RSF.-
infected bovine tissue into meat and bonen1eal fed to
cattle. BSE soon reached epidemic proportions in the U.K.
The epidemic peaked i11 1992- 93 \.vitl1 37,280 cases diag-
nosed in 1992. Disease eradication and prevention pro-
gi-ams that '"'ere established (see "Treat1n e11t and Control,"
below) have been successful In greatly reducing the num-
hers of cases. 'fr;:insn1ission of RSF. to felicts ;:ind exotic 11ng11-
lates is thought to have occurred through feeding of in-
tected animal protein, and although the source tor vl;JD
(human) is not proven, ingestion of contaminated animal
products is considered the most likely source.
BSE has a long incubation period with a mean incuba-
tiu11 veriuu uf 4-5 year::;. Cli11ic..:aI ::;ig11::; i11c..:lude a Ju::;::; uf
conclitio n or weight loss, couplect vvith one m o re CNS
signs tl1at include behavioral changes (apprehension, fear,
easily startled, depression). hyperesthesia, hyperreflexia,
muscle fasciculations, tren1ors, myoclo11us, ataxia, 11yper-
metria, pruritis, and autonomic dysfunctio11s (reduced ru-
1nination, bradyca rdia).
Etiologic Agent
BSE is causcd by PrPBSE prion with physicochcmical prop-
erties comrnon to all abnorn1al prions. Only one strail1 of
BSE (PrPllSE) has been identified. Unlike scrapie, suscepti-
bili Ly to BSE 11a::; 11ut yet l.Jet:11 G1s::;uciatt:d witl1 a11y cattlt: PrP
gene polymorphism s. There are no detec"table differences
in age susceptibility or incubation periods reported for var-
ious cattle breeds. PrJ>BSE has not been found in sheep nat-
urally infected with sera pie, although the pote11tlal risk of
BSE-infected sheep is recognized. No strains of PrPSc have
been found that are similar to PrPllSE, although the nurn-
1;0:::1 uf :>lla.i.u 1..uUlJJ<1li::.utl::. l1 a::. l;o:::o:::u liu1ilo:::LI.
Host-Prion Relationship
While polymorpllisms in the PrP gene of cattle have been
identified, there is no evidence suggesti ng thP~P pnly rnnr-
phis111s 11avc a11 affcct on d isease susceptibility. Unlike
scrapie, PrPBSE is not readily detected in lyn1ph tissues
prio r to t he onset of CNS infectivity and cllnical discasc.
PrPBst has been detected only in the CN:> (brain and spinal
cord) and retina of naturally infected cattle. Experimental
studies indicate tl1at it does track through Jymph tissues,
but it is detected inconsistently and in relatively small
arnour1t::; as curnpared tu scrapie. Tl1c followi11g is a brief
sumrnary of experimental studies in c.attle.
Following oral inoculation of cattle, PrPRSE is first de-
tected in lymphoid follicles of the distal ileum at 6
months postinoculation (PI) . It can be occasionally
found in the ileum at later PI dates. lt also has been de-
tected in the t onsil between 6 - 14 months PI in one study.
It is first detected in the CNS and dorsal root ganglla at
32 months PI, followed by trigeminal ganglia at 36
n1011 Lhs PI. Prf>BSE has i1ol bee11 delecled in ei ll1er
retropharyngeal, mesenteric, or popliteal lympl1 nodes
of naturally infected cattle. Within the CNS, lt progres-
sively accumulates and is associated with degenerative
CNS lesions t hat are gene rally sim ilar to other TSEs. As
\'\1'itl1 oth erTSEs, there is no immunologic response to the
PrrBSE by the host.
Laboratory Diagnosis
·rhere is no ;:intemortern test c.urrently availahle for diag-
nosis of DSE. Postmortcrn diagnostic tests on the brain are
available that are similar to those tor scrapie (immunohis-
tochernistry, ELlSA, a11d vvestern blot tests on CNS, not
lymph tissue). These tests are utilized in BSE-infected
countries as part of their national eradication programs.
Rouline h islopalhology of ll1e brail1 of cli11ically affccted
anima Is will show lr.sions compatible •vith BSE, hut confir-
mation of the diagnosis requires confirrnatory testing. In
the United States, a national surveillance program tor HSE
is conducted by Animal Plant Health Ir1spection Service
(APHIS) of the United States Departme11t of Agriculture
(LTSDA). The surveillance samples include field cases of cat-
tle exhibiting neurologic tlisea::;e, c..:attle cu11de11111ed at
slaughter for neu rologic. reasons, rahies-negative cattle
submlttcd to public hcalth laboratories, neurologic cases
submitted to veterinary diagnostic laboratories and teach-
ing hospitals, cattle that are "nonambulatory" ("down-
ers"), and cattle dying on farms.
 Chapter 66 Transmis'>ihlP Spongiform Enccphalopathies
Treatment and Control
There is no treatment for BSE. Countrics with confirmed
BSE cases are attempting to eradicate 1t, and countries V\1 here
it is not present institute preventative/surveillance pro-
gran1s Lo prevenl ils occurrence. l11 Ll1e U.K., a series uf key
regulatory measures were taken by the govern ment to stop
thc food-bornc transmission of BSE. 'fhese measures, cou-
plcd with detection, diagnosis, and cradication programs
have provento be very successful in stemming thc cpidcmic
an<.1 urasticany redudJlg the number or bovine cases. lhe
first of thP'>P measures was a ban on the use of meat and
bonemeal in run1 ina11l feed in July of 1988. Tl1i:. \.vas ful-
lowed in September of 1990 by a further ban on the tL'>e of
specificd bovinc offal in fccd stuffs of any species; these were
bov1ne tlssues thougl1t to harbor the highcst concentration
of infective material. Whlle these measures resulted in a re
ducliu11 i11 new cases, eviden ce suggested that new feed-
borne cases c:ontin11ecl in i'lniméllS, and in March 1996, thc
feed ban was extended to a total ban on the use of 111a111-
n1alian proteins in feed produced for any farm animals.
( HRONIC WASTING DISEASE
Disease
Chronic Wasting Disease (CWD) is a fatal ch ronic progrPs-
sivc dcgcncrative CNS disease of mule dccr, white tail deer,
and eJk that is tound primarily in North Amcrica. ·rhere
are no reported human 1'SE cases associatcd '"'ith CWD.
CWD was ñrst diagnosed in 1967 in Colorado. The disease
was subsequently recognized asan endemic disease in deer
a11d clk íu <111 area that encompassed portlons of Colorado,
Wyoming, ancl Nehri'!ski'I. ThP rangP and incidence of the
disease began to increase after 1990. Thc rcasons for lhis
suddcn sprcad are not clear, but both unique features of
thc discasc and thc husbandry of captivc cervids likely
contributed. CWD can spread laterally vía direct contact
transmission, in distinct contrast to BSE where contact
trausulisslon <loes not appear to occur. ·rhe lateral trans-
1nission of C:WI) i'lppi>i'lrS to be even more efficient than
that of scrapie. 'l'he source of the shcdding P1rcvvo fru1u
infected animals and mode of inoculation of uninfected
animals are not known, although oral inoculation is con-
sidered the n1ost likely route of natural intection.
Modeling studies suggest that lateral transmission
through shedding of Prrcwn probably begins even before
the onsct of clinical signs in infcctcd deer and elk. CWD
sprcads fro111 iJ1fecled Lo u11i11fe1...Leu ue1::1 u1 elk whe11
comingled in confined surroundings. To addition to dircct
contact transmission, indircct transxnission from contam-
inated pastures or paddocks occurs, and this indirect trans-
mission also appears to be more effícient than is reported
wil11 :iLT<t¡,>ic. Tllis relative ease of both direct and indirect
contact transmission likPly Pxpl11ins the very high preva-
lence of CWD in sorne infected captivc hcrds (n1ore Lh<:111
90o/o of mule deer over a 2-year period in one herd). The
prevalence of CWD in frcc-ranging ccrvids from endemic
areas can also be high (<1- 1.)%).
433
Aside from the relative ease of transmission, geographi-
cal sp1ead of Lite Llisease 11as alsu been facilitated through
Lransportation of farmed dP.<'r ;incl Plk hi>tween. farms . In
North America (as of 2002), the disease had been detccted
in tree-ranging deer ir1 Colorado, Wyoming, Nebraska,
Wisconsin, South Dakota, Ncw Mcxico, Illinois, and Sas-
katchewan, Canada, and in farmed cervids in South
Dakota, Nebraska, Colorado, Oklaho1na, Kansas, Minne-
suta, Montana, Wlsconsin, and the Canadian provinces of
Alhf'rta i'!nc1 Si'!skatchewan. lt has also been diagnosed in
farmcd cervids exportcd to lb.e Republic of Ku1ea. Mal1::1-
nal transmission may occur (there apparently is no dircct
evidence whether maternal transmission docs or does not
occur), although modeling studies suggest that CWD epi-
demics cannot be explained solcly by maternal transmis
sion. I11 Ll1e wild, it is pussible thattransmission might also
occur from clecay of \.WD-infi>ctPrl c;ircasses, with release
of thc Prrc:wn into the environment through scave11ging,
and maggots, exposing foragin g cervids.
Experimental CWD infections havc bccn documented
in cattle, sheep, and goats, but only following intracerebraJ
inoculation. There appears to be a substantial species bar
ric1 belwee11 Llte:.e ho:.1: speLies and lnfection with PrPcwo.
1 atura] transmission from clPPr or Plk to these species has
not bccn documented. Therc is no evidence of experin1en-
tal transmission to cattle either by direct contact or oral in-
oculation more than five years following cxposurc.
Based on cxamination of natural cases, cw lJ is though t
to have a m.inimum incubation period of 16- 17 months,
which i:. a11 apµre ciauly shurter tncubatlon periocl than ei-
ther scrapie or RSF.. Thi> clinici'll '>iens in cleer or elk with
CWD include loss of body condition and 011e or n1ore of
the tollowing CNS abnormalities: behavioral changes
(changes in interac..1:ions with handlcrs, walking pattcrns,
depression, lowered head and ears), polydipsia, polyuria,
increased salivation or drooling, incoordination, ataxia,
head tremors, wide-based stance, and hyperexcitability.
HowPvPr, because clinical signs may not be observed in
free-rangir1g deer, the salic111 findings al 11ecrupsy u1ay iI1-
clude Joss of condition and dcath dueto aspiration pncu-
rnonia, possibly duc to dy~phag i a or ptyalism.
Etiology
CWD is caused by ll1e P1Pc"vo priu11, wllich has physico-
chemical properties similar to other ahnormal prions. l 'he
origin of PrrDVDwill probably nevcr be known, but might
include a spontaneous mutation in a cervid or cross
spccics adaptation following initinl cxposurc of deer or elk
ro scrapte. Strain-typ1ng stuc11es 1n mice indicate tt1at
PrPCWD is unique frotn severa! strains of scrapie, BSE, and
Transn1issible Mi11 k EuccphG1lopat~1y (TME) agents. ·rhere
is no currcnt information rcgarcling PrPCWD strains.
Host-Prion Relation ship
Multiple deer PrP gene polymorphisms have been de
sc1ibed, buL the affelts uf these polymorphisms on discasc
susceptibility is unknown . 'rhe rathcr uniform CWD sus-
434 PA irr TTI Vi ruses
ceptibility noted in captive <leer would suggest that resist-
ant gcnorypes, if p resent, are infrequent.
Lik.e scrilpie, Prrcvvo is rt>ilc1ily c1Ptt>ctahle in lym p hoid
tissuc:s throu ghout t h e body of infcctcd dccr a nd clk, nn d
the pathogen esis of tissue distribution shares many simi-
larities to scrap ie. Following oral exposure, Prrcwo is de-
tected first in lymph tissucs prior to detection in the brain.
Experimentally it has b een found in the Peyer's patches of
the lleum, ileocecal lymph node, tonsll, anc.l retropl1ary11-
geal lymph node by 42 days ;:iftf'r inff'rtion. lt is first dc-
Lecled in the brain within the dorsal nucleus of the vagus
nerve locatcd in the caudal brain stem (obex), similar to
sera pie. In onc study, Prrcvvo was first dctected in the obex
:~ months touowing n rst aetecnon in the GALT. Lesíons
specific for CWD are con fined to the CNS and are similar
in overall nature to other T.SEs.
Laboratory Diagnosis
There is no antemortem diagnostic test for CWD. Postmor-
tem diagnostic tests available are similar to those used for
scrap1e, and sorne of the monoclonal antibodies used for
dctection of scrapie in the United States also are utilized
for dete<.:tion of PrpC\\'D. A pre:.u111plive diag11osis ca11 also
ht> mac1P hasPrl o n routine histopathology in clinically af-
fected anin1als if sufficient CNS lesion dcvclopmcnt h as
occurred . Confirmation of the diagnosis through ad di-
tional tests con firms the diagnosis.
Treatment and Control
There is no trcatmcnt for CWD. The long incubation pe-
riod, resistant etiologic agent, absence ot reliable ante-
mortero diagnostic tests, and incomplete unciPr<;tanc1ing
uf t lie 111ude of Lrans1nission all co111plicate effo rts to con-
trol oreraclicate CWD. Quarantine and depopulat ion of af.
fcctcd hcrd s ¡¡re prim¡¡ry mcont. for control in fa rmed fa cil
itlcs. Eve11 with depop ulation of infcctcd farmed facilities,
thcrc is concern of reinfection follo,.ving placement of n ew
uninfected sto<.:k, due to possiblc cnvironmental cor1tarni-
nation . There also are concerns for spri>acl from in fectPcl
fd1111eu facililies Lo co111iguous free-ranging populations
across fence lines or, conversely, from endernic-free popu-
lations to farmed facilities. An active, cfficient surveillance
program is theretore recommended in far med facilities,
not only to identify and remove infected animals, but also
to prevent movem ent of tnft::<.:tt::cJ au1 u1al:. Lu uLhe1 fcu1ueu
facilities. ManagemPnt of frPP-rangi ng populations is even
n1orc problen1atic. Cu rrent management involves active
surveillance to determine p revalence, coupled with con-
tai n ment and reduction of prcvalcncc tl1rough culling in
focusect end ernic areas. Human-fac1litated relocation, or
feeding of free-ranging cervids, is banned in endemic
areas. Selective cu lling of cUnlcal animals l.Jy itselI lla:. 11u1
significantly affected prevalence in endi>mic ar~as. A pro-
graIU uf localiz.ell populalio11 reduction in Colorado has
hPPn instituterl, hut the effectivencss of this program has
yet to be determincd. Tt is thought that a n aggrcssivc pro-
gram of selective culling or general popu lation rcduction
may be effcctive in regions where ncw cases of free-ranging
infect1on are detected early, and prior to establish1nent of
endemic p opulation infections. 'fhc potential for trans-
port of h unter-killed in fected <.:<tr<.:a~~e~ to u1liufec led areas
and subsequcnt co11tamin;:ition of t h esc n ew environ-
1ne11Ls vía discardcd infected offal also re presents a signifi-
cant concern for potential spread of CWD to n ew regions.
Thcrc is a USDA program currcntly in place to eradicate
cwu from farrned elk operat1ons 1n the United Statcs.
 PART IV

Clinical Applications
 Circulatory Systeni and
Lyniphoid Tissues
RICHARD L. WALKER
The circulatory system includes the blood-vascular and
lymphat1c systems. The blood-vascular system is composed
of t h P. hP.ílrt, ílrtf'ri f'~. c;:ipillaries, veins, <1nd components of
blood itself. Infections involving the pericardial sac, wl1ich
enclo:;es the heart, are also included in this chaptP.r. ·rhf'
lymphatic systcm includes lymphatic capillaries, afferent
lvmphatic vessels that drain interstitial fluid and cells from
tissues, lymph nodes, and efferent lymphatics that recircu
late lyrnph and cells (primarily lymphocytes) from lymph
nodcs to the blood-vascular system. Because of the func-
tional associalion wilh a11d cellula1 lraffil.:ki11g tl1rough tl1e
drculatory system. infections involving othcr lymphoicl
tissues (splccn, bonc marrow, and thymus) are included in
this chapter. Mucosa-associated lymphoid tissues, al-
though less organized, are included with other lymphoid
tlssues. 1·he role of mucosa-associated lymphoid tissues in
imrnune surveillance and as potential cntry sites for sorne
palhogens in LO lile hOSl art:: Sj..lt::Cifil.:ally ui:;cus:;e<l ill tl1ose
chapters on systems where they are found or chapters cov-
ering specific agents involved.
While functionally similar, there are anatomic differ-
ences in the organization of lyrnphoid tissucs of animals
and b1rds. Bí rds possess a Bursa of Fabricius, a lymphoep-
ithelial organ, which is dorsal to and comm unicates with
the cloaca. It IS a primary lymphoid organ serving as the
ni;i in ~itP nf H lymphorytP rliffprpntirition Fr.r t h e lnost
part, poultry do not have lymph nodes but rat her rely on
cecal tonsils, Peyer patches, and Meckel's diverticulum in
thc intcstincs; lymphoid follicles in various organs; t he
spleen; anda special paranasal concentration ot lymphoid
tissue (Harderian gland) for secondary immune functions.
Antimicrobial Properties
Phagocytic cells in the spleen and livcr providc thc pri-
mary defense for the vascular systcm by removing poten-
tial pathogens from circulatio11. Depending on the loca-
lilH 1 .l11 tl 11.: vascular systern anc.I severtty of lnl ury caused
directly hy an infectious agP.nt or its toxin oras a result of
the subsequent inflammatory response, ability to repaiJ:
damage is variable. Cardiac muscle has Jittle regenerative
capability following infectious proccsscs that rcsult in rny-
o cardial cell death. Such injury leads to scar formation.
Injuries to small vessels that destroy cndothelial cells can
resull iI1 tllro111l>us forrnation in those vessels. Unaffected
vascular endothclial cclls at thc pcriphcry of the lesionare
able to proliferate and reendothelialize those areas that af-
fcctcd vessels supplied.
T l11.: ly1nphoid tissue plays a n1ajor role in defense of the
hody from infection. It is central to the development of
the anin1al's inunune syste111 (priu1ary lymphoid tissues)
and plays an ongoing role in immunC' survf'ill;:incf' t1nrl rlP-
fense. Details on mcchanisms of immune defense pro-
vided by lyrnpho1d tissues are covered in Chapter 2.
Beca use of the surveillance role it plays, lyrnphoid tissuc is
exposed to numerous potentially pathogenic microbes,
which if nnt ront;:iinPrl m;:iy involve the lymphoid tissue in
the diseasc process or cause a systen1ic infeclion.
Transient and Persistent Microbes
As a rule, the clrculatory system and lymphoid tissues are
not rf'cogni7ed to have a normal roicrobial flora. Transient
bacteremias do occur, oflen as a 1esull of Lrau111alic or il1va-
sive events (e.g., dental extractions, endoscopic proce-
durcs of thc digestive tract, and urethral catheterization)
or subsequent to treatments that compromise mucosal
barriers (e.g., chcmothcrapy, radiation thcrapy). This al-
lows normal mucosal inhabitants to enter t he blood-
stream. Chronic condition s, such as severe gingival dis-
ease, Ll1a Lcon1p10111ise Lhe 11vru1al hv:>t l>arriers rnay al::;o
lead to spontaneous bacteremia. In sorne inst;:inc.P.s, no
predisposing event or conditioi1 is recognized Lo accounl
for a bacteremic episode. The resulting bacteremia is usu-
ally ttansicnt, lasting only a short pcriod of time (less than
30 minutes) before removal by phagocytes in the liver and
spleen. Not ali microorganisms appear to be removed that
rapítlly and may persist in the vascular system for longer
periods. for example, a pPrsistf'nt h11t <:ubclinical bacte-
rcmia with Dartonella species occurs in son1e cats. Baclere-
mias can serve as a source of microorganisms for serious in-
fections of the circulntory systcm (c.g., infectious
endocarditis, bacterial sepsis).
Microscopic and molecular-based studies suggest that
lhe bluvu:.lrc::arn rnay in fact be colonlzed with specific mí-
crobes rather than just hf'ing pf'riodically exposed to organ-
isms through transient bacteremias, and ll1al lhese "rt::si-
dents" persist in the blood in a benign fashion. However,
evidence that a true normal rnicrobial flora of the blood-
stream exist remains to be conclusively demonstrated.
437
438 PART IV Clinical Applications

Sorne viruses, particularly retrovlru:>e:>, persisteutly iu-
fect cells in the lymphoirl ti-.o;11 p ;:ind hlooci for tht> Jife. of
lhe anin1al.
lnf ect ions
Access of microbial pathogcns to tl1e clrculatory systern
occ.:urs through a number of mech;:inio;m<;, incluciing direct
i11uculalíon inlo l11e blood (e.g., insect bites, contami-
nated needles, blood transfusions) or by spread from ini-
tial site of infection via thc vascular systc1n or the lym
phatics draining that site.
Microbial agents that enter via the lymphatic system
may be ell1n1nace<1 or, ac leasr, arresrcd at regiu11al ly111plt
nades, or, if they avoid cont::iinment ::it the lymph nodes,
they sprcad lo tbe bloudstream and can dissen1inate t o
othc>r sites in the body. 'fhe circulatory system, thereforc,
provides a means for delívcry of many microbial age11ts
from their site of entry to thcir ultimate target organ(s) in
the body. Depending on the agent invo!ved, the circula-
tory system itself may oc may not be affected . For many
viral infections a viremia is the p ri mary means of dissemi-
natlon in tl1e host l>ut is ufte11 a clulically li1apparent
event. Similarly, sorne hacteria and fungí in animals reach
their primary or sccondary target organs via thc circula-
to ry system without obvious or major clinical signs of cir-
culntory systcn1 involvement.
Thc focus of this chapter is on infeceions in which the
circulatory system an d/or lymphoid tissues are one of the
pri1na ry si tes affected in the infectious ¡;roce~~. !11 addi lion
to specifir lt>~ions or rliniral signs directly related to the
circulatory system and/or lymphoid tissues, many of these
agents also cause systemic, nonspetific signs that include
fcvcc, anorexia, depression, prostration, and weight loss.
Sorne 1nfeetions of the blood-vascular system present as
ra viuly fatal iufeclio11s \'Vilh few orno premonitory sign~
(c.g., anthrax, hlackleg) . Sorne of the most comn1on
and/or important infectious agcnts affccting thc circula-
tory system and lymphoid tissues of domestic animals and
poultry are listed in Tables 67.1-67.7. Specific chapters on
each of these pathogens should be refer red to for more de-
tails on pathogenesis, spectrum of clinical signs, ::ind thf
¡Jathulugy Lliey L-duSe.
Bacteria! Sepsis
Bacteria! sepsis and septic sh ock are characterized b y vas-
cular collapse and multio rgan failure. Tn addition to tht-
signs specifically related to altered organ pt>rfusion, clin -
Ldl :>i!)ll:> vf :.epsis include f~r, tachypnea, and hypothcr-
mia. The most common agents involved in sepsis are
gram-ncgativc bacteria bclonging to the fan1ily En terobac-
l'eriaceae, altho ugh other gram-negat1ve path ogens (e.g.
Pseudomonas aeruginosa, m embers of the Farnily J>asteurel-
laceae) and gram-positive coccl (c.g., staphylococci, stre¡.r
tococci) also cause sepsis élnd SPptic shock. Sepsis fre-
yueutly uccurs in neonates, and is especially important i:-i
proc111ction animals, horses, and poultry. In veterinary me-
dicine, the mcreasing level of mtcnsivc carc affordcd an.-
mals with debilitating conditions has inc.reased the risk fo:
acquiring nosocomial infections and thus the poten tia! foz
bacteria! sepsis to ctevelop in those patients.
Central to the development of septic shock is lipopoly-
saccharide in gram-negative urg<J1üs111s or other n1a jor ce!..
wall co111.ponents in gram-positive organ isms (e.g., peptido-
gylca11, lipoteicl1oic acid). 'J'hese pathogen-associated mole-
cules bind to receptor signaling complexes on monocytt:"I
and macrophagcs. Toll-likc rcccptors, transmembrane sig
nal transducing coreceptors, play a central role in thL
process. Exposure of cells to these rnicrobial products resul~
in a dysregulation of the lmmune re~pu11~e a11<l tlic vrutluL-

Table 67 . 1. Common and/or lmportant lnfectious Agents of Circulatory System and Lymphoid Tissucs of Dog$

Agent Dlsease

Vi ruses
Canine adenovirus 1 lnfectious cuninc hcputitis Disseminated intravascular coagulopathy, oral petechial hemorrhages, lymphadenopathj
Canine distemper virus Canine distemper Leukopenia, myocarditis in neonates
Canine herpesvirus 1 Canine herpesvirus disease Generalized ecchymotic hemonhages in neonatal puppi~. lymphoid necrosis
lanine parvovirus Canine parvovírus disease Leokopenia, lymphoid necrosis, myocordilis
Bacteria
Bortonc/fo spccics" lnfectious valvular endocarditis Heart murmur, valvular endocarditis
Borrel1a burgdorteri Canlne Lyme borreliosis cardlac arrhyttimias, myocarditis
Ehrlich;a canisb Canine ehrlichiosi~ Anemia, bleeding tendencies, limb edema. lymphadenopathy. splenomegaly
Eryslpefu!/11 ix )µµ. 1t 1 r~,Livu> volvulo1 e111.lvcanliti5 l leort murmur, cmboli formotíon, volvulor endocarditb
Leptospira spp. Leptospirosis Generalized hemorrhages, icterus, septicemia
Neorickettsia he/minthoeca Salmon poisoning Lymphadenopathy, splenomegaly
Rickettsia ricketts;; Rockv Mountain spotted fever Edema, hemorrhages, fymphadenopathy, myocarditis, vascular obstruction

•1nchJdes B. ~insonii subsp ber'mofli and B. damdgeiae.

tioiher Ehrfichia 5J)ecies cause dinical siqns related to anemia, leukopenia, .:nd thrombocytopenia.
 Chapter 67 Circulatory System and Lymphoid Tissucs 439

Table 67.2. Common and/or lmportant lnfectious Agents of Circulatory System and Lymphoid Tíssucs of C.:its

Agent Diseese Circulatory/Lymphoid Associated Findings

Viruses
Feline immunodeficiency virus Feline immunodeficiency Anemia, leukopenia, lymphadenopathy, secondary infcctions
Felíne infectlous peritonitis virus Feline lnfectrous peritonitis lmmune complex vasculitiS/perivasculitis, lymphadenopathy, pericardial ettusion
Feline leukemia virus Feline leukemia AnPmia, lymphoid depletion, lymphosarcoma, myeloproliferative disease, secondary
infections
Feline panleukopenia virus Feline panleukopEnia Leukopenia. mesenteric lymphadenopathy
Fclinc s;ircoma virus ~line sarcoma íibrosarcomas
Bacteria
Mycoplasma haemofelis Feline infectious anemia Anemia, icterus. splenomegaly
fr;¡ncisclfo tuforcnsis Tularemie Leukopenia, lymphadenopathy. lymph node abstt!))~), )¡il~11om~galy
Streptococcus canis Cat stranqles Cervical lymphadenitis. lymph node abscesses
Yersinía pestis Plague Ccrvícal/submandibular lymphadenitis, lymph node abscesses, septicemia

Table 67.3. Common and/or lmportant lnfectious Agents of Cirrul;itory <iystem and Lymphoid Tissues of Horses

Agent Oise¡se(s) ~~~~~~~~

Circulatory/lymphoid Associatcd Findfugs
Viruse)
African Horse Skkness virus• African Horse Sickness V;i1ruliti~ with pulmonary, subcutaneous and eyelid edema
Equine infcctious anemia virus Equine infcctious anemia Ane111id, l 1e111or1 lidye), itlerus, splenomegaly
Equine viral arteritis virus Equine viral arteritis Edema, hemorrhages. leukopenia. vessel infarction
Vene2uelan equine encephalitis virus• Venezuelan equine enccphalitis Ccllular depletion of lymph nodes, spleen and bone marrow
Bacteria
Actinomyces spp. Actinomycosis Mandibular lymph node abscesses
An;;p/Jsma phagocytophila Equine ehrlichiosis Anemia, edema of the legs, hemorrhages
Agents of neonatal septicemiab Neonatal septicemia Septicemia, hypotension, organ failure
Burkholderia maller Farcy, glanders Lymphangitis, lymphadcnitis, splenk absccsses
c.orynebacterium pseudotubercu/osis Ulcerative lymphangitis lymphangitis
Neorickettsia rirticii Potomac horse fever Leukopenia, mesenteric lymphadenopathy
sueprococcus equl subsp. equl Strangles PharyngeaVsubmandibular lymphadenitis/abscesses
Purpura hemorrhagica lmmune-complP.X v;i1ruliti~, PrlPm;i
Fungi
Hi~'toplasma farclmlnosum• Eplzootlc lymphangltls Lymphangitis. regional lymphactenitis
Sporothrix schenckii Sporotrichosis 1ymphangitis

ªClassified as a foreiqn animal disease aqent fn the United States.

btndudes Actinob~ci/11~ Pquulí, Ell'hPrkhi11 rnlí, ~lmnnPll~. 5treptococcus equi subsp. zooepidemicus.

tion of excessive levels ofpro1ntlam1natory cytokines ('l'N1~­
alpha, IL-1, IL-6), which are largely responsible for the sys-
Le111i<.: 1;:ff1::<.:ts ulJservec.l in sepsis. Toll-llke receptors are also
found on endothelial c.ells lining hlc'inrl vessels.
Sorne gram-positive organisms produce superanUge11s
that nonspecifically actívate largc populations of T lym-
phocytes to produce excessive an1ounts of proinflamma-
tory cyrokines. Coagulation abnormalities, specificaUy
disseminatcd intravascular coagulation, can also be a con-
seque;:11<.:c uf se::¡.>sis.
Duri ng a hac.tP.rial Sf'ptiremja, rarely are organisms pres-
ent in high enough numbers to be detected by direct mi-
croscopic examination. Sorne bacteria are, however, pres-
ent in large enough numbers in thc blood in the terminal
stagcs of disease to be detected in direct smears. Bacillu.s an
lftrui·is, t!Je etiologic agent of anthrax, causes an over-
whelming Sf'pticf'mi;i, predominately in run1inants, and
resuJts in largc numbers of organjsn1s beiug fuuutl lrI tl1c
blood during the time just prior to death (Fig 67.1 ). Pasteu-
rella n1ultocida, thc agcnt of fowl cholera, and Dorrelia anse-
rina, the agent of avían spirochetosis, can also be found in
large numbers in the blood of affccted birds.
440 PA1rr IV Clinical Applications

To ble 67 .4. Common and/or lmportant lnfectious /\gents of Circulatory System and Lymphoid Tissues of Cattle

Agent Disease Circulatory/lymphoid A5'0Óated Findings

Viruses
Alcelaphine herpesvirus-1ª Malignant catarrhal fever Hemorrhages. let1kopenia, lymphoid prnlifPration, lympharlimopathy
Bovlne leukemia virus Bovine leukemia Lymphosarcoma
OvinP herpPwin 1<- / Malignant catarrhal fever Hemorrhages, leukopenia, lymphoid proliferation. lymphadenopathy
Rlft Valley fever virus• Rifl Vdlley feve1 Splenomegaly, widespread hemorrhages
Rinderpena Rinderpest leukopenia, destruction of lymphoid orqans
Bacteria
Anapfasma margina/e Anaplasmosis A11emia, kleru~, ~leuomegaly
Arcanobacterium pyogenes Traumatic reticulopericarditis Pericarditis (often polymicrobial)
lnfectious valvular endocarditis 1leart murmur, heart failure, valvular endocarditis
Bacillus anthracís Anthrax Edema, septicemia, splenomegaly, bleeding trom orífices
Clostridium chauvoeí Blackleg Myocarditis, pericarditis
C/ostridium haemofyticum Bacillary hemoglobinuna lcterus, intravascular hemolysis, hemorrhages
Ehrlichía ruminantíumª African heartwater disease Edema, hemorrhagPs, hydroperirardium, splenomegaly
Leptospira spp. Leptospirosis A11errrid, ille1u~, intravascular hemolysis
Myco.bacterium avium subsp. paratuberculn<i< lohnes disease Granulomatous lymphangitis of mesenteric lymph vessels
Myu1/Jdlle1iu111 /Juvi> Bovine tuberculosi> Cronulomatous tr<>chcobronchioVmcdiortinol Jymphodcniti>
Pasteuref/a multocida serotypes 8:2 or E:ia Hemorrhagic septicemia Edema, hemorrhages, hemorrhag1c lymphadenopathy
Salmone/111 spp.h Su lmoncllosis Septicemia, splenomegaly

•Clo»ified os a fo1ei9n animal disca:c ogent in the United States.

bcommuu ~rulype> inlluu.s Dublfn •nd Typhimurium.

Table 67.5 . Common and/or lmportant lnfectious Agents of Circulatory System and Lymphoid Tissues ot Goats and Sheep

Agent Circulatoryllymphoid Associated Findings

Viruses
Bluetongue virus Bluetongue Edema ot head and neck, hemorrhages, hyJ)Erem1a
Peste des pctits virus' Peste des p€tits Generalized lymphadenopathy, leukopenia, splenomegaly
R1ft Valley fever virus• Rift Valley Íever Splenomegaly. widespread hemorrhages
Rinderpest vir~ Rinderpest Leukopenia, de<trurtioa of lymphoid organs
Dacteria
Anaplasma ovis Anaplasmosls Anemia
Bacillus anthracis Anthrax Edema. septicemia, splenomegaly, bleeding from orifirP.~
Corynebacterium pseudotubercu/osis Caseous lymphadenitis Lymphadenitis, lymph 1101.le óU)lf!))f!)
Myroplasma haPmnvi< Eperythrozoonosis Anemia
Mdm1f1dmid /Jaemolytic (S) Septicemic pasteurellosis llemorrhagic septicemia in lambs
Mycoplasma mycoides subsp. mycoides (G) Septicemic mycoplasmosis Septicemia, pericarditis
(large colony type)
Pasteurella trehafosi (G) Septicemic pa,"teurellos1s Hemorrhagic septicemia in lambs
Staphylococcus aureus Tick pyemia of lambs Lymphadenopathy, septicemia

(G) =goats. (S) =sheep

G(l311ified as 3 foreign animal agent disease in the United States.

lnfections lnvolving the Heart and Pericardium

The major infectious processes of the heart are infcctious
valvular endocarditis (usually bacteria!) and myocarditis
(bacteria) or viral). Infcctions of thc pericardium occur ei
thcr as a result of a systemic infectlon, trom a focus within
thc hcart (e.g., endocarditis) or by extension of an infec-
tious process from adjacent tissues (e.g., pleuropulmonarv
infcctions).
lnfectious Valvular Endocarditis. Valvular endocarditis results
from bacteria from another site in tl11:: IJuuy =>eedi11g one
of the heart valves. Prr.exi<:ting injury ora functional ab-
 Chapter 67 Circulatory Systen1 and Lymphoid Tissues 441

Table 67.6. (0111111011 and/or hnportant lnfectious Agents of Circulatory Systen1 and Lyn1phoid Tissues of Pigs

pisease- Circti.latoi:y/Lymphoid Associated findings

lliruses
,., African'SWine feyei 11iru.5" Afrrcan swine fever Generalized .edemalhemorrhageS/infarctioos, hemorrtiagic [ymph nodes, ,
pericarditis, skin cyanosis, splenomegaly
Enccph¡¡Jomyocarditis virus Encephalomyocarélitis Hydropericardium,. myocarditis" pericarditis
HOQ' cholera virus" Hog cholera, Classical swine fever Generalized hemorrhages/in1arctions, llemorrhagic Jymph nodes,
lympnoiffi depletion; skin qanosis, splenic infarcts
Lelystad virus Porci11e reproductive and respiratory l)'ndrorne Secondary ihfectioos due to ma~rophage dep!etion
•. Pordne Circovirus 2 eost-weaning multisystemir wasting syridrome lymphadenopathY, myocarditis, poof grbli.1h.
Bactaria
Baci/lus anthracis Anthrax Pharyngeal Iymphadenopathy and edema, septi(emia
BurkholdP.ria psP.11domalleiª MeJk1idosis Lymrih OC\de ~nd splenk ~hsce<<el
Erysipelothrix rhusiopathiae Erysipelas Hemorrhages, splenornegaly, skin cyanosis, infectious valvuld1 e11UUld1dili~
E5cherichía ·co/i Septicemia Septicemia in unweaned pigs. skih cyanosis. c0ngested organs.
lymph:idcnop¡¡thy
fsche.richia co/i-(Shiga-like toxin positive) Edema disease Edema in subcutaneous tissues/stomach mue.osa due to vasculitis
Haemophilus parasuls Glasser's oisease Pericarditis, septicemia
•
Mycobacterium a'viíJmb Swine·mycobaaeriosis Granulomatous <ervi(af, pharyngeal, and mesenteric lymphadenitis
Myrnrfa<ma <rr.' Myropl~<m~ pol~Prn<iti< PPrir~niití< with nthPr <Prn<itirlP~
Mycoplasma hae.mosuis Por,cine epérythrozoonosis Anemia, icterus, splenomegaly
SalmoneJlad Salmonellosis Lymphadenopathy, skin cyanosis, septicemia. spleoomegaly
Streptococcus porcinus Jowl absc-ess Ccrvic¡¡J Jymphudenitis
Strep:tecoccus suis: StFeptococcal septicemia PericarditiS, sepficemia

•c1ass1l!ed as a·fore19n ammat drseas.e agent~o'lbe Unrted ~tates.

bpfO{>OSed tbat swine straíos of Mycobac:terium avium be induded in the subspecies hominissuis. Other-mycobacterial species involved incJode M. kansasii, M. xenopi, allQM. forliiitum.
Rhodococcus equi is also occasionally involyecf.
'lncludes Mycopfasma hyopneumoniae, M. hyprhinis, ano M. hyos.ynoviae.
dMost common ser0types are Choleraes!li.s var Kunzendorf and Typhimurium.

normal.ity of he;irt Vil lve ;i llows for platelet anrl fihri n rlep-
osition. These deposits provide sites for attachment for
bacteria that are present in the circulation, often as a re-
sult of onc of thc mcchanisms o.f t(ansient bactere.mi.a
previously described. Attachment to the heart valve is
mediated through a number of bacteria! surface adhesins
that include surface glucans ar1d fibronectir1-l>inding pro-
teins. F.xposed extr;ic.el111lar matrix on the valve m;iy also
serve as a receptor favoring bacteria expressing fibrino-
gen- or laminin-binding proteins. A preexisting heart
valvc lcsion is not an absolutc requircment for infcctious
valvular endocarditis to develo p. Other cardiac abnormal-
ities (e.g., subaortic stenosis in dogs) or invasive vascular
procedures, including catheterization, also predispose to
he;irt v;ilve infec.tions. Sequelae to v;ilvu lilr enclocaT<iitis
include embolization, multiorgan infarction, and sudden
death.
Bacteria involved in infectious endocarditis are prc-
dominantly, but not exclusively, gram-positive organisms
and include streptococci, enterococci, staphylococci, Cory-
nebac.Leríurn, and Ar<.anobacteríurn spp. ·rhe genus Erysipe-
lothri.x is specifically associated with valvular endocarditis
i11 sorne a11il11als (swine, dogs, poullry). When gra111-
negative organisms are involved.. they are usually memhers
of the families Erztcrobactcriaccac or Pscudomon.accac. Bar-
tnnella, another gra rn-negative organism, is inc.rcasingly
being recognized as a cause of infectious valvular endo-
carditis in dogs.
Myocarditis. Myocard.it1s, an infla1nmation of the heart
muscle, usually is the result of systemic infection with
foci of infection in the heart. Darr1age occurs directly to
myoc.ytes o r to v;isc.11lilr enclotheli;il VPSSPls s11pplying
heart tnuscle. 'fhe mechanisms of in jury to the heart n1us-
cle vary and include 1) direct toxic action of an agent on
myocytcs, 2) cffccts of circulating toxic products, or 3) im-
mune-mediated mechanisms. A Wide variety of infectious
agents have potential to cause myocarditis. Sorne com-
mon microbial age11ts speclflcally associated with my-
oc;irciitis in ani1nals inclt1de canine parvovirus, en-
cephalomyocarditis virus i11 pigs, Clostridiutn chauvoei
in cattle, and Listeria 1nonocytoge11es in ruminants and
poultry.
lnfections lnvolving the Pericardium. T-Tydropericardiun1, a serous
fluid accumulation in the pericardial cavity, in con¡unc-
tion with other systemic signs is a characteristic of certain
i ufectiou:; <.lisea:;e,s (e .g., heartwater in cattle, Af.rlcan
Horse Sic.kness, c.hirkP.n ilnPmi;i virus infPction) rind i<; thP
rcsult of vascular damage. Fluid accumulations il1 the
442 PART IV Clinical Applications

Table 67. 7. (0111111011 dlltl/ur lr11µorld11l lr1fe1.liou) Ayer1b uf Cir1.uldlory Sy)ler11 d11tl Lyr11µhoid Tissues of Poultry

Agent Disease Circulatory/Lymplloid Associated Findings

VirusR~

Avid11 i11 flu~11Ld viru)·HS or H7•
(highly pathogenic)
Avian leukosis viruses (C)
Chicken anemia virus (C)
Exotic Newcastle disease virus•
lnf1>rtin11< h11r<.>I rli<P;i<P vinK (C)
Marek's disease virus (0
Reticuloendotheliosis virus
Turkey adenovirus 2 (i)
Bacteria
But ttdíd dll)t:t ina
Chlamydophila psittací (T)b
Erysipelothrix rhusiopothiae
Escherichia coli
A4ycobamrium ;wium
Pasreuretta multodda
Sillmnnp/l,lb
Avian influenza, fowl pla'iJue Generalized hemorrhages; edema of head, wattles, and como; lymphoid
necrosis; myocarditis
Lymphoid leukosis Anemia, hemangiomas, lymphoid tumor5, SJrcoma5
Chicken anemia Anemia, hemorrhages, hydropencard1um, hypoplasia of lymphoid and
hemopoetic tissues
Exotic Newcastle disease Generalized hemorrhages (especially intestinal), edema
lnfPrtin11< h11r<nl rii<Pa<P. <iumborn rii<ea<P. Enlarged/hemorrhagic bursa of Fabricius. lymphoid necrosis. B lymphocyte
deficiency. immunosuppression
Marek's disease Lymphoid tumors of heart, bursa, thymus. spleen
m Reticuloendothcliosis lymphoreticular neoplasia, lymphomas
Hemorrhaqic enteritis of turkeys lmmunosuppression, intestinal hemorrhage, splenomegaly
Avían spirochetosis Anemia, hemorrhages, splenomegaly
Ornithosis, chlamydiosis Pericarditis, splenomegaly, fibrin exudates
Erysipcl~s Generalized hemorrhages, splenomegaly with infarcts, valvular endocarditis
Colisept1cem1a Pericarditis, omphalitis, septicemia, splenomegaly
Avían tuborculosis ~fl!Pnit !Jr~n11lnm~~
Fowl cholera hemoni1ages, pericarditis, septicemia
G~11~1 d!it~tl
Salmonellosis< Myocarditis. omphalitis. pericarditis. splenitis

(0 =chicken. (T) = turkey

ªClassified 05 a forei9n •nim"I rii<P•«> •9•nt in th• tlnit•cl ~tate<.
b1nc1ud~ scrovar. Pullorum, Gallinarum, and Typhimurium.
'lnduJes Pullo1um disease, Fowl Typhoid, and Paratyphoid.

F 1G U R E 6 7. 1 . Blood smc<Jr from v cow with anthrax. Blood Pericarditis and pleuritis in a goat caused by a
F 1G U R E 6 7 • 2 .
was collected 1 hour prior ro the cow's dearh. Numerous, /arge, Mycoplasma mycoides subsp. mytoitle) (ldrye to/uny Vdtidnl) }ep-
squared-end rods typical of Bacillus anthracis are present in the ticemia.
smear. Wright's stain.

...

,,

pPrir;:i rc1i;:i 1 s;:ir ;i lso res u lt fro m dama ge due to immune-
comp lex deposition in vesscls (c.g., fel ine infectious peri-
tonitis).
Pericarditis denotes an inflammation of t he peri
cardium. Like myocarditis, a number of viral and bacteria]
agents can be involved. Pericarditis is often part of a sys-
tcmlc lnfection involving other :.eru:.al :.urface:. a11u cavi-
ties (e.g., Gl;:iss1>r's c1is1>;:isp in pigs, mycoplasma septicemia
in goats) (Fig ó7.2).
Tl1c most common traumatic pericardial infection in
animal:; is t raumatic rcticulopcricarditis (hardware disease)
in cattle. ·rhis is m ost often assoc1ated with the extension
of an ingested linear metallic object (e.g., wire, n ail
through the reticulum and dtaphram intu the peri<.:aruial
sac. lt provides bacteria thf' nf'Cf'S<>a ry ;:iccess to the pericar-
dial space il1 order for infection to establish. Such infections
are usually polymicrobial, with Arcanobacteium pyoge11es
and Fusobacteriurn necrophorurn fccquently involved.
 Chupler 67
lnfections Affecting Blood Vessels
In general, the endothelium of blood vcssels plays an in-
tcractivc role in most infla.m matory rcactions through
endotheliaJ-leukocyte interaction, pro-coagulation activ-
ity, and release of mediators (cytokincs, chemokines) .
Microbes that specifically infect endot11elial cells of small
vessels and capillaries can cause necrosis with increased
vascular pcrn1eability resulti11g fron1 dirccl vascular e11-
dothelial injury by an agent or its toxin (e.g., Shiga-like
toxin) or by immune-complex deposition andan ensuing
inllammatory response. Disruption ofthe endothelial bar-
rier leads to edema and/or hemorrhages in affected organs.
Cllnlcal s1gns depend on the agent ano vascular Síte(s) 1n
the body primarily affected (e.g., African Horse Sickness
and the puhnonary vasculature wi.th an ensui11g pul-
monary edema). Central nervous system sign s in dogs as a
result of bleeding il1to the bruin are attributcd to Rickettsia
rickertsii, t h e Rocky Mountain spottcd fcver agent, and the
vascular damage it causes. Similarly, damage to the in-
tegrlty of vessels >'Valls dueto immune-complex deposition
in feline infectious peritonitis in cats résults in the fluid ac-
cumulations in serosal cavilies, as is fou11d i11 tl1.t: "wct
form of the disease. In addition to severe hepatic necrosis,
11
Rift valley fever virus causes massivc gcncralizcd cndothc-
lial damage resulting in widespread hemorrhages. Loss of
endothelial anticoagulant activity and platelet activation
ru<1y cause thrombosis of blood vessels and lnfarction in
<;omP <>ystPmir infections ultimately le<iding to tissue
necrosis at the affected site (e.g. 1 skin lcsions of erysipela::.
in swlne). Details on the pathogenesis of these and other
infcctious d iscascs that affect thc b lood vasculature andas-
sociated pathology are covered in greater dctail in chapters
on the specific agent or system(s) involved.
Omphalitis, an tnflammation of the umbilicus of
neonates, deserves special attention. Both the umbilical
artcric:-. a11LI vci11s car1 l.Jc ir1vulvcu. ll i:-. ~pt:cially irnpur-
tant in farm animals anrl horst>.'i. ThP h;ir.teria rP_<>ponsihle.
are either of enteric origin, inl1abitants of mucosa! sur-
faces, or environmental contaminants (e.g., Actinobacillus
sp, /\rcanobacteriun1 pyogenes, E. coli, streptococci). Umbil-
ical infections can lead to local abscess development or,
somctimcs, serve as a site for establishment of Clostridiinn
leluni a11LI ll1t: Llt:vt:luµ111t:11 l uf Lcla11us. A scµticcruia ruay
also develop from umhilical infections (nave! ill), whir.h
can lead to infections elsewhere in the body, including
polyarthritis and meningitis. Failure of passive transfer is a
common predisposing factor in these cases. Tn poultry,
yolk sac infcction and omphalitis is also a serious problem.
A variety of organisms are involved, E. coli, Salmonella, and
PseucJu1nurtus l.Jt:i11g µrurui11t:11l t:tiulugit.: age11ts.
lnfections lnvolvl ng Red Blood Cells
Anemia is a common finding in many in fectious processes.
A numbcr of mechanisms lead to anemia <tnd includc sup-
pression of erythropoiesis, sequestration of red blood cells
(RBCs), antibody-mediated hemolysis, erythrophagocyto-
sis, dliecL ly:.is ufRBCs, ar1LI altcratíuus in RBC rnembranes
that decrease overall lifespan.
A11aplas111a 111arginale specifically infects RBCs in cattle
Circulatory System and Lymphold T!ssues 443
FJ G U RE 6 7 . 3 . Blood smear from a cow with anaplasmo~i~.
Anisocytosis <ind polychrom<isi<i <irc evident. A number of Ana plasma
organlsms are present in red blood ce// margins. Wrighr's srain.
(Fig 67.3). Infections elícit an immune response that re-
Sltlts in removal of both infected and uninfected RBCs and
n1ay lead to a packed cell vulu111t: a:-. luw as 6%. The anerni<1
observed with chicken anemia virus disease and Fel.V in-
fcctions in cats are cxamples of anemia due, at least il1 part,
to decreased erythropoiesis. ·rhe bacteria! hemoparasitc,
M)'coplasn1a haemofelis, causes anemia in cats by multiple
rnechanisms including RBC scqucstration and antibody-
mediated hemoJysis. Irnmune-mediated hemolytic ane-
111ia is also inq.Jorli:url ilr Fe LV iofl!ctiun:; anu canine el1rli-
chiosis. lmmune-mediated hemolysis can he. the. re.sult of
1) prcscnce of microbial antigens on the RilCs, 2) cross-
reactions between antigens of normal RBC proteins andan
ínfectious agent, o r 3) exposure of usually unexposed RBC
ant1gens during the infeetious process. Intravascular he-
molysis resulting in anemia can be the result of action of
certain l.Jacterial toxin:; (e.g., phospholipase C). Leptospira
spp. ano (./nstridíum hen1n/ytir11n1 (ITP notable agents that
destroy RllCs by this mechanism. Anen1ia in these cases is
often accompa11ied by 11emoglobinuria.
lnfections lnvolving White Blood Cells
A uu1nl.Jer uf viruses affect cells of the myelold and lym-
phoi<i si>rii>s. Vi>nPznf'lan Pquine encephalitis vin1s is a
prominent example of a virus that destroys 11emopoelic
and lymphoreticular cells Jeading to cellular depletion in
bone murrow, lymph nodcs, and thc splccn. Manyviral in-
fections that target cells in these series predispose animals
to secondary infections consequent to the immunosup-
press1ve effects that occur. One of the main targets of infec-
tious bursal disease virus in chickens is the cloacal bursa,
which inilially is eI1largeu ar1LI euernatous but eventually
bccomes atrophied (Fig 67.4) . ThP. re.sulting R lymphocyt<'
deficicncy lcads to sccondary infcctions. Feline leukemia
virus, feline immunodeficiency virus in cats, and porcine
circovirus in pigs similarly make animals more su sceptible
to secondary lnfections through tmmunosuppresive ef-
fi>rt~ . These effects can be long-term; however, other viral
infections (e.g., disLeo1pe1 vir u:-., l1ug cllulera virus, par-
444 PART IV Clinical Applications
F1G U R E 6 7 . 4 . Enlarged edematous bursa in a chicken with
infectious bursal disease. (Courtesy Dr. H. Shivaprasad.)
voviruses) cause temporary leukopenlas and the immuno-
suppressive cffcct is short-term.
Ba<.:teri<d <tg1::1 1t::. 111ay also infecl white blood cells. Cer-
tain members of the genera Anaplasrna, Ehrlichia, and
Neorickettsia are pathogcns of cclls bclonging to thc myc-
Ioid or megakaryocyte series. Fungal agents are not com-
monly associated \.vith infections of the myeloid or lym-
pho1d cell series. liowever, the syscemic fungal agent
flistoplasrna capsulaturn specifically infects macrophages
and, therefore, hi:>tuµ la:>1110::.i::. i::. co11side1ed a disease of
thP monocyt·c-macrophage system.
lnfections of Lymph Nodes, Lymphatics, and Other Lymphoid
Tissues
The lymph nodcs play a major role in filtering primary
sitt>s of i11ft><.:tiou vía tire ly111viraliL v1::ssels and, ll1erefore,
art as p rincipie sites for containment of potential patho-
gens. Many viral infections are dispersed to oth cr parts of
the body (target organs) by this route.
Lymphadenitis, an inflammation of the lymph nodc,
can occur in a single lymph node or multiple lymph nodes
draining a comroon region (regional lymphadenitis) o r
manift>!>"t as <:t gt;:111:raliL.1;:1.i ly111phadenilis il1 systenlic infec-
tio ns. 0Ppe.nding on the agents involved, the inflamrna-
tory response can be non-supp u rative, suppurativc, n ccro-
tizing, or granulomatous. In sorne cases, dependin g on the
agent and the host response, lymph n ode abscessation oc-
curs. Microbial agents causing lymph node abscesses are
usually but not cxclusively bacterial or fungal. Partin1l;ir
organisrns con::.istc1llly a:.sociaLed with lyn1pl1 node ab-
<;ct>sS<-''\ inrlu<IP r:nrynebacteriurn pseudotuberculosis in sheep
and goats (caseous Jymphadenitis) and Streptococcus cqui
subsp. equi in horses (stran gles). Although less com mon
nowadays, Streptococcus porcinus was an important cause of
cervical lymph n ode absccsscs in swine (jowl abscess).
Yersinia pestis should al-.-vays be con sidered when mandibu-
lar ly1nph notlt> au:.ce:.:>c:> (vft1;:11 bilale1al) are detectcd in
F1G U RE 6 7 . 5 . Swolfen mandibular Jvmph node in a cat with
Streptococcus canis Jymphadenitis. (Courtesy Dr. P. Blanchard.)
cats from geographic regions whcrc plague is endcmic.
Francisella tularensis cau ses lymphactenopathy with absces-
sation in cats, along with generalized signs of infcction..
Streptococcus canis also causes puruJent inflammauon of
lymph nodes on the head and neck in cats (Fig 67.5)
Outbreaks, prt;:sumauly vi<:t oral 11ausn1ission, have been
described in cats kept in roloniPs.
Ge11eralized or regional lymphadenitis is encountered
v.'ith tl1e systemic mycotic infections (blastomycosis, coc-
cidioidom.ycosis, cryptoco ccosis, histoplasn1osis) and typ
ically results in a caseous-type necrosis in the affected
lymph node.
Lymphangiti:. i:s a11 a<.:uli;: u1 ch1011ic üúla1n111alion of
thP lymphatic: channels that results when infections are
not contained locally. lnfections mostly involvc subcuta-
neous lymphatics. Lymphangitis is not common in ani-
mals, but whcn it occurs t hc ctiologic agents are most
otten bacteria! or fun gal. l'arasitlc agents sh ould also be
consid ered as causes of lymphangitis in an imals b ut are
not within the scope o f t h is booi<.. lr1fla11u ualio11 of l1 1t
lympha tic w;ill~ ra n rPs1 1lt in lymphatic obstruction and
persistent lymphedema in sites drained by thc affcctcd
lymphatics. Lymphatic vessels may be swollen (corded1
with sporadic discharging abscesses occurring along the
lymphatic tracts. Lymphangitis is most commonly recog-
n ized in horses. Sporothrix sch.enckii, a dimo rphic fungus
and Corynebacterium pseuduluberc..ulu~fa a1e classical causes
of equine lymph;ingitis. Although considered foreign ani-
mal disease agents in t h e United States, Burkholdcria mallci
(agent of glanders) and Histoplasrna farciminosum (agent ot
cpizootic lymphang:itis) should also be considered as po-
tential causes of Iymphangitis in horses in cou ntries where
t hese agents are fou nd. Mycobacteri11rn avium su bsp. paratu-
berculosis, the agent of Joh11t;:s l.ii:;c.::a:.e iu rurn ü1a11ts, causes
a gra11ulom;ito11s lymphangitis in the mesentery of the in-
testines in conjunction with granulomatous enteritis.
The spleen plays an active role in antigen trapping as
wcll as rcmovnl of defective erythrocytes. Splenitis com-
 Chapter 67
F 1G U RE 6 7. 6. Splenomegaly in a calf that died from
Salmonella Dublin septicemia. (Courresy Dr. M. Anderson.)
• can occur. Thc bovine leukemia virus, also a retrovirus,
monly occurs as a consequence of generalized infcctions
due to acute congestion and/or reactive hyperplasia.
Saln1011ella septicemia is a common cause of splenomegaly
in many animals (Fig 67.6). In cattlc, anthrax and anaplas-
u1osi:. al:.o shoultl lJc cousiucrcu. lu µigs, African swine
fPvC'r anct C'rysip<'las, along with Salrnnnt>lla, arf> potr.ntial
agenls Lo consider wl1en splenon1egaly is deLected.
·rhe thymus is an u ncommon site for infections to
occur in animals. Viruses that infected ·r lymphocytes
Circulatory System and Lymphoid Tissucs 445
(P.g ., fplinP lP11kPmia vin1<;, fplinP imm11nodeficiency
virus) cause thymic atrophy. A lymphohistiocytic thy-
musitis with depletion of cortical thymocytes is a major
pathologic fcaturc in cpizootic bovinc abortion, a discasc
limitcd to the western United States. lt is caused by an in-
fectious agent of, as yet, unknov;n etiology.
Neoplasias of the Hemopoetic and Lymphoid Tissues with lnfectious
Etiologies. Various viral-induced neoplasia involving he-
rnopoetic and lymphatic tissues occur in animals. Marek's
discasc virus, a hcrpcsvirus, causes a lymphoprolifcrativc
discasc that affects predominately nervous tissues but also
causes lymphoid htmors in a number of other tissues in-
cluding thc heart, bursa, thymus, and spleen. A number of
animal rctroviruses exist and are able to integrate proviral
v-v11<.. l:)CI u::::. i11 Lu l 1u:,L 1..ell ula1 l)f-JA. TlH; al.Jilil y uf feliue
leukemia virus to infect different hcrnopoctic cells ac-
cou11ts for thc varict y of disordcrs of thc hcmopoctic sys-
tcm sccn in infected cats. In addition, salid neoplasias in-
cluding lymphosarcoma (e.g., thymic lymphosarcoma)
c;iuse-: lymphosarco1na that can involve a number of or-
gans including hcarl, kidncy, splcen, ly n1pl1 uodes, auu
brain. Avían retroviruses of thc lcukosis/sarcoma group
cause various neoplasias of hcmopoctic origin (crythrob-
lastosis, myeloblastosis, myelocytomatosis}, as well as lyin-
phoid Jcukosis and endothelial tumors (hen1angiomas).
Viruscs of rctlculoendothellosls group cause lymphoid
lcukosis and reticuloendotheliosis in turkeys.
 Digeslive Sysle111 a11.d
Associated Organs
RICHARD L. WALKER
'!'he primary function of the dlgestive system is to p rocess
food in order to providc nutrition for thc body. ·T his is pcr-
tormcd by a series of complex physical, secretory, and ab-
sorptive processes. An in-depth descriptíon of all of the
varied and interactive functions of the dlgestive system is
well beyond the scope of t his chapter. Because the díges-
tivc ::.y::.tc111 i11 il::. ::.i111vlesl le1n1 1ep1esenls a11 open tube to
thP Pnvironment. the opportunity for exposure of thc di-
gestivc systcm to potcntial pathogens is grcat.
·rhe anatomy ot the digestive svstem vartes markedly
among different animals. In carnivorous animals, it in-
cludes the oral cavity, esophagus, stomach, and srnall and
large intestines. The digestive systexn of 11crbívores is sub-
stanttally tlifferent frorn carui vure::. l.Juth <:111<1tuulic<:1ll y <:1utl
f11nctio na lly. Among thP hPrhivorous anima Is the re is also
great variation in the digestive system makeup depending
on the digestive mechanisn1s cmployed (e.g., rumination,
cecal digcstion). Tn poultry, further specialization of the
d1gest1ve system 1s seen by the presence of a crop (a diver-
ticulum of the esophagus for storage of food) and the divi-
sion of t!Je ::.to1nacl1 i11to a gla11tlular ::.tu1uacl1 (pruveu-
trin1 l11s) ano muc;c:ular <;tom;ich (vPntrin1Jus OT gizz;irci).
Sorne of these anato1nic differences selectívely predispose
to specific infectious processes. Infectious diseases of tl1c
acccssory organs of tl1c digcstivc systcm are also covered in
this chaptcr and ínclude comn1on dlseases of the liver, gall
bladde r, and pancreas.
Antimicrobial Properties of the Digestive System
'fh<'r<' are a number of anatomic, physiologic, and im-
munologic mechanisms in place to protect the digestíve
system from infection by potentia!ly pathoge11ic mi-
crobes. Following are the 1najor p rotective features of the
dígestive system.
Gastric Acidity
Acid production in the stomach provides a major protec-
tive barríer agaínst patbogens reaching dist al sites in the
digcstive system. The normal acldlc environmeot in the
stomach effectively inactiva tes sorne viruses and kills most
e11lt: riL l.Jacleda. Ils il11po1 Lance is evídc11l in l<un1ans by
thc increased risk for enteric infections observed in indi-
446
viduals with achlorhydria or in individuals when stomach
acid l1as been neutralized.
Peristalsis
Peristaltic activity in the digestive system is a mechanism
whcrcby nonadhcríng microorganisms are swept distally
In the small bo\vel, per1staltic act1vity plays a major role iP.
host defense. Likelihood of discasc is directly related to size
of a populatiun of a patl!ugc11ic ::.µecic::. vf bacle1 ia i11 Lht:
small intestine. The most important rPg11lator of thP si7P o ·
ll 1is populalion is perislallic activity, since there are fe\·
other regulators sucl1 as thosc that exist in the Jarge bo\ve1
(Eh, fatty acids, and pH). Pcristalsis also has an indircct
protective role by maintaining distribution and numbcrs
of the normal bacteria! flora. ·
Mucus and Mucosa! lntegrity
'fhe mucus !ayer and thc integrity of mucosal surface are
important factors in providing a barrier to ínfection in th1:;
d1gestJve system, as well as to systemic infections that orig-
i nate through the digestive system. The mucus barrier.
compo~ec.J of mucin glycoprutein.s .secreted l.Jy gul.Jlct cell::.,
bine!~ o rg<inisrr1s thPrPhy hlocking intt>rac:tion with under-
lying epithelial cells a11d 1 along with peristalsis, promotes
tl1cir rcmoval. The n1onolayer of epithelial cells lining the
digestive syst em provides an addítíonal barrier to entry by
luminal o rganisms. 1I1testínal enrerocytes are zippered to-
gether by intercellular junctional complexes. Damage to
inte!)tinal epitlleliun1 alluw::. fui i11le1cellu1ar lrai1slocalion
of potPntially pathogenic microbes. Sorne microbes are
able to translocate across intestinal mucosa by il1traccllu-
lar mcans.
Bacteria( lnterference
Once the nor1nal flora is established, it gives thc animal a
very pote11t defense against microorganisms that might
cause disease, if they establish. An example of the effec-
t1ve11ess of this "colonization resistancc" is the exclusion
of Saln1onella from the intestinal tract of poultry by "cock-
taJls" containing r1ur1nal ílura 111il:1uu1garúsn1s. Disrupt-
ing colonization resistance puts the animal at risk by ex-
posing receptors on potcntial target cells and eliminating
a mechanism for regula ti ng the population size ot taculta-
 Chapter 68
tive. organisrns, including species or strains with patho-
genic potential. Produci:s of normal bacteria! flora, espe-
cially the anaerobes that make up the majority of the oral
and colonic flora, are important in controlling pathoge11
establishment (see "Microbial Flora of the Digestive
System," bel<>'A').
The 11ewborn anirnal is pi:lrticulcirly susceptiule to e11-
t eric disease hecause, besides being im1nunologically
n<live, it is devoid of established flora. The most vulnerable
area is the midjejunum and distal ileum.
lmmune Defense
Passive protectlon ls afforded the neonate by colostrum.
Tmrn unoelobul ins in colostrnm, Spf•cifir fnr rin t ieenic de-
ternlinants on adhesins used by pathogens for attach -
ment, combine >"lith tbese structures to block attachment
of the pathogen to its target cell . Failure of p assive transfer
anct tnus t he absence of tnese protective immunoglobu-
lins is one of the major factors Ieading to the in creased sus-
cepLiblily of neonales lo enleric i11feclions.
·rhe active imrnune defense mechanisms in the diges-
tive systen1 rely on patrolling phagocytes and humoral
and ceH-mediated immunity. "lhere is a normal back-
ground of neutrophils, inacrophages, plasma cells, an.d
lym.phocytes found in the lamina propria indicating a
continuous surveillance activity. When stin1ulated by po-
te11 LiaJ ]JaLhoge11s, iufla1nn1atory n1edialors a11d cheu co-
tacf'ic agP.nts are produc:ed and result in an influx of adcli -
tional inflammatory cells. As part of the body's mucosal
immune system, thc gut-associated ly111phoid tissue
(GAI.:r) is composcd of ly1nphoid tissuc in Peyer's patchcs
and ly mphocytes in the lamina propria. The n1icrofold (M)
cells overlying follicles of Peyer's patch play a role i.n anti-
gen samplir1g for the immune system but n1ay also provide
a portal of entry for sorne pathogens. Tl1e benefits of GALT
i:tr1<.l i:H ttige11 sa111vling in Ll1e d igeslive syslen1 are nol jusl
local hut rather henefit the entire host through the com-
n1on n1ucosal in1111une systen1. Secretory lgA and associ-
ated secretory piece, "\-Vhich provides resistance to lumiI1al
dcgradation, has a role in osponization and neutralizing.
spec1f1c IgM antibOdies are a1so invo1vea.
Commensal bacteria play an important role in the de-
vt:lü]JJ r1er 1Lof Lile n1ucosal ünn1une syslen1 of lhe gastroin-
testinal tract by promoting lymphoid follicle develop-
ment; however, the immune syst em responds more
vigorously to pathogenic organisms than it does to com-
mensal organisms. This is possibly dueto the closer attacl1
inent of patl1oge11s to n1ucosal cells or that commensal or-
ganisms, being more permanent residents, temper the
in11ntu1e response directed to1'vard Lhern by blockillg ]JrO-
inflammatory responses. lnappropriate inflammatory re-
sponse to normal commensal bacteria is hypot hesized to
be an underlying cause for ínflammatory bowel disease in
human s.
, In parts of the digestive tract, the normal flora is inte-
gral to maintaining an active innate host surveillance
sysLen1. In lhe n1oulh, for inslance, Lhe ]Jerio<.luntal rni-
crobiota stimulates the formation of an interleukin-8
(IL-8) gradient tl1at pron1otes migration o f neutrophils to
bacterial/epithelial interfaces. 'fhus these commensal mi-
D.igestive Systetn and Associated Organs 447
crobial con1munities in t he oral cavity provide for an ac-
tive surveillance in the gingival crevices against potential
oral pathogens.
Asan associated organ of the dige.stive system, t he liver
plays a major role in rernoval of pathogens from the blood-
stream.1.his innate host defense is accomplished by a com-
pl<::x ueutruphil-Kupffer ct::ll (resitleut Jiver rni:lcrophage)
interaction.
Other Antirnicrobial Products
Saliva, along with provid ing an important flushing effect,
contains a number of potential antimicrobial agents, in -
clud1ng antibodies, complement, lysozyme, lactoferr in,
peroxidases, and defensins. In t11e intestines, bile salts and
antimicrobial peptide:s contribute to lin1iting and influ-
encing t he microbial makeup. Bot h alpha and beta de-
fcnsins are produccd by cclls of t hc intestinal tract (c.g.,
Panetl1 cells). Lactoferr1n and perox1dase from tne pan-
creas 1nay also affcct bacteria! growth in t he intestine. In
a<.ldi.Uun tu tl1e i:IIltilJodies in colostrum, the p resence of
other factors such as lactofe.rrin ano JysozymP providP ad-
ditional protection for the digestive system in neonates.
Microbial Flora of the Digestive System
A ru icruuial llora tl1at is pi:lrt of a cou1plex t::cusysterr1 in-
hahits the oigestiVP. t ract. l n aooition to its rolf> in prOtf>C-
tion against the establishment of pathogens, the normal
t1ora plays an important role in physiological health of the
}1ost througb fu11ctio11s that includc promoting fu11ctional
intestinal villi formation, synthesis of nutrients (e.g., vita-
min K), and contributing to the establishment of a func-
tional il1testinal mucus consistency by degradation of the
secreted glycoproteins.
Establishment
The result of i11teractions bet>"leen host and microbe is an.
ecosystem comprised of many thousands of niches, each
innabitect by tne species or straíns of microbes most aptly
suited to that location, to the exclusion of others. 'fhe host
conlribuLes Lo lile establisl1rnen Lof a norinal llora by fui:-
n ishing receptors for adhesins on t he surface of prospec-
tive niche dwellers. The niche dweller is the one that has
successfully competed for t hat particular site.
The fetus is microbiologically sterile as it starts down
the birth canal. Microorganisms are acquired from th·e
b.i rth canal and, after b irth, from t he environment. 'fhe
iuuue<.liatt: t:11virourueut uf the newtJorn is populated
with microorganisrns excretf'.d hy t he. oam ancf othPr ani-
mals. These microbes are ingested, compete for nich.es,
and ;vith t ime becorne established as part of the normal
flora. During the first days to 1nonths aftcr birth , thc flora
is in a state of flux c.tue to the interplay between the vari-
ous microbes, the n iches of the host, and the changing
uiet. Diet ir1fluence~ the nutrittonal envlronment at the
leve! of thP n ir.hP, whirh in turn influences the kinds of
microbes that will successfully con1pete for tl1ese i1ulri-
ents. Throughout its life the bost's normal flora is influ -
448 I>ART I V Clinical Applications
enced by a number of other factors (e .g., aging of the host)
and adapls accordi11gly.
Members of the normal flora establish themselves in
a particular nlchc utilizing various bacteria! and host
properties in tl1e process. A powerfut way for bacteria to se-
curc a particular niche against other species is to secrete
antibiolic-like substances such as bacteriocins (cattonic
mcmbrane-active con1pounds that form pores in the tar-
gc::l 1..c::ll~) a111.l 111iuuci115 (si111ila1 lll bac le1iocil1s bul sn1allcr
than 10 kDa and active against gram-negative microorgan-
isms). 13oth of these substances are signilicant, cspccially
in the communities living in the oral cavity. Microcins
probably play a significant role in rcgulating the popula-
tton composition in the gastrointestinal portion of rhe di-
gcstivc system. The role of bacteriocins in this region is less
clear.
An i111port;int merhanism for rcgulating population
size and e11suring niche sccurity is fatty acid excretion by
obligate anaerobes. In the gingival crevice, dental plaque,
and the largc bowel, the obligate anaerobes in tl1is 1nan-
ner play a central role in regulating the siZe and composi-
tion of the normal facultative flora, the members of
whlch may include potentlal pathogens. Under tl1e cu11-
<litions of the bo~vel (low Eh {<500 mv} ancl pH of .'\to 6),
liul y1 ic, acelic, a11d la clic acids are extremely t oxic to fac-
ultative anaerobes, especially members of the fa1nily
J.!,nterobactcriaccac. Anothcr wuy for bacteria to compete
successtully is to acquire nutrients more successfully than
competltors.
Disruption
Antimicrobial drugs are the single most efficient agents at
decreasing colonization resistancc. Most antimicrobial
agents allect the microbial tlora ot the oral cavity by cte-
pleting the nun1ber of streptococci that inhabit the surface
of the cheeks and tongue. As a result, these areas are usu-
ally repopulated with resistant (to the antimicrobial agent
1Jci11g ad111i11isle1ed) n1e1n be1s o[ the fa111ily r.:nterobacte-
riaceae within 24 to 48 hours. Resistant members of the en-
vironmental flora are also found. Me1nbers of the genus
Pseudornonas are notorious examples ot this group.
J\ntimicrobials also affect the members of the obligate
anacrobic comn1unities that inhablt the gingival crcvtce
and dental plaque in the mouth and the large boivel.
OvergruwllJ uf va1iou:> 1111::111be1s of Lhe fanlily Erzterobac-
tt>riaceae rC'_'iult'i hecause of decreases in levels of fatty acids.
Colonization by potcntial pathogcns (e.g., Salrnonclla) is
enhanced by antibiotics affecting obllgate anaerobes liv-
ing in the bowel.
Composition
B;irterial spec:ics as "''cll as sorne protozoal and fungal
species constitut c tbc bulk of thc microflora in the alimcn-
tary canal o! animals. 'l'he overwhelming ma1or1ty of the
normal flo ra is obligately anaerobic bacteria (up to 99.9%).
V1ruses are typically only transicnt residents of the ali-
mcntary canal.
The mi<:robial flura uf tl!c 111uulh i:> 1ougil1y u1liiorn1
;imong <lomestir man11nals. No information is available
F 1G U RE 6 8. 1 . f'haryngeal swab from a dog. Simonsicllil,
showing characterisric monoseriate fílaments, are attached to an
epithe/ia/ ce//. Other bacteria/ rods are a/so present. Wright's stain.
•
fo1 fowl. Tl1e description that follows is general and applies
to carnivores and herbivores. The bucea! surface, tongue,
an<l teeth (plaque) are inhabite<l by facultative and oblí-
gate aerobes. ·rhese include streptococci (alpha and non-
hemolytic), xnembers of the family JJasteurellaceae, Actino-
rnyces spp., enterics (Escherlch ia coli belng the n1osr
common), Neisseria spp., CDC group Ef-4 ("eugonic fr.r-
menter"), an<.l Simunsiellu (a uuilfUt: 01al co1nn1e11sal lhal
forms ch;ir;irteristir monos0riatC' fi lan1ents) (Fig 68.1). 'rhe
flora of the gingival crevice is com posed almost entirely of
obligate anaerobes, the most co1n mo n genera being Bacte-
roidcs, Fusobactcriu1n, Peptostreptococcus, Porphyron1011as,
and I'revotella. Sa!Jva conta1ns a mixture of facultative and
obligate species of anaerobes and aerobes. The esophagus
does not possess a norrnal flura but i~ cu11ta11Jl11ated wilh
org;in isms fo11ncl in saliva.
In ruminants, rumen flora is composed of a complex
microbial community that includes bacteria (eubacteria
and archaea), fungí, and p rotozoa. The rumen flora is in a
dehcately balanced symbiotic relationship witl1 tl1e llost
that is necessary to maintain rurne n health for its proper
fermentative fu11Ltiuu. Th e uulk uf lhe nora are obligate
;in;ierohes, with Prevntella spp. and Bulyrivibrio spp. being
among the most common genera found. Also includcd
are bacteria (Riuninococcus, Fibrobacter) specitically re-
quired for digestion of cellulose-rich foragc. Disruption!>
in the normal rumen flora can lead to serious metabolic
and physiologicai problems (see "Tnfertion .~ of th<>
Stum<1ct1, Rurr1iI1a11t fure:.lo111acl1, a11d Abon1asum,"
helow). 'í h e. flora of t he rest of the alimentary canal varíes
substantially a1nong different anin1als, as is shown in
·rables 68.1- 68.5.
Digcstive system-associated organs (liver, gall bladder,
and pancreas) are not generally considered to llave a uur-
mal llora but may be transiently seeded as a result of
asymptomatic b<1ctc::n::111ia. Clu:.l1iuial spoces are readily
fo11nc1 in the live.r of many animals but remain dormant
unless tissue oxygen tcn sion becomes low cnough to allo"'
spores to germinare and vegetative cells to proliferate.
 Chapter 68 Digestive System and Associated Organ s 449

Table 68.1. Microbial Flora of t he Chicken

NUmber of Viable MlcroorganlsmS/uram of U>nten~

Stomach Small lntestine Cecum Feces

Crop Gizzard Upper Lower

Total 6 6 8-9 8-9 8-9 8-9

AnaProhM 3 'i-6 <] <] ~q 7-il
Enterobacteriaceaeb 6 <2 1-2 1-3 5-6 6-7
Streptococci/Enterococci 2 <2 4 3-5 6-7 6-7
lactobacillus 5-6 2-3 8-9 8-') 8-9 8-9

•Expr~scd .is logm of thc numbcr of orgonisms cultured.

~M.i11ly E. <uli.

Tabl e 68.2. Microbial Flora of t he Bovine

Number of Viable Microorganisms/Gram ot Contents"

• Small lntpc;finP

Abomasum Upper lower Ce<:um Feces

·1otal 6-8 >7 6-7 8-9 9

Anaerobes 7-$ NA' 5-6 8-9 &-9
Enterobacteriaceaeb 3-4 >7 5-6 4-5 5-6
~frPptn<orci/ FntPrn<orri 6-7 7-3 3-4 4-5 4-5
Yt!d)~ 2- 3 <3 2

"l:xpremd a> log1o of the num~ of organisms cultured.

tMalnly E. col/.
<Not available.

Table 68.3. Microbial Flora of t he Horse

Numb'J!r of Viable Microorganisms/Gram of Contentsª

Small lntestine

Stomach Upper lower Cecum Feces

Total 6-8 NJ\< 6-7 8-9 8-9
Ana erobes 3-5 3-4 4-6 3-4 j-)
Fnterobacteriaceaeb 6-7 5-6 5-6 6-7 5-6
Sl• t!¡Jl.o<.Ullil Elllt!IUlUld <3
Yeasts 6-8 NA' 6-7 8-9 8-9

1Expressed as log
10 of the number of organisms rultured.
l>Mainly f. coli.
'Not •v•íl•bfe.
450 PAnT rv Clinical Applications

Table 68.4. Microbial Flora of the Pig

Number of Viable Mlcroorganlsms/Gram of Contenb•

Small lntestine

Stomadt Upper lower Cacum kces

Total 3-8 3-7 4-8 4-11 1(}-11

Anaerobes 7-8 f..-7 7-8 7-11 10-11
E11le1uúdtle1 Ídtedeb 3-5 ~ 4-5 6-9 6-9
Streptococci/ Er1terococci 4-6 4-S 6-7 7-10 7-10
Yeasts 4-5 4 4 4 4
Spiral organisms NA' NA' NA' NA' 8

4Exp1essed as log of the number of organisms cultured.

10
bMainlv E. coli.
'Not availablP.

Table 68.5. Microbial Flora of the Dog

Number of \fiable Microorganisms/Gram of Contents•

Small lntestinc

Stomach Upper Lower Cecum feces

Total >6 >6 >7 >8 10-11

Anaerobes 1-2 >5 4-5 >8 11>-11
Enterobacteriaceaeb 1-5 2-4 4-6 7-8 7-8
Streptococci/ Entcrococó 16 56 s7 89 ~10
Spiral organisms {relative amounts) 1+ 1+ 1+ 4+ u

'l:Jlpressed as 10910 of the numlJer of organisms rul1ured.

0Mainly E. coli. •

lnfections of the Digestive System and
Associated Organs
lnfections of the digestive systen1 and associated organs
are in1portunt in all domcstic animals. Sorne digestive sys-
te1n pathogen s (e.g., enterotoxigenic E. coli, rotaviruses)
are specific for a particular animal family '"'hile others af-
fect a wide variety of animals (e.g., Sallnonella enterica
serovar ·ryphimurium). ln addition to differences in
anin1al susceplibility-agc, in1n1une status, and genetic
susceptibili ty-individuals \Vithin a species may also pre-
di:;po:;c to infcction by spccific pathogcns. Sorne of thc
most common and/o r important pathogens of the diges-
tivc systcm of n1ajor domestic animals and poultry are
listed in Tables 68.6-68.12.
lnfections of the Oral Cavity
'fhe oral cavity of ani1nals is susceptible to infcction by a
various endogenous (usually bacteria! or fungal) and ex-
ogenous 1nicrobcs (usual ly viral). Viral pathogens are
often contagtous and may, tl1erefore, affect large pupula-
tions of ;:in im;:ils ;:it ;:¡ timr. lnfrctions fro m endogenous mi-
crobes tend to involvc onc ora limited number of animals.
A nu1nber of viruses cause diseases of the oral cavity.
'fhc viruscs that cause the vesicular sto1natides variously
affect ruminants, horses, and s>V'ine. Included in this
group are foot-and-rnouth disease virus (picornavirus),
vesicular stomatltls virus (rl1aulluviru~), ~1>vi11e vesicular
disease vir11'> (PntProvir11s), and vesicular exanthema of
swine virus (calicivirus-now believed to be extinct).
These are contagious viruses. Lesions initially present as
vcsiclcs that cvcntually cuptuce and leave painful ulcecs in
oral mucosa. The coronary band and hcel ¡unctions of the
digits may also be involved, causing moderate to srvrrP
lameness. Thc exotic na.ture uf ~u1ue u( tire vesicula1
viruscs makes thPm of s11hst;:intial economic importance
in countries free of these diseascs. Other viruses are impor-
tant causes of crosivc stomatitis and inciude bovine virai
diarrhca virus in cattle, fe line calicivirus, rinderpest viruc
in sheep and cattlc, blueton~ue virus in sheep, and the ma-
lignant catarrhal fcver viruses in cattle. Clinical signs and
 Chapter 68 Digcstive System and Associated Organs 451

Table 68.6. Common and/or lmportant lnfectious Agents of the Digestive System of Dogs

Agent ----------~~-~--~......
MajorCllnltal..ManifestatiOns (Commón Disea~e Njl(Tle)_______________ A~ Group{s) Commonly Áffecteél
,.,._..,..,.,,..-
Viruses
Canine adenovirus 1 Diarrhea. jaundice, vomitiog Typically less than 6 mos
(anine coronavirus Di~rhea, vomrting Any age, typical!y in puppies
Cariine distemper virus Diarrnea, vomiting, dental enamel hyoplasia (distemper) Any age; most suS:Ceptible at ~ mos
Canine oral papillomavirus Oral cayity warts (oral papillomatosis) Typiéally less than 1 yr
Canine parvovirus Dianhea, vo¡niti.ng Any age, most susceptibl.e at 2-4 mos

Bacteria
C<1mpylob<1ctcr jcjuní!co/i. Diorrhco with or witnout blood Any age, typicalty less than 6 mos
Leptospira spp.• Hepat1t1s, vom1tmg (1eptospiros1s) Any age
Neorickettsia helminthoeca Diarrhea, vomiting (saJmon poisoning) Any age
Sa/monefla spp. Diarrhea, vomiting Any age, young ¡¡nd old mostsusceptible
Fungí
Histoplasma capsulatum Diarihea with or without blood, oral ukers, weight loss (histoplasmosis) Any age, typically less than 4 yrs
Algae
Prototheca spp. Bloodyodiarrhea (protothecosis) Any age

ªlncludes teptospira serovars canicola, gtippotyphosa, and icterohemorrhagiae.

•

Table 68. 7. Con1111on and/or hnportant lnfectious Agents o f t he Di y.,1Liv" Sy1Le111 o f Cob

Maiet CliniEal Manife5!j!tions \Common Oisease Name} A~ Grot¡ s} Commonly Affectett

VjruSl!S
feli11e talidvir u1
. Fe!ioe immunodeficier:icy virus
Feline infectíous peritonitis virus
Feline Jeukemia virus
~eline panleukopenia vir.us
Feline rotavirus
~==='""'""'""
Uk e.c1!ive ~lomalitis Typically le11 Litan 1 y1 oí aye
Secondary gingivitis/stomatitis. diarrhea Any age
lleal or colonk granulomas with vomitíng or constipation Any age, typically less tlían 2 yrs of age
Seconcfary gingivitis/stomatitis, diarrhea, vomiting/diarrhea dueto Any age
alimentary lymphoma
Diarrhea, vomiting Any age, typically kittens 2-12 mos
Diarrhea 1-8 wks of age

Bacteria
Campyiobaeter jejunifcoli lJiarrhea Any age, typiEally less than 6 mos
Safmonella Diarrbea Any age, young and old most susceptible

Table 68.8 . Common and/or lmportant lntectious Agents ot the Digestive System ot Horses

Agent

Viruses
---------
Mafor.Oinkal Maniffitatlons (Common Dis~ase ~ame)

Equine rotavirus Diarrhea 1-8 wks of age

Vesicular stomatitis virus Oral ulcerslvesides Anyage
Bacteria
Clostrídíum perfdngens (Types A.B.C) Bloody diarrhea {hemorrhalJÍC ente¡ocolitis) Less than 1 wk of age
Clostddium diffjcil,e Di¡¡rrbea Adults and foals less than 2 wks
C/ostridium piliforme Diarrhea, hepatitis, sud den death (1y2zer's disease) 1-8 wks of age
Neorickett5ia risticii Di,arrhea (Potomac horse fever) Typical!y in adult anlmals
RhqúutULlU5 e4ui Dia1 rh~a. mesente1 ic \ymphadenitis 2~ .roos ot age
Salmone/la spp.4 Diarrhea with or without blood Any age

•commonserofypes include Sa/monel/a s_erotypes Typhimurium, Anatum, and Agona.

452 PART IV Clinical Applications

Table 68.9. Common and/or lmportant lnfectious Agents of the Digestive System of Cattle

Agent Major ainkal Manifestations (Common Oisease Name) ___.....,.. __ Age Group(s) Commonly Affected

Viruses
Alcelaphine herpesvirus 1ª Oral ulcers, diarrhea (malignant catarrhal fever) Any age
Bovine coronavirus Diarrhea 1-4 wks of age
Bovine papular stomatitis virus Oral ulcers Less than 6 mos ot age
Bovinc rot;ivirus Diarrhea (white scours) 1-3 wks of age
Bovme viral d1arrhea Virus Ural/esophageal ulcers, diarrhea Typically 6 mos-2 yrs of age
Ovine herpesvirus 2 Oral ulcers, diarrhea (malignant catarrhal fever) Any agP
Rlnderpest virus• Oral ulcers, diarrhea (rinderpest) A11y dye
V~irular viru~b Vesicles/ulcers on tongue. oral mucosa Any aoe
Bacteria
Actinobaci/lus /igniersii Oral pyogranulomas (wooden tongue) Adull d11imdb
Actinomyces bovis Granulomas of mandible or maxilla (lumpy jaw) Adult animals
Arcanobacterium pyogenes Liver dU)le))e), weiyli Llu)I Adult animals
Clostridium haemolyticum (C. novyí Type O) Hepatic necrosis, sudden death(bacillary hemoglobinuria, red Any aqe
water disease)
Clostridium perfringens-Types B&C Bloody diarrhea (hemorrhagic enterocolitis) Less than l wks of age
E. co/í-enterotoxigcnic< Diarrhea (ffiC diarrhea} Less than 1 wk of age
E. co/i-anaching and effacing Uiarrhea (AttCdiarrhea} calves
Fusob<icterium necrophorum Liver abscesses, weight loss Arlult animals
Necrotic lesions of oral cavity (necrotic stomatiti5) CdlV~
Mycob<icteri11m avium ~11h~p parnh1hPrr11ln~i~ Diarrhea, weight loss (Johnes disease) Greater than 2 yrs of age
5d/111u11elld ~pp.d Cholecystitis, diarrhea with or without blood (salmonellosis) All ages affe<:ted, calves 2 wks 2mos most
susceptible
Yersinia pseudotubercu/osis Diilrrhca, wcight loss Calves, adults

Fungl
Agents of mycotíc rumenitise Decreased appetite and weight gain (mycotic rumenitís) Ruminating animals

•considered a foreign animal disease agent in the United States.

llindudes foot-and·mollth' aJld vesicular stomatitis virmes.
<tndudes fimbrial types K99 {also dosignatad fS) and fd 1 •
dcommon serotypes indude Sa!mone/lil serotypcs Dublin, Montevideo, Ncwpon. anó Typhimurium.
' ln<ludes Abstdla, Asperglllus, MucOí, and Rhlzopus specles.

pathogcncsis of infections are described in detail in spe-
citic chapters related to t hese viruses. Uther common viral
infections of the oral cavity are bovine papular stomatitis,
whlch causes papules 011 varlous structures throughout
the oral cavity and canine oral papillomatosis, \.\lhich in
turn presents \.vith cauliflu"''er-like g1vwlhs (papillon1as)
th;it c;in hP widespread throughout the oral cavity.
Bacteria! causes of oral infcctions typically origina te en-
dogenously from normal oral flora. Infections such as acti-
nobacillosis (woody ton gue) and actinomycosis Oumpy
jaw) 1n cattle are initiated by sorne preceding traun1a that
breaches the normal mucosa! barrier and a llows the intro-
c.luction of Ac:linubuttllu:; lirt~rtier~ii aotl Ac..Linornyces bovis,
res pectivf> 1y.
Gingival and periodontnl di:scn:ics urc more common
problems in dogs and cats than other anin1als. Multiple
bacteria! species, predominately gram-negative anaerobcs,
are involved. Spirochetes also make up a large percent of
the bacteria) population found in gingivitis and periodon-
tal dlseases, but their role in disea:.e patl1ugeue~i::. i:. u11-
clear. Tn cats sPcon<l;iry h;ictPri;iJ gingivitis can be the re-
sult of undcr]ying immunosupprcssivc viral infections
ll' eLV, FIV).
Mycotic oral infections a re uncommon in most ani-
1ual:.. Oral ca11JiJia::.i::. (ti 11 ush) caused by Gandida species
(usllally C. olbicans) is thf> most common mycotic oral in-
feclion encountered. Prior antibiotic treatment, stressful
conditions or debilitating diseases that disrupt normal
oral flora ali prcdisposc to infcction. Jt occurs in all ani
mals but with varying frequency. Candidiasis is especially
common in poultry, where it frequently also involves
other parts of tl1e digestlve systern. Lc:.iu11:; i11 the 111oulh
appear as ulcer-likf> pl;iq11Ps. In dogs, oral granulomas as a
result of dissen1inat ed llistoplas111a capsulatun1 infections,
one of the systemic mycoses, occur with enough tre-
qucncy to be worth noting. Other fungal infections of thc
oral cavity are rare.
lnfections of the Esophagus
húeclio11s of the esophagus are relatively uncommon,
probably due to the rapid passage of material through the
 Chapter 68 Oigestive System and Associatcd Organs 453

Table 68.1 O. Common and/or lmportant lnfectious Agents of the Digestive System of Goat.~ and Sheep

Major Olnical Manifastations (Common Disease Name) Age Group(s) Commonly Affected
Agent
-------
Viru~e~
Bluetongue virus (S) Cyanosis of mucous membranes. oral ulcers Any agP.
Nairobi sheep disease virus• (S) Dloody diarrhea Any age
Peste des petits virus~ Diarrhea, necrotic stomatrtis Any age
Rota virus Diarrhea Typically 1 8 wks of agc
Rift Valley fever Virus• Hepatic necrosis, diarrhea Any age
Rinder¡ipst virusª Oral ulcers. diarrhea {rinderpest) Any age
Veiluldl vi1~b V~itl~uker~ on to11gue, oral mucosa Anyage

Bacteria
C/ostridium haemolyticum (S) Hepatic necrosis, sudden death (bacillary hemoglobinuria, red water disease) Any age, usually adults
C/ostridium novyi·Type B Hepatlc necrosis, sudden death (lnfectlous necrotlc hepatitis, Black disease) Any age, usually adults
C/ostridium perfringens-Type B Bloody diarrhea (lamb dysentery) 1ess than 2 wks of age
C/ostridium perfringens·Type C Dloody diarrhea (necrotic enteritis) L~11 ll 1a11 1 wk uf age
Clostridium perfringens·Type D Diarrhea, sudden death (enterotoxemia) Rapidly growing animals
Clostridium septicum (S) Hemorrhagic abomasitis (braxy) Usually young animals
E. coli·enterotoxigenic' Uiarrhea Less than 1wk of aqe
Mycobacterium avium subsp paratuberculosis Hypoproteinemia, weight loss (Johnes disease) Greater than 2 yrs of age
5d/111011e/ld ~l'I'· Oiarrhea All ages affected

•
G - goats, S - shttp
•consldered a forelgn animal disease agent in Unit.ed 5tates.
~lncludes foot-and-mouth virus• and »esicular stomatitis virus.
qndudes fimbria! types K99 {also designated FSl and f4 I.

TahlP 6R . 11 . Common .and/or lmport.ant lnfectious Agent s of the Digest ive System of Pigs

Agent Major Clinical Manifcstrtions (Common Discasc Name) Age Group(s) Commonly _____
_,__Affected __,
Viruses
African swine fever virusa Diarrhea. vomiting (african swine fever) Anyage
Hemayyluli11ali119 entephalomye!itis viru> Vorniting (vorniting and wasting disease) up to 3 weeks of age
Hog cholera virusª Diarrhea. vomrting (classical swine fever) Any age
Porcine circovirus type 2 Diarrhea, icterus Nursery and growing pigs
Porcine epidemic diarrhea virus Diarrhea, vomitinq Typically piglets postweaning
Swine rotavirus Diarrhea 1 8wks of Dgc
Transmissible gastroenteritis virus Diarrhea and vomiting (transmissible gastroentent1s) AIJ ages, most severe in piglets
Vesicular virusesb Vesideslulcers in oral cavity Any age

Bacteria
Brachyspira hyodysenteriae Bloody diarrhea (swine dysentery) Growers and finishers
Brachyspir~ pilosico/i Di•rrhea (colonic spirochetosis) Wc0Jnc~1 growc~, ond finishcrs
Cfonridium perfringens (lype·A) Diarrhea ~ucklings, weaners, and growers
Clostridium perfringens {Type O Rloorly rliarrhPa (necrotízing enteritis) Typically less than 1 wl: of age
E. co/i-attaching and effaci119 Diard1ea 1-a wks of age
E. co/i-enterotoxigenic:' Diarrhea (colibacillosis) 1 day to 8 wks of age
E. coli Shiga likc toxin positivc Dinrrhea, edema of stomach wall (edema disease) Typically re<ent postweaning
Fusobacteríum necrophorum Necrotic ulcers in oral cavity (oral necrobacillosis) 1-3 wks of age
L¡¡wsonii intracel/u/aris Diarrhea (intestinal ad.enomatosis) 6 20 wks of Dge
Bloody diarrhea {prolfferative hemorrhagic enteropathy) Finishers, breeders
Salmonella speciesd DiarrhPa with or without blood. rectal s1rictures Typically postweaning

•considered a foreiqn animal disease aqent in United States.

btncludes foot-and·mouth.• v~rular rtnm~titi<. "'""" ~icular disease•. and vesicular t>Xanthom• of <wine viru~.
qndudes fimb<~I types KSS. K99, 937P (also desigll3ted F4, FS, ~nd F6, rcspcctivcly), and F41. F18 ¡, as>Ocieted with ~aning diarmea ami edema di>ease.
"usual serovell are Typhimurium varCopenhagen or Cholttroui> vo1 kuuteuuoif.
454 PART IV C:linical Applications

Table 68.17. Common and/nr lmrnrtant lnfPrtinu< AgPnt< nf thP OigpqivP <;yqpm nf Pnuftry

Agcnt
""""'"""".........-
Major Oinical Manifestations (Common Disease Name) Age Group(s) Commonly Affected

Vfruses
Hemorrhagir enteritis vin1s (T) Blnody droppings 4-9 wks of age
Avid11 1vlavirus Watery droppings Up to ~ wks of age
Turkey coronavirus ffi Watery dropping, weight loss (bluecomb disease) Any aqe, typicallv 1-6 wks
Exotic Newcastfe disease virusª Stomatitis, esophagitis, nccrohcmorrhagic enteritis Any agc
Bacteria
8orre/ia aruerina Greenish diarrhea (avian spirochetosis) Anyage
Chlamydophila psittad (O Green gelatinous droppings, hepatitú (ornithosis) Anyagc
C/ostridium colinum Watery droppings, liver necrosis (ulcerative enteritis) 3-12 wks ot age
Clostridíum perfríngcns Typcs A&C (C) DiJrrhea, sudden death (necrotic enteritis) 2-16 wks of age
Salmonella serotype Pullorumb D1arrhea, hver ne<:rosís Typlcally less than 3 wks
Sa/monella serotype Gallinarumb Diarrhea, liver necrosis Mnrp rnmmon in adult birds
Fungl
r11nrlirl11 11/hir11n~ Diphtheritic membrane on crop, esophagus. and mouth Young birds more susceptible
(thrush, sour crop)

C - diickens, T - turk~
• Consldered a toreign animal disease agem m Unlted States.
bOther Salmonella serot}-pes {paratyphoid inte<tions) are usuail'y asymptomatK except rn very young birds (<.l wks of age).

f'soph;.ig11.~
;incf thP tough str;itificd squa1nous epithelium lnfections of the Stomach, Ruminant Forestomach. and
that lines it. Sorne viral infcctions, typically as part of a sys- Abomasum
te1nic infection, cause esophagca.l crosions or ulcers. Most
notable among thcsc are bovinc viral diarrhea virus in cat Bccause its contractile nature causes relatively rapi<.l transit
tle and exotic Newcastle d1scasc virus 111 poultry. Mucocu- of il1gested 1naterial, as well as its mucus coatine ;incl <iciclic
taneous candidiasis in birds 1nost commonly involves the t::r1vicvr11 111::11t, Lile sto111ach does nol providc a very hos-
crop (so ur crop, crop mycosls) but can also involve tl1e pitahle environment for pathogens. Recently, 1nuch atten-
l:'soph;igu~ propi>r ancl provi>ntrin1h1~ . A psi>11clomi>mhrani>
tion has been paid toHe/ícobacter spccics, an organism that
composed of necrotic material ovcrlying n1ucosal surfaces has adapted to live in the stomach, as the cause of gastritis
is the typical presentation (Fig 68.2). and gastric ulcers in humans. Many Helicobacter species
have been identified in animats; however, their role tn dls-
casc ren1ains to be clearly established, in part duc to their
fr1.:4ut..:11l cvlv11izalio11 of clinically i1orn1al anin1als. Tn
sheep, a severe hemorrhagic aboxnasitis (braxy) caused by
Clostridiu1n septicu111 occurs and is rclatcd to particular fccd
F 1G U RE G8. 2. Crop mycosis in a turkey caused by Candida types. A Sarcina-likc organism has been associatcd with
alb1cans. 1-'seudomembranous patches and necrotic material on the abomasal tympany in lambs and goats.
mucosa/ surface are evident. (Courtesy Dr. H. Shivaprasad.) Tn ruminants, damage LO mucosa! surface uf tl1e fon~­
sto mach or disrt1ption in thc ru mi>n flor:i can le:icf to si>ri-
vu:> d11d polenlially fatal consequences. In dairy cattlc, L
integrity of the reticulum is penetrated by forcign linear
objects such as ir1gestcd wirc (hardware disease), peritoni
tis and pericarditis may develo p. fhese infcct1ons are poly-
microbial with Arcanobacterium pyogenes and Fusobacte-
r1L11n 11ecropt1urum corn111ouly l11volvel..l.
Sncfcfen clii>t:iry c.hangcs to carbohydra te rich feeds.
which are easily fcr1nentable, leads to a dccrca:;c rumcn pH
'T'he lower pH kills acid-sensitive run1en tlora and damage~
rumen mucosa. Bactcrial rumcnitis inay develop and can
serve as a source ot organisms for embolic hepatitis tha¡
eventually result in Jiver abscessalion. Common agents re-
covered from liver abscesscs are the familiéir a!J:.cc!>:. pru-
ducers of ruminants, A . pyog<'nP.~ :inci F. necrophorurn
M y LvliL iu111e1úlis also dcvcl o p:s subsequent to ruminal aci-
dosis or prior antibiotic treatment that has disrupte'-
 Chapter 68 Digestive System and Associatcd Organs 455

ruJ1 1t:11 flora. l'he fungal age11ls involved in n1ycotic ru-

1nenitis are often angioinvasive, causing asevere vasculitis, F1G U R E 6 8 . 3 . Transmission e/ectron micrograph ot teces
which leads to further tíssue necrosis. Thc zygomycctcs from a 10-day-old Holstein calf with diarrhea. Both rotavirus (arrow)
(Mucor, Rhizopus, and Absidia) and Aspergillus spp. are the and coronavírus (arrowhead) particles are present. A phage particle
is a/so present. (Courtesy R. Nordhausen.)
fungí most frcqucntly involvcd. Mycotic rumenitis may
a1so serve as a source ror hematogenous disseminanon or
fungal elements leading to mycotic abortion.

lnfections of the Small and Large lntestines

Microbial intect.ions ot the small and large intestines affect
all do1n~~stic animals. Effective vaccination against many of
the major Viral enteric pat hogens (e.g., parvoviruses, dis-
temper virus) in dogs and cats has substantially decreased
lhe inciLience of conlagious enteric diseases in con1panion
animals. When dogs and cats are held in el ose confinement
such as kcnncls, shovvs, and animal shelters, the risk for
contracting cont agious e11teric diseases increases.
Intestinal infections of both bacteria! and viral origin
remain of great clinical and economic significance in
horses, production animals, and poultry c1ue to intensive
iearil1g co11dítio11s, n1anagen1ent factors (e.g., failure to il1-
sure adequate passive transfer or proper nianure Tnanage-
mcnt), and thc absencc of cffective vaccines against sorne
of the major pathogens. Host age susceptibility exists for
sorne enteric pathogens (e.g., Clostridium perfringens I'ype
C, enterotoxigenic E. coli, rotavirus). Neonates, in general,
are most susceptible.
Tl1e 1uajur cli 11ical 1na11ifeslalions of Inicrobial infec-
t ions of the intestinal tract are diarrhea and vorniting.
VomitiI1g oft.cn occurs in enteric infections as part of the
enteric defense response and is regulated by the vomition it helial cells resulting in loss of absorption and resorption
ccntcr ill tl1e brain. Diarrhea is defined as increase in fre cnpncity nnd disruption in osmotic cquilibrium is mnnifcst
quency, fluidíty, or volu1ne of feces due to an increase in by diarrhea. Rotavirus and coronavirus are common
water content . h1sults to the intestines that cause in- causes o f viral enteritis in many animals (Fig 68.3) and af-
creased secretion or decreaseci absorption can result in di- fect predominantly vUlus epithelial cells. ·rhe time course
arrhea. Severity, duration, and characteristics (watery, from exposure to manifestation of clinical signs is usually
bloocly, ele.) of diarrl1ea differ depcndi.ng on t he agent in- shorl. O Lher viruses affecl crypl epilhelial cells (e.g,
volved. Whether diarrhea is actualiy a benefit to the host, Rinderpest virus in sheep, canine parvovirus) and cause
pathogen, or both is unclcar. Diarrhca not only serves the more severe tissue damage. ·rhe spectrum of clinical signs
host. as means to elimina te a buildup of pathogens but also and details on pathogenesis associated with specitic viral
serves the pathogen by providing a ineans for dissemina- agents that affect the jntestines of domestic animals are
t ion and, thereby, maxlmlzes potential for the pathogen to found in the specific chapters on those agents.
infect other hosts. Bacteria are also major pathogens of the s1nall and large
Vi1al infeclio11s i11volving tl1e small and large il1testines i11lesti11es. Tu µruuuce disease il1 ll1e intestinal tract, µu-
may be li1nited to the digestive tract (e.g., rotaviruses, tentially pathogenic hacteria rnust fi rst adhere to target
coronaviruses) or be part of a multisystem infcctious ce/Is . Tf the target ccll is part of a n iche occupied by normal
process (e.g., Atrican swine fever virus, canine distemper flora. the microorganisms \l\ ill encounter "colonization re-
1

virus, and parvoviruses). Intestinal viral infections may be
acquired directly vía the oral route or as a result of a
viremia with localization to intestinal epithelial cells.
Son1e viruses acquired by tl1e oral route are acid resistant,
V\rhic.h allows their passage through the stornac.h, while
others are acid-sensitive but can be protected by the b uffer-
ing action and fats in milk or in feedstuffs with a rapid gas-
tric transit time. Attachment to intestinal epit helial cell re-
ceptors (e.g., sialic acid-containing oligosaccharides) via
viral attachment proteins is the initial step in establishing
infeclion. This is follovved by uplake of Lhe vi1us inlo Lhe
cell, often through receptor-mediated endocytosis, and
replication within the ccll. Subscquent destruction of ep-
sistar1ce," which they must overcome before adhe.rin.g.
Adherence results from the interaction (selective adsorp-
tion) of microbial surface structures (adhesins) with recep-
turs un target cells. Allhe:sins are con:stder<:!d v.i ru.l ence fac-
t ors becau se most pathogens cannot produce disease
will1ou l firsL adhering Lo a largel cell. Fi1nbrial adhesi11s
are protein in na tu re and protrude from the surface of the
bacteria! cell. Thcy are rcsponsiblc for ndherence of sorne
bacteria to carbohydrate moieties that are part of glycopro-
teins on the surface of host cells. The most commonly
found fimb.riae (Type 1 fi.n1briae) on the surface of gran1-
negritive bricteria have affinity for mannose-containing
glycoproteil1s 011 the surface of cells. BacLeria expressing
456 PART IV Clinical Applications
·rype 1 fir111Jriae, wlleu 11lixeu with red IJluud cells, aggluti-
nat<> th<>s<> ci>ll~; this agglutination is inhibited by mannose
(mannosc-sensitivc). Intestinal cpithclial cclls display
structures that serve as receptors tor timbriae exrpressed by
entcropathogenic strains of bacteria.
Other strucrures on rhe surface of batterial ceJls influ-
ence how the bacterium will interact with host cells. These
structures are carbohytlrares and influence the interaction
by rcndcrine th<' ~11rf;:ic·p nf th<> hacti>rial ci>ll ri>latively hy-
drophilic. This hydrophilic property imparts a rep ulsive
force relative to the host cell surface, since the host cell sur-
facc is somcwhat hydrophobic. On thc othcr h and, pro-
tein receptors on the surface of sorne host cells h ave affin-
ity for these surface carbohydrates. ·rhe o utcome o f th e
latter interactlon IS a<lheslon .
;\ftcr ad h erence, the pathogen may produce disease by
1) ::.t:crt:Lio11 l Jf <111 exoLoxin, res ulling, for cxan1p le, in dis-
ruption of fluid and electrolyte regu lation of t h e target
cell; 2) invasion of thc targct ccll, cuusing its d cat h , usually
by t he action ot a toxin (a cytotoxin); or 3) in vasion of t h e
ta rget cell and thc lymphatics, resu lting in a system ic in-
fection . Tne mecnan1sn1s by whtch the host Is affectetl l>y
d ifferent bacteria! enteric pathogens ar.e varied and cnm -
µlex . T11 ~01111:: Lases, as i11 lhc c11lcroxigenic E. coli, t he diar-
rh<>a is sol<>ly a ri>s11lt of the production of enterotoxin and
little to no pathology is observed (fig 68.4). Other patho-
gens (c.g., Saln1011ella) use complex communication sys-
tcms to translocate effector proteins into host cells (e.g.,
Type TTT secrction systems) as \vell as produce both entero-
toxic and cytotoxic effects. Still other pathogens, such as
Mycohacteriu111 aviurn subsp. paratuberculusis, exert their ef-
fect on thP int<>~tinal tract hy tr;inslocating through mu-
co:sal epithelial cells and existing in macrophages in thc
lamina propria and regional lymph 11odes. A granuloma-
tous enteritis results, however, the severity of t h e lesion
does not necessarily correlate with tne severity of clinical
signs. Specific details on the mechanism of p athogenesis
a 11tl rt:~ ul li 1 1g pall1u lugy uf Ll1t:: 11upu1 La ul !JaLLt:::1ial
pathoge ns of the intestines of domestic an im als are cov-
ered in the respective chapters on each agen t .
F 1G U R E 6 8 . 4 . Enterotoxigenic (K99) Escherich ia coli infection
in a 1-day-old calt. Numerous gram-negative rods are adhered to
intestinal villus. Brown & Brenn stain.
Orgaui~u1~ k11uw11 tu IJc )J<lll1ugt:u:> uf Ll1e i11Leslinal
tract in ccrt.ain animals may be found as normal flora in
others. Their role in causing disease in sorne animals, if
any, remains to be established. As an examplc, c;an1py10-
bacter jejuni is the most common cause of diarrhea in hu-
mans and is commonly found 1n the Intestinal tract of
companion and domestic ani1nals- but its ability to cause
disea.se in sorne a11irual:. i:> urrclt:ur. TI 1t:rt:: i:. a h igh ca11iage
rat<> of C. jejuni in chickens with no apparent adverse ef-
fccts on birds greater than 2 weeks of age.
Various factors predispose or make more likely that an
enteric pathogen will establish and cause disease. The im-
po rtance of antimicrobials on the disruption of normal
fl o ra and in allowing pathogens to establish has already
been dlscusst:!d. Other factor:> tl1at <.: n.:alc ::.lress for Lhe h ost
will alsn r<>s11lt in ch;:i ng0s in t h c intestinal flora, 1nainly rc-
sultin g in a drop in th c a naerobic com p onent . Thc lcvcl of
coliform bacteria w il l be h igh er atter a decrease in con cen-
tral'ion of fatty acids produccd by anaerobic flora. '!'h e ac-
tual reason for the decrease io t he number of obligare
anaerobes is not known. In addition to these cha11gcs, the
itlllUUJI l uf fl1.J1 UI lt:Llill (d glyLup1ul1;:ü1) CVdling epilhelial
r.ells in t he oral cavity decreases. Since this glycoprotein
possesses rcccptors for gram-positive specics in t hc oral
cavity, dccrcasc in this population occurs with a corre-
sponding incrcasc in gram-ncgative species, especially
members ot the family J:.nterobacteriaceae.
Fungal infections of the intestincs are not com mon . Of
those cncountered, granulomatous enteritis in dogs
causcd by Histoplasma caps11/ah1n1 is among the most com-
111011. Pyll1iurn i11:;ic./iu:;urfl, au oou1ycele, causes gra11ulon1as
in thc suhmucosa or muscularis mucosae of tl1e small
intcstinc (and sometímes stomach) in dogs, and it is man-
ifest by vomiting, diarrhea, and weight loss. Rare alga! in-
fections caused by Prototheca spccics result in an in tract-
able diarrhea as part of a 1nore generalized p rocess (usually
with ocular involvcmcnt) in dogs.
lnfections of Digestive System-Associaled Organs
lntections o t t he liver occur through a numbet of routes
ancl include tl1e 1) portal vein, 2) h epatic artery, 3) ascen-
sion thro ugh the b il iary system, an d 4) con tiguous spread
fro m adjacent infectious processes (e.g ., reticulitis).
TIJt::: live1 111ay be a la1gel for a 11lu11ber of viruses, often
as part of a systemic infection. Livcr damage can occur by
infection of endothelial cells (c.g., caninc adcnovirus 1)
causing vascular stasis and hypoxia as well as t !1rough in-
fection of parenchymal cells resulting in hepatocellular
necrosis.
A number of bacteria! genera and speciP'\ can affi>rt th<>
livt:1. Clo:.l1 idial species are an1ong the most importan t .
C.lostridial spores of C. hae1nol,vticun1 (C. novyi 'fype D) and
C. novyi Type B, the agcnts of bacillary hcmoglobinuria
and intectious necrotic hepatips, respeetively, are present
in the liver of cattlc and sheep. They germinate when local
liver necrosis rcsults subsequent to immature flu ke migra-
tion or when sorne other event da m ag<>s th<> liver (e.g.,
livt:r IJiuµ:.ie:.). Upon ge1111l11alio11 under this anaerobic cn-
v iron ment, a number of cytolytic toxin s are 1)roduced that
fur th er dan1age the livcr. Th c agent o f 'l'yzzcr's d isease,
 (;haprer 68
FIGURE 68.5. Modified Steiner si/ver stain of a liver le~ion
from a foal that died of Tyzzer's disease. Silver-stain positive
Clostrid tum p iliforme organisms are evident in the typica/ "pickup
sticks" arrangement. Modified Steiner si/ver stain.
Clostridium piliforme, causes a .rare but ac"l!te and h1ghly
fatal infection in a number of animals. It is most important
in laboratory animals. Foals are 1nost commonly affected
among domestic éll1imals. Characteristic spindle-shaped
bacilli are fou11d in tl-ie liver alo1-ig th.c cdgc of cireas of 1-ic-
patic necrosis (Fig 68.5). Clostridium colinu1n causes hepatic
necrosis along with ulccrativc lcsions in the intcstincs of
chickens and turkeys (ulcerative enteritis) (Fig 68.6).
Certain serovars of Leptospira will cause a nonspecific
reactive hepatitis. severity of the lesions can vary from se-
vere chronic hepatitis to mild diffuse hepatocellular vac-
uolalion. Oflen renal involven1ent also occurs.
As already mentioned, rumenitis provides a source of
bacteria tt1rough the portal system that can result in liver
abscesses. Miliary liver abscesses, most commonly in rumi-
nants, rcsults from h.e matogenous seeding by a number of
differcnt bacteria. Common agents include Yersinia
pseudotuberculosis and Rhodococcus equi. In poultry, liver
granulomas are periodically reporteu to l>e cau:scu by a
gram-positive anaerohic rod, Rubacterium tortuosum, and
are thought t o be of intestinal origin.
Tl1e gall bladder infections can be viral (e.g., canine in-
fectious 11cpatitis virus, Rift Vallcy fcvcr virus in shccp)
and bactcr.ial (e.g., Salmonella enterica serotype LJublin in
Digestive System and Associated Organs 457
FIGURE 68.6 . lnfP~tinal
11/rPr in ;¡ rhirkPn with 11/rPrativP
enteritis caused by Clostridium colinum. Liver necrosis was a/so
present. Clostridium colinum was iso/ated from the /iver.
calves) in ctiology. lnfectio11s of the pancreas are rarely re-
ported in domestic animals.
Diseases of the Digestive System of Unknown but suspected
lnfectious Etiology
lt is not unco1nmon for the cause of diarrhea in domcstic
a11imals to go undiagnosed. ·111ere are a number of impor-
tant conditions of the digestive tract of do1nestic a11imals
that are believed to be infectious in origin; however, no
specific agent has bee11 conclusively demo·nstrated to be
tl1e cause. 111 son1e cases n1ultiple age11ts are likely iJ1volved
and/or sorne interplay betwee11 host and cnvironment is
necessary for clinical signs to devclop. Sorne major dis-
eases of suspected intectious etiology, but where the spe-
cific agent(s) has not been conclusively identified, include
poult enteritis mortality syndrome in turkeys, colitis X in
horses, he1norrhagic gastroenteritis in dogs, and winter
uy:selllt!ry Íll c.:alllt! (pü:S$ibly cau:st!tl by 1.Juvi11e c.:oru11a-
virus).
 Integunientary Systeni
RICHARD
·rhe integument 1s the largest organ of the body. It plays a
major role in temperature regulation, se~sory per~eption,
and protection against fluid loss, and prov1des a b.arr1~r from
externa! insults, including potentially pathogen1c m1croor-
gaui:>1u:>. It b <.:u1uposed of epidernlis, dernlis, subcu1is, h.air
foil ir.les, and glandu lar structures. Glandular structures in-
clude sweat and sebaceous glands as wcll as spccializcd
structures such as anal sacs. Hair type and density vary on
thc body according to functional need, including sensory,
thermoregulatory, and protective functions. Birds have
evolved feathers, probably originating from scales, in plarP
uf 11air. Fuulpalb, l 1un 1:>, hooves, 11ails, and beaks are ali spe-
cialized keratinized struchues of the integumentary system.
The epidermis is in continual contact "''.ith t hc sur-
rounding enviro11ment and providcs a home tora res1dent
flora. 'J'hc outcr layer of the epidermis, keratinized stratum
corneum is held togetl1er by a lipid cement, anct together
they for~ the major physical barrier of the skin. Epíder-
rnal tl1ickr1e:>s varíe:> ¡.u11u11g a11i111al:> 1 a1 lll>llg l1reeds, aud al
difff'rf'nt sites on individual animal~;. The dermis, through
collagcn and clastic fibers, provides tensile strength and
elasticity to the integumentary system and also varies sub-
stantially in thickncss throughout thc body. The hypoder
ffilS prov1des additional ncx1b1hty along \vith insulation
through adipose tissue.
'fhe externa! ear canal is included in this chapter be-
cat1sc the external surface is covi>rPcl with <;kin (pinna) nr
epilheliun1 and glandular structures (externa! auditory
rneatus). Overall, the lesions found in otitis externa are
similar to thosc found in skin infcctions, and sorne of the
important pathogens respons1ble for ot1t1s externa ~re the
same oncs that cause infections elsewhere on t he skin.
'fhe mammary gland, although technically nut part uf
the integumcntary system , isª'"º incl11c1ec1 in this chapter
because of its dircct con1n1unication ~vith the skin.
Mammary gland pathogens, sorne of which are resident or
transicnt flora of thc skin, primarily cntcr tbrough the
streak canal of the teat. ·rhe teat sph1ncter and a keratin
plug produced by epitl1elial cells lining the teat canal pro-
vide the prllnary physical barrler for the rnamrnC:try glauü.
Antimicrobial Properties of the Skin
The skin provides a less favorable environment for micro-
bial growth than do mucous membranes of the alimen-
tary, rcspiratory, and urogenital systems owing to proper-
ties presentcd 1n the following sections.
458
L. W ALKER
Dryness
ThP norma 1 o ryness of the skin surface limits the ability of
n1any microbes to survive and establish. Cond·itions th.at
interfere witl1 the norn1al evaporative process cause prohf-
cra tion of resident and translcnt skin flora th.rough Jn-
creased moisture retention and changes in temperC:tture,
pT I, and C0 2 tensio11. Excessive folding o~ tl1e s~in in cer-
tain anirr1al tJreeü:. a11<.l ubese individuals is a prime exam-
ple of an anatomic condition that increases hydration and
tcmpcrature of the stratum corn~um, providing a more
hospitable environment tor bacter1a1 proliteratíon.
Dcsquamation
Continuous she<.l<.li11g uf :;u¡.¡e1fiLial skin layers eli111i11ates
t r;.insiPnt organisms. Residcnt flora numbers are also re-
duced but are p romptly replcnishcd from thc residual
population.
Secretions and Excretions
Hoiocril1e sebaccous glands secrete lipids, including long-
chain fatty acids, many of which inhibit bacteria. They
and thc apocrinc swcat glands con tribute toan intercellu
lar seal in superficial epidermal layers, limiting microbial
access. Apocrine and eccrine sweat glands excrete lactate,
propionate, acetate, caprylate, aod htgh conce.11trations of
sodittm chloride. lnterferon, lysoi.y1ne, transff'rn n, ;inc1 ali
classes oI in1n1unoglobulins are also present. Keratinocytes
synthesize the antimicrobial peptides-cathelicidins and
bctu-dcfcnsins. AH thcsc substances contribute to the self
sterilizing action of thc sk1n-that is, its resistance to colo-
nization by transient mjcroorganisms.
Microbial lnteractions
Resident bacteria exclude intruders by excreting in-
hibitory mctabolit cs (c.g., volatile fatty acids) and bacteri
ocins, and by occupying available niches.
lmmune System
A ski11 in1mune systen1 responds to local antigenic stimuli
including microbial ones, and comprises cell types corre-
sponding functionally to those operating on mucosa! sur-
taces. Langerhans cells, an antigen-presenting cell, and in-
traepithelíal T lymphocytes are prominent constituents
the system. Keratinocytes partll:ipate in ilwuu11e defe11~e L.

producing immune-modulating substances. 'fhe interac-
tions between these cells constitutes tl1e skin-associated
lymphoid tissue. Complement, cytokines, and irnmunoglo-
bulins are found in the emulsion layer of the skin and are
important to overall lntegumentary lmmunocompetence.
Microbial Flora of the Skin
·r:p.e microbial flora of the sl<in plays an important role in
defense and is probably acquired at birth from the dam.
These mlcroorganisms are limitec.l to the superficial epider-
mal layers, where intercellular cohesion is relaxed prior to
desquan1ation, ancl to the distal portions of gland clucts
and hair follicles. "fhe microbial flora exists predominately
in .microcolonies, ratl1e.r tban being evenly distributcd
over the skin. Bacteria! ad.hesion, througt1 Jipoteichoíc
acicis in gram-positive cocci, is critica! to t he establish-
n1enl a11d persistence of resident llora. Kerali11izalion de-
fects, as found in seborrhca, provide additional attach-
mcnt sites and allow for increased nu1nbers of resident
tlora, as weU as clJanges in overall tlora makeup.
Bacteria and yeast variably colonize sites on the skin of
animals. 1'he greatest numbers are found in 1noist, pro-
tected areas such as the axilla, inguinal region, intercligital
spaces, and ear canals. 1'heir concenlralion is Jov.7 er than
on colonized mucous membranes, rarely exceeding
10'/cm2 and,. in sorne areas, 102 /cm 2 .
Gram-positive organisms predominate among resident
flora . Among the gram positives, coagulase-negative
st aphylococci constitute the majority. Certain strains of
coagulase-positive staphylococci are also considered to be
.resident organisms in sorne anirnals. Facultatively anaero-
bic diphtheroids (Corynebacterium and Propionibacterium
spp.) are conslstenUy presenl, as are i'vficrococcus spp. and
virirlans streptococci. ()f gram-negatives. only Acinetobac-
ter spp. are co11sidered a major part of thc rcsi.dcnt flora.
·rhe main fungal resident is a Jipophilic yeast, Malassezia,
which resides on the skin and in the externa] ear canal.
Viruses are not generally considered part of the normal
skin flora. Viral agents that directly infect the skin are
n1ainlaí11cd in a11in1al populati.ons by persistently infected
individuals but are also capable of surviving for extended
periods in thc cnviro11mcnt, which can serve as an altcr-
nate source tor infecting other animals.
Various trans.i ent microbial flora can be encountered.
Beta-hemolytic streptococci (generally Lancefield group
G) on cat or dog skin are usually associated with abnormal
condilions. Men1bers of Lhe fan1ily Erzterobacteriaceae, pa1-
ticularly Escherichia coli and Proteus rnirabilis, and entero-
cocci are common transicnts. Thc fcct of farn1 animals
carry fecal bacteria, sorne of t-vhich participate in toot in-
fections, most notably Fusobacteríum necrophorum and
Prevotella melanínogenica. The agent of ovine footrot,
Llíchelobacternodosus, although hardly a normal commen-
sal, is lin1ited to epidern1al tiss ues.
Transient fungal flora of tl1e skin represent contami-
nants of airborne or soil origin . Many genera can be iso-
lated from the skin . Con11non transient fungal genera
íound on the skin include Aspar3íllus, Chrysosporium, Cla
ctosporium, Penicl//íum, a11d Scopulariopsis.
Chapter 69 Integumentary Systen1 459
Sterilization of the skin is impossible due to the pbysi-
cal inaccessibility to mttch of the skin flora. 1'horough
clea11sing of shaved skin " 1ith soap and water, followed by
soaking with 70% alcohol, will remove 95% of the flora.
More tha11 99o/o removal of skin flora is claiined for re-
pP;:itprl povirlonP iorlinP ;:ippl ir;:ition' ;:inrl rin'"'' with
chlorhexidine (O.So/o) in alcohol. Following such treat-
ment there is rapid repopulation, usually by tl1e saine
organisms.
lnfections of the Skin
Susceptibility of sk.i11 lo ü1feclion is inversely relaled lo
thickness and co1npactness of tl1e stratum corneum.
Spccific animal brccds, bccausc of anatomic conforma-
tions, physiologic factors, or genetic factors, are more pre-
disposed to skir1 infections than others.
lI1Leguu1eI1tary irrfectiuus are uften secundary, rec1uir-
ing a disruption in the host's innate clffP.nsP rnech;inisms.
ractors such as trauma, excessive 1noisture, irritants, insect
or animal bites, and burns are all predisposing causes for
skin infections to clevelop. Undedying discascs, including
preexisting sl<in conditions, and immunological disorders
also predispose to secondary skin infections. Deep dermal
a11d sulJcuLa11euu:; ir1fectiun:; typically require sorne fonn
of traumatic in1plantation that allows for t hP introrlurtron
of microbial pathogens that \<Vould otherwise not persist
on the externa) epiderrnal layer.
Systemic u1fections somctiincs involvc thc intcgumcn-
tary systen1. Agents with trophisrn for vascular endothe-
lium or epithelial ce!ls can cause focal or gen.eralized le-
.sion.s in the skin.
Viral lnfections of the Skin
A numbcr of viruscs gain cntry to thc host via thc skin, ci-
t her tl1rough abrasions, insect or a11imal bites or by expo-
sure to contaminated equipment (e.g., needles, harnesses).
Sorne of these viruses simply use t he integum entary sys-
ten1 as a rou te for entry into the host and cause either
systemic infections or 11ave prin1ary Largels ll1al are organs
or systems other than the skin (e.g., rabies virus). Details
011 mcchanisms used for entry, replication, and spread via
t h e integumentary syste1n tor specitic viruses are covered
in chapters on those individual viruses.
Por so1ne viruses the skin is the primary site of infection
or is one of the principal sites where clinical signs mani-
fesl. The papillu111aviru:se:s allu ¡;uxviruse:; are prurninent
víral pathogens of the integumentary system in animals.
1"he papillomaviruses are inu·oduced through skln abra-
sions and infect epithelial cells. Infected epithelial cells be-
come hyperplastic. Hyperkeratosis is the rcsult. 1'hc lc-
sions (papillomas) are typically raised and filiform, and
may be pedunculated. The papillo1naviruses are fairly
l1ust-specific1 alt11uugl1 bovine papillomaviruses 1 and 2
are associaterl with eq11 ine sa rroirls.
Men1bers of the fan1ily Poxviritlu.e also affe<.:t a 11u1111.Jer
of animals and birds. Many viruses in this family have a
limitcd host rangc, altl1ough sorne are zoonotic. Transfer
occurs through contact with skin Jesions, scabs trom in-
460 PART IV Clinical Appllcations
tected ani1nals. or mechanically by biting insects. ·rhe
parapox virus that causes sore mouth in shcep and thc
fowlpox virus i11 poultry can be transmittcd by rcspiratory
d roplcls, as well as by direct contact with lesion mat erial or
<.:011lar11 i1 ialeLI eyui¡.1111e11 l. Depe11Lli 11g 011 lhe vil us in-
volved, clinical disea.se can he 111ilcl to severe. Generalized
signs include fever ar1d anorexia. 'fhc skin lesions are ty-
pically papulonodular and become pustular or prolitera-
tivc, cvcnlually resulting in the production of scabs that
Icavc scars.
Cutaneous manifestations are part of the overall clini-
cal presentation of sorne generalized ur ruultisysteu1 viral
infection;;. Vir11s1>s that ca11<;1> vC'c;icular ctiseases (foot-and-
mouth <lisease, S\vine vesicular disease, vesicular exan-
thema of swine, and vesicular stomatitis viruses) produce
vcsicles that variably affcct thc tcnts, coronnry bnnds, and
intcrcligital areas of the skin. 'the vesicles readíly rupture
lcaving ulcerative lesions that may bccome secondarily in-
fectect wlth bacteria. In addltlon to the Iyrnpt1oreti<.:ular
and neurological lesions it prochrcPs, thP MarPk's diseasc
vi1 us of cl1icke11s causes nodules to develop in the feathcr
follicles and uses fcather follicle dander as a primary
means of spreading virus to other birds. Caninc distempcr
virus causes nasal and tootpad hyperkeratosis in dogs as a
late clinical manifestation.
Bacteria! lnfections of the Skin
Bacteria associated with skin infections primarily origi-
natc from the cnvironment or director indirect contact
with carrier animals, orare resident flora of the skin, oral
cavity, genital tract, or lower digestive tract of the affected
an1 mal. Bacteria! skin infections are classified as superficial
pyodermas (e.g., impetigo, superficial bacteria! folliculitis)
or tleep pyuder1na:> (e.g., fulliculiti:>, furuu<.:ulu:>is, celluli-
tis, and suhcutaneous ahscesses). Coagulase-positive
staphylococci (e.g., S. aureus, S. intennedius, S. hyicus) are
thc primary agents of superficial pyodermas. They pro-
duce a myriad of enzymes and toxins that contribute to
the disease process. Along with cell wall components,
sorne of these microbial products have potent chemotactic
effects that accuunt fur tl1e µyogc1ü<.: ir1fla11uuatory re-
sponse observed in staphylococcocaJ infec.."tions. Staph_vlo-
coccus hyicus, thc cause of grcasy pig tliscasc, produces nn
cxloliative toxin that specifically causes separation of in-
traepidcrmal layers resulting in focal erosions in the epi-
dermis. In neonaral pigs, this generalized epiderrnitis is as-
sociatcd with substantial mortality.
Derrnatophilus congoll?lsis, an ac..tinuruycetc, alsu cau:,e:,
" s11p1>rficial ciPrmatitis, most oftC'n in horses and run1i-
nants. lnfection stimulates an intense inflammatory re-
sponse that is characterized by alternating waves ot in-
flnn1mntory cclls followed by newly generated epider1nis
(Fig 69.l). In contrast to the abunctance of products pro-
duced by coagulase-positive staphylococci, few virulencc
factors are rccognizecl in Dermatup/Jilus. lt produce:. au ex-
traccUtil11r serine protP<iSP that may contrihute to the over-
all disease process; however, environmcntal factors are key
to the development of disease. Persistent wetting of the
skin or inscct bites are nccdcd for Derrnatophilus to initially
invade the epidermis. Once 1nvas1on occurs, an inflamma-
F 1 G U RE 6 9. 1 . Waves of inflammatory exudate and
regenerated epidermis from a cow with dermatophilosis
(hematoxylin and eosin).
tory reaction is induccd, which rcsults in profuse cellular
exudation. li predisposing conditions remain to promote
invasion of newly gencratcd epidermis, the infection/in-
flammatory cycle repeats, giving rise to the characterlstlc
histopathology; otherwise, thc infection resolves.
Deep bacteria! pyuLler1ua:, i11volve lhe der111is and
<;om1>tim1>s the s11hc.11taneous tissue. lnjury or trauma al-
most always precede tl1c development of deep pyodern1as.
In son1e cases, deep skin infcctions occur as a result oi the
cxtension of a superficial pyoderma. J\gain , coagulase
positive staphylococci are frcquently involved, although
other bacteria may be secondarily involved. Furunculosis
(lnflammation of the dermis and subcutis) and folliculitls
(inflammation of the ha ir foil iclt>) are most c'ommon ly r1>c-
og11iz.cd ir1 dogs but also occur with some frequency in
horscs, goats and sheep.
Subcutaneous absccsscs nre typica!ly thc result of a bite
wound or penetrating foreign body. ·rhe bacteria intro-
duced usually represent rnembers of the oral flora and
cause an accumulation of purulent exudate in the dermis
and subcu taneous tissue. Subcuta oeous absc:PSSPS most
con1n1011ly occur in cals and n1ost frequently involve Pas-
teurella multocida and obligate anaerobic bacteria. ~ubcu­
taneous abscesses are also co1nmon in ruminants, with
Arcanobacrerium pyogenes frequently being recovered.
Corynebacterium pseudot11berc11/osis causes subcutaneous
auscesses iu sl1eeµ aud hur:>e:>. 111 callle il n1a11ifests asan
ulcerative dermatitis rathPr th<in forming ahscesscs.
Cellulilis is a loose purulent inflammation of the sub-
cutaneous tissue. It is generally poorly cootained and ex-
tcn<ls rapidly along tissuc planes from the site of initia
tion. A variety of bacteria! agents cause cellulitis. Among
the most serious forms of cellulitis is the anaerobic celluli-
tis caused by one of a number uf llistutoxi<.: <.:luslriLlidl
species. LJnder anaerohic Pnvironments the.se specie<.
produce potent and highly destructive toxins. Horses, ru-
minants, swine, and poultry are most commonly affected
by clostridia1 ccllulitis. lnfcctions have a high fatalir
rate, even in the face of aggressive treatment. An E. co/:-
associated cellulitis in broiJer chickens is ar1 especially ec ....
nomically important dlsease for the poultry indu:>try, a-.-

counting for a substantial percentage of condernnations at
slaughter plants. Staphylucuccus aureus causes a11 acute ccl-
lulitis in horsf>s that sprPacls rapi<lly an<l causes necrosis of
the overlying skin. 'fhoroughbrcd racehorses are most
commonly affected.
Othcr bactcrial infcctions of the skin include mycobac
terial granulomas. Cats are most commonly affected by
tl1ese organisms, although infections also occur in swine,
canle, an<.I <.logs. ·rhe sapropl1ytic u1yco1Jacteria rcspuusi-
l)lt> arf> intro<lnc:ed hy sorne trau1na. Jf they estahlish, infec-
tions are characterized by chronic, nonhealing wounds
'\-Vith draining fistulas. A specjfic entity in young cats re-
fcrrcd to as feline leprosy is caused by Mycobacteriu1n leprae
n1urium and presents as cutaneous nodules of the head and
extremities that sometimes ulcerate. A regional lympha-
<.leuopat!1y is ufte11 µreseu t. A secu11Ll, 111ure geJJeralíz.eu
form of the disP;:isi> c;:i11sPcl hy ;in nnidf>ntifipcf mycoh;ic-
terium has been described in older cats. Pyogranuloma-
tous dermatit is caused by Actinomyces spp. occurs in dogs
(A. viscosus or A . hordeovulneris associated with plant awns)
and cattle (A. bovis). Draini11g tracts and the presence of
yellow gr<u1ules composed of bacteria] colonies, some-
tiu1.es surruu.1uJe<l by a hu111oge11ous eosi11o¡;liilic 111alerial,
0
are fo11nd in th f' clr;iiningm::itf'ria l (Fig 69.7.).
Son1e bacteria! skin infections involve two or more bac-
teria working in concert. In contagious ovine foot rot,
Fusobacterium necrophorurn causes an interdigital dermati
tis in macerated skin, which allows Dichelobacternodosus to
establish by fim.brial attachment and proliferate. Produc-
tion of a nu1nber of serine an<.I basic pruteases 1.Jy D. nu-
dosus results in an undf>rn1nning of thf> solf> an<l, thf'rPhy,
allows for additional opportun istic bacteria to invade, fur-
ther aggravatingthe condition.
A substantial intcgumcntary componcnt is part of cer-
tain systemic bacteria! infections. ·rhe urticaria! plaques
and diffuse erythematous lesions in the skin of swine, re-
sulting from cutaneous infarctions, are characteristic of
erysipelas. ·rhe effects on vascular endothelium of bacter-
ia! toxins or l1yµcr:se11silivily reaclio11s lo bacterial anli-
gP.ns may also n1anifest in the skin. Subcutaneous swelling
of the eyelids, lips, and forehead occur in edema discasc in
pigs, caused by Shiga-like tox.i n producing strains of H. coli.
F 1G U RE 6 9. 2. Numerous granules from Acti nomyces bovis
lesion in a cow (hen1atoxylin and eosin).
(~1zapter ó9 Tnt<>.gumentary Syste1n 461
"fhe dependent ede.n1a resulti11g from tl1e vasculitis that
uccurs i11 ¡;ur¡;ura l 1eI11orrhagica i11 horses is a sequelae lo
infection with Streptococcus equi suhsp. equi.
Fungal lnfections of the Skin
Fungal infections of t he skin are classified as superifical cu-
t aneous mycoses or subcutaneous mycoses. In addition,
fu11gal age11ts lltal cause sysleulic ruycu:ses (BlusLurnyc.es
derrnatitidis, Coccidioides imrnitis, Cryptococcus neofbrmans,
Histoplas111a capsulatu111) cause pyogranulomatous lesions
in tl1e integumentary system subsequent to clisseminated
infection.
The most important of the superficial mycotic infections
is dermatophytosis or ringworm. lt denotes a specialized
uer111at<.1111ycusis causeü 1.Jy fu11gi tl1at sµecifically attackker-
;itinizPd stn1cn1res. ThPse fi.1ngi (Mícrosporum spp, Trichophy-
torz spp.) produce enzyn1es (keratinases) that digest keratin
and infect growing hair and stratum corneum (Fig 69.3).
Lesions are inflamed, crusty, or scaly and often circular to
oval in shape due to the centripetal p rogression of the le-
sion. Affected hair is brittle, leaving areas of alopecia. Der-
111atupliytes are uut always patl1ogens anti can represent
tr;:insif'nt flor;1 of the skin (usually geophilic dermatophytes)
orbe carried inapparer1tly (zoophilic dern1atophyte:;).
Malassezia, a lipohilic yeast, is the other main fungal
agent involved in superficial rnycotic infections of the skin
(Fig 69.4). It causes an erythernatous, scaly Iesion. Since it
can be found i11 low numbers on normal skin, clínica! rel-
evance of its isolatio11 rnust !Je <.leci<.led 1.Jaseu on clirlical
signs.
The subcutancous mycoses involvc thc dermis and sub-
cutaneous tissues and may be localized lesions or spread via
the lymphatics. Most of the fungal agents involvecl are
from an environmental source (e.g., soil, plant material)
a11d gain entry by traurnati.c introductiOl1. A number of dif-
ferent subcutaneous mycotlc infections are recognized and
classified by their gross and histopathologic characteristics.
Tl1ese different conditions include chron1oblaston1ycosis,
phaehyophomycosis, and eumycotic mycetoma. They are
dcscribcd in more clctail in thc chaptcr on agcnts of subcu-
F1GU RE 6 9. 3 . Funqaf hyphae and arthroconidia on the haír
shaft from a goat with dermatophytosis caused by Trichophyton
verrucosun1. Gomori methenamine si/ver stain.
462 PAJtT IV Clinical Applications

F 1G U R E 6 9. 4. (A) Skin of pig with Malassezia dermatitis
(hematoxy/in and eosin). (B) Numerous broad-based budding yeast,
characterirtic of Malassezia are found in stratum corneum. Periodic
acid·Schiff reaction.
F 1G U RE 6 9. 5. Grdin fru111 d eu111ytulit 111ytelu111d in llie >ki11
of a horse. The qrain, composed of aberrantly shaped fungal
hyphae, is surrounded by a mixture of neutrophils. macropha9es.
plasma ce/Is, and giant ce/Is. Gomori methenamine si/ver stain with
hematoxy/1n countersta1n.

F 1G u RE 6 9. 6. Numerous yeast forms of Histoplasma

capsulatum are present in skin from a dog with disseminated
histoplasmosis. Gomori methenamine si/ver rtain with hematoxylin
counterstain.

taneous mycoses. More than one h1ngal age11t may be re-

sponsible for each of these conditions. For example, eumy-
cotic mycetomas-which are characterized by 1) localized
swelling at the infcction sitc (tumcfaction), 2) draining
sinus lrac.Ls, and 3) lh.e prcsel1.CC: o( gJanulcs or grains con1-
posed of colonics of the causative agent (Fig 69.5)- are
causcd by over 14 species of dematiaceous (pign1ented)
fungi and over 9 species of hyaline (colorless) h1ngi. 'l"l1ey
produce dark and white grained mycetomas, respectively.
Sporotrichosis is a subcutaneous mycosis caused by rhe
dimorphic fungus, Sporothrix schenckii. Lesions of sporotri-
chosis are ulcerative 11utlult0~ ur rct.:urrcut tlrdi11i11g LracLs in
thP skin. !nfí'rtions rnay also involve the Jymphatics caus-
ing a lymphangitis, in additíon to the skin lesions. Cats,
horses. and dogs are most commonly attected.
Cutaneous pythiosis is caused by an oomycete, Pythium When one of the systemic mycotic agents involves the
insidiosu111, and therefore ís nota true fungal infectíon. It is lntegumcntary system, the lesiuns are typically ult.:erali\t
found in swamps and ponds, and the dísease is associated nodules that may dPvi>lop clr;iining tr;icts. These pyogran-
with exposure to tlio!>t! t!11víro1uue11t~. Sk.i.r1 ilúecLio11s a1e ulomatous skin lesions (Fig 69 .6) may be the initial clinica1
char;irtPri7.Pc1 hy firm or <;pongy, ulcerated lesions that manifcstation of an infection even tbough organisms re-
often have draining fistulous tracts. Necrotic masses of tis- sponsiblc for thc skin lcsions almost always origina te from
suc (kunkcrs) may be tound in the lesion. Althougl1 un- 01ssem1natea resprratory Lract infections.
conunon, horses and dogs are most frequently affected. A.n Common and/or important agents of the inte¿,'l.1men-
addirional genus, Lagenícliurn, has also been reported to be tary systern uf tlu1111;;~tit.: a 11i111<:il~ aud poulti:y are listed ir.
involvcd in such lcsions. 1'ables 69.1-69 7
 ChuJ1ler 69 I11 lcguu1cutary Systeru 463

Table 69.1. Common and/or lmportant lnfectious Agents of the lntegumentary System of Dogs

Agent Major Oinical Manifestations (Common Disease Name)

Viruses
canine dlstemper virus Nasal and footpad hyperkeratosis (distemper}
Canine papillomavirus Cutaneous papilloma~ CraninP papillnmato~i~)

Bacteria
Aaínomyces víscosus, A. hordeovulnerls Orainin91raas, subcutaneous absce~s
Bruce/la canis Scrotal dermatitis
Staphylococcus intermedius' Ce!lu!itis, fo!liculitis, furuncul05is, impetigo
Fungi
M;i/assezia p~chydermatis Exfoliative dermatitis
Microsporum Cdllfa, M. yyµ)eu111 7iilhuphytu11 Cirtulij1, ~toly, t•u~ly, alorrettic skin lesions (dermatophytosls, rtngworm)
mentagrophytes
Pythium insidiosum Ulcerative and pyogranulomatous skin lesions (cutaneous pythiosis)
Systemic mycotic agents~ Paputes, nodules, abscesses, draining tracts

'Other co•gulase-posit1ve Staphylococcus sp. mvotved mpyoderma mclude s. auteus and S. scllleiteri subsp coagulans.
b1ndudes 8/astomyces dermatitidit Coccidioides immitis, Cryptococcus neoformans, and Histopfasma capsulatum .

•
Table 69.2. Common and/or lmportant lnfectious Aqents of the lntegumentary System of Cats

Agent Major Oínical Manifestafion~ (Common Oio;pao;p NamP}

Viruses
Cowpox viru~ Macules, papules, nodules (cowpox virus infection)
Feline sarcoma virus Cutaneous and subcutancous nodulcs
B<lcteria
Mycobacterium sppb Chronic nodular dermatitis. draining tracts. panniculitis (atypical mycobacteriosis)
Mycobacterium lepraemurium Noduloulcerative skin lesions with lymphadenopathy (feline leprosy)
Obligate anaerobic bacteria' Subcutaneous abscesses
Pasteure/la multodda Subcutan~us abscesses

Fungi
Cryptococcus neoformans Draining tracts. nodules. ulcers {cryptococcosis)
Microsporum canís Alopectic annular skin leslons (dermatophytosis, ringworm), pseudotnycetoma
Sporothrix schenckii Draining tracts, lJlcerafive nodules (sporotrichosis)

'Not seen in the united States.

btncludes M. fortuitum, M. chelonei, M. xenopi, M. smegmatis, and M. phlei.
'lncludes Peptortreptococcus spp.. Fusobacterium spp., Porphyromonas spp.. and Clostridirrm ~pp.
464 PART IV Clinical Applicatioi1s

Table 69.3. Common and/or lmportant lnfectious Agents of the lnt egumentary System of Horses

A ent Ma·or Oinical Manifestations (Common Disease Name)

Viruses
Bovine papillomaviruses 1&2 Verrucous, fibroblastic or flat and thickened skin lesions (equine sar(oid)
Equine papillomavirus Cutaneous papillomas of lips and nose (equine papillomatosis)
Equine viral arteritis virus Edema of distal limbs, scrotum, and ventrum
Vesicular stomatitis virus Vesicles/ulcers on coronary band
Bacteria
Bacillus anl11rati~ Diffuse subcu:taneous and dermal edema (anthrax)
Burkho/dería ma/fe¡a Subcutaneous nodules that ulcerate. lymphangitis (farcy)
C/ostridium perfringensh Ccllulitis
Corynebacterium pseuáotuberculosis Pectoral (pigeon breast) or inguinal abscesses, lymphangitis (ulcerative lymphangitis)
Dermatophi/us congolensis Exudative dermatitis (dermatophilosis, rain rot)
Rl1oáococcus equi cutaneous abscesses, cellulltls
"Staphylococcus aureus Celfulitrs, folliculitis, furunculosis

Fungí
Histoplasma farciminosumª Nodutes of head, neck, and leg; lymphangttis (histoplasmosis farciminosi)
PythitJm insidiosum Ulcerative pyogranulomatous 1esions (cutaneous pythio,js, ~w~mr canrer)
Sporothrix schenckii Ulcerative nodules on feys, lympho11yilb (;pot ulridru>i>)
Trirhnphytnn er¡11in11m, T. mentagrophytes, Crusty skin lesions; hair loss often involving head, shouldets, and back (dermatophytosis. ringworm)
M. equinum

'CoosÍdéred a forei911 a·níJnal disease agent in the Uníted States.

bOther closiridial species include c. sepricum, e sordellii, alld c. sporogenes.
'é?ther ctfagulase·positive species affecting horses include 5. intermedius and 5. hyicus subsp. hyicus.

Table 69.'1, Common and/or lmportant lnfectious Agents of the lntegumentary System of Cattle

Agent Mejor Ciinicel Menifestations (Common Disease Name)

.....,,,...,..,...,..,.,....,,....,...,,.....,.....,...,.._,......,.....,.,...........,.,.....,..,..,_,,,-,..,....__,"'=,....,

Vlruses
Bovine herpesvirus 2 Mammary vesides and ulcers (bovine mammilitis)
Bovíne papillomavirus Cutaneous papillomas (bovine papillomatosis, warts)
Lumpy skin disease virusª Generalized or local papules and nodules that ulcerate (lumpy skin disease)
Pseudocowpox virus Mammary vesícles, papules, and scabs (pseudocowpox)
Pseudorabies virus Uncontrolled pruritis (pseudorabies)
Vesicular virusesb Vesicles and ukcr; on coronory b<ind and intcrdigital arcas
Bacteria
Actinobacíllus /igniersii Head and neck abscess. draining fistulas
Actinomyces bovis Draining tracts, subcutaneous abscesses (actinomycosis, lumpy jaw)
Arcanobacterium pyogenes Subcutaneous abseesses
Clostridium s.epticum Cellulitis (malignant edema)
Corynebacterium pseudotuberculosis Ulcerative dermatitis
Dermatophi/us congolensis Exudative epidermiti1 (derm>itnphilosis)
Fusobacterium necrophoruml Prevotel/¡¡ hrlerúiyildl de1111dlili>, celluli tis (interdigital necroba<illosis, footrot)
melaninogenicuf
Sa/mone/la Dublin Gangrene of distal extremities, ears, and tail duc to terminal cndartcritis

Fungl
Trichophyton verrucosum Round to oval. crusty skin lesions with alopecia (dermatophytosis. ringworm)

'Considered a foreign animal disease agent in the United States.

bindudes foot·and-mouth rli<P><P viru<' anrl ve.<indar <tomMítis virus.
<Agents act synergístically.
 Chu¡1ler 69 Tuteguu 1eulary Sysle111 465

Table 69.5. Common and/or lmportant lnfectlous Agents of the lntegumentary System of Sheep/Goats

Agent Major CUnlcal Mallifestations (Common Disease Name}

Pr.ions
scrapie prion PFUfítus, exc0riatíons, self-mutilation (scrapíe)
Viruses
Bluetongue virus Erythema, edema of ears.and n:iuzzle, corc<>nitis (bluetongue)
Goat pox, ~heep pox víruses• Papules, veslcles, pustules (goat pox, sheep pox)
'l'arapoxvirus Crusting proliferative mucocutaneous lesions, teat lesions (contagious ecthyrna, soremouth)
Vesicular virusesb Vesicles a11d úlcer~ on teats, coronary bands, and i11terdí9ital areas
Bacteria
Clortridiun¡ novyi Edema of the head, neck, and tnorax (bíg head in Farns)
corynebacterlum pseudotuberculosis Skin abscesses (caseous lymphadenítls)
Dermatophilus congo/ensis E~udative dermatitis (dermatophilosís, lumpy wool rliseasE>, !ttrawherry fo¡:¡trot)
Dichetobactet nodosus/Fusobacterium necrophorum' lnterdigital dermatitis, underrunning of the sale .(contagious footrot)
Stap/Jylococcµs aureus<! Pustular dermatitis of face, udder, teats, and ventral abdomen (staphylo~occal dermatitis)
Fungí
Trichophyton verrucosum, T. me11tagropl1ytes Crusting, circular skin lesions witli alo¡.¡~cio (<le1rmitophytosis, ringworm, club lamb fungus)

3 Considercd a for~ign animal dise;ise agent in the tJnited States.

blltLludtt~ foul·d11U·1noull1ilb>eC!:ie vj1 U).;. d11U-v~it.uld1 .)to111a'litj~ virus.
'Agents act synerg\stically.
dStaphylococcus aureas subsp. ana8fobíus isassociated with subcutaneous abscesses in sheep.

Table 69.6 . Common and/or lmportant lnfectious Agents of the lntegumentary System of Pigs

MÍ!jor Clínica!·Manifestátions {Common Disease Name)

......................................................__,,.............."!!!'"...........""""i'

Viruses ·.···
AfriGan swine fever virus" Reddish-purple discoloration of si<in (African swine fever)
Hog Cholera virus• Erythema, purple discoloration of skío, necrosis of ears and tail (hog cholera)
Swinepox vtws Macules, papules, pustules (swlnepox)
Vesicular viruse.sb Vesídes aorl ulre~ nn cnronary banrl anrl intPrnigital arpas

Bacteria
Bacif/us anthracis Diffuse suticutaneol.is and dermal edema of neck and thorax (anth,raX)
Erysipelothrix rhusiopathiae Ccingested raisP,d skin lesions, rhoml\nidal,shapPd nec;rotk skin lesinns (ery,sipela~
fatl1e1it/Jid to/i (Shiga-like toxin positive) S·ubcutaneous edema of lips a~1d eyelids (edema disease)
Salmone//a Choleraesuis Reddish-purpfe discoloration of the skin
Streptococcus equi subsp. zooepidemicus, P1.1stular dermatitis, subcutaneous abscesses
S. dysga/adiae subsp. equisimilus
Staphylococcus hyicus Generalized exudatíve dermatitis (exudative epidermítis, greasy pig disease)

fungí
Microsporum nanum, Trichophyton spp. Reddish circular skir:i lesions with crusting at periphery (dermatophytosis, ringworm)

'Considered aforeigo aoimal disease agent in the United States.

b1ncJudes foot-and-mouth.'vesicular stomatiti~virus, swine vesicular disea1e. and ~esicular exanthema of swine viruses.
466 PAHT IV Clinical J\pplications

Ta b 1e 6 9. 7. (0111111011 a11d/01 lmportant lnfectious Agents of the lntegumentary System of Poul try

Agent
-- Ma" r Oínical Manifestations (Common D1sease Name)

Viru~~
Fowlpox viro) Pdpul~ dlld nodul~ uf be.ik, co1nb, .:md wdttles (.ivicln pux)
Marek's disease virus (Q Nodular lesions of feather follicles (Marek's disease)
Bacteria
sraphylococcus aureus Plantar abscesses (bumblefoot), gangrenous dermatitis
Clostridium septicum, C. perfringens Gangrenous dermatitis
Erysipelothrix rhusiopathiae Turgid/red·purple snood, erythema (erysipelas)
Escherichia coli (C) Cellufitis
Fungí
Microsporum galfinae White, powdery le;ion; oí ~omb, Wdltle), dlld l~y) (u~1111dlu¡.ihylo)Í5, íovus)

Table 69.8. Common Agents of Canine Otitis Externa and Key Organism Characteristics

Typical Colonies On: Aridll;iry TP'it~

Agent Blootl A!Jdí {244311~) MocCorikey Agar Gram stain Oxidase

Staphylococcus intermedius White or off·white, often with No growth Positive cocci NA Posítive
double-zone hemolysis
Staphylococcus schletfert Whrte, hemolytic No growth Positive cocci NA Positive
subsp. coagulans
Proreus mirabills Swarms. no discrete colonies Colorless Negdlive rod; N~yotive NA
PsPudomonas aerugino.!il Gray to greenish, fruity odor, Colorless, sometimes pigment Negative rods Positive NA
hemolytic deteded
Streptococcus canis Small, white, 6 hemolytic No growth Positive cocci • NA Negative
Eschcrichiu coli Smooth, grey, some strJins ore Pink to red with red hilzc NcgJtivc rods Negative NA
hemolyt1c
Klebsiella pneumoniae Mucoid, whitish-grey Mucoid, pink without red haze Negative rods Negative NA
Malassezia pachydermatis No growth or tiny colonies• Nogrowth variable staining, NA NA
budding yeast

NA= not appticable

iMay rPquirP prnlnng•rl inrnh>linn RP<t rPrnv•ry on ~ahouraud'< dP.xtmse agar at 37'( under microaerophilk rondition<.

Otitis Externa
Otitis externa in dogs is one of the most cornmon derma-
tological problcms cncountcrcd. ·rhcrc is a dircct rclation
between ear conformation and otitis externa, 'v1th pendu-
lous-eared dogs being predisposcd to infection. The L-
shaped configuratlon of the external meatus is a ftuther
complicating factor bccause it limit~ at>ration ano orain-
age. An abundancc of lipid-rich ear•vax and heavy ear
canal hair additionally contribute to development of otitis
externa. A brccd prcdilcction (c.g., cockcr spanicls) for dc-
velopmcnt ot otitis externa is aJso evident.
The agents of canine otitis externa are endogenous in
origin and <tre not thougllt tu play tlic irlilialíng role iu the
disease process, but rather actas opportunists once otht>r
faclo1~ a1t'. in place. lnfeclions are frcqucntly polyn1icro -
bial. c:ommonly recovered agents fron1 c;ininf' otitis P.X-
te1 na aie listed in "la ble 69.8.
Mastitis
Ma::.tili::., a11 iníla11uualiu11 of lhe n1an1mary gland, occurs
in ali animal species. lt is most common in dairy cattle a11d
is of greatest economic significancc to thc dairy industry.
For mastitis to develop, the innate physical barriers must
he breached. Once that occurs, both innate immunity
(lactoferrin, complcmcnt, resic.ient immune cells) anc.1 spe-
cific immunity play ;i rnlP in prPventing microbial patho-
gcns fron1 establishing. lf a pathogcn does establish in thc
mammary gland, chemotactic gradients formed by in-
flammatory cytokines and cl1emokincs rcsult in rupid re-
 Chapter 69 Integumentary System 467

cruitmen ton i nl"lamn1atory cells in an ettort to control tl1e

F1G U R E 6 9 . 7 . Lactophenol aniline blue wet mount infection. Most of the clinical sign.s associated with masti-
preparation of a Prototheca iso/ate from a cow with mastitis. tis are a con sequen ce of the inflan11natory respo11se.
Sac·ltke structures (thecae) containing variable numbers of daughter Bacteria are the main infectious agents of mastitis, al-
ce/Is (autospores) are seen.
lhough v iruses aie iniportaul u1a11 uuary patl1ugeus iI1
.. sorne animals (Maedi-visna, sheep; c:AE, goats). Fungí
(e.g., Candída, Aspergíllus, I'seudallesclu~ría) andan acbloric
algae, J>rototheca (Fig 69.7), are rare causes of mastitis in
cattle but can occasionally cause 11erd outbreaks.
Different organisms predominate as mastit.is pathogens
depending on the anim.al involved. Coliforms (dogs,
swi11e), Stuphylococcus uureus (slteep, guats), l.H::ta-J1e1nulytic
streptococci (horses), Mannhehnia haen1olytica (sheep), and
lvtycoplasnta species (sheep, goats) are the n1ost commonly
encou11tered bacteria.
The age11ts associated witl1 mastitis in cattle are exten-
sive and are classified as either "contagious" pathogens
where the mammary gland is the source or as "environ-
n1enlal" palhogens when lhey origi11ate fru1n tra11sient
skin flora or environmental reservoi rs. C:ow agP., p;:irity,
stage of lactation, milking operation management meth-
octs, anc1 environmental sanitation control (housing and
bedding) are underlying factors in most cases of mastitis.
Common agents ofboVine inastitis are listed in Table 69.9 .
•

Table 69.9. Agents of Bovine Mastitis

S eciflc Featúres.

:Arcanobacterium pyogenes (){casionaJ Associated Wlth teat inju¡y or cannulafdilator use, poor treatment resp0nse.
Clostridium peffríngens Rare Causes gangrenous mastitis
Coagulase-negative Staphylococcus spp. frequent Source is skin, many infections transient
1
Escberichia có/i Frequent Environmental source, acute infections, may cause systemic illness
Klebsíella pneumoniae Occasional Severe mastitis, asso~iated with sawd!Jst/wood shaving beddíng
Mycoplasma spp,b,c Frequent Cow is source, spread by milkinc:¡, destructive mastitis, cow not usually systemically ill, cah
be eradicated
Mycobacteaum spp Ka~e ~pread by con¡aminated treatment equipment oc materiats.
Nocardia spp. Rare Spread by (Ontaminated treatment equfpment or materials
PiJsteu11,d/¡¡ multutÍÚil R~re Sputodit i11lecliu11~. ~uyye'l' uu~~.,_u11lamimilio11 úwi11y lsedlmenl
Prototheca spp. R:are Achloric algae, associated with poor environmental hygiene. noftreatable
Staphy/ococcus aurec{fl rrequent Source is infected udder, spread by milking, rar.e cause of gangrenous mastitis, can be
eradícated
Streptococcus 39~/;,cti;,eb OccJ~lonJI CaU$C$ hígf:i bulk tank $Omatic Gel! count, can be cradiC'.ltcd
s. dysgalactiae subsp. cl'{.sgalactiae Frequent Bovine origin, survives in environment
'>trepto(ocn1s uberis Frequent Fom1d on bovine skin and environment

ªAlso consider Corynebaderium bovis, Serratia. Bacillus. f'seudomonas; andvario¡¡s othe~ Enterobaderiaceae.
b(ontagious p;ithogens. '
<f.Aost common spccics-rccovcrcd \JfC M. bovis, M. c::iJifomicum( ~r.d M. cana.cJcnsc.
 Musculoskeletal System_
RICHARD L.
·rhe musculoskeletal systen1 provides a structural fra1ne-
work for the body, protects vital organs, and provides the
capacity for locomotion. It is composed of the axial and
appendicular skeleton and associated Iigaments, muscles,
ctnc.l ten<.1011::;. Juiut ::;pace::; a11d b ursas and their sy11ovial
me.rnhranes are included in t his system.
The musculoskeletal svste1n , does not havc a normal
flora per se, although seeding trom transient bacteremias
or "trafficking" through supposed sterile sites may occur.
Bacteria! spores (Clostridium sp.) rnay be found dormant
in muscle, particularl y in ruminants, subsequent to en-
try through the c.ligestive trctct an<.l l1eu1cttuge11uu::; <.li:>-
tribution.
Antimicrobial Defenses of the Musculoskeletal
System
The antimicrobial defenses of the musculoskeletal system
predominately rely on the circulating imn1une defenses.
Normal healthy bone is considered falrly resistant to in -
fectiu1i. Eve11 <.lirect iuuculatiu11 uf lJuue wi Lh paLltogeuic
hacte.ria does not usually lead to infection unless predis-
po::;i ng factors are present. 13one undergoes constant re-
modeling, ~tnd injured/infected bone can be resorbed -and
replaced with new bone.
Muscle has a rich blood supply and, therefore, benefits
from its intimate association with circulating in11ate in1-
rnune <.lefen:>1;:s. Skel(.!tal u1u::;cle al~u Ita~ great alJiliLy Lv re-
ge.nerate necrotic muscle segments resulting from intlam-
matory/infectious processes.
Jn synovial membrane-lined sites (synovial joiI1ts, bur-
sas, and tendon sheaths), the sy novium lining is com-
posed of a thin cellular surface layer of predominately
m.acrophages and fibroblast-like synoviocytes and an un-
<lerlying ricl1 vascular layer. Pru<.luctiur 1 uf p rui11flau u11a-
tory cytokines hy cells at these sit es (chondrocytes, syn-
oviocytes, synovial macrophages) promotes a strong
inflammatory response in the face oí infection. ·rhe rich
blood supply to synovial membranes predisposes to local-
ization of microbial agents but also allo,\ls for rapid recruit-
ment of vascular im1nune defenses. Wl1ile the inflamma-
Lu1y re~puu~e i~ itupur laul fur cuul1vlli11g i11fe1..liv11, i l ca11
also lead to degradative cha11ges to articular cartilage in
synovial joints by stimulating production of cutabolic
rnetalloproteinases and inhit)iting synthesis of collagen
and proteoglycans. As inflamn1ation resol ves, fibrosis may
lead to decreased functionali ty.
468
WALKER
lnfections of the Musculoskeletal System
Tnfections of t he musculoskeletal system are initiated by
introduction of microbial agcnts through 1) dircct inocu-
lation from traun1atic or iatrogenic events, 2) extension of
infectious processes from contiguous focuses, or 3)
hematogenous seeding from distantly infected ::;ites ur
during septicemia. Within the 111usc:11loskP.lP.tal systern
sites o( Lrauinalic injury, arcas of active growth vvitl1 in-
c:re.aserl vascularity or sites with spccific vascular features
(e.g., discontinuous epithelium in capillarics in vertebral
end plates and 1netaphyses) are predisposed to infection.
Viral, bacterial, and fungal agents can be involved.
Parasite infections of muscle are also in1portant but are
not witb in the context of t his book. As a rule, bacteria are
the most com1non micru1Jial ctgents iuvulve<.l a111011g Lhe
three groups of age.n ts disc11sst>rl ht>re.. Spe.cific bacteria!
faclors are i111porta11t for infection to develop. Adherence
factors (e.g., fibrinoge11 or fibronectin -hinding proteins),
toxins that promotc inflammatory response and tissue
damage (e.g., superantigens, cytotoxins), and factors tl1at
aid in evasion of the ilnmune system (e.g., capsules, pro-
tein A) ali provide an advantage to establishtnent and
persist ence.
The 111ajor infeclious processes ir1 tl1e n1usculoskeletal
system are 1) infections of bone including vertebral body
and associated intervertebral disk infcctions; 2) infections
involving articular surtaces or bursas including their syn-
ovial tnen1branes; and 3) infections of skeletal muscle, te11-
dons, and surrounding fascla. l'l1esc lnfectious p rocesses
do not necessarily occur as distinct enti tit>s (t>.g., inft>c:tion
of n1elaphy.seal bon.e and associated joint infcction:; in
11eonatal septicemia in sorne animals). Diseases of neuro-
logic origin that affect muscle activity as a result of inhibi-
tion ot neurotransmitter release (tetanus and botulism) are
covered in chapter 71 . Microbial agents causing cellulitis
may overlap with musculoskeletal infections and ctrt! al::;u
covered in chapter 69. Co1nmon ¡:¡nd/or important agents
associa led wilh n1usculoskeletal infections of domestican-
imals and poultry are listed in 'fables 70.1- 70.6.
lnfections of Bone (lncluding vertebral Body and lntervertebral
Disk lnfections)
Osteítis is an inflammatio11 of the bone. Osteomyelitis and
periostitis denote involvernent of the medullary cavity and
periosteu1n, respectively. Osteomyelitis can be furtl1er di-
videc.l into t1erncttuge11uu::; ur pu::;t-trau1ualic osteon1yelitis.
 Ch.apter 70 Musculoskeletal System 469

Table 70.1 . Common and/or lmportant lnfectious Agents of Ta b 1e 7 O. 3. Common and/or lmportant lnfectious Agents ot
t hP M11~n 1ln~kPIPt;il <;y~tPm nf (;ittlP

A'.genl Major Clinical Manífestatíons Agent ,Majc¡r (ijnical Manifestafiom;

•J€í mmor¡ J)isease Name) (Common Disease.Name)
•

Viruses
feline sy11cytium·fu1miug vi1u1 (Cl
Bacteria
Actinomyces spp.ª
Beta·hemolytil 5úeptocottU)
spp. (D)
Dorre/ia burgdotf~ri (D)
Bruce/la canís (O)
Leptospira spp
Oblig¡¡te anaerobesb
PastP11rpJ/;¡ m11/tncid;i (C)
StaP.liy/ococcus intermedíus
Fungi
Aspergillus spp. (D)
Blastomyces dermatitidi~ (D)
Coccidioiqes immitis (D)
(C) = cots, (D) = dog<
' lndui;les A yíscosus and A bordeovuln.eris.
b1ncludes rasobacterium, Bacteroides, f'orphyromonas, and Peptostreptococcus.
Bacteria
Aflhritis Actit1obad/lus lignieresii Myosítis (actinobacillosis)
Actinomyces bovis Osteomyelitis (lumpy jaw). myositis
Arconolx>ctc(ivm pyogcncs Arthrtti:;, di$l<O$pondyliti::;, f.::i:;cíiti:;,
Oisko,rnndylitis. oiteomyelitis
myositis, ¡>steomyelitis
Arthritrs, diskospontlylitis, myo,sitis,
G:lostridium chauvoei Myositis (l:ilackleg)
necrotizing fasciitis
Escherichia coli Arthritis, osteomyelitis
Arthritis (Lyme disease)
Fusobacterium necrophorum Arthritis, diskospondylitis. osteomyelitís
Diskospondylitis, osteomyelitis
Histotoxic Cfostridium spp' Myositis (malignant edema)
Polymyositis
Mvcopfasma spp.b Arthrüis; bursitis, tenosvnovitis
Myositis
5almonella spp . Arthritis, osteomyefitis
.Myositis
Arthritis, diskospondylitís, myositis,
•1ndude~ e perfringens, e nollj'i, c. seplicum, and e sordellii.
osteomyelitis bincludes M. bovis, M. calífornícum, M. a/kalescens, and M. arginini.
Diskospondylitis, 01teomyelitís
Osteomyelitis
Osteomyelitis Ta b 1e 7 O. 4. Common and/or lmporta nt lnfectious Agents of
t he Musculoskeletal System of Sheep and Goats
•
Agent MafoJ Clínic.al f>l@nífestations

Viru.ses
(Comrnon Dise.ase Name)
-------
Bluetongue virus {S} Muscle intarEtion
Ta b 1e 7 O. 2. Com mon and/or lmportant lnfectious /\ge nts of Caprine arthritís-encephalitis virus (G) Arthritis
Muscu loskeletal system of Horses
Bacteria
Arcanobactenumpyogenes (S) t>iskospondy[itis, my:ositls
Agent Major Ch"nlcal Manífestátíons Chfamydophila percorum Arthritis
{Co'mirton Dísease .Naroe)
----- corynebacrerium pseudotubercu/osis
Frysipeiothrix rh11~inp;ithii1P (S)
Myositis
Arthritís (P.rysirP.las)
Bacteria
Actinnbaciflu~ eqüuli ssp. equali Arthritis, osteomyelitis Goinf ill) Hblutoxic C::Joslridium spp• Myosítis
Bruce/la a.bortús Atlantal or supras~ínous liursitis {poli Mycoplasma spp. (G)b Arthritis. tenosynovitis
evillfístulous withers), osteomyelitis
EschcrichiJ coli Arthritis, osteomyelitis (joint ill) {G) := goats, (S} =sheep
'lnclud~ C perfringens. C novyi. C septicum. and C sordellii.
Hístotox1c Clostridium spp.• Myositis·(clostridial myositis) btncludes M. mycoides subsp. mycoide; Qarge colony o/P•), M. capricolum sub~P- capd-
Salmonel' a spp. Arthrítls, osteomyelitis fjoint ilf) colum, and M. patJ;~fadens.
Staphylococcus aureus Artflritis, atlantal or supraspinous
b11~itís (poli P.villfi~ulo11s wither'},
myosilis, osteomyelitis, tenosynovitís
Streptococcus e.qui subsp. equi Muscle infarctíon- immune complex-
mediated organisms encountered include enterics (E. coli, J>roteus
Streptococcus eqw subsp. Arthrttis, osteomyelitis úoir.i¡ ill) spp.) and obllgale a11aerobes. 111 l1orses, Lhe agents n1osl.
•
zooepidemicus commonly isolated from osteomyelitis il1 neonates are Ac·
tinobacillus cquuli subsp. equuli, F,. coli, Streptococcus equi
•1ncludes C. perfringens, C sordellii, and (. septícum. sut)Sp zooepidernicus, and Salmonella, while coagulase-
positive stapl1ylococci predomll1ate il1 .a dults. In rurrúnants
an d pigs, Arcanobacterium pyogenes a11d Sal1nonella are
major causes of osteomyelitis. Others of note in production
ai1in1als ai·e E. coli ar1d Fusobucleriurn neLrophorurn.
Most bone infections are bacteria! in nature. While a vari- 'I'he rnicrovasculature (discontinuous epithelium and
ety of bacteria can cause bone infectio11s, a select number lack of basement membrane) and possibly the slow blood
predomin.ate wittlin anini.al groups. ln compa11ion animals tlow through capillaries in areas of active growth favor the
and poultry, coagulase-posítive Staphylococcus species (S. in- establishment of infections (hematogenous osteon1ye-
terrneáius, s. aureus, s. hyicus) are frequently involved. Other litis) . Macrophages associated With vascular endothelium
470 J>ART IV Clinical Applications

Ta b 1e 7 O. 5. Common and/or lmportant lnfectious Agent s ot Use ot sy11thetic material in reconstructive or replace-
Mu~culoskP.IP.till SystP.m of Pig~ ment surgeries (e.g., hip replacements) 1nay also disrupt
the innate resistan ce of bone to infection by providing an
fíllajor C{inical Manlfestlltions avascular surface and protection from in1mune defenses.
(Common Disease Name) Bone ce111e11-l used in so111e replacen1ent surgeries n1ay it-
self inhibit phagocytosis and co1nplement activity. I-Iost
Bacteria fibronectin deposited on implant n1aterial allows for bac-
A_rcanobocterium ,oyogenes Arthritis, osteo¡nyelitis teria! attachment, '"'hich is tollowed by p roduction of
Beta-hemolytic Streptococcus spp. Arthritis exopolysaccl1arides (glycocalyx) by bacteria. Along with
Bruce/J¡j suis Arthritis, diskospondylitis (bruccllosis) host products, the glycocalyx forms biofilms that provlde
C/ostridium sépticum Myositis (malignant edema) bacteria protection from host defenses and killing by
Etysipelothrix rhusiopathiae Arthrítis, diskospondylitis (erysipelas) anli biolics.
Jiaemophilusparasuls Arthrltls (Gtasser's disease) Bite wounds, pe11etrating fore ign bodies, orthopedic
11Aycnpfasm;i hynrhini~ Arthritis surgical procedures, and trauma tic in jurics are possible ini-
MyLupla~ma lryosynovia~ Artbritis tiat ing events for develop1ne11t ot osteomyelitis in compan-
Pasteurella multocida AtrÓ'phy of nasaf turbinate bones ion animals. 'fhe long bones are most commonly affected.
(atiophic rhioitis) In productio11 a11in1als, hematogenous osteomyelltls ls
Streptococcus suis (Type 2) Mhritis a frequent event. Hematogenous osteomyelitis in nf>On.1-
Lal ru1ui 11a1 1ls is associaled vvitl1 failure of passive t ransfer.
Infections begin at the metaphysis or the epiphysis be-
neath articular cartilage and oftcn occur in conjunctiOll
with or subsequ ent to intectious synovitis. fn neonatal in-
Table 7 O. 6. Common and/or lmportant lnfectious Agent s of fections, vessels that cross tl1e growt.h p lates are impor-
Musculoskeleta 1System of Pou ltry
tant for spreading infection to the metaphysis from
joints. Ep.iphyseal osteomyelitis in conjunction with arth -
Agcnt Major CfiniCal Manifestatfons ritis in calves is commonly cau:;eu uy Sulrnuriellu serovar
....,....,....;._,..__,.,,_..,_......,,.............,.,,...-. (t:ommon Disease 'Name} D11blin. Arr:annhar:terium pyngenes osteon1yelitis in older
Viruses
calves and adults n1ore commo11ly begins on t he meta-
Reovirus Arthritis, bursitis, tenosynovitis physeal side of the growth plate. Arcanobacteríurn pyogenes
also causes a hcmatogcnous vertebral osteomyelitis in
Bacteria pigs associated '"'ith t ail biting o r foot lesions. Commer-
Arcanobacterium pyogenes (T) Osteomy;elitis cial turkeys develop focal areas of osteomyelitis caused by
Ety>ipefu(/j1Jx rl1u>iupal11ide (T) Ar l1 uífb {er~ipel•>l' s. aureus or E. cou. The p roximal tltJlutarsus a11u pruxilual
Escherichia coli Arthritis. bursitis, osteomyelitis, ff>m11r .1rf> most oft f>n ;iffecte.d ;ind infections are associ-
tenosynovitis at ed ·with a green discoloratio11 of the live.r in adole::;cen.t
Mvcoplasma meleac¡ridis (T) Bowinc¡ of tibiotarsal bone, cervical 1nale turkeys (turkey green-liver osteon1yelitis complex)
vertebrae deformation (Fig 70.1). Hcmatogcnous ostcomyclitis is uncommon in
Mycopla~ma synoviaé Arthritís, bursitis, tenosynovitis dogs a11d cats.
(infectious synoviti>) Post-traumatic osteomyelitis is also common in produc-
staphylococcus aureus, 5: hy:iéus Arth¡jtis, bursitis, osteomyelitis, tion ani mal~;, usually in adults. In cattle a chronlc pyo-
tenosynovitis

ffi=turkevs.

F f G U RE 7 O. 1 . O~leo111yelítís in l/Je feo1ur of a 17-week·old

turkey wíth qreen-líver osteomyelitis complex. Staphylococcus hyicus
was isolated from the bone lesion. (Courtesy Dr. H Shiv;;1pr;;~;ui.)
are the main defense in tl1ese areas. Infections also occur
in areas of bone with poor or interrupted vascular supply
fruu1 trauiua (pu:;t-trau111alic u~leu111yel i lis) 01 when adja-
cent tissue infections result in ischemic ínjury. lf both the
medullary and periosteal vascular supply to bone becomes
comprornisect cturing a11 in!ectious process, a sequestra-
t ion of necrotic bone may result. In son1e cases, persistent
drainage fron1 sinus tracts develops. U11der these condí-
tions, bacteria are better able to cstablish and more diffi-
cult tu eli1niI1ate ue<..:ause tl1ey uecou1e irraccessible LO iin-
m11ne defcnscs and rcfractory to antibiotic t reatment.
Host ccllular products, and pcrhaps sorne bacteria! prod-
ucts, stin1ulate 1nonocytes and fibroblast to produce oste-
olyti.c cytokines and stirnulate osteoclastic activity, caus-
ing greater separation of living from dead bone.

granulomatous intlammatJon lovolvlng th.e mandible
("lumpy jaw") or maxilla is caused by Ar:tínnmyr.P.s hovis.
Viral agents rarely cause inflani.mat ory bo11e disease.
Distemper vir11s in dogs may damage osteoblasts and cause
growth rctardation. Canine hepatitis virus can cause meta-
physeal t1emorrhage and necrosis.
Fungal bone infections occur but at a low frequency.
fungal osteomyelitls ls usually the result of d.i ssen1ination
from another si te, most often the h1ng. Many of the sys-
ten1ic 1nycotic agents are capable of causing fungal os-
teomyelitis. Coccidioides immitis is notable for disseminat-
ing to the appe.ndicular skeleton in dogs. Disseminated
Blastomyces dermatitidis intections in dogs can involve the
bones in up to 30Qk> of t hc cases '"it h vertebrae, and long
bones are most commonly affected.
Diskosponclylitis, an inflammatory proc.ess of the inter-
vertebral disk and adjacent vertebrae, is a particularly com-
mon site for bone infections to occur. lt usualJy begins at
the vertebral end plates. The discontinuous capillary ep-
itheliurn and slow-flowing venous channels predíspose
this area to bacteria! seeding. Diskospondylitis is most
conunor1 in dogs <1nd rurnir1ants and often results via
hematogenous <lissemination. The l.7-Sl region is;¡ c.om-
monly affected si te in the dog but any vertebral body can
he affected. Staphylococcus intermedius is the most common
agent identified.
Vertebral infections of'fl3- L3 in the dog are associated
with migration of foreign bodies (e.g., plant awns).
Actinon1yces spp. are the usual agents involved. Diskospon-
dylitis associated with Bruce/la species deserves particular
alle11lio11. Tl1e persiste11t bacteren1ia that occur.s with
sorne Brucella species predisposes to infection at extragen-
ital sitcs and Bruccl/a should always be considcrcd as a po-
tential agent in diskospondylitis in dogs (H. canis) and pigs
(B. suis). Calves with di.skospondylitis caused by hemato-
l{enous disseminatio11 of either A. pyogenes or Fusobacte-
riu111 necrophorum present vvith paresis or paralysis.
(;erman shepl1erd dogs are particuldrly proJJt: tu Jevt:l-
oping diskospondylitis 1n1cl osteomyelitis of fungal origin
caused by Aspergillus .spp. Aspergillus tcrrcus and A. dcflcctus
are the most cornmon species recovered.
lnfections lnvolving Articular Surf¡¡ces, Bursus, and Synoviul
Membranes
Arthritis denotes a11 inflammatory process of a joint space.
lvfost joint infc:ctions an: l:ntctcrial, but viral and fungal in-
fections do occur. Monoarticular infections typically arise
from direct inoculation or spread from a contiguous site
(e.g., distal interphalangeal joint infection in cattle follow-
ing sole abscessation) . Hen1atogenous infections fre -
qu1::11lly rt:sull iu pulyarlhrupdlhies. Iu in:u11a les, tl1e uu1-
hilicus or gastrointestinal tract are common cntry points.
Arthritis is a common scquelae to septicemia, especially in
young animals and especially where there is failure of pas-
sive transfer. In aduit animals, joint abnormalities, im-
munosuppressivc diseases, infections at other sites in the
body, intra-articular injections, surgery, and joint prosthe-
~es ali preubµu~t: tu juiI1t iI1fectious.
Infection of the joint usually he.gins in synovia 1 tissue-
in part, because of the rich vascular supply and lack of
CIIapter 70 Musculoskeletal Sy.ste1n 471
basement membrane. Tnfection results in expression of a
c.ascarle of inflammatnry mP.cliatnrs (TNF, TT.-1, TT.-6, ancl
NO) that result in increased blood flow to the area, in-
creased capillary permeability, and an influx of inflamma-
tory cells. Tl1c agcnt involvcd dictates the typc and intcn-
sity of inflammatory response. Synovitis in unchecked
infections results in increased synovial fluid containing
cellular exudates that subsequently progresses to involve
thf' artir11lar s11rfaces. Alnnf' nr in c'nmhin;ition, bacteria]
products, products resulting fron1 the inflan1ni.atory re-
sponse, and proteases already present or produced by cells
in the joint damage articular cartilage. Once damaged, ar-
ticular cartilage 11as limited capacity for repair.
Current evidence suggei:ts that when viable bacteria are
no longer detectable ln cases of septlc arthritis, the inflam-
matory respnnsf' contin11Ps ancl furthe.r destruction. to
articular cartilage ensues. This is likely dueto residual bac-
teria! products such as peptidoglycan-polysaccharide
co1nplexes that continuc to promotc an infla1nmatory re-
sponse. Even bacteria! I>NA, specifically unmethylated
CpG motifs, appears to stilnulate a variety of cell types to
produce pro-inflamm.atory cytokines leading to further
tiss11e clestr11ction . Joint fun ctionality may be further af-
fected by fibrosis that results during resolution of the i.n-
flammatory process. ln sorne cases ankylosis of the joint
occurs.
Post-infective art hr1tis, which. is associated with micro-
bial fragments localizing in t he joint during a septicemia
but vvithout microbial replicat ion in the joint ltself, is less
wellrecognized in veterh1ary medicil1e than it is in human
n1edicine. When it occur.s, joint dan1age resulls solely fron1
immune rnechanisms.
Morphologic variants of bacteria huvc also bccn associ-
ated with joint infections. Hacterial L - forms, which are
wall-less bacteria, 1AJhich have "turned off" the genes re-
sponsible for cell ~vall synthesis, and small colony variants
ofbacteria, which have decreased growth rates presurnably
due Lo defeclive respiralory 111eLabolis1n, have been associ-
áted with persistent joint infections.
In spe.cies where transphyseal vessels provide a direct
connection between the metaphyses and the epiphyseal
cartilage (e.g., ruminants, horses), both acutc ostcomyc-
litiS and joint infections are commo11. This is also true
when metaphyseal bon e is included within thc joi11t cap-
sule. Sornetimes synovitls represents only one of a number
of clinical 111an ifestations of sy stemic iI1fectious processes
(c.g., polyscrosi t is i·n pigs Ciluscd by Mycoplas111a 1zyorhi11is
or Haemophilus parasuis).
Bacterial arthritis is uncommon in companion animals.
When it occurs, coagulase-positive Staphylococcus species
a11d Streptococcus species are the most common agents in-
vul ved. lnfections result frorn direct inoculation from
trauma or s11rgery. 'l'he ranint.> stifle joint appears particu-
larly predisposed to post-surgical infectio11s. Recurrent
lameness, sometimes involving multiple joints, is associ-
ated wit h a cl1ronic and progrcssivc arthritis duc to Borrelia
burgdorferi, the Lyme d.isease agent.
Arthritis is co1nmo11 in production animals and horses,
anda variety of organlsms ca11 be involved. In neonates,
Salmonella and E. coli are tl1e main agents. Mycoplasma
arthritis in run1inants is also an in1porlanl enLily in (eed-
472 PART IV Clinical Applications
F1G U R E 7 O. 2 . Pofyarthritis invofving the carpa/ joints of a
Holstein dairy calf. Mycoplasma bovis was isolated from the joint
fluid. (Courtesy Dr. P. Bfanchard.)
lot cattle and dairy calves. In addition to arthritis, which
usually involves carpal and hock joints (Fig 70.2), tenosyn-
ovitis and bursitis occur. Mycoplns1na bovis is the usual
:>pecie:> 1ecove1ed. In guals, n1ycoplasma arll1rilis affecls
both kids and adults. Mycoplasn1a 1nycoides subspecies rny-
coides (largc colony typc) and M. capricolurn subsp. capri-
colu1n are principal species isolatecl. 1\1ycoplasma arthritis is
also important in pigs. Chlamydial joint infections (Chfa-
mydophila percori11n) are commonly recognized in goats
and sheep ("stiff lamb disease"). Infections can p resent in
coll j ur 1clio11 wilh cunj u11cli vi lis. ·rhe chlan1ydial species
in volved is different from the species associated with goat
and sheep abortions. ln horses, joint infcctions in nco-
natcs are causcd by organisn1s also associated with neona-
tal ostco1nyelitis and include E. cofi, Sc1/monella, .4ctinoba-
cillus, and Streptococcus spp. In adult horses, Staphylococcus
or Streptococcus spp. are usually involved.
Bur:.ili:> 1efe1:, lO an inflan11naUon of lhe sy11ovial li11ed
hursas and can be affected in a manner similar to synovial
membranes in joints. Of specific importancc are Bruce/la
abortus and S. aureus inlcctions ot the atlantal and
supraspinous bursas in horses (Fig 70.3). These infections
may spread to lnvolvec.l $plnuu:s pruce:s:se:s uf C1djace11t
vertebrae
Although bacteria are responsible for most cases of in-
fectious arthritis, viral agents are someti.rnes involved.
Fclinc syncytium-forming virus infcctions cause both pro-
liferative and erosive forms ot joint involvement in cats.
Caprine arthritis encephalomyelitis virus is of substantial
importancc in goats causing a hyperplastic polysynovitis,
usually in goats oldcr than 12 months. Avian reovirus
cause::; arthriti:, a11d lcJJu:,y11uvili:, iu lu1keys a11d chickens.
FIGURE 70.3. Gram stain of exuda te from a fistufous withers
Jesion in a horse. Clusters of gram-positive cocci are present.
Staphylococcus aureus was isolated.
The d lgltal flexor an<.I rnetCJtCJr:sCJl exLe1 1::;ur leuúou:> duú
hock joints are most rommonly affected. The stability of
Ll1e virus, polcntial for horizontal and vertical transmis-
sion and high-dcnsity rearing practices used today make it
a potcntially scrious flock problcm.
lnfections of Skeletal Muscle, Tendons. and Fascia
lnfcctions of skeletal muscles are uncommon. While viral
and fungal agcnts occaslonally cause musde infectiu11:s,
bacteria again are the most fretjuent of the thrPP gronp.~ in -
volved. Myositis can be in the forn1 of a localized abscess,
granuloma or diffuse inflammatory process that spreads
along fascial planes. Prcccding cvcnts includc trauma, in-
jections, bite wounds, or a contiguous infectious process
(e.g., cellulitis, subcutaneous abscess, osteomyelitis). Pyo-
genic bacteria cause locallzed abscesses (e.g., Pasteurelfa
nn1/tocida in cats). Granulo1na tous and pyogran11 lomatis
n1yosilis are assocla led wit b bacteria! species known to
evokc g ranulon1atous pyogranulomatis int1ammatory re-
sponses (c.g., Mycobactcrium bovis, Actinomyccs spp.,
Actinobacill11s li~1ieres1í). Clostr1dial n1yos1t1s is typically a
more diffusc infcclious proccss ancl spreads rapidly along
fasclal planes. Jt Is the most severe anc.l aggre:ssive furu1 uf
thc .infcctious myo<;iti<IP'\ rl11P to thP procluction of potent
histotoxins. Myositis n1ay occur subsequent to or in con-
junction \-vith clostridial cellulitis. Most a11imal species are
affcctcd; howcvcr, clostri.dial myositis is most common in
ruminants and horses. Clostndial agents usually reach af-
fected si tes by direct penetration (e.g., penetrating wounrl,
i11jecliull:>) u1 1 i11 lhe case of Clostridiutn chauvoei, the agent
of hlackleg, are already present in muscle as dormant
spores. When tissue becomes dcvitalizcd, producing an
anaerobic environment, the spores germinate and vegeta-
tive cells proliferate (Fig 70.4). Numerous cytotoxic exo-
toxins produced by thcse clostrictial spectes contribute co-
agulative muscle necrosis.
Hemolytic Streptococ<-11S spp. l1C1vc lJee11 associaled will1
severe necroti7.ine fa<>riiti<> in ctogs. In horses, immunc
con1plcx vasculitis is the supposed mcchanism by which
 Chupler 70 Musculu:.keletal Sy.steru 473

muscle damagc due to vcsscl infarction and hemorrhage

F 1G u R E 7 O• 4 . DirPrt f/11nre~cent antibody test using
fluorescein-labeled, anti·Clostridium ch¡¡uvoei antiseril on un
impression smear from muse/e of a cow wirh a fibrinohemmorhaglc,
necrotizing myositis. Fluorescent C. chauvoei organisms are
apparent.
results post-Streptococc11s eq11i subsp. eq11i infections.
Te11uu11 :.lteall 1:. l 1ave ;111 Lt 111er :.yuuvial 1ue111bra11e a11d
may hecome infected as a result of hematogenous dissem-
ination, trauma (e.g., bite wounds), treatments (e.g.,
sheath injections in horses) or spread from a contiguous
focus. Tenosynovitis is especially important in horses,
most commonly invo!ving digital tendons. lnfections typ-
ically result from a wound with a variety of bacteria being
i11vulvet.I ur :.uu:.e4ue11l lu :.lteallt i11je<..:Liu11, wl1ere S. uureu~
is usually the cause .

•
 N ervot..1s Systern
RICIIARD
Thc ncrvous system is <.livitletl iutu tl1e ce11tral autl vc::ri vil-
era! ncrvous systems. ThP cPntr::il nPrvous system includes
Lh.e braLn ¡u1d spinal cord, the cerebrospil1al fluid that
h::ithPs it, an d th e meningeal layers that cover it. The pe-
riphcral ncrvous system is comprised of ncrvcs that a risc
from the central n ervous system (cranial or spin al nerves)
and innervate muscles or cffector organs. The peripheral
nervous system is furthcr divlded into somatíc-sensory
and autonomic divisions.
Ne1 vous syslen1 ilúeclions n1ost commonly involve the
central nervous system. In sorne cases, peripheral nerves
are the targeted site for infcctious or immunological
processes or microbial toxin action, or serve as the site tor
cntry of infcctious agcnts or toxic products that act on the
central nervous system . The nervous system does not have
a normal mlcroblal flora . Sorne viruses (e.g., herpesvir uses,
dlstempcr virus) can cau se latent infectiuu:;, autl il1 :.01ue
cases viruses integrate intn thP hnst gPnomP <is p rovi ruses
(c.g., visna virus).
Antimicrobial Defenses
Spnsitivity of the nervous system to injury makes exclu-
sion of microbial pathogen:; or their toxins of paramount
importance. Anatomic and immunological defenses are
thc prün¡1ry defense mec.:han isms available; the following
scct1ons present these ctefen ses.
'rhe skull and vertebrae provlde rlg1d cuverings that pru-
tect the braLn and spinal cord from traumatic or penetrat-
ing injuri~:; tl1at llligl1l lc::atl Lu i11l1oduclion of rnicrobial
p::ithogPns. ·rhc meníngea] layers (pía, arachnoid, and
dura) providc further anatomic barriers that act to contain
or prevent infectious processes trom progressing into t he
nervous system parenchyma.
The blood brain barrier provtdes the ma¡or anato1nlcal
separation between the ncrvollS system paren chyma and
cu111pu1 1e11l~ uf Lile va~L ulc11 :>y:>Lo::111. fl v1olec ls lhe central
nervous syste1n frorn agcnts d isseminated via the blood
stream from other sitcs in thc body. Central to tl1e blood
brain barrier are the capillary endothelial cells, which
form tight intercellular junctions that prevent movement
of blood constituents into thc central nervous system.
Movcment of substances across thc cndothelial cells is fur-
thcr controlled by specializctl c.:arric::r lra11spor l syslen1s.
4 74
L. WALI<ER
AsL1ocyle processes and pcricytes, a subclass of microglial
cells, that surrou11d capillaries andan extracellular matrix
contribute to th e ovcrall makcup of tl1c b lood b rai n bar
rier. Not all areas are prot ected ])y t he bloo<i brain barrier
(c.g., pituitary, choroid p lexus). ·rhe secretory selectivity of
choro1d p lexus epithelial cells an d epenctymal cclls con-
tribut e to a barrier betwcen the blood and cerebrospin::il
fluid. Nerves of tl1c veri J.Jheral nervous systen1 are pro-
tected from inflammatory reactions and immune re-
sponses by a blood ncrvc barricr, although it is not as re
strictive as the bloo<i brain barrier.
lmmunol ogical Defenses
Much of o ur knowledge of immunc defense of t he nervous
system is based on stu<iies in rodents a11d humans. '["he cen-
tral nervous system h as lo ng been considered an immuno-
loglcally prlvileged slte bec<1use it lacks a11 urgau izetl 1y JJ1-
phatic system for antigen rlPlivPry to ly mph nodes and
norn1ally h.as greatly reduced expression of major histo-
compatibility complex determinants. Current evidence in-
dicatcs that thc ncrvous systcm has a much more advanced
immunological defense system than was previously
thought. Antigens from the cerebrospinal fluid are able to
access lymph nodes (cervical) by lymphatic drair1age alu11g
cranial nerves. Major h istoromp::iti hility <intigens are cx-
pressed by cells i11 th c central nervous system pare11chyma
(e.g., astrocytes anti microglial cells) under proper con d i-
tions. Thc blood bruin barrlcr con tributes to the relative
immu ne privilege of the healthy central nervous systc1n by
excluding en try of large molecules from the blood anc1 hy
r1;::.trlclü1g il un1 une cell enLry. 111ls restriction is not ab-
solute anci, alt ho11gh fpw immune cclls are normally found,
both activated and naive 'f lymphocytes can penetratc thc
blood brain barrier and are present in the absence ot in-
flammation, suggestLng a "patrolling" function. While an
active immune system 1s in place, it is geared to provide a
degrce of protection and yet minimize in nocent bystander
damage. This is rnea11t tu avuitl i11flar11n1alory responses
th;it lP<irl to profo11nrl or i rrcversible d istu rbances in n eu-
ron al functio n . Local in flammuto ry rcspon:>cs nrc rcgulatcd
by im mu nosuppressivc mechanisms. Subclasses of glial
cells express cytokir1es (e.g., TL-6, TGFE2) that h elp restrict
the inflanunatory response. An abillty to Ln<iuce T Jympho-
cyte apoptosis is also present.
The central nervuus sy:.tc111 vu:.sesses cerlail1 il1r1ate in1-
mune capabilitiPs. C:omrlPment plays an esse11tial role in
innate in1mu ne defense. Doth neuronal cells and astro-

cytes have ability to produce complernent components. A
number of nervous systcrn patl1oge11s w ill i11duce co111ple-
mPnt c.omponent synthesis (e.g., exotic Ne\.YCastle disease
virus, Listeria 111011ocytogenes) which plays both a direct
1
role in pathogen killing by membrane attack con1plex for-
mation, as well as in recruitn1ent of leukocytes. Uncon
trolled complement activation, resulting from progressing
infectious processes, contributes to pathology whe11 host
cell rnerr1lJr<1ne co1uple1ueul l ul 1J.l1i to.r:s a.re overwl tel u 1.ell.
Various cell types are also critica! to central nervous sys-
tcro defense. Microglia cells are rr1yeloid bone marrow-
derived cells. When activated, they produce various
chemokines and cytokines, actas macrophages, and poten
tially llave a role in antigen presentation. Dendritic cells,
which are potent antigen-presenting cells, have also been
den1onstrated in the brain. Astrocytes have tJeen shown
to be involved in antigen presentation and chemokine/
cytokine production. ln the face of an inflarnmatory re-
sponse, astrocytes becomc activatect anct can torm glial
scars to insulate damaged areas of the brain parenchyma.
Nerve cells, themselves, are capable of producing certain
cytokines (interferon y).
Duri11g i11feclious processe:>, 111igralio11 of i11fl<1111111a-
tory cells from the vascular system to affected sites is im-
portant for controlling progressing infectious processes.
lnflammatory cell migratio11 is mectiated, as elsewhere in
the body, through productlDn of chemokines and expres
sion of adhesion ligands (selectin and integrins).
lnfections of the Nervous System
Formicrohial pathogens to affect the nervous system. they
or thcir products must rcach thc ncrvous system (route of
intection'), be able to penetrate or interrupt anatomic bar-
riers, and establish and persist by evading or subverting
in1mune ctefenses. Clinical signs resulting from infection
depend on whether and wher.e the infection is Iocalized,
wl1at age11t i:s i11volveLl a11Ll Lite lype and degree of inflan1-
1natory response induced. Most nervous system infectious
processes have a rapid progression, requiring timcly intcr-
ve11tion. Most infections involve the brain or meninges;
l1owcvcr, other areas can be involved concomitantly or
serve as the pr1nc1pa1 targetea sire. The aecreased fre-
quency of spinal cord involvement is most likely dl.1e to re-
duceu l>loou íluw Lo ll1e spi11al cord ralher Lhan sorne
greater inherent resistance to infection than the brain. In
sorne cases, the major or sole clinical 1nanifestation is re-
lated to spinal corct lesions. For example, equil1e her-
pesvirus 1 infections may result in immune con1plex-
related vasculitls causlng necrosis that affects both the
brain and spinal cord. Lesions in the spinal cord are some-
liioes lile source for Lile predon1ir1a11l cliuical sigus.
Péripheral nerve involvement is less frequent but is the site
for important neurological diseases (e.g., Marek's disease
in cl1ickens).
The presence or growth of a microbial agent in th.e
nervous system is not necessary for signs to develop.
Microbial toxins ingested or produced at another site i11
Lhe hosL (e.g., (~. perfring·e ns epsilo11 LOxlr1, l!oluliuuu1
toxin, and tetanus toxin) and imn1une-mediated events af-
Cha¡>ter 7.1 Nervous System 475
fecting vessels supplying the central nervous systern (e.g.,
feline infeclious perilo11ilis virus, equine herpesvirus 1)
are responsihle for important nervous system diseases.
'l'he role of infectious agents in autoimmune diseases is
still poorly understood. ln humans, cross-reacting path-
ogen and host antigens results in molecular mimicry and
has been associated with certain nervous systen1 diseases.
Notable is the mi.Jnic(y between lipopolysacchadde struc-
ture~ of ::;elected serul yµe::, uf CurnpylubuLler jejurti a11d gar 1-
gliosides (e.g., GMl, GDla) of motor neurons. 'fhis is
thought to account for the association between antece-
dent Campylobacter infections and sorne cases of c;uillain-
Barré syndrome, an acute demyelinating polyneuropathy,
in humans. Some cases of acute canine polyradiculoneu-
ritis (coonhound paralysis) may similarly be associated
with im1nune reactions to viral or bacteria! lnfectlons.
Sorne of the most comn1on and/or important microhial
agents associated with nervous system infecti.ons il1 do-
mestic animals and poultry are Jisted in Tables 71.1-71.7.
Routes of lnfection
Tlie liernatogenou:; ruute is tl1c n1ost coIIunon route of
entry for microhial pathogens. Othcr important routes in -
clude retrograde moveme.nt within neurons or extension
of the infectious processes from contiguous sites. Sorne
pathogens appear capable of using more than one of these
rou tes (e.g., Listeria).
Hematogenous Route. Systemic infections or infectious
processes that involve multiple organ systems typically
.reacl1 ll1e nervous syslen1 by Ll1is roule. InfecHon occurs
wl1en pathogens enter through vessels of the choroid
plexus, meninges, or paren,chyma, or from septi.c: emboli
that Iodge in vessels and result in ctirect da1nage to vascu-
lar endothelial cells. Sorne viruses are able to cross the
blood brain barrier themselves or are carried· across ln in-
fected imrnune cells, while others directly infect the en-
doll1eliun1 of capilla.des or cells of tl1e cl1oroid plexus or
ependyma.
Studics with E. coli shO\.Y that 1) a high dcgrcc of bac-
teremia, 2) invasion of brain microvascular endothelial
cells, 3) host cell actin cytoskeletal rearrangement, and 1)
specific signaling mecnanisms promote trans1ocation of E.
coli across the blood brain barrier. Different bacteria use
differenL signali11g ruechanis1ns. Cy Loki11es ¡;roduced by
astrocytes and microglial cells and nitric oxide from in-
flammatory cells in response to insults contribute to dis-
ruption otvascular barrier anct further loss of ability to ex-
clude entry of pathogcns.
Retrograde Movement Within Neurons. Some infections result
whe11 au ageut iufects peripl1er<!l nerve::; anti rnoves in a
retrograde fashion to reac:h the central nervo11s systPm
(e.g., rabies virus, pseudorabies virus) . Ilinding to specific
cell receptors is necessary for entry. For exa1nple, rabies
viruses use nicotinic acetylcholine and low-affinity
nerve- growth-factor receptors to attach and penetrate
cells. In sorne cases cell-to-cell junctions, ·including synap-
tic junctions, are crosseu. 5orne herpesviruses Iatently in-
fec.t <a~nsory g;ingli;i ilncl ;ire la ter activated and may extend
476 PART IV l.linical Applications

Table 71 . 1 . Common and/or lmportant lnfectious Agents of the Nervous <iystem of nogs

Agcnt Disease(s) Neurological Sign5"

Viruses
C;ininP ildPnnviru1 1 lnfP.rtio111 r~ninP. hP.p<ititis Seizure.~
Cdl lÍll~ uill~lllfl~I
virus C<1nine dislemper Ataxia, seiiures
Canine herpesvirus Canine herpes virus disease Depression. opisthotonus. seizures
Pseudorabies virus rseudorabies Intense pruritus, seizures
Rabies virus Rabies Temperament change, aggressive behavior, paralysis
Bacteria
Agents of otitis externa' Otitis media·intema Vestibular dysfunction
Ehrlichia úlnis Ehrlichiosis Ataxia, cerebellar and vestibular dystunctions, seizures
Clostridíum botulínum Botulism Flaccid paralysis, paresis
Clostridium tetani Tetanus Upisthotonus, seizures, tremors
Rickettsía rickettsii Rocky Mountain spotted fever Ataxia. depression, seizures, vestibular clysfonctinn1
Fungí
Crvptococcus neoformans Cryptococcosis Ataxia, head tilt, paresis, seizures

~lncludes E. coli, Proteus spp.• Pseudomonas spp., Staphylococcus spp., and Streptococcus spp.

Table 71.2 . Common and/or lmportant lnfectious Agents of the Nervous System of Cats

Agent Oi<P~SP

Prions
BSE prionª Feline spongiform encephalopatl\y Ataxia, behavioral changes, muscular tremors
•
Viruses
Fellne panleukopenia virusb Cerebellar hypoplasia Ataxia
Feline immunodeficiency virus Feline acquired immunodeficiency Aggre55ive or psychotic behavior. seizures
syndrome
Feline infectious peritonitis virus Feline infectious peritonitis Ataxia, paresis, seizures
Fclinc lcukcmia virus Epidural lymphoma Posterior paresis
fe/me leukemia Abnormal vocalization, hyperesthesia, paresis
Pseudorabies virus Pseudorabies Hyperexcrtability, paralysis, par~is
Rabies virus Rabies Aggressive behavior, paralysis
Fungi
Cryptococcus neoformans Cryptococcosis Ataxia, paresis, craniafnerve deficits, seizures

~aassifi~ as a forei(¡n animal diSs>""' ;ig•nt in th• tlnitPrl ~t~IP<.

beongcnit~I infcction.

in to thc central ncrvous systcm. Tctanus toxin moves in a spinal cord by direct extension or as a result of pressure
retrograde fashio11 in the penpheral nerves to reach the from epidural abscessation.
central nervous system .

Extension of lnfectious Process from Contiguous Sites. Pare.nc:hy.
inal or n1e11i11geal il1fections niay result from extensio11 of
infectious proccsses involving paranasal sinuses, tooth
roots, or thc middlc car (c.g., otitis media-interna in
calves). lntect:J.ons of the ep1dura1 and subdural spaces usu-
ally result by direct invasion of pathogens subsequent to
trauma or :.urgery (e.g., tail-uuckl11g i11 :.heep). Baclerial in·
fections of vertebrae or intervcrtcbral disks can involvf> thf>
lnfections of the Central Nervous System
Once a pathogen establist1cs, injury results from either di-
rect cytotoxic cffects by the pathogen or as a result of in.
1ury due ro rhe mflammarory rc:.puu~e üi1e1...leü c1L 1li1.:
pathozen, ora comhin;:ition of hnth. l.linical signs associ-
alcd with infections are varicd and typically progrcssivc.
Sp<'c:ific clinical signs may aid in localizing the infections
 Chapter 71 Nervous Systern 477

Table 71.3. Common and/or lmportant lnfectious Agents of the Nervous System of Horses

.. '
l\gent Dlsease
Viruses
~quine encephalornyelítis vrrus~s Equine encephalomyelitides Ataxia, drowsí¡iess, head·pressing, paralysis
(WEE, EEE, VEEª)
Equine herpesviru~ 1 Myeloen(ephalílb Ataxia, tetnr ór pdi'ápfegia
Rabies vin,1s Rabiep Ascending paralysis, ataxia, depression, vocaJization
West Nile·virus West Nile encephalitis Ataxia, muscle tremor, paresis, seizures, somnolence

Bacteria
Clostridium.botulinumb Botulism (shaker foal disease, forage disease) Flacdd paralysis, muscle fasciculations, pa esís
Clostcidíum tetani Tetanus Muscle spasms, prolapsed third eyelid, rigid "sawllorse• stance, seizures
Streptococ.cus equi subsp. equi Brain abscess, meningitis Blindness, cirding, coma, head-pressing, seizures
Guttural pouch infectíon Dysphagia, fiead-shakíng

fungí
Aspergíl/usJumígatus Guttural pouch mycosis Dysphagia, head-shaking

•classifled as a foreign animal dis.éase agent ih tlle<Uílited States.

bMost commonly lypes B and (.

Ta ble 71.4. Conirnon and/or lrnportant lnfectious Agents of Lile Ne1 vous Sy>Le111uf Cdllle

Neurological Sígns
.........
A:geri.~
•
P.iions
BSE p1ionº

Viruses
Akabane virus•,b Hydran'tnCefl.hafy, neurogenic arthrogryposis Sénsory and motor defiéits at óirth
(Akabane diseas~
Ak.P.phaline herpeSVírus 1ª Malignant catarrliál fever (wildebeest) Ataxi'a, head-presslng, paralysis, tremors, seizures
Bovine'viral diarrhea virusb Cer.ebelfar hypóplasía Heaq tremors, inGoordioation
lnfecti9us bovine rhinotracheitis IBR encephalitis Ataxia, hyperexdtability, tremors
t!Jvine herpesvírus 2 Malígnant cataaha/ fcvcr (shccp) Ataxia, head,pressíng, paralysis, tremors, seizures
Pseudorabies virus Pse.uilorabies Intense pruritus,
,
salivatlon, seizures; vocalization
Ralilcs virus Rabies Ataxia> paralysis

Bácteltct
Areanobaeterium pyogenes Brain/pituitary afucess Ataxía, blindness, depression, tascial paralysis
Hhtophi/us "omn~ 1'.tiromboembolic meningoencephalitis, otiti$ A:taúa, blindnQ'" qpi,tliotonu<, 'ttsp.o r
media-interna
<:Jostridium botulinumd Botulism Flaccid paralysis, loss of tongue wifhdrawal a11d blirik responses, m1:Jscle
fa5ciculations, paresis
Clostridium tetani Tetanus !)pisthotonus. seizures, tremors
Eschedchra co/i Meningitis Blindness, head.-pressing, ·seizures, somno1ence
Fusobacterium necrophorum Bra!rilpituítary abs€ess Ataxia, blindness, depression, tacial paíalysis
listeria monocytog,cnes Listerio1is Ataxia, cirdíng, facial.palsy, head tift
Mycoplasma bovis Otitis media-Interna Ataxia, droopy eat, head t ilt
Pasteurella multocída Otitis media-interna Ata~ia, droopy ear, nead tilt

'ClassifiecJ.aS'a foreign ánima! disease ageot in.t~e \111itell $tates.

b(ongenital:iofectí~n. · · •·
,.Hae¡nopSilus ;omtfus"·-i~ tlle obsólefe nome for this, ocganism.
dMost commcmlyTyp~.s B,..C, aml D.
478 PART IV Clinical Applicatíons

Table 71.5. Common and/or lmportant lnfectious Ac¡ents of the Nervous System of Sheep and Goats

Agent Di sease Neurological Sigos

Prions
Scrapie prion Scrapie Ataxia, exaggerated nibbling retlex, intense pruritus, muscle tremors
Viru1Ps
AkdUdll~ virus•·b Hydranencephaly, neurogenic arthrogryposis Sensory and motor deficits at birth
(Akabane disease)
Bluetongue virus• Ccrcbcllür hypoplasia, hydrancnccphaly Blindness at birth, inability to walk
Border disease virus' Hypomyehnogenes1s Ataxia, tremors
Caprine arthritis-encephalitis virus (G) Caprine arthritis-Encephalitis Paralysis. paresis. tremors
Louping ill virusb Louping ill Ataxia, muscle tremors
R~hiPI virus Rabies Ataxia. constipation. paralysis
Visna virus (S) Visna Abnormal gait, ataxia, paralysis, paresis
Bacteria
Arcanobacterium pyogenes Cerebral abscess, pituitary abscess Ataxi~. he~ci rrP11ing, hP~ci tilt, hlindness
Clostrld/um botulinum Botulism AldXid, flaccid paralysis
C/01tridi11m perfringens Type D Focal symmetrical encephalomalacia Coma. depression, head-pressing, opisthotonus
Clostridium tetani Tetanus Opisthotonus, seizures, tremors
Escherichia coli Meningitis Blindness, head-pressing, seizures, somnolence
Lirteria monocytogenes listcriosis Ataxia, cirding, facial palsy. head tilt

(G) • goat::, (S) = sheep.

'Coogenital disease.
bClasslfied as a foreign animal disease agem In the Unlted States.

Table 71.6. Common an d/or lmportant lnfectious Agents of the Nervous System of Pigs •

Agent Disease Ncurological Signs

Viruses
Hemagglutinating encephalomyelitis virus Vnmiting anci wastin9 cii..,.asP O?pre11inn, jerking movements. paralysis, seizures
E11te¡.>hdlumyU1.dl di lb vil U) E1 lltphalo1 nyocarditis Ataxia, depression, tremors
Porcine enterovirus 1 Porcine polioencephalomyetit¡s (Talfan/Teschenl Ataxia, paresis, coma, paralysis, seizures
1log cholera virus•,h Cerebellar hypoplasia Ataxia, seizures
Nipah virusª Nonsuppurative meningitis Paresis, tetanic spasms, se1zures
Pseudorabies virus Pseudorabies Ataxia, tremors, seizures
Rabies virus Rabies Aggressive behavior, ataxia, paraly1i1, 1eiLu1e1
Bacteria
Clostridium tetani Tetanus Opisthotonus, mu~le rigidity, seizures, stiff gait, tremors
Eschench1a co/1 (Shrga toxm pos1tJve) ~dema disease Ataxia, edema of tace, paralysls, stlffness
Haemophi/us parasuis Meningitis (Glasser's disease) Ataxia, paddling, tremnrs
Lisrerla monocyrogenes Usteriosis Aldxid, hypeiexuldbility, trembling
WrPptn<nrr111 1ui1 (Type 2} Meningitis Ataxia, depression, paralysis. seizures. tremors

•c1assified as a foreign animal áJSease agent in the United States.

b(ongenit>I infPrtinns r;iu<P rnngPnit~I trPmni< in pigleK

to the n1eninges (nuchal rigidity, depressed mental status),
cerebrum (circling, behavioral changes, seizures), brain-
stem (cranial nerve deficits, head tllt), cerebellu1n (ataxia,
trcmors), or spinal cord (tetra or paraplcgia).
Mechanism of Central Nervous System lnjury
Vascular Oamage. Damage to blooct vessets may be the initi-
ati ng factor in disease. Microbial toxins can cause vaso-
genic cerebral ede1na tl1rough effects on the blood braln
barrier and resultant lcakage of proteins into extr;icPlh1 J;:ir
spact!S. Tllis i:; tl1c µruµuse;:d 1necha11isn1 of action of C. per-
fringe1i~ 'fypc D epsilon toxin, which produces a focal sym-
mctrical encephalomalacia, cspccially involving thc tha la-
mus, hippocampus, and midbrain in sheep (Fig 71-1) and
the Shiga like toxin of toxigenic Escherichia coli strains as-
soc1ated with edema disease In swine_ Viral effects on the
vascular system include immune-mediated vasculitis ::inrl
 Chapter 71 Nervous System 479

Table 71.7. Common an d/or lmportant lnfectious Aqents of the Nervous System of Poultry

Disease Neurological Sígns

Viruses
Avian encephalomyelitis virus
Avian influenza virusª
Eastern equine encephalitísvirus
Exotit. Neyvr.astle disease virusª
Ma1el\\ úi,ea'e vi1u' (Q
Avian encephalon¡yelitis Ataxia, paralysis, tremors
J\vian influenza Ataxia, depression
m Eastern equíne encephalitis Ataxia, depresslon, paralysis
t=xot ic Newr.astle disease Depression, paresis, torticol liS; tremors
Ma1ek's tlisease Aldxid, l~y 01 winy pot~)is/por aly)b

Bacteria
Cíostridi1.1m botu{inumb Botulism (limberneck) Flaccid paralysis, inability to support head
Listeria monocytogenes tisteriosis Ataxia, opisthotonus, tortlcollls
Sa/monella enterica subsp. arizonae (T) Meníngitis/encephalitis (Arizonosis) Ataxia, paralysis, seízures

Fungi
Aspergí/lus fumlgatus Mycotic eocephalitis Loss of equflibrium, torticollis
Ochroconis ga//opavum' Mycotic encephalitis Ln" nf eq11ilihri11m, torticollis

m
(12) "'mickens, =turl<eys.
ªOa«ifiP.rl a< ;i for.Pign animal rli<P!!<P •gen¡ in the llnit•d State<
.bMost commonly Ty~ C.
' Dactylaria ·gallopava is the obsolete oame for this organjsm.

mation results in sorne cases (e.g., distemper, visna).

F1G U RE 7 1 . 1 . Focal syn11netrical encephalon1alacia in a sheep Movement of lnfectlous agents may occur within the
brain. Lesions are associated with enterotoxemia due to Clostrídíum cerebrospinal flu.\d or interstitiu m, orwithin different cell
perfringens Type n . (Cnurtesy nf nr. F. /17¡¡ /.) t}'pes .
•

Viral lnfections. Viruscs wtth. neurotrophic properties affect

perivascular inflammation (e.g., feline infectious peritoni-
tis, equine herpesvirus 1). R.ickettsial agents (Ehrlichia and
Rickettsia) induce endothelial damage and vasculitis that
lt::a<.l to blet::<.ling i11 tl 1t:: braír1. Tliror11bo:si:s uf vt:::s:st::l:s rnay
cause malacia of hrain parenchyma (e.g., Histnphilus snmni
in calves, Saln1011e/la. enterica subsp. arizonae in turkeys).
Septic emboli may resu lt in brain abscesses.
lnjury to Brain Parenchyma or Meninges. Injury to cens in the
central nervous system is by direct action of the pathogen
or fr~J111 tl1t: re::;ulti11g i11lla111u1atory re:spou:se. 'fhe inflarr1-
m;itory rPsponsf> c;in be suppurative, nonsuppurative,
granulon1atous, or son1e con1bination of these a11d is pre-
dominately influenced by the agent involved. I11flamma-
tory processes of the brain parenchyma (encephalitis),
rneninges (meningitis), or spinal cord (myelitis) occur
independently or in combination. Disorder i..n 1nyelin for-
al! anim al species (see ·tables 71.1-71. 7). Sorne viruses
specifically affect the nervous system (e.g., rabies virus in
all species, pseut1orabies in most species), and ot her
viruses involve the nervous system as part of a multisystem
d isease process (e.g., n1alignanL calarrhal fever v irus in caL-
tle, exotic Nevvcastle disease virus in poultry, distemper
virus in dogs). Viruses usually reach the central nervous
systern via the blood strearn, often as the result of a sec-
ondary viremia. Entry is v ia one of the methods described
previously (see the section "Routes of Infection," above).
Effects on specific cell types in the nervous system are ei-
Ll 1er due Lo <.lirec.:L viral cytuc.:i<.lal effect:; ur <.laiuagt:: re:;ult-
ing fo r t he ensuing inflammatory response. Gliosís,
perivascular cuffing, and 11euronal degeneration typically
characterize viral encephalitis. Viral infections in the ce11-
tral nervous system only rarely serve as thc primary mech-
anism for transmission between. hosts. Typica1ly transmis-
sion relies on infection at other si tes in the body.
So1ne viral infections in the tlam affect the fetal nervous
system ;incl Jp;icJ to cli>vi>lopmi>nta 1 p roblems, includiI1g
cerebellar hypoplasia (bovine viral diarrhea virus, pan-
leukopenía virus), hydranencephaly (bluetongue virus),
and hypomyclinogcncsis (Bordcr discasc virus).
Bacteria! lnfections. Bacteria! meningitis in dogs and cats is
relatively u n.common and often associated with primary
infections at other sites (e.g., urinary tract infection , endo-
cardilis). I11volve1111::ut uf the r1ervuus system rnay also
arise from local extension of infectíous processes (e.g., ear
infcctions, tooth root abscesses, sinus infections). Organ·
isms involved are usually endogenous and include aerobes
(Staphylococcus, Streptococcus, l'asteurella, Actinornyces) and
480 PART IV C:llnical Applications
anaerobes (Bacteroides, Porphyromonas, Fusobacterium, J1ep-
tostreptoroccus).
Bacterial meningitis is more common in neonates, with
horses and production animals most commonly affected.
T11 Llie:>e a11i111al:>, Lht: t:liolugy age11l is ofle11 ai1 e11leric or-
ganism (e.g., F.. coh) and is associated with failure of pas-
sivc transfcr of maternal immunoglobulins. J-Jaemophilus,
Pasteurella, Saln1onel/a, and Streptococcus are other genera
cncountcrcd with sorne frequency in bacterial meningitis.
Prolongcd bacteremia and the total bacteria! numbers ap-
pear directly related to the likelihood that the blood brain
barrier wi ll be crossetl. In ln1cteri<1l 111e11i11giti:>, a fil.iri110-
purulPnt rPspnnsP typira l ly rr.su 1ts.
Brain abscesses are also more commoo in horses and ru-
n1iI1ants than in companion animals and develop as a re-
sult ofbactc.:rc.:mia, trauma (dircct imp lantation ), or sp read
.trom a conti~ruous site. Streptococcus equi subsp. equi is the
inost common cause of brain abscesses in h o rses, a form of
so-called "bastard stranglcs," and is relatetl to a bact ereruic
event subsequcnt to rhi noph;:iryngiti.s ancl lymph node ah-
scessalion (slranglcs). Brain abscesses in cattle are also as-
sociated with primary extraneural infections (e.g., hard-
ware disease). 'fhc pituitary gland is an cspccially common
site in ruminants, possibly due to the anatomy and close
association of the rete mjrabilis with the pituitary gland.
Common ruminanr pyogenic agents (A rcanobaaerium pyo-
genes, J:usobacteri111n necrophor11n1) are the usl.ial suspects in
tl1e:;c ca:.c:.. 111 íecliv1 t:> i11volving verlebrae a11d interverte-
hral disks, especially in dogs, ruminants, and swine, may
involvc thc spinal cord and manifest as posterior paraly-
sis/paresis. Sorne bacteria! species exhibit a greater degree
of neurotropism than others (e.g., Listeria in ruminants,
ffistophilus sornni ["Hal>rnopflilus somnus" is the obsolete
name] in beef cattle). Lcsions associatcd with Listeria en-
c1::µl1aliti:. tyµically are in Lhe fvr111vf111icrvausct:s:.es iu Lhe
hrainstem (pons, medulla oblongata) and are almost
pathognomonic for Listcria cnccphalitis (Fig 71.2).
As already mentioned, bacteria! toXins rather than the
agent itself may be responsible for clinical signs an d
pathology. Sorne bacterial toxins directly affect th e vascu-
latu re of the central nervous systen1 (e.g., epsilon toxin of
F1G U R 1: 7 1 . 2. Micro1Jbscess in the brainstem of a cow t hat
died from Listeria encephalitis. Listeria monocytogenes was isolared.
Hematoxylin eosin stain.
e;. perfringens in enterotoxemia and :)higa-like toxin of E.
coli in edema disease). On the other hand, tetanospasmin
(tetanus toxin), the toxin produced by Clostridiurn tetani,
binds to peripheral nerves and travels to thP CPntral nPrv-
ous systen1 where it block:; release of inhibitory neuro-
transmitters from presynaptic inhibitory motor nervc
endings.
Fungal lnfections. As a general rule, fungal infections of the
central nervous system are infregue11t. When they occ.ur,
the inflammatory response is typically gran11lnmatou.s.
MosL of lhe syslen1ic n1ycotic agent s (Blasto111yces,
Cocddíoídes, Cryptococcus, flistoplasma) have the poten ti al
to be involved in ncrvous systcm discasc, b ut this usually
occurs secondarily and late in the cottrse o f th e infection .
Most fungal infections result from h ematoge11ous dissem-
in ation. Crypcococcus neofi>rmans is the n1ost con1mon fun-
gus encountcred in nervous system discase ancl mn.st con1-
1uv1 1ly aífecls dogs and cats (Fig 71.3) . It produces a
number of virulence factors, inclu ding a large polysaccha-
ride capsule, which allows it to pcrsist (see Pig 45. l ). Evi
dence suggests it uses monocytes and end othelial cells to
cross the blood brain barricr. Granulomatous e11cephalitis
wíth focal caseonecrotic lesions caused by Aspergíllus sp.
has been described in poultry. Outbreaks of mycotic en-
cephalltls in poultry flvck:. cau:.t:l.l liy a the1n1ophilic fun-
811s, Orhrnroni~ gallnpavum (Dactylaria gallopava- obsolete
name), have also been reported.
•
Prion Diseases
Transmissible spongiform encephalopathies or prion dis-
eases (bov1ne sponglform er1cepl1alvpatl1y [BSE), :>crapie,
feline spongiform encephalopathy) are rare bnt important
nervous syslen1 diseascs because t h ey are largely untreat-
able, may cross spccies barriers. and can have su bstantial
economic impact bec~use of public health con cer11s. An
abnorrnal conformer of normally p resent prion protein
(PrPc) <lesignated PrrRcs (for "resista11t" PrP) is b elieved to
cause conversion of normal PrPc into tbe pat hological iso-
F1G u Rt: 11 . 3 . Cryptococcus neoformans meningítis in a cat.
Numerous yeast ce/Is are present. Spaces around stained yeast bodies
:irc thc rcsult of shrinkage of the po/ysaccharide capsule duríng the
stainlng process. Mayer's mucicarrnine st'ain.
•

F1G U R E 7 1 . 4 . Sponqiform encepha/opathy characterized by
vacuolation of neurons and neuropil in the brain of a sheep with
scrapie (H&E). (Courtesy Dr. 8. Barr.)
form . Acquired principally through ingestion ot PrPR~~­
contarninated material (e.g., meat and bone mea! in BSE
a11d possibly infectec.1 placenta or feces in scrapie) p rions
are able to pass from the digestive tract and are potentially
a.111plifit:cl i11 Lhe ly1nphorelicular sysLen1 before n1ovi11g up
the pt~ripheral nervous system to the brain. ·rhe accumula-
tion of PrPRes is responsible for the pathology (neuronal
intracellular spongiosis) associated with t ransmissible
spongiform cncephalopathies (Fig 71.1).
lnfections of the Peripheral Nervous System
As 1101.ed previously, the peripheral nervous systen1 is less
frequently involved in infectious processes than the cen-
Chaptcr 71 Ncrvous Systcm 481
F1G U R E 7 1 . 5. Botulism in a cow. F/accid paralysis of the
tongue has resulted in an inahility nf thP rnw tn retr;irt it~ tnnguP.
Clostridium botulinum type C was the cause of botulism in this case.
(Courtesy Dr. R. Moel/er.)
tral nervous system, however sorne important diseases
specificall.y or predominantly involve the peripheral nerv-
ous systen1. Th.e toxi n of Clostridiurn botulinurn affects pe-
ripheral nerves, specifically components of the synaptic
vcsiclc docking and fusion complcx at pcriphcral motor
nerve terminals. l'his blocks the release of acetylcholine
resulting in the flaccid paralysis characteristic of the dis-
ease (Fig. 71.5). Involvement of peripheral nerves, usually
sciatic and brachial nerve plexuses, is a characteristic fea-
lure of Marek's disease virus. Grossly enlarged 11erves with
both inflammatory and neoplastic histologic characteris-
tics, as v'lell as myelin degeneration, are found. Mycotic
and bacteria! infections ot the guttural pouch ot horses
may involve the glossopharyngeal and vagus nerves, re-
sulting in s•vallowing difficulties or laryngeal hemiplegia.
 Ocular Infections
RICHARD L. WALKER
'rhc primary function of thc cyc is vision. Factors that af-
fect vision impact overaJJ animal well-being. 111 this chap-
ter, the ocular system includes the eyelids, lacrimal appa-
rarus, con¡unctiva, the eye itself, and the surroundlng
fascia. The eye includes tl1e cornea, sclera, lens, uveal. tract,
relina, oplic nerve, and aqueous a11d vltreous cl1an1bers.
The majar sites far infectious ocular disease to develop are
t!1c conjunctiva, cornea, and uveal tract .
Antimicrobial Properties of the Eye
Consiclering the frequent exposure to environmental ele-
ments, the eye is remarkably rcsistant to il1fcction.
Mechanical, anatomical, antimicrobiai, and imrnunologi-
cal factors all play roles in protectlng the eye from infec-
tion. Tl1e followi11g sectio11s. detail these spccific factors.
Mechanical and Anatomic Factors
The eyelids, including the cilia, and blink (menace) and
cornea! reflexes provide a barrier to externa! insults that
n1ay Lraun1alize Lhe eye and p1edispose lo infeclion by en-
dogenous or exogenous microbes. Intact conjunctiva and
cornea epitheliu1n provide addit ional barriers to i11fection.
Precorneal tear tilm, a complex multilayered tluid, contin-
uously coats ex posed surfaces of tl1e eye without irnpairing
v.ision . The tear filin has a nuLnber o.f functions including
lubrication, retarding evaporation, and nutrient trans-
po1 l. Meibo1n iar1 and lacrin1al glands, lhe conj uncliva,
and cornea all contribute to the composition of the tear
filni. 'lears provide overa11 protection for tl1e eye surfacc
through the unitorm coating ettect and by rnechanicaJ ty
rinsing the eye of i1oxious n1aterials and rnicrobes.
l'rotectio11 far i11ternal structures oí the eye comes fron1
tight junctions of endothelial and epit helial cells that form
llie bloucl-aqueous ancl blood-reli11al barriers. 'fhe blood
aqueous barrier is forxned by ciliary epithelial cells betwee11
capillaries in the ciliary stroma and aqucous fluid in thc
postedor chambet ot the eye. 'fhe blood-retinal barrier is
composed of tight junctions of endotl1elial cclls of retinal
capillaries and cells of the pigmented reti11al ep.itheliun1.
'fhese barriers afford protection to intraocular structures of
tire eyt'. froJI1 Hli<.:ro!Je:; uf l1e111aluge11ous origi11. When nli-
crobes from systemic infections do involve intraocular
sites, it is these areas \-vhere infectior1 typically initiates.
BreakdO\~·n of blood-ocular barriers is largely tl1e result ot
inflammatory p rocesses that disrupt tigl1t junctio11s.
482
Antimicrobial Factors
In addition to serving asan ir1terface to auieliurale lhe ef-
fects of PxtPrn;il sti muli; tPars c.ontain non.specific antimi-
crobial substances that includc:
1. Lactoferrin. Lactoferrin is a substa.n tial protein com-
ponent of tears (up to 25%). By b.i nding free iron,
lactoferrin xnakes this esscntial enzyme component
unavailable to bacteria, thereby lirniting bacteria!
gro>vth . In adllition, laLtoferriI1 ruay l1ave a role in
enhancing natural kiUer cell function ancl inb.ibit-
iug for1uaUou uf C3 converlase.
2. Lysozyme. Tears are rich in lysozyme (up to 4091> of
tear protein) . The enzymatic action of lysozyrnc on
the glycan chain ot the peptidoglycan ot bacteria!
cell walls provides nonspecific protectio11 frorn exo-
genous and resident bacreria. Variat ion in lysozyn1e
concentration occurs a.m ong differen t animals and
may, in part, account for variation in susceptibility
of different animals to externa! oc11l;ir infPctions.
Decrease in the lysoz:yn1e concentration in tears
correlates with increased ocular infectio11s.
3. Antitnicrobial Pcptídcs. Broad-spectrurn cationic an-
timicrobial peptides are innately produced by ocu-
lar surface tissues. These peptides act as natural an-
tibiotics through interactions with bacteria! cell
su:rfaces. They lnay also play a rolP in sign;:iling that
activates host cell processes in volved in immune de-
fense. Antimicrobial peptides can be detected in the
co11junctiva, cornea and tears.
lmmunological Factors
The conjunctiva is part of t he common inucosal immune
system, which includes gastrointestinal, respi ratory, uro-
genital, and marnmary n1ucosa. It is unclf>ar if antigen-
prese11tiug capabilities exbt in t he conjw1ctiva or lacrimal
gland lyn1phoid tissue. It is plausible that ocular imn1uniza-
tion occurs by passagc of antigcn through the nasal lacri
ma l duct to GA.i.:r or JJAL:l sites. Plasma cells in the lacrimal
glands are derived by clona! expansion a11d d.ifferentiation
of IgA committed B lymphocytes that localize in tl1e
lacrimal gland. lgA is produced, which in h 1rn c.omhines
will1 secrelory pleces. Secretory IgA is resistant to prote-
olytic enzyn1es in tears and constitutcs the major immuno-
globulin in tcars. lts role in microbial defense includes pre
venting bacteria! attachme11t and neutralization of viruses.
A functional complement system is also present in tears.

Beca use of its rích vascular supply, an aggressive inflam-
n1atory response, prcdomínately con1posed of !leu-
trophils, occurs in the conjunctiva. The cornea, dueto its
avascular naturc, is supprcsscd or dclaycd in its inflamrna-
tory response.
The intraocular immune response is programmed to
prevent an overexuberant response that may irreparably
damagc intraocular structures and ultimately vision.
Subsets ofT lymphocytes that cause subsla11Ual bysla11de1
injury are suppressed, and the immune response is more
localized.
Competitíve lnhibition Effect of Microbial Flora
Tt i<; po~sihl1> th;it thl' norm¡il flora, as elsewhere in the
body, plays a protective role in the ocular syslen1 by in-
hibiting cstablishmer1t of more pathogenic species.
Microbial Flora of the Eye
A norn1a l ronj11nrtívíll floríl i.~ present but varíes by ani-
mal, brced, geography, housing conditions, and lime of
year. The most commonly recovered flora are gram-
positive organisms and includc staphylococci, micrococci,
streptococc1, d1phthcro1ds, and Bacillus spp. Less fre-
quently nonenteríc, gram-negativc bacteria are isolated
and include predomlnately Moraxella, 1Vetsseria, and Pseu-
dotnonas species. In rumiñants, Moraxella species may be
tl1c predomina11l bacle1ial type in no11nal eyes. Myc.u-
plastna spccics are also found as conjunctival flora in sorne
animals. Fcw studics havc been done to actually quantitate
the relative numbers ot cach ot the ditterent species that
constitutc normal flora. Not ali conjunctival specimens
from normal animals yield microbial growth, indicating
that the conjunctiva is not heavily populated by normal
flora. Interna! strucrures of the eye are normally sterile.
Ocular lnfections
()cu lar infections can be primary infection s or part of mul-
tisystem infectious processes (e.g., upper respiratory tract
infections). ·rhe organisrns involved are often contagious
and may afff'rt pop11lation.~ of ilnimílls rather than indi-
viduals. ln other cases, ocular infections are secondary to
insults that comprise the intcgrity and innate defenses of
the eye. Compromising factors in thcsc cases includc dc-
creased tear productlon, excess ultrav1olet radiation, im-
munosuppressing diseases, trauma or penetrating injuries,
a11atu111ic tlcfc1..ts (t!.g., entropions), or surgical interven-
tions. Jn such sítuation<:, normíllly hf'nign ocular or other
endogenous flora can cause serious infections once t11ey
gain entry to unprotected sites. A number of systemic in-
fectious diseases also include ocular manifcstations as a
consequence of disseminaaon from the initial focus of
infection .
Deµe11tli11g 011 1l1c 111icro1Jial agent involved, the agent's
tissue trophism, routP. of f'xpos11rf', ílnc1 the host's ability to
contain infi:ction, ocular infections n1ay or n1ay ilOL be
limited to specific parts of the eye. The inflamrnatory
C:ha¡1ter 72 Ori1l;ir lnff'ctions 483
F 1G U RE 7 2. 1 . Endophthalmitis in a chicken caused by
tschench1a coh. (Courtesy Dr. H. Shivaprasad)
process can and frequcntly does involve structures in tl1e
eyc adjaceul lo llit: ~ile uf i11itial lnfection or, especially In
uncontrolled infections, hecomcs widespre;id to involvf>
intraocular cavitics and surrounding structures (endoph-
thalmitis, panophthalmitis} (Fig 72.1 ). Route ot exposure
of the eye to infectious agcnts is through surface contact
with endogenous or exogenous microbes or vía the blood
stream or lymphatic systcm and, perhaps, by extension
fro111 11e1 vou~ li~~uc~.
c:ommon and/or important ínff>rtío11s agf>nts of thf' oc-
ular system in domestíc mammals and poultry are listed in
Iables 72.1-72.7.
lnfectious processes of the eye frequently includc thosc
presented in the follow1ng sect1ons.
Eyelid and Lacrimal Apparatus lnfections
Bacteria are the n1ost con1111on c¿tusc of in fcclions of eyelid
margins (blepharitis) and lacrimal glands. The source for
bacteria is cndogcnous, with staphylococci an d strcpto-
cocc1 be1ng the most common agents. In dogs, a purulent
blepharitis occurs in conjunction with ju venile pyoderma.
Dermatophyte lnfectlons may extend to invo.lve eyelids.
Conjunctival lnfections
lnfcctions of thc conjunctiva induce an inflammatory re-
sponse characterized by hyperem ia, chemosis, and cellular
cxudate. Conjunctivitis occurs both as a local infection
(e.g., Chlarnydopl1ila lnfections in cats) oras part of a srs-
tcmic disease (e.g., distemper in dogs).
Viral-i11duced co11ju11c.:livili~ (c.g., alphatJerpesviruses
conjunctivitis) often occurs in conjunction \-vith npp¡>T
rcspiratory or digestive tract infections when virus specifi-
cally attaches lo and replicates in surface epithelial cells.
Cytopathic effects causcd by viruses and induction of an
inflammatory response account for din1cal s1gru ob-
served. Conjunctival infcctions may spread to or concur-
1enUy in vol vt: ll 11:: curuca (keratocon¡unctivitis) .
Bacterial infections of thf' ronj11nctiva may begin in the
484 PART IV
Ta b 1e 7 2 . 1 . Common and/or lmportant lnfectious Agents of
the Ocular System of Dogs
Agent
Viruses
Canine adenovirus 1
canine distemper virus
Cánine papillomavirus
Bacteria
Beta-hemolytic streptococci
Bruce/la <ai:Jis.
Coagulase-positive staphylorórri
fl11/it/1/<1, S!Jfl·
Leptospira spp.
Rickettsia ríckettsíi
Fungí
8/astomyces dermatitidís
Cryptococcus neoformáns
Algae
Prototlíeca spp.
Ta b Je 7 2 , 2. Cu111111ur1 et11u/u1 lr11pu1 ldll l
the Ocula r 5ystem of Cats
Agent
Viruses
Feline herpesvirus 1
FelinP imm11norlefiriPnry vinll
Feline infectious peritonitis virus
Fe!ine leukemia virus
feline panleukopenia virus
Bacteria
<Shlamydophila fe/is
Mycoplasma fefir>
Fungí
Cryptococcus neoformans
' Role asan ocular pathogen is uncer:tain.
(]inical Applications
Ta bl" 7 2 . 3. Common anrl/or lmportant lnfectious AgPnt s of
the Ocular System of Horses
Major Clinical M¡¡nifeg¡¡tions :Ayeot ~jor ,Glinital ~anifestatións
(Common Dis~ase Name) {Commort Disease'llame)
Viruses
Cornea! edema, immune-complex Atricail Horse Sickness virus• Conjunctiv1t1s, eyelid and periorbital
uveitis, keratitis (bhie ey'e) edema
Choriore¡ioil:ls, conjunaivitis, cptic Equine ar¡eritls vlrlis Conjunctivitís, períorbita! edema
neuritis (di>tempP.r) Equine herpesvirus 2 Conjunctivitis, keratitis
Pdpillun1d~ of eyelids and conjuoctiva Equine influenza virus Conjunctivitis
Bacteria
Coojunctivitis, dacryocystitis Leptospira spp. Panuveitis (equinP rP.rurrent uveitis)
Anterior uveitis, endophthalmitis Pseudomonas aeruginosa Kerdlilb, cornea! ulcer
Rl~pharitis, coojunctivitis, dac¡yocystitis
fungí
Anterior uveitis, conjunctival hyperemia,
Aspergil/Jus spp. Keratitis, cornea! ulcer
chorioretinitis
Fusarium spp. Keratitis, cornea! uker
Anterior uvcitis
Anterior uveitis, cborioretm1tis,
conjunctival hyperemia, retina!
hemorrhage
Antcfior uvcitis, chorioretinitis,
T~ b I o 7 2. a. Common a nd/or lmportant lnfect ious Aa ent < nf
endopntt1alm1t1s
t he Ocular System of Cattle
Chorioretinitis, optic neuritis
Agent Mator Olnlca! Manlfestations
Anterior uveitis, chorioretinit1s
Víruses
(<;ómmon Disease Name)
------..a
Alcelaphine herpesvirus '1ª Anterior uveitis, conjunctivitis, cornea!
edema, eyelid edema, kerntitis
ln feLliuu~ A\Jell b o[ (málignant catarrhal teverJ
Bovine herpesvirus 1 Conjunctivitis, cornea! edema/opacity
(lntectious bovlne rhlnotracheltis)
Major C!inical Manifestations Bovine papi!lomavírus Papilloma<-of P.yeliil ;mn ronjunctiva
(Common Disease Name.) Bovine virar dia1rhea virus Cd laracts, retina! atrophy, optk neuritis
(bovine virus diarrhea-in utero
infection)
Conjunctivitis, cornea! ulcer, stromal
Ovine herpesvirus 2 Anterior uveitis, conjunctivitis, cómeal
keratitis (feline viral rhinotracheitis)
edema, eyelid edema, keratitis
Anterior 11veitis, chorinrntinitis
(rnalignant catarrhal fever)
Anterior uveitis, chorioretinitis, keratic
precipitates, keratitis (feline Bacteria
infectious peritonitis) Arcanobactcrium pyogenes Orbital fjellulitis
Anterior uveitis, uve.al lymphosarcoma, Ristophilus somnib Ketinal hemorrhages, retinitis
retín.al hemorrhage (tbromboembolic meningoencernaliti~)
Retina! degeneration, retina! dysplasia Listeria monocytogenes Conjunctivitb, ker alilb, uveitb
(teline panleukopenia-in t1tero Moraxella bovís Conjunctívitis, kératitis, corneal ulcer,
infection) panophthalmitis (infectious bovine
keratoconjunctivitiS' or pirikeye)
Mycoplasma bovoculi Co:níunctivitis
Conjunctivitis {feline pneumonitis)
Conjum:tivítis ªClassified as a forei9n animal dlseas• agent In the United States
b•Haemophilus somnus' is the obsoletc n~mc for this orgoni.sm.
Chorioretinitis, optic neuritis
(cryptococcosis)
 r:hapter 72 Oc.u lar Tnfec.tions 485

Ta b 1e 7 2. 5. Common and/or lmportant lnfectious Agents of Ta b 1e 7 2. 7. Common and/or lmportant lnfectious Agents of
the Ocular System of Sheep ;;ind Go;;its thc Oculi:ir Systcm of Poultry

Agent' Majot-€1inifal Mánifestatioos Ayerit Ma}qr ClínícaLMapifestati.oris ,

(Common Disease Name) ________..._ _ _ _,.. _mon Disease N~'l?!!.l
(c_om
...._""""',_.,.~.,......~~='!'.......,...,..,,

Prions Viruses
scrapie prion Retina! deta.chment Avían encephalomyelitis virus {C) Cataracts; uveitis
lnfectious laryngotracheitis virus Conjunctivitis, keratitis
Bacteria Marel<.'s di;ease virus (C) less of ifis pigrnentation, panuveitis
Chlamydophila peco.rum• Conjunctivitis, kerutitis
Newcastle disease virusª Conjunctjval edema, hemorrhage
Listeria monocytogenes CoojunctiVitis, keratitis, uveitis
Moraxefla spp. Branhamel/a ovísª Conjunctivitis, keratifis Bacteria
Mycoplasma conjunctívae· Gonjunctlvltis, keratltis (lnfectious Bordetella avlum (i) <;onjunctivltis
ker;,toconj1inctivitis) Chfamydophíla p.~íttarí Conjunrtiviti~
fscherichia coli Conjunctivitis, endophthalmitis
'Role asan °'ul¡¡r pathogen is uncertain. f1aemophilus paragailinarum Conjunctivitis, periorbital edema
fAycop/asma gallisepticum Conjunctivitis,
.Pasteureila multocida Conjunctivitis, eyelid edema, orbital
cellulitis
Salmonel/a spp.b Endophthalmitis
Ta b 1e 7 2. 6. Common and/or lmportant l nfectious Agents of
the Ocular System of Pigs Fungi
Aspcrgil/us spp. Endophthalmitis, keratitis
M:.jnr rlinir;il ManifM>f;itinn<.
(Common Disease Name) ((} = Chicke~. (T) = Tutl<ey
oC!aosificd o; a forcign animal di:;casc agcnt in thc Unltcd St<i\cs.
Viruses blndudes Salmonellaarizoi11Je.
African swine fever virus<' Conjunctivitis (African swine fever)
Classical swíne fever virus• Coníunctivitis (Classical swine fever,
•
hog cholera)
Porcine rubulavirus Cornea! opacityledema, keratitis (blue inflammatory response in tl1e cornea results from migra-
eye disease) tion of neutrophils from the conjunctiva or limbic sclera.
fseudorabies virus Conjunctívitis, keratitis ln chronic ctisease the cornea. becomes vascularizect anct
Swine influenza virus Conjunctivitís directly participates in tl1e lnflammatory respo11se.
Bacteria Among the most co1nmon causes of viral keratitis are
Chlamydia suis Conjunctivitis members of the herpesviruses. Cats and cattle are most
E1rhPrirhia rnii (Sniga toxin positive) Palpebral edema (edema disease) frey_uently affectetl. In so1ne herpesvirus iufectious, recur-
l'<istetirella multocida <::onjunctivitis, nasolacrímat duct rt'nc.P somPtirnPs re.su lts from rPac.tivation of latPnt i nfec-
occlusíon (atrophic rhinitis) tions in sensory ganglia (e.g., trigeminal ganglia) follow-
ing stress.
ªClassified as a foreign animal d.isease ageot in the Unjted States, Primary bacteria! keratitis is rare. However, once the
cornea is l)reached a numt)er of bacterial species will read-
ily establish and spread to involve the cornea! stroma.
'fhese opportunistic bacteria inctu<.le staphylococci, strep-
tococri, ;in<l Psf'udornonas species. Darnage to th.e cornea
conjunctiva or n1ay resu1t from extension of eyelid or dueto bacteria! toxins and enzyrnes (e.g., proteolytic en-
lacrin1al gland infections. Chlarnydia/Chla1nydophila con- zymes of Pseudomonas) is further exacerbated by enzymes
junctivitis occurs in a numbcr of animal spccics and can be from rccruitcd ncutrophils (c.g., collagcnascs, clastascs).
a primary con¡unctivitis or, il1 addition, involve other f'seudornonas aeruginosa ca11 be an especially virule11t
sites. Bacteria! conjunctivitis inay develop secondary to cornea) pathogen once it is established and is associated
pri1n.ary viral con¡unctival infections. As with viral con- with so-called "tnelting ulcers" but stlll requires a break in
junctivitis, conn1rrt'nt cornt'<'ll involvt'mt'nt may occur. the cornea! epithelial barrier in order to establish. Mor-
Fungal infections of the conjunctiva are rare. axe/la bovis is 011e of tl1e few bacleria in velerinary 111edi-
cine that cause a prin1ary bacteria! keratitis. lt produces
spccific virulcncc factors including adl1esins (fimbria) for
Cornea! lnfections
adherence to epithelial cells a11<.1 toxins that cause necrosis
Cornea! inflammation (keratitis) with or without loss of of epithelial cells (fig 72.2).
the epithelium anti part of the stroma (cornea] ulcer) is a Mycotic keratitis (keratomycosis) is of greatest signifi-
common conclition in most ;inim;ils. Kt>ratitis can begin cance in horses. Exposure of the eye to plant material
externally on the epithelial surface or inte.n 1ally at tl1e often i11Lroduces Lhe fungus, allhougli :;uu1e :;tutlie:; llave
leve! of the endotheliun1. Because it is avascular, the initial found various fungi on the conjunctiva from normal
486 PART IV Clinical Applications
F 1G U ll E 7 2 • 2 .Cornea/ u leer in a cow with "pinkeye" caused
by Moraxella bovis. (Courtesy Dr. J. Angelos.)
equine eyes. 1·11ese most likely represent transient flora as a
l l:!~Ull ur ¡¡:u1don1 env ironn1enlal exposure. The intact
corneal epithelium provides an cxccllent barrier to fungal
infections, lhus rcquiring trauma to thc corncal cpithclinl
as a preceding event to the development of keratomycosis.
Corticosteroid use enhances the likelihood of fungal ínfec-
tions of thc cquil1e cornea and exacerbates the condition
once present. Typically, mycotic infcctions of the cornea
úo not havt> a co11currt>I1t coIJjuuclivili~.
Intraocular lnfections
Intraoc ular infections are frequently the consequence of a
systemic inft>ction with exogenous organisms that localize
in the uveal tract (iris, ciliary hody, choroid). The infection
111ay iuiliale aud/or predonlinale al a paJlicular sile in tl1e
nvPíl 1 trílrt; howPvPr, involvPmPnt of othPr sitP~ in thP trílrt
is co1nmon. At sorne leve!, at lcast histologically, wide-
sprcad involvement of the uveal tract occurs in most infec-
tions. Uveitis can be divided on the basis of the anatomic
site most prominently involved in the inflammatory
process (e.g., anterior uveitis) and whether adjacent sites
are also involvec.l (e.g., chorioretiuitis). lr1 i11traocular iIJ-
fPction~, PXtPnsion to othPr parts of thP PYP oc.c11rs clue to
proximity of other structures (e.g., retinal involvement),
Lhe fluid 11ature u1side lhe eye, a11d Lhe ope11 con1n1unica-
tion between intraocular chambers. Depending on the
agcnt, stagc of thc inflammatory response and cvcn thc
an1ma1 invotvect, tne 1nflammatory response in tl1c uvcal
tract can be suppurative, lymphoplasmacytic, granu-
lomatous, or sorne cornbinatlon uf these re:;puu:;c:;.
The pathogenesís of viral-induced uveitis is either by
di11::cl infeclion of Lhe uveal Lracl a11d subsequent inflan1-
matory response or through dcposition of irnrnune
complcxcs rcsultillg ill immune-mcdiatcd Typc III hypcr-
sensitivity reactions. Likewise, bacteria! uveitis occurs sub-
sequent to bacteria! (e.g., Bruce/la) localization to the uveal
tract or in sorne cases from immune complex cteposition
(e.g., Leptospira). Nonspecific bacteria! uveitis c;:in rPs11lt
::.ulJ~eyue11l Lo 0Lhe1 p1eexisling bacteria) co:nditions (e.g. 1
gingivitis, prostatitis). All of the systemic mycoses agents
(Dlasto1nyces, Histoplas1na, Coccidioidcs, Cryptococcus) hnvc
t!1e capability ot causing a panuveitis. Most prescnt clini-
cally as a chori.oretinitis, with dogs and cats predomi-
nately affected. In dogs, Prorotheca, an achlorophylluus
alga, causes a granulomatous chorioretinitis in conj11nr-
tiu11 witl1 utlJcr sy:;Le111ic 111anifeslalio11s (e.g., bloody diar-
rhPíl, pí!rPsis).
Congenital defects of ocular structures in utero infec-
tions, usually viral in origin. occur in sorne animal species.
l3ovine viral diarrhea virus infections in pregnant cows
have been hnked to retina! atrophy and cataracts in calves.
Panlcukopenia in cats is associated 1.vith dysplastic ocul;ir
<.lt!v~lop111c11t iu kitte11~.
lnfections of the Orbit
Tnfcctions in the orbit can be thc rcsult of a forcign body,
penetrating wound trorn the oral cavity or hematogenous
dissemination. Purulent infections in the form oí orbital
celluHtis and retrobulbar abscesses most often result. Ali
animal species can develop orbital infections, although
n1osl occur if1 dogs and cats. Thc etiology is usually a mix-
ture of bacteria! agents, oftcn .including Pasteurelfa species.
 Respiratory Systelll
RICHARD L. WALKER
The primary function of the respiratory system is gaseous
exchange. The structure of the respiratory tract ts such
that nnxiou.~ s11hstanc:P-~, partic:u latP matPrial, and m icro-
bial pal11ogc11s are prevcntcd fro1n entering and con1pro-
n1ising t he distal portions of respi ratory t ract where
gascous cxchangc occurs. Innatc protcctivc propcrti.c:; urc
present at all Jevels of the respiratory tract.
In most vertebrates the respiratory system is composed
of tl1e n<1sctl cC1vity, sinuses, Jarynx, pl1arynx, trachea,
h ronc:h i and hronc:hioles, and lungs. Tn hirds, thP rPspir;i-
tory syste1n is more complex and markedly different from
other vertebrates. Most notably, birds possess large, subcu-
taneous, infraorbital sinuses that com1nunicate with tl1e
nasal cavity and are especially p redisposed to infection-
in part, because of poor drainage. 'fhe lungs of birds are
fairly rigi.d comparect to other vertebrares. Air sacs are pres-
ent that co1nmunicate with the lung-s and are located in.
ll1e coelon1 a.n d n1cdullary cavity of son1e bones.
Antimicrobial Properties of the Respiratory
System
'fhe act ot breathing involves exposure of the respi ratory
tract to airborne microorganisms, including potentially
pathogenic ones. Resident microorganisms are present in
most upper parts of the tract, while various defense mech-
cu1isn1s operale lo exclude or t~lin1inate then1 fron1 other
sites.
Diffcrcnt protcctivc mcchanisms opcratc in thc na-
sopl1aryrrgeal, tracheobronchial, and puln1onary portions
of the respiratory tract. Aerodynamic filtration operates
through c!ifferent forces at these Ievels in depositing vari-
ously sized airborne particles. Inertial forces deposit larger
parlicles (>5 µn1 ii1 dian1cter) in the 11asophary11geal a11d
upper tracheobronchial sections through impaction. In
small bronchi and bcyond, whcrc air vclocity is reduced,
gravity acts to sediment particles 5 to 1 µin in size. ln the
smallest bronchioles and alveoli, particles measuri11g less
than 1 µm gain contact with membranes through Brown-
ian movement.
111e ruucus lirli11g coveriI1g airwCJy epithelium contains
numerous suhstances •vith antimicrohi;il propPrtiPs or
that provide protective effects. Lysozyn1e, which is selec-
tively bactericidal by its action on peptidoglycan, is pres-
ent in varying quantities throughout the respiratory tract.
Broad spectrum antimicrobial peptides, beta-defensins,
produced by ciliated epithelial cells are active against
viruses, bacteria, and fungí. 'fheir expression is increased
upon exposure to microbial components (e.g., lipopoly-
saccharide) . The antimir.robial reactive nitrogen species,
nilfic oxide, is also l'roduce::u l.Jy ciUatt:u e¡.>ithelial cells,
primarily by inducible nitric oxide synthetase (íNOS).
Buctcrinl product:i modulntc thc cxprcssion of iNOS. Nitric
oxide plays an important role as a biological mediator i11
the regulation of host d.efense and inflammation, produc-
·1ng both pro- and anti-inflammato ry effects ..Also present
in the mucus are imn1unoglobulins and interferon and
lactoferrin, which by binding iron, n1akcs il unavailable Lo
most bacteria. Alpha-1 antitrypsin, an enzyme inhibitor
that reduces the dcstructivc cffcct of inflamn1atory rcac-
tions, plays a protective role.
Nasopharyngeal Co1npartment
l1rotective 1nechanisms in the nasopharyngeal compart-
ment include vibrissae (guard hairs) around the nostrils of
so1ne anlrnals that arrest the largest inhaled particles (15
pm in diameter) and th.e nasal r.oncl1ae. Tl1e nasal r.onchal
arrangen1e11t creares a turbulent airflow thal increases Lhe
chances that particles will impact mucosal surfaces. Once
impinged on t he mucus-lined nasal turbinates or the na-
sopharyngeal wall, they encounter m ucociliary action (see
"Mucociliary Apparatus," below) and are transporte.d to
tl1e caudal pharynx to be swallovved and eliminated via
the digestive tract.
111 Lhe huinid, wan11 nasal vas::,agt::>, ¡.>arU<.:le:> :>well
through hydration, hecoming more likely to impinge on a
mucous men1b.rane. Warming of air in the nasal passages
also benefits coJd-sensitive clearance 1nechanisms in the
lovver tract. Pharyngeal lymphoid tissues act in thc filtra-
tion of microorganisms and initiation or the í1nmune re-
sponses as a constituent of the mucosa-associated lym-
pl1oid ti:;:;ue.
The resident flor;i p rovidPs coloni;1ation resistance as
well as production of antibacterial substances. 'fhe sneeze
reflex aids in clearing ínfectíous particles from tbis area.
Tracheobronchial Compartment
The tracheobro11chial compartment includes the larynx,
trachea, bro11chi, and bro11cl1ioles. Closure of Lhe glolli~
during S\vallowing protects this area from contami11ation.
C~oughing removcs gross accumulations of fluid . Th.c tra-
cheobronchial co1npartment is lined by mucociliary epithe-
lium, which traps particles and transports them cranially to
487
488 PART IV Clinical Applications
the phnrynx (see "Yfucociliary Appaiatus," below). Dcpo-
sition of particles on airway membranes is favored by
bronchial bra11chi.ng dueto direc."tional alrflow changes.
Bronchiolar-assoclated lyinphoid tissue (BALT) is dis-
tributetl Cllong the aiJways ::inrl is rnnrf'n tr::iterl ::it hrnn-
chial bifurcations, which corresponds to sites where t he
greatest trappil1g of inl1alcd particles occurs. BAI;r in-
cludcs both ccllular and humoral immune responses. Epi
thellal cells med1atc the active transport of IgA from the
lamina propria to the air,.vay lumen.
Pulmonary Compartment
Clearance mechanisms of the pulrnonary con1partment
(alvcoli) co nsist of pulmonary alveolar macrophages
(PAMs), neutrophils, and monocytes recruited from the
blood. Particles are d isposed of by phagocytosis. Suscep-
tible rnicroorganisms are kllled and dlgested. Phagocytes
migrate to si tes 1-\lhere n1ucoci)iary transport occurs or via
the lymphatics tu r<::u1uvc ul111:1 e11gulfed parlicles. Tl1c
'\::ime prott>rtive sul1stances as in tracheobroncl1ial secre-
tions operate at the pulmonary level, supplcmcntcd by
those derived trom alveolar macrophages.
Mechanisms
Overall, tl1c n1ucociliary ::ippar::it11s ;i nct PAMs constitute
Ll1e 111ain cl<:'.arance 111echanisn1s of the respiratory tract
and are described in greater detail belo1~1.
Mucociliary Apparatus
The mucoclllary apparatus Is composed of clllated and se-
cr<>tory cclls. C.iliated cells are pseudostratified in the nasal
and cranial tracheobron chial portions of the tract, simple
columnar in the smaller hronchi, and simple cuboidal in
thc smallcst b ronchioles. Thc cilio, sorne 250 pcr ccll,
measuring ~.o x 0 .3 µm, resem ble eukaryotic nagella and
beat up to 1000 times a m inute. 'fhe density of ciliated cells
<.lecreases grauually frurn t!H:! prux11ual tu dl~tal lJru c1<.;J 11-
nl e.~. Altrratinns in cilia activity or <leciliation of the ep-
ithclial cells hjnder clearance activity and promote inva-
sion by opportunistic pathogens. In addition, Joss ot
ciliatcd cpithclial cell function rcsults in dccreased pro-
duction ot antimicrobial substances and cytokilles that
mcdiate the inflammatory response.
The secretory components of the mucociliary appara-
tus are goblet cells, intersperscd with ciliatecl cells, ::in<I, in
Lhe nose, lracl1ea, and largcr bronchi, submucosal serous
and mucous glands. Serous fluid bathes tl1e cilia, while a
viscid mucus layer engages their tips. Mucus is propelled,
along with particJes trapped in it, caudally in the na-
sopharynx an.d cranially in the tracheobronchial compart-
ment toward the pharynx by cilla beatlng ata rate of up to
20 mm/n1in. The particle clearance rate is fastest in the tr;i-
<.:hta au<.l slowesl i..n Lhe sn1allcs1 airways, vvhere goblet cells
are abscnt, mucus is sparse, and cilia beat xnore slowly- an
arrangcmcnt t hat preven t.'> logjams in thc largc airways.
'J'he trachca (e.g., cat) can be cteared witlun an hour, and
ali airways witl1in a day.
Mucociliary clearance is inhibited by temperature ex
tren1es, respiratory viruses, sorne bacteria (e.g., Bordecella),
dryness, general anesthetics, dust, noxious gases (sulfur
diuxíc.le, carbun dioxide, au11uuuia, tulJacco =>111oke), a11d
hyrnxia. Mucus prod uction through disrupt ion of goblct
cell integrity increascs in rcspon~e to irritant exposure.
Pulmonary Alveolar Macrophage
Thc pulmonary alveolar macrophage (PAM) is a monocytP
adapted to the lung enviru11111c11t a11d lucated in Lhe alve-
ol::ir sp::ice. lt is rerr11ited from the hlood when needed. ·rhc
pulmonary alveolar macrophage is a pleomorphic cell, 20
to 40 µm in diametcr, with man y tysosoma1 granules con-
taining nun1erous bioactívc substances. Also produced by
PAMs are mediator substances-complernent con1po-
nents, interleukin l, a nd tumor necrosis fa ctor- which f'n -
alJI<:: auuitiu11al <.:cll ula1 aud l1u1110.cal dcfcnses to be mobi-
li7<'rl. Cn1nplement ::ind JgG rcccptors on PA1v1s enhance its
phagocytic capability. PAMs are motile and usually cxist in
the alveolus less than a wcck. Energy is obtained mai n ly by
oxidative phosphorylation. The absence of ciliated epithe-
hal cells and mucus-producing cells in the alveolus rc-
q uircs that PAMs remove particles that reach the alveoli.
Parrlcles ingested by PAM~-utln:r ll1a11 =>uscepüble bac-
tcri<1 killed 11pon ingestion-;i rc removed via the mucocil-
iary escalator or via interstitial centrípeta] or centrifuga!
lymphatics. The centrípeta! routc Jcads directly to the hilar
lymph nodes and may requlrc two wccks. Thc centr ifu ga!
route goes via t h e pleura and may take n1onths. Agents
thnt cannot be removed are sequestered by inflammatory
processes (abscesses, granulo m as).
PAM activities are inhibited by sulfur dioxide, ozone,
n!trogen oxíc.les, anc.l respiratury viiu=>c~. Baclcrial leuko-
toxins and hemolysins dcstroy PAMs and are majar viru-
lcnce factors produccd by sorne important bacteria! rcspi-
ratory pathogens (e.g. , Mannlle1mía haemolytica in cattlc,
Actinobacillus pleuropncun1oniae in swine).
Microbial Flora
1'hc dcnsity and constitucncy of thc microbial flora of the
respiratory tract varíes an1ong an1mals and wit hin the res-
piratory tract itself. Resident flora are limited to the nasal
cavity and pharynx wherc a highly diverse flora can be
found. For example, ovcr 30 diffe rent gram-positive bactc-
rlal speLies alon<:: ca11 ve rc<.:uv1::1 ed IJ.0111 lhe i1asal conchae
;ind tonsils of unweaned and weaned piglcts. Overall, the
nasal ílora consistently includes viridans streptococci and
coagulase-negative staphylococci along with potent1a1
pathogens that vary w ith thc a nimal host. Altl1ough not
usually considered a respiratory tract patl1ogen, coagulase-
positive staphylococci can colonize the 11ose and b1' c::ir-
rled ata higl1 rate i11 ~uu11: J.JV.lJUlalions. ·rhere tl1ey serve as
::i source for infections elsewhere in the body (e.g., integu-
mcntary infections). Sorne of thc rcsident flora of thc
upper respiratory tract and orophar ynx represent ma1or
respiratory bacteria! pathogens (e.g., members of the
Pasceurellaceae and Streptococcus and Mycoplas1na species) if
they are able to establish in lower parts of thc tract. M::iny
 Chapter 73 Respiratory Syste111 489

potentially pathogenic mycopl;ismas are normal residents Ta b 1e 7 3 . 1 . Common and/or lmportant lnfectious Agents of
of lhe upper respiratory tract of the host they affect orare t he Respiratory System of Dogs
carried there by persistently infected ind ividuals. They
play a promincnt role as pathogens at most levels in the Major Clinkal Manifestations-
respiratory tract, contributing to "respiratory disease con1- {Eommon o· easa Name)
plexes," or under the proper circumstances are by them-
Viruses
seJves significant pathogens.
As with the digestive tr;ict, the resident flora of the res- caníne adenovirus .2 N.asal dlscharge, tracheobronchitis
piralory systen1 confer colonization resistance tl1at is re- (keFmel cough syndrome).
duced by antibiotic treatment and environmental changes bronchointerstitial pneuinonia
that alter its composition . Caoine diste¡nper virú~ N.asopharyngitis, laryilgitis, bronchitis,
Nonresident organis1ns 1nclude both potential patl10- bro1wh0interstítial pneumonia
gens and har1nlcss tran sients. 1"ransient flora are com- (dístemper)
prised of 1nlcrobes t hat enter lluring the !Jreatlli11g Canine parainfluenza virus 2 Nasal·di·scharge, tracheoj:lronchitis
process anrl, thf>rf>forf>, reflec.t the environmcnt in v. hich 1 (canine kennel cough syndrome)
the anin1al is maintained. f.nviron m ental factors, such as Bacteria
dry, dusty envi ron ments or confined environments Actinomyces spp. Pyogranulomatous pneumonia, pleuritis
where ventilation is poor, increase the microbial load and Borderella br.onchisep,tica Tracheobronchitis (infectious
types of transient flora an animal is exposed to. It is not tracheobronchitis, kennet cou!J.h),
uncommon to isolate E. coli a11d otl1er enteric bacteria bronchopneumonia
from t he uppcr respiratory tract as t ra11sie11l flo ra. Tl!e sig- Escherichia co/i Br-0nchopríeumonia
nifir;ince o f the.ir presence in the nasopharynx is difficult Nocardia spp. l'yogranulomatis pleuritis
to assess without corresponding clinical and pathologic Oblígate. ana.erobesª Bronchopneumonia
information.. Pasteurel!a multocida Brondiopneumonia
Th e Jarynx, trachea, bronchi, and lungs lack a resident
flora. H.ovvever, the lower portion of the respira tory tract is Fungi
con tinually being exposed to microbes that are present in Agents of systemic mycosesb Granulomatous pneumonia
the upper portion. In tl1c uucu111pruuliseu respiralory Aspcrgíflus fumigatus Rhinitis, sinúsítis (nasaf asp~rgillosis}
tract, these organi.srns are q11ickly remove.ct hy th e natural Cryptococcus neoformans Ciranulomatous nasal masses
• (cryptoco,¡cosis)
hosl defense n1echanisms. Fluid from the distal tract may
contain up to 103 bacteria/mi in normal animals (e.g., Protist
cats). Rhinosporidium seeberi Nasal granulomas (tare)

•!ncludes Bacteroides, Peptostreptococws; Fusobacterium, and Porphyromona~species.

lnfections of the Respiratory System b¡ncJudes Blastomyces dermatitídis, Coccidioides immitis; Oyptofoccusneoformans, and
Hi<topla<m;i rap.1tilatum.
Respiratory tract iufcctiu11s are of subsLanlial in1porlance
il1 all anim;ils. Sorne. of the 1nost common and/or ímpor-
ta11t agents responsiblc for respiratory tract diseases in Ta b 1e 7 3 . 2. Co1n n1on and/or ln1portant lnfectio us Agents of
major domestic animals and poultry are listed in Tables the Respiratory System of Cats
73.1-73.7. Agcnt chnractcristics, route of infection, host
susceptiblity, and host ímmune response determine the Agent Major Clinical Manifestations
location(s) in the respiratory tract affected, severity of the {Common Oi~ease Name)
infection, and associated pathoiogy. Pathogens of ttte res- -------.,,-~
pi.ratory tr::ict covPrPcl in t his chaptf>r inch1clf> viral, h;irtf>r- Viruses
ial, and fur1gal agents, as well as one aquatic protistan p ar- Feline Ealicivírus Rhinitis, inte~stitial pneu((ionia, tracheitis
asite, l~hinosporidium. ffellne calici.virus disease)
Potential respiratory viral pathogens belong to a range Feline hefpesvirus l Rhinotracheitis (feline viral rhino.tracheitis)
of families (e.g., Adenoviridae, Caliciviridac, l-:oronaviridac, feline infectious peritonitis virus Pleural·effusion, pyogranl!l1omato.us
Herpesviricla.e, Paramyxoviridae, Orthomy:J(ovíridae). 'fhe ma- pleuritis ·
jority of thc l.JC1Lterial respiralory lJ'acL paLhogens belong Lo Bacteria
the family fJasteurellac:eae or to thf> gener;i Rnrdl'.tt!lla, Bordetella bronch/septlca Tr.acheo.bronchitls, br.oochopneumonia
Mycoplasrna, and Streptococcus. So1ne in1portant respiratory (felinP. horcietello1L1)
tract pathogens are associated with specific. well-defined Chla111y:dophi!d f.:lis l'neumo11ia {felh)e pneumonitíl), rhinitis
clinical entities (e.g., Rhodococcus equi and pyogranuloma Obligate anaerobic bacteria Pleural empyema {pyotnorax)
tous pneumonia in foals). Under th.e proper conditions, a Pasteurella multocida Ple1:1ral empyema (pyotnorax)
number of opportunistic bacteria (e.g., Actinon1J1Ces spp.,
IIH.:rn!Jer~ uf tlte fa111ily EnlcrubuLletiuccue, o!Jligale auaer- Fungí
ohes) from the oral cavity and lower digestive tract can Cryptococcus neoformans Rhinitis,.granulomatous nasal masses.
cause o r contribute to respiratory disease (e.g., aspiration sinusitis, pneumonia
pneumonia) . Fungal agents of the respiratory tract are pre-
490 P ART IV (]inical Applications

Ta b 1e 7 3. 3 . Common and/or lmportant lnfectious Ag ents of Ta b 1e 7 3. 4. Common and/or lmportant lnfectious Agents of
the Respiratory System of Horses the Respiratory Syst em of Cuttlc

Agent Major Cllnlcal Manifestations

(Common Disease Name)
..........-
Age11t Major Clinical Manifestations
(Common Disease Name)..._...,. ____
Viruses Viruscs
African Horse Sickness virusª Pulmonary edema (Atrican horse Bovme herpesvirus 1 Khinotracheitis (infeaious bovine
sickness) rhinotracheitis)
Equme adenovirus 1 Bronchiolitis, interstitial pneumonia Bovine respiratory coronavirus lnter~lilidl pneumuuid
(equine adenovirus di<;?ase) Bovine respiratory syncytial virus lnterstitial pneumonia (bovine respiratory
Equine herpesvirus 1 Rhinili~, p11~u111unili> (equine syncytial virus disease)
rhinopneumonitisl Parainfluenza virus 3 Rhinitis, interstitial pneumonia
Equine herpesvirus 4 Rhinitis, pneumonitis (parainfluenza virus 3 infection)
Equine influenza virus Rhinitis, tracheobronchitis, interstitial
Bacteria
pncumonia (equine influenza)
Arcanobacterium pyogenes Embolic pneumonia, lung abscesses
Equine viral arteritis virus Rh1mt1s, interstitial pneumonia
fusobacterium necrophorum Nccrotic IJryngitis (calf diphtheria)
Hendra virusª Pulmonary edema with respiratory
Histophi/us somniª Bronchopneumorna, otitis media
distress
Mannheimia haemolytica Bronchopneumonia (enzootic
Bacteria p11eurriu11id, ~ltippiny feve1 )
Actinbadllus equuli ssp. haemolytica Bronchopneumonia, pleuritis Mycnbacterium bovís Granulomatis pneumonia, pleuritis
Burft:holderia mallei" Rhinitis, pyogranulomatous nasal (bovine tuberculosis)
nooules (glannPT1') Mycoplasma bovis Bronchopneumonia, otitis media
Burf<hofderia pseudomallef' A!x.~ Íll lldXSI lllUlUXI, e1nbolk Mycoplasma dispar l'neumonia-alveolitis
pneumonia. pulmonary abscesses Mycoplasma mycoides subsp Bronchopneumonia, pleuritis (conta91ous
Escherichía coli Dronchopneumonia, pleuritis mycoidcs smatl colony typeb bovine pleuropneumonia)
Mycop/asma fe/is Pleuritis Pasteurel/a mu/toada Bronchopneumonia tenzootic
ObligJtc JnJcrobcsb Bronchopneumonia, pleuritis pneumonia. shipping fev~r), ntiti~
•
Rhodococcus equ1 l'yogranulomatous pneumonia media
Streptococcus equi subsp. equi G11tturñl rnurh Pm[lyP.mo, Salmonella Dublin lnterstitial pneumonia
1l1i11uµl1d1 y119itis, retropha1y11geal
Fungí
lymph nodes abscesses (strangles).
Morrierella wo/fii Embollc pneumonia
sinusitis
Streptococcus equi subsp. Bronchopneumonia, pleuritis, sinusitis
•·memophilus somnus- Is me obsolete name ol lhis organism.
zoocpidcmicvs
bConsidered a foreign animal disease agent in the Unrteil States.
Fungi
Aspergi/lus species Guttural pouch mycosis
Protist
Rhino~µoridium ~eeberi
nants, is the role playcd by p ulmonary intravascular ma-
crophagcs in rcrnovin.g bloodborne pathogens.
'Considertd • foreign animal disease ogent in the United Stotes.
~lncludes fusobacterium, l'eprosrreptococcus, and Prevoie//a. lnfections of the Nasopharyngeal Compartment
The major infectious dist>;i<;P<; of tht> nasopha ryn geal com-
pa1 l1nenl a1e rllli1itis and sinusitis, which can occur indc-
pendently or concomitan tly. 1U1initis is an inflammation
uun1inately l11e age11ts of systemic mycoses (fllaston1yces of the nasal mucosa. Common signs of rhinitis are sneez-
dennatitidis, Cocddioides hnn1ilis, Cryptococcus neoforrnans, ing and nasal discharges of vary1ng composition. The
Histoplasrna capsulatun1) and Aspcrgillus spp. Individual characteristic of the exudate in rhinitis is th e result of
chapters on particular respiratory tract pathogens sl1ould serous or m ucus sec retions, dlt«::ri:ltiuu:. i u vascular pern1c-
be consulted fo r details on pathogenesis and pathology ability (fibrin ogen dt>po<:ition), ilnd influx and type of in-
spcclfl<.: to d p drttcular dgeu L. n an1n1atory cclls.
Many inft>\tious respiratory diseases are m u ltifactorjaJ, Viral rhil1itis can be caused by a n um ber ofviruses (e.g.,
rcquiring environn1ental, host, and agent factors to b e in hcrpcsviruses, adenoviruses, in íl uen za viruses) and oc-
play. Respiratory infections commonly involve seque ntial curs, to sorne degree, in most animals. ln general, ciliated
infection ;vith diffcrcnt pathogcns (c.g., viral p n eumonia cpithelial cells are infected, sloughed, and subsequently
lcading to secondary bacteria! pneu1non1a). Respiratory replaced. ClinicaJ slgns refl«::<:t tite a:.:.o<..:ialed i11flan1n1a-
tract infections can initiate cithcr by the aerogenous or tory response. Second;iry harterial infections can be a
hcn1arogenous route. A factor predlsposing to ir1fe::<..tiu11 of co111plicatio11 of a primary viral rhinitis (e.g., rhi n otra-
hematoe<'no11s origin, espt>rially in cats, pigs, and rumi- cheitis virus and calicivirus intcctions in cats predtspose
 Chapter 73 Respiratory System 491

Ta b 1e 7 3. 5. Com mon and/or lmportant lnfectious Ageots of Ta b 1e 7 3. 6. Common and/or lmportant lnf ectious Agents of
the Respirat ory System of Sheep and Goats thc Rcspiriltory Systcm of Pigs

l'flajqr Clinkal !:/loniféstalions Agent Majorflinical Manlfestations

.....
~~~~ .....
~~.._~
(Cóiinnon 01sease Nlíme)
............. (Comrilon Disease Name)
~~~,...,,...-...-.-----...,..,.....

Viruses
Caprir:ie arthritislenceplíalitis
virus (G)
Jaagsiekte slíeep fetrovirus (~)
Maedi/visna virus (S)
Paramfluenza vtrus 3
Bacteria
Arcanobacterium pyogenes
Fusobact.erium neaophorum
Mannheimia haemolyUca
Mycoplasma caprkolum subsp
capripneumoniae fG)ª
Mycoplasma mycoidcs subsp
mycoiaes-large colony Jype (G)
Mycop/asma ovipneumoniae (S) lnterstitial pneum:onia (ovlne
Pasteurella treh~losi
(S) =sheep. (G) =goats.
'Considered a fc¡reign animal disea~ agent ih the United Statiis.
Viruscs
lnterstitial pneumonia Lelystad virus lnterstitlal pneumonia (porcine repro-
ductive and respiratory syndrome)
lnterstitial pneumonia, pulmonary Nipah virus• Alveolitis, bronchointeF~iitial pneumonia
carcinoma (ovine·pulmonary Porcine herpesvirus 1 Rhinopharyngitis. tracheitis
adenocarcinoma} (pseudorabies, Aujeszky's disease)
lnterstiiial pneumonia (ovine proqres:slve Porcine h.erpesvirus 2 Rhínitis (inclusion body rhinltis)
pneumonia, maedi} Swine influenza virus RhinÍtis, trachcobronchitis, bronchoin
lnterstitial pneumonia terstitiaf pneumonia (swme influenza)
Bacteria
Pulmonary ilbsc0Sscs, traumatic Actinobacillus pleuropneumoniae Bronchopneumonia, pleuritis (por,cine
pllaryngitis pleuropneumonia)
Necrotic laryngitis, traumatic pharyngitis Bordete//a bronchisepticab Rhinitis (atrophic rliinitis),
Bronchopneumonia, pleuritis (pneumonlc bronchopneumonia
pasteurellosis) Fu~oh;irtPrium nerrnp/)()n1m NPaotic navil cellulitis (necrotic rhinitis,
8rond1opneumonia, pleuritis (contagious bull nose}
caprine pleuropneumonia) Haemophi/u5 parasuis Bronchopneumonia, polyse-r-ositis
Pncumoniii, pleuritis (Glasser's disease}
Mycoplasma hyopneumoniae Broncliopneumonia (enzootic
. pneumonia)
nonprogressive pneumonia) Mycop/asma hyorhlnls Polyserositis
Broocnnpneumonia PiÍsreureff;¡ m11ltocid;ib Rhinitis [atrophir rhi"nitis),
• bronchopneumonia
Salmoneila species Bronchointerstitial pneumonia
Streptococcus sws Brond'.lcpneumonia, pleuritis, embolic
pneótnonia

•considered a fore1gn animal d1sease agent mthe Umted ~tates.

hB.bronchiseptica and P. multocida sometimes act synergistiC3lly.

to bact eria! rhinitis and sinusitis). Latently infected ani-

1nals that periodically shed virus (e.g., infectious bovinf>
rhinolracheilis virus) are <t con1111on source for infections
in naive animals.
Bacteria! infcctions of thc nasopharyngeal compart-
ment, while notas frequent as Viral infections, ar.e still sig-
nifican t . Notable among these are atrophic rhinitis in. pigs,
contagious rhinopharyngitis (strangles) in horses, ancl
sinus infections in poultry (see below). Tn atrophic rhinitis
ir1 pigs, the dern10.necroti.c toxin of Pasteurella n1ultocida
Crype D) induces osteolysis of nasal turbinates and distor-
tion of thc nasal cavity (Fig 73.1). To a lcsscr cxtcnt, thc
dennonecrotic toxin of Bordetella broncfliseptica is also i11-
volved. Pigs present ivith sneezing and a clear to cloudy
nasal discharge. The dlsease can be a mild, nonprogressive,
ar more active, progressive form . Reducc~d weight gain,
poor feed conversion, a11d il1creased su sceptibility to otl1er
respiratory infec"tions are the main consequences of at-
rophic rhinitis.
Streptococcus equi subspecies equi, tl1e cause of conta-
gious rhinopharyngitis (strangles) in horses, typically also
involves submandtbular and/or retropharyi1geal Iy1npl1
nodes. The res11 lting intf>nse infl;:immatory response pro-
duces a tl1.ick, bilateral, purulent nasal discharge. Stra11gles
is considered .highly contagious. Serious consequences can
result from strangles infections includi11g guttural pouch
empyema (see belovv), "bastard strangles," and purpura he-
morrr1agica.
Sinusitis is an inflammation of one of the nasal sinuses.
It ca11 result fro1n extet1siou of a J1asa.l cavity infection into
sin1 1ses or be rel;ited to othf>r problf>n1s of the or;il-n;is;il
cavities (e.g., extension of infection fron1 an infected
toot h). It is an occasional occurrence in most animals.
Agcnts involvcd are typically rcsidcnt flora of thc nasal cav-
ity or those involved i11 rhinitis.
Tn poultry, sh1usitis is an especially common problem
that has majar economlc conseqnences due to the conta-
gious nature of sorne of the agents involved and pot ential
for large i1un1bers ofbirds Lo be allecled (Fig 73.2). A nun1-
ber of m icrobial agents can infect the sinuses of birds, and
typically sinusitis is found in conjunction with clinical
signs in other areas of the respiratory system (e.g., Gallid
herpesvirus 1 in chicke11s, avían influenza viruses). 1'vfyco
plasma gallisepncurn in ru rKeys (ir1fecrtous Sinusitis) and
Haemophilus paragallinarum in chickens (fowl coryza) are
an1ong lhe n1osL econon1ically irnporLar 1.L cau~e~ of ~ü1u~i­
tis in poultry. Poor growth performance and decreased egg
492 PART IV Clinical Applications

Ta b 1e 7 3. 7. Common and/or lmportant lnfectious Agents of

the Respiratory System of Poultry F 1G U RE 7 3. 1 . Atrophic rhinitis in i1 pig. Cross scetion of
snout showing severe arrophy of nasal turbina tes. (Counesy Dr. M.
Agent Majur Oinic.al Manifestations Anderson.)
(Common Disease NameJ

Viruses
Avían infeaious bronchitis virus (Q Air sacculitis, tracheobronchitis (avian
Avlan influenza virus
Avían paramyxovirus 1•
Avían pneumovirus
Avían poxvirus
Gallid herpesvirus 1 (C)
infectious brond1itis}
Air sacculitis, sinusitis, tracheitis (avian
influenza)
Hemorrhagic tracheitis (exotic Newcastle
disease)
Rhinotrochcitís, sinusitis (turkey rhinotra-
cheit1s), penorbital and mfraorbital
sinus swelling in chickens. (swollen
heacl syndrome)
f)iphthPritir IP.1ions of nare_1, pharynx.
larynx and trachea (pox-díphtheritic
form)
Laryngotracheitis (infcctious loryngo
tracheltis)

Bñrtf!ri<l F 1G U RE 7 3. 2. Sinusitis in iJ 5 wcck old turkey Sinus cavity is

Botú~lella avium Rhinotracheitis, sinusitis (bordeteUosis, greatly enlarged. (Counesy Dr. H. Shívaprasad.)
turkey coryza)
Escherichia coli Coli~ptíccmío, secondary pneumonia
Haemophilus paragallinarum (CJ Rh1mt1s, smusrtis (fowl coryza)
Mycop/asma gallisepticum Aír sacwlitis (chronic respiratory disease),
m
rhinitis, sinusitis (infeaious sinusitis)
Mycoplam'l<I synovi;¡e Aír sarrulitis. sinusitis
Or11il11ul.idtle1 ium //1irHJtr oúreole Air sacculitís, bronchopneumonía,
sinusitis
P.:istcurd/;:i multocid;:i Air sacculitis, pneumonia
Funyi
Aspergillus fumigatus Tracheitis, air sacculitis, pneumonía
(brooder pneumooia)

(C) ~ chickcm only.

'Con>i<lt•w • ío1tiy11 •ni111•I di>••>• •genl in lhe United Stales.

production are among the main reasons for econor111c

losses.
Fun8;:il in fPC'tion~ of th<> nasopharyngcal compartment F 1G U RE 7 3. 3 . Rlrino)µoriúiu)i) in d doy. Section o f a nasal
polyp showing the variable·sized spherules. Mature spherules (up to
C1re iufre4 u1::11 t a1 rd when Lhey occur Lhey evoke a granulo-
300 µm in diameter) contain numerous endospores. One large
matous inflammatory response. Aspergillus rhinitis and si- spherule fa µre)enl 11ed1 !11e )Ut face of the lesion (H&E).
nusitis in dogs and nasal cryptococcosis in cats are among
the most important tungal intections ot the nasal cavity.
Rhinosporidiu111 seeberi, a protist in the class ~1esomyce­
tozoca, is a rare cause of granulomatous nasal masses con-
tain ing large spherules and endospores of Rhinospnridi11m
(Fig 73.3). Gru::.::.ly lile nasal lcsio11s resemble multilobed
granular polyps. Although any animal including poultry
can be infccted, most cases are rcportcd in dogs and
horscs. Infections are associated with exposure to freshwa-
ter ponds, lakes, or rivers.
Si tes that communicate with the upper respiratory tract
may be affcctcd by the direct extension of <111 infectio11s
process iu thi..: llCJ::>UIJI 1a1 yugeal con1par ln1cnt or becon1e
inft>rtt><l ín<lPp<'ndent of other nasopharyngeal disease by

residents of thP na<:opharyngeal compartrnent. Guttural
pouch empyema in. hor.ses, caused by S. equi suhsp. equi,
develops as a sequel to primary rhinopharyngitis or rup-
turc of an abscessed retropharyngeal lymph node (stran
gles). Unilateral nasal <1.ischarge, especially whe11 the head
is down, is a common way for guttural pouch infections to
prese11t. Tncomplete c.lrainage frorn the guttural pouch,
sc;irring of thP. pharyngeal opening, and development of
concretions (chondroids) on the floor of the guttural
pouch interfere with resolution of the infection. Fungal
infections also occur in the guttural pouch of horses (gut
tura! pouch mycosis), typically iI1volving the dorsal wall of
the medial compartrnent. Aspergillus species (especially A .
nidulans) are tl1e u1ost cu11u1.1u11 t::tiulugic agt::nts. Tlit:: i 11 iti-
ating factor(s) for guttural pouch mycosis has not heen
clearly elucidated. When guttural poucl1 infections in-
volve neural or vascular structures, serious (dysphagia, la-
ryngeal 11emiplegia and Horner's syndrom.e due to nerve
injury) or even fatal (rupn1re of the interna! carotid artery)
consequences may result.
Olilis n1edia in calves is con1111only caused by respira-
tory pathogens (e.g., A1ycoplasrna bovis, Pasteurella multo-
cida, Histoplúlus so1n11i) harbored as resídents of the upper
respiratory tract. ·rhe likeJy route oí intection is vía the au-
ditory tube into the middle e.ar. Calves present with a head
ti.lt, nystagmus, and c.1roopy ear(s). The tympanic bullae are
typically partially or completely filled with caseous debris
and ::;erosa.11guineous Cluid. In so1ne cases Lile dísease p10-
grt>ssc~s ancl results in otitis interna and meningitis, vvhere
calves may exhibjt severe neurologic sig.ns. Other species
of animals are silnilarly but less commonly affected.
lnfections of the Tracheobronchial Compartment
·rhe ma¡or diseases of the tracheobronchial compartment
are laryngitis, tracheitis, and broncl1itis. Viral ancl bacter-
ia! age11ls are lhe prin1ary causes of i11fectious diseases of
the tracheobronchial compartment. Aspergillus infections
in poultry can, however, involve thc trachca and bronchi.
Viral intections ot the trachea and bronchi (e.g., equine
influenza, infectious bovine rhinotracheitis, ínfectious
laryngotracheitis in chickens) da1nage resplratory cplthe-
lial cells and interrupt the mucociliary apparatus. Tracheal
lesions caused by n1any dífferent víruses n1ay not have par-
ticularly distinguishing features; however, inclusions in
some infectíons asslst in idcntifyin.g thc particular virus
involved (e.g., intectious laryngotracheitis in chickens,
avían pox viruses). Viral tracheitis is often sufficiently de-
structive to predispose to seco11dary bacterial infections.
Necrotic laryngitis in young feedlot cattle is one of tl1e
!UU~l LUUUUUU UaLle1ial }a1y11t;t::a) Ui~t::d~t'.~ (Fig 73.4).
Fusobacterium necrophorurn, the etiologic age11t, establishes
in preexisting contact ulcers found on the laryngeal mu-
cosa, which are suspected to originate trorn sorne preced-
ing trauma (e.g., reflex coughing). Once established, F.
necrophorurn causes a severe necrotic laryngitis that can be
fatal if un.treated . Other run1inants are affected but less
frequently.
In dogs, the kennel cough syndrome, an infectious tra-
chcobronchitis, involvcs rnultiple potential etíologic
agents, sometimes working in concert. Kennel cough in
Chapter 73 Respiratory System 493
F 1G U RE 7 3. 4. Necrotic laryngitis in a ca/f. Fibrinonecrotic
material is covering the arytenoid cartilages. (Courtesy of Dr. M.
Anderson.)
dogs is considered contagious and is associated with dogs
maintained in confir1ed, close-contact_environments (e.g.,
kennels, a11imal shelters). Viral agents include canine ade-
novirus 2 and paraínfluenza virus 2. Mycoplasn1a species
11avc bee11 in1plicated, bul ll1eir ínvolven1enl is unproven.
The bacterium, Bordetella bronchiseptica, is also associated
with the ken.nel cough syndrome. It is considered a pri-
mary pathogen o! the respiratory tract because of its abil-
ity t o adhere to ciliated epitl1eli.al cells and affect epithelial
cell functi.ons. Infections invo!Ving B. bronchiseptica tend
to be more productive than those caused by viral agents.
Evideuce is growil1g ll1al B. brur11.J1iseplic,u is also a siguifi-
cant respiratory pathogen in cats.
In poultry, flordetella aviu111is a common cause of upper
respiratory infections (rhinit is, sinusitis, tracheitis) 1n
turkeys and to a Jesser extent in chickens. Sneezing is the
most common sign, along \-Vith an oculonasal discharge.
After colonization, damage to ciliated respiratory epithe-
lial cells anll ciliustatic effects uccur. Cullapse uf the tra-
chi>a ci11P to softrning of trachP.al rings may also ri><:ult.
Infections v, ith B. aviuni predispose birds to ot her infec-
1
tions, such as colibacillosis.
Somc microorganisms that affcct thc trachcobronchial
compartmcnt concurrently involve other parts of the res-
piratory system (e.g., felinc viral rhinotracheitis, ínfectious
bovine rhinotracheititis, Bordetella avium ln turkeys).
Additionally, systemic infections n1ay involve epitl1elial
cell:; Íll lhe li<tCI 1evl11 ~JI ILI li<tl LUUl¡JaJ L1 Ut::Ul (t::.g., fiuli uu-
hemorrhagic tracheitis in exotic Newcastle disease in poul-
try) (Fig 73.5), along \vith sitcs in thc host outsídc the res-
piratory system.
lnfections of the Pulmonary Compartment
The 111ajor infeclious dísease of ll1e pulrno11ary con1parl-
ment is pneumonia. Pneumonia can be classified 1norpho-
logically as a(n) bronchopneun1onia, interstitial pneumo-
nia, granulomatous pneumonia, eml)olic pneumonia, ora
494 PART IV Clínica! Applications
F 1G U R E 7 3 • 5 • Exotic Newcastle disease in a chicken. Exposed
!d1y11x dll<Í lrdtlit!dl lu1fft!fl dft! tUVt!Ft!<Í wil/1 d (iurinuliernorrhagic
exudate. (Courtesy Dr. H. Kinde.)
mixture of these types. The agent, ro11tP of infpction, and
i111111u11e 1esponse delern1inc the type of pneumonia that
develops. Viruses, bacteria, and fungi are alJ potentialJy
important pathogens of thc pulmonary compartment.
Chapters on specitic pathogens shoulct t)e consulted for
details on pathogenesis and pathology associated "1-Vith
that particular agent.
lnfcctlons of the puhnonary compartment often rc-
quin:: a vrc1.:cLli11g evenl(s) Lhal i1npairs the innale anlin1l-
crohial factors. Confined housing environment, ammonia
build-up (noxious gas), and tra.nsportation stress would
typify the type of factors that sufflc1ently 1mpair defe11ses
and allow pathogens that would otherwise be rapidly
cleared by innate host defense mechanisms to establísh in
the lower respiratory tract.
Su111e of Lhe saine viruses found in Lhe upper respiralory
tract cause mild lovver respiratory tract disease (e.g.,
parainfluenza virus, adenovíruses). Serologic evidcncc in-
dicates that a large percent ot populations are inappar-
ently or subclinically infected by thcsc organisn1s. Other
viruscs are substa11tial pathogens of the puln1onary com-
partment in their own right, sometimes causing fatal in-
ft:~Liu11:> (e.g., Africa11 Horse Sickncss virus, He11dra virus).
Viral respiratory pathogens in animals with the potential
to be transmitted from animal to humans (c.g., influenza
viruses, Hendra virus, Nipah virus) are ot substantial pub-
lic health concern.
While sorne bacteria! pathogens are consictered pri-
mary pathogens of the pulmonary compartinent (e.g .,
R/iuclu1..ut1..u.~ equi in horses, Actinobacillus pleuropneunzoniac
in pigs). viral infections often precede bacteria] infections
and create an environment that allows bacteria to cstab-
lish by damaging the mucociliary escalator system or alter-
ing the i1nmune response by impairing pbagocytic func-
tion. Once predisposing facrors are in play, it is not
uncommon to find multiple bacteria! pathogens involved
Íll luw1:1 n.::.pi1dlV1y lidLl ii1f1:LliOJl~.
The bovine respiratory discasc complex is an example
of thc complcx a.nd, as yct, incomplctcly understood in ter
actions of environmental factors along Wlth specific infec-
tious agents and host response. It constitutes one of the
most cconomically sígnificant disease complexes of cattle.
Env1ronmental or management-related stresses (weather,
transportation) and/or viral ínfcctio11s (e.g., aden.ovirL1s,
l.luviut: 1t::>püatu1y :.y11t.ylic1l vi1 u:., i11f1:clio u 5 bovine
rhinotracheitis virus, parainfluenza virus 3) are believed to
alter respiratory tract cpithcliun1 and innate defenscs.
Possible immunosuppressive ettects relatect to bovine viral
diarrhea virus may also contribute to increasing suscepti-
bility to bacteria! infections. These factors allow one of rhe
primary bacteria! agents in bovine respiratory disease
complex, Mannhein1ia hal?nulylku, tu c:;talJlish in Lhe pul-
mon;iry compartment. Armed with a number of virulcnce
factors, lvf. haetnolytica is able to engage the host defcnscs.
Endotoxin activatcs pulmonary intravascular 1nacro-
phagcs, ncutrophils, and lym ph()cytes, and precipita tesan
inflammatory response tllat ultimately determines the de-
gree of pulmonary injury and whether the pathogen is
conralncd. In addition, leukutux..i11Iru111 M . ftaernolytica de-
stroy~ ph;igocytic cells and, at lower concentrations, fur-
ther enhances the inflammatory respo11se (Fig 73.6). If
pulmonary defenses are compromised sutticiently, more
opportunistic bacteria! pathogens are established a11d fur-
ther aggravate the condit1on. It 1s common to recover
Arcanobacteriurn pyogenes, a major abscess-former of rumí-
nants, from n1ore chronic bovine respiratory disease cu1u-
plex lesions (Fig 73 .7) .
TJ 11;: syste1níc n1ycoses (coccidioidon1ycosis, cryptococ-
cosis, blasto1n ycosis, and hi'l;toplastnosis), for the most
part, begin in t he lo\-ver respíratory tract t.C1using a granulo-
matous pneumonia and an associated lymphadenopathy.
Severity of respiratory infection varíes based on individual
animal immune status, breed, and animal species. Dogs,
horses, and cats most commonly devclop clinical disease.
InalJility of tl1e cu1iinal to cu11tai11 til~e i11feclions Lo the
rt>spiratory tract leads to disseminated infections involv-
ing other systems in the body. Other than the systcmic
mycoses, fungal poeumonias are not common in animals.
The exception is aspergillosis in poultry wl1ere pneumo-
nia, often with concurrent tracheitis and a ir sacculitis, is a
major respiratory tract discase. Th is is a disease of su bstan-
F 1G U RE 7 3. 6 . BronchnpnP11mnni;¡ in ;¡ r;¡/f r;¡11sed by
Mannheimia haemolytica. Cranioventral lung lobes are predomi
nantly 1nvolved. (C.ourtesy ur. M. Anderson.J

F 1G U R E 7 3 • 7 • Chronic bronchopneumonia in a calf. Mu/tiple
areas of abscessation are apparent. (Courtesy Dr. M. Anderson.)
tia! economic significancc to thc poultry indu5tr y, cspc-
cially the turkey industry. Closed environments that allow
for increased concentration of aerosolized asexual repro-
duc live :sl1uclu1es (co11idia) a11d/ur r11oldy ft:t:ds are corr1-
n1on predisposing factors. Grü\Vth in aerated air sacs,
bronchi, and trachca pcrmit the production of large num-
bers ot conidia that are not typically produced in solid tis-
sues (Fig 73.8).
Aspiration pncumonias in most cases are polymicrob1al
in nature, and bacteria are usually the microbial agents in-
volved. füpiration pneumonii:l'in animals results \<Vhen im-
pairt=>rl airway prott=>ction allow<: fluirl<: or matf>rial to enter
the lower respiratory tract, initially causing a chemical
pneumonia. Common events leading to aspiration pneu-
monia are ilnpropcr trcatmcnt proccdurcs (c.g., drcnch-
Chapter 73 Respiratory System 495
F 1G U R E 7 3 . 8 . Aspcrgillosis in iJ turkey Conidiophores,
vesic/es, and phia/ides are evldenr In material from lumen of the
trachea. Numerous conidia are dispersed throughout the field.
Hematoxylin-eosin stain.
ing, impropt=>r <:tom::irh tubing), bottle or bucket feeding of
young animals, poor sucking reflcxes, nasopha1yngeal
tube feeding, choke, o r aspiration of gastric or rumen flu-
ids that might occur during thc ancsthcsia recovery pe-
riod. In newborns, aspiration of meconium may also lead
to aspiration pneumonia. ·rhe bacteria most frequently in
volved lo asplratlon pneumonia typically reflect organ-
isms found in the upper respiratory or digestive tracts and
include E. coli, Bordetella, Klebsiella, Pasteu1ellu, Pseudurnu-
nas, Strcptococcus, and oblígate anaerobes (Fusobactcrium,
Pcptostrcptococcus, Prcvotclla, l'orphyromonas).
 Urogenital Systelll
R ICHAH.U L. W ALKER
·rhc urin;iry an11 genital tract.s a re included togetl1er in this
cl1apter because of tl1eir anatomical proximity, sl1arcd
structures (urethra in rnales), and sorne overlapping dis-
ease proccsscs. Common and/o r important urogenital
tract agcnts o f domestic animals and poultry are listed in
Tables 74.1-74.7.
The Urinary Tract
The urinary tract has a numbcr of important functions,
including eliminatioo of metabohc wastes, aeid-base
regulation, maintenance of the cxtraccll ular potassium
ion concenrration, and entlucri11t fu111.: Livn:s (viLan1i11 D
convcrsion ;ind prod11ction of erythropoietin and reni11).
'fhc n1a1nn1alian urinary system includes the kid11cys 1
11rr.·t c rs, hladcler and urethra. T he functional filtration
unit of the kidney is the n ephron, which includcs tl1c
glomerulus, p roximal and distal tubules, loop of ttenlc,
and collecting duct. In birds, the urinary tract differs
from mammals. The kidneys are dlvided into lobcs. A
bladder is absent and ureters enter the cloaca, medi;il to
tlH.: tlefe1e11l ducl in Ll1e 111ale and dorsal to the oviduct
in the female. Urine, concentratcd as a slurry, is voided
with feces .
Antimicrobial Defenses
Becausc it is p rim;irily an f>xcretory system, the urinary
lracl is not subject to n1assivc microbial expo~ure but !1as
developed specific an ti1nicrobial defenses to counter occa-
sional cxposurc to potcntial pathogens. Protective fcatures
include:
l. Washout by Urine. 1'he flo'"' of urine- its direction,
diluting cffcct, and frcquent periodic removal-
discourages the cstablrshment of microorganisms
in the normally sterile portions of the tract-that is,
tl1c klu11ey:, 1 un:Le1:., 1.Jlauu1::1, <11 1u J.J•VAi1nal 111ale
urethra. Urine retention is correlated with increascd
urinary tract infections.
2. Bacteria/ lnterference. Colonization of the distal ure-
thra by no rmal flora may block attachment sites for
colonization o f the lower unnary tract by poten-
tially pathogenic organisms.
3. Glycoprotein Slirne Layer. Mu~ln cuvcring Llu: epilhe-
lium may inhibit bactf>rial t1cihe-~ion.
4. Hpit11elial Desqua111atio11. Exfoliating epithelial cells
promote shedding of uropathogens.
496
S. Local and Systemic ln1n1une lJefenses. Cysteine-r1ch
antimicrobial peptides play a role in inhibitiI1g bac-
teria from establishing. lmmune response to url-
nary tract infections has been studied mostly in h11-
man.s and laboratory a11 i111 ctl~ . Sludies i11dicate that
sen1m an<111rinary antibody titers tend to be low in
cystitis and asympton1atic infcctions and high in
pyelonephritis. Secretory lgA (slgA) tends to be
most promiJ1ent in urine, but IgG and lgM antibod-
ies also occur regularly. Serum IgA, IgG, IgM, and
sigA antibodies are produced in renal infections.
The protectivc functlon of tl1ese a r1Liliuliies is un-
rlP;ir ·rhP ;ihility to rf>aclily mohilize Jeukocytes pro-
motes rapid clearance of uropathogcn s from thc
urinary system.
6. Antirnicrobial fJropert.jes o( Urine. Urine itself has
properties tha t may p lay a role in lirniting bacteria!
growth and include:
a . High osn1olalir.y: Urlne osmolality (1000 rnC)sm/
kg) reduces growth, particularly of rod-shapf>cl
bacteria. It 1uay, l 1uweve11 depress lcukocyte ac-
tivity and preserve bacteria with cell walls dam-
aged by immune reactions or antibiotic thcrapy.
Combined with high concentrations of ammo-
nia, which is anticomplementary, tuine osmo-
lality may contributc LO the susceptibility of thc
renal rnedulla to infection.
l.J. Urine pH: WI 1i lt t:X Lren1es in pH discourage mul-
ti plication of sorne bacteria, ranges bactericida!
to common urinary tract pathogcns are unlikely
to be reached.
c. Urine constitucnts: Urea imparts to tuine an unex-
plained bacteriostatlc effect, which is di111ilti:.ht:Ll
by rem oval of urea from urinf> ancl enhanced
liy dieLa1 y suppleo1c11tation . Methionine and
hippurtc and ascorbic acids produce an antibac-
terial effect largcly by acidifying the urine. Uri
narv ammonium nitrogcn also has antibactcrlal
properties.
Normal Flora
For most of the urinary tract there j:, 110 reside11t flora. The
distal urethr;i dof>s ht1ve a rcsident flora and is colonized by
bacteria that generally are not associated with urinary tract
infection.1'he flora is predominantly gram-positivc and 1n-
cludcs coagulase-negative Staphylococcus spp., Streptococcus
spp., c;orynebacteriun1 spp., and Enterococa1s spp. but varies
 Chapter 74 lTrogenital System 497

Ta b 1e 7 4. 1. Common and/or lmportant lnfe ctious Agents of Ta b 1e 7 4. 3. Common and/or lmportant linfectious Agents of
the Urogenita 1 Systen1 of Dogs the Urogenita 1 System of Horses

··.. IVlªi~Oiní~al Ma.nifestations ~gent Major- Qinicat Má'nifestatíoQs

.· (~6fülíl9f\ D$li.$e Ná~I (<?Ommon Dise~~~aml?) ·

Vir.uses Viruses
Canioe adenovirus l lmmun~-(omplex gl0merulonephrltrs Equine herpesy.irus 1 Abortion (equine viral rhinopneumonitis)
(infectious caníne l'lepatitis) Equine herpesvirus 3 Vesicles/erosions on external genit.alla
eanine h.erp·esvirus Abortion¡ balanoposthitis,infertility in females (coital exanthema), ba)anoposthit1s
Equíne infectious anemia lmmune<omplex glomerulonephrit1s
Bacteria
fa¡oine v.iral arteritis virus Abortfon (equine viral arteritis)
Brucellu cu.nis Aoortion, epididymítfs (caninc brucellosis)
Escherichia coli Cystitis, epic!füymitis, ordtitis, prostatitis, Bacteria
pyometra, va$initis Actin0bacil/us equuli subsp. eqvulf Glomerulonephritfs(sleepy foal dlsease)
Leptospira spp. lnterstitial nephritis, renal failure fscheric/iía coli Abortion
(leptospir.osisl Leptospirif spp. Abortion
Other agents ot UTI Cyrtítis Pseui:Jomonas aeruginosa Pyometra
(see Tapie 74.8.) St¡¡phylococcus aurevs Post-castration spermatic cord infection
(sdrrhous cord)
Streptococcus equi subsp. Abortion, endometritis
zooepldemlcus
Ta b 1e 7 4. 2. Common and/or lmporta nt lnfectious Agents of fayJorella ·equigenita/ísa Cervicitis, endnmetritis (rnnt~ginu'
the Urogenital System o f Cats equine metritis)

Fungl
'Maj9r Glinica.1ManifeStittions
A¿pergil/11.1spp. Ahortion
tCcwmon 9iseasé NameJ Candida spp. f ndonietrítis
Viruses
Felin.e infectlous peritonitis virus lmmune-mediated pyogranulomas of 'Considered a foreign animal disease agent in the United States.
•
kirlney.
Felifle leukemia vírus lmmune-complex glomerulonephrttis,
ietal absorption, abortion, renal
lymphoma
Feline panleukopenia virus Abortion, c,ongenital abnormalities terial involveme11t in feline urinary tract disease is
(panleukopenía) uncommon. lnfections, particularly cystitis and
~eline rhinotracheitisvirus Abortion pyelonephritis, are important in cattle and swíne.
Urinary tract infections are less common in goats,
sheep, poultry, and horses.
2. Anatumic and J>hysiulugic Facturs. lnterferer1ce witl1
the free flow of urine ano with c.omplete ernptying
of the bladder predisposes to UTT. This can be dueto
according to animal, housing, and hygiene. Small numbers
tumors, polyps, calculi. anatomic anomalies (e.g.,
of bacteria rnay enter the lJladder via tl1e urethra, especia Uy
ectopic ureters, patent urachus), and neural defects.
in the fema le, l111t are normally removed during urination.
Vesí.co-ureteral reflux, tl1e reentry of urine i11to the
·rhe significance of asymptomatic bactcriuria is unclear,
ureters during urination, causes bladder urine to
however, when detected investigation into potential un-
reach the renal pelvts, posstbly carrylng bacteria
derlying diseases is warranted.
in to a susceptible area. R.e flux is aggravated (perhaps
c:ertain viruses that persistently infect tubular epithelial
inítiated) by infectior1 and con1plic<ttes existing in-
cells, although not normal flora, may cause a prolonged
viruria (e.g., equiue rhiI1itis A virus a11u areuaviruses). fections by increasing the likelihoocl of renal in"
volvcmcnt.
3. Other Host ractors. ()ther host factors incluáe en-
Diseases docrine disturbances such as diabetes mellitus and
Host Factors. /\. num ber of host factors can prcdisposc an hyperadrenocorticism (Cushing's áisease). Long-
animal t o urinary tract infections (U'l1): term use of corticosteroids appears to predispose
dogs tu UTI.
l . Animal Suscepti.bilily. Urinary tract infections are
most common and of grcatcst significancc in dogs. Routes of lnfection. The primary means by which uropatho-
In cats, 1<11opatl11c lower ur1nary tract a1soraers are gens reach the urinary tract are the ascending and 11ema-
fr~~qucntly encountered. Viral, nutrit ional, and togenous routes. The ascending route vía the urethra is
1uetalJolic f¡¡ctors llave tJeen in1plicated in the etiol- most common. The presence of potential pathogens near
ogy, particularly of ohstruc.tive forms. l-lowf'vf'r, bac- the uret hral orífice and the usual Jocalization of infection
498 PART TV Clinic':il Applir:itions

Ta b 1e 7 4. 4. Common and/or lmportant lnfectious Agents of Ta b 1e 7 4 . 5 . Common and/or lmportant lnfectious Agents of
the Urogenital System of Cattle the Urogenital System of Sheep and Goats

Agent Major Cllnlcal Manlfestatlons Agent Major Clínica! Manifestations

Viruses
Bovine papillomavirus
Bovine viral diarrhea virus
lntect1ous bovme rh1notrachert1s
virus
Rlft Valley fever virusº
Bacteria
Arcanobacterium pyogenes
Bruui/liJ abur (U)
Campylobaeter fetus subsp.
venerealis
Corynebacterium rena/e group
tpizootic bovine abortion agent
(unidentified)
Escheríchia coli
Fusobacterium necrophorum,
othcr anaerobes
Leptosp1ra spp.
Listeria monocyto9enes, L, ivanovii Abortion
Mycoplasma spp., ureap!asma spp. Granular vulvitis
Fungí
Asper9illus sp.
Morrierella wolfii
(Common Dísease Name) (Common Disease Name}
= ...,..-
Viruscs
Penile and vaginal tibropapillomas Akabane virus• Abortion, dystocia <fue to arthrogryposis
Abortion, congenital abncrmalities of fetus
Abortion, mfectious pustular Bluetongue Virus (S) Abort.ion, congenita¡ abnormalities
vulvovaginitis, balanoposthitis (hhtPtongue)
Abortion (Rift Valley fever) Congenital abnormalitíes, stillbirths
(Border disease, hairy shaker disease)
Cache V.:illcy virus Abortion, congenital abnormalities
Abortion, metriti), pyomelra, seminal Rift Valley fever virus• Abortion
vesiculitis
Abu1 tion, epididymitis, orchitis, seminal Bacteria
vesiculitis "Actinobacillus seminis"(S) Epididymitis
Early embryonic de~th (bovinc venere~! Bruce/la abortus, B. melitensis<1 Abortion, orchítis
campylobacteriosis, vibriosis) llrucel/a ovis(S) Epldidymitls, rare abortions
Pyelonephritis Campylobacter fetus subsp. fetus Ahnrtion
Abortion (foothlll abortion) Campylobacter jejuni Abortion
Chlamydophila abortus Abortion (enzootic abortion of ewes)
lrtle!)lilidl 11~pl11 ilh (white-spotted Corynebaeterium rena/e group Dalanoposthitis (ukerative posthitis,
kidney disease). pyelonephritis. pizzle rot), pyelonephritis
pyometra Coxiella burnetti Abortion {Q-fever)
Post·parturient metritis Histophilussomnf> (S) tp1didymitis
Leptospíra sp. Abortion
Abortion Lfsterla monocyrogenes, L. ivanovtl 'Abortion
5almonPllil 'fl- Abortion, metritis
(G) =goats predominately. (S) =sheep predominately.
•Ú>nsider@d a foreiga animal disease agent in the United St~tP'
Abortion b•Histophilus ovis• is !he obsolctc Mmc.
Abortion

"Co~ide1ed 6 foreign animal tf1>ease <>gent in 1111:' Vnil<ll Sl•l~

Ta b 1e 7 4. 6. Common ;:ind/or lmportant lnfectious Agents of

the Urogenital System of Pigs

Agent Major Clínica! Manifestations

in the bladder point to it as the portal of e11t rance.
(Common Disease Name)
Pyelonephritis is a conscqucncc of retrograde extension of
infcction from tl1e bladder. Viruses
lnfcction of the unnary tract via the hematogenous African swine fever virusa Abortion, immune-complex glomeru·
routc occurs secondary to bacteremia/viremia and prima- lonephritis (African swine fcver)
rlly affects the kidney, causing glomt>ruloueplllitis ur iJ1- Classical swine tever vir!W Abortion, embryonic death, immune-
terstitial ncphritis. Tt occ11r<; lP<;<; commonly than lo\.-\rer uri- complex glomerulonephritis (dassical
nary tract infectious, due probably to the high resistance swine fever, hog cholera)
of the renal cortex, where infection bcgins. Young animals lelystad virus Ahortion (porrinP reproductive and
are cspccially at risk bccausc of the greater Jikelihood for cespiratory syndrome)
septicemia to occur in that age group. Pseudorabies virus Abortion (pseudorabies)
Swine parvovirus Embryonic dcvth, mummification
Glu111t'r ulullt>JJI 11 ilb dllU 111 lt'r~ Li lial Nt>fJI 1ri lb. G Juu11::1 ulu11t: ¡;Ju i-
ti .<; is an inflammation of the glomerulus dueto localiza- Bacteria
tion of an infcctious agent in the glomerulus and cnsuing Actinobacufum suís Cystitis, pyclonephritis, ureteritis
inflammatory response or from deposition ol i1nn1 une Bruce/la suis Abort1on, orch1t1s
complcxcs. Viral glomcruloncphritis is the result of se Eschcrichi.;i co/i Vaginitis (discharging sows)
lected viruses (e.g., infect1ous can1ne hepatitis virus, Leptosp1ra spp. Abortlon (leptosplrosls)
equine arterltis virus, n ephrotoxic infectious bronchitis
virus In chickens) replicating in glorn~ru1C:1r <..:C:1pillary 1::11- •considered a foreign anmlal disease agent In the Unhed States.
dothelial cclls, usually as ;i con<;rqurnrr nf ;i systemic vi ral
 Chaptet 74 Urogenital System 499

Ta b 1e 7 4 . 7 . Common and/or lmportant lnfectious Aqents of

the Urogenital System of Poultry
Agent
Viruses
Avian adenovir\Js (C)
Avian encephalomyelitis virus
Avian influenza virus
Avian leukosis virus (C}
Avían pneumovirus
lnfectious bronebitis virus .(C)
Newcastle disease virusª
F 1G U RE 7 4. 1 . Embolic nephritis in a foal caused by
Acli nobacillus e4uuli su/J)p e4uuli. MictOdU)(e))e) dre )tdlleteú
Major Clióital Manifestatil>ns. throughout the renal cortex (A). Bacteria/ microco/onies anda
suppurative inflammatory response are present (B).
'(Cammo1r Dtsease:Namer
Soft -shelled or shell-less egys (egg drop
syndrome)
Decreased egg production
Decreasec:f e,Qg production, oophorítis
Nephrqblastoma, renal ca;rcinoma
Decreased egg production
Decreased egg productron, decreased
haltltaüili Ly, 11eµl11 ili1 (infetliuus
bron(hitis-nephrotoxic straíns)
Decreased egg pr.oduction

Bacteria
Gsch~richia coli (C) ~olpingitis
Gallibacterium analis (Qb Oophoritis, salpingjtis
Haemophilus parag;¡/linarum {C) Decreased egg production (infectious
coryza)
Mycoplasma ga!lkentirum Oecre~sed IO!J!J production, salpingítis
Mycoplasma iow&e (T) Reduced hatch~bility, i1;Ued1ed e1111í1yo
mortality
5almonella Pullorum Oophor·itis, salpingitis {pullorum disease)
Salmonel/a Gallinarum Oophoritis, sálpingítis (fowl typhoíd)
5almonel/a Enteritidis Oophoritis; salpingitis
(phage fype 4)
•

(Q= chickens predominately, (T) = turkeys pred.ominately.

•co.nsidered a foreigh animal diseaie agent in !he United .States.
btncludes organisms previous!y identified as •Actinobacillus salpingitidis,• "Pasteurella-
.haomolytica.like: or "Pasteurella haen¡olytíca-Actinobaci!lus sa/pín9itídis-<omplex,•

i.n fcction. In bacteria! glomcruloncpl1ritis, bacteria from a

septicem.ia/bacteremia settle i11 tl1e glomerulus (e.g., A.cti-
nobacil111s equuli subsp. equuli in foa ls). The ensuing in-
flaIIl!Halury re::;¡.iu11se is Ly¡.iically suppuralive. Tlle re:>ulL is
an emholic nephritis (Fig 74.1).
In sorne hematogenous infections, the renal tubules
rather than the glomeruli are the primary target. Intersti-
tial nephritis is commonly seen with E. coli septicemia in
neonatal ruminants (whitc-spotted kidney) and leptospi-
rosis in a J1umber of different an imals.
Depo~iliou of iu1111u11e cou1plexes i11 Lhe glu111eruli i11
the case of persistent viral infections (e.g., infectious ca-
nine hepatitis, equine infectious anemia, feline infectious
peritonitis), specific bacteria! infections (e.g., Borrelia
spp.), or chronic bacteria! infections at other sites in the
body may result in immune-complex glomerulonephritis.
pathogen that subsequently reaches t he blat1der through
rnultiplication, extension along the epithelial surface, or
n1igration tl1ro ugl1 aclive n1otilily or randon1 n1oven1enl.
The resulting infection, after further multiplication, is
inarkcd by bacteriuria (fig 74.2) . Pyuria and low-grade p ro-
teinuria may be prese11t. lntlanunation is triggered by the
in teraction of lipopolysaccharide (gram negatives) or mu
ramyl dipeptides (gram-positives) wi.th transitional cells of
the hladder that secrete proinflammatory mediators,
V\l'hich a llracl polyn101 pl1011uclear 11eulrupliil leukoc.:ytes.
Signs, when present, include clysuria or urinary frequency
or urgency. 1'here may be hematuria and. incontine11ce.
'l'he prevalence of the comn1on agents impUcated in ca-
n ine urinary tract infections and characteristic features are
summarized in ·rabie 74.8.

Cystitis, Bacteria! cyslilis (i11flarr11nalio11 of L!Je uri11ary
bladder), especially in dogs, is the most common u rinary
tract infcction encountered byveterinarians. 1'he ability of
bacteria to a<ihere to epitheliwn is considered a prerequj-
site to establishment of cystitis. 'fhe infection may begin
with colo11lzation of the urethral orífice by a potential
Pyelonephritis. Tl 1c lllU~t :><::riou::; c.:u1nplication of lower uri-
nary tract infection is pyeloneph ri tis, wh ich is ca11sPc1 hy
an ascending infection vía th.e ureters. Pyelonephritis is es-
sentially an int1ammation of the renal pelvis and renal
parenchyina. Once bacteria rcach tl1c renal pelvis thcy are
hard to elin1 inate dueto the poor vascular supply and in-
500 PART IV Clinical Applications

n1ost common cause of pyelonephritis in animals. Other

F 1G U RE 7 4. 2 . Gram stains of urine sediment from a dog with bacteria} agents that cause cystitis have the potential to
cystitis. Stain in A is fron1 a dog with Escherichia coli cystitis, and the cause pyelonephritis, .if they reach the renal pelvis.
stain in B is from a dog with Enterococcus sp. cystitis. Diphtheroid bacteria, belonging to t11e Corynebacteriurn
renule group are specifically associaled wlth pyelonephritis
in cattle. They colo11ize the lower genital tract and pass be-
tween anima1s by direct and iI1direct co11tact. Many clini-
cal cases are probably endogenous. "111e process is an as-
j
{ cending urinary tract infection, beginning with cystitis,
which proceeds to ureteritis and pyelonephritis. An a11aer-
obic diph.theroid, .Acrinohnc11l11rn ~11i~, c'::t11<;i:><; 11 r in;:iry tr;irt
.i.l1fection of sows (Fig 74.3). Like bovine pyclonephritis,
the d isease is an apparently ascending infection caused by
un u rcalytic diphthcroid agent, limited to females and
/ often related to breedi11g operations, pregnancy, and par-
turition.

Uroliths and Urinary Tract lnfections. TJroliths of dogs a rf' p rf'-

hibitory effects of urine osmolarity and ammonia on im-
mune defenses, as mentioned pTeviou sly. Signs of pyelone-
phritis are vague. Fever is t ransient even during the acute
phase. Pain in the thoracolumbar area is not specific un-
less directly associat ed with kidney palpation. Urinalysis
may revea! lowered specific gravity a11d casts. In adva11ced
l..C:l:>t::., ulvuu U ll;:d ulllV~t:ll i:. t:k:vctled.
<.lorni11a11tly (70%) Lhe result of infectio11 and consist of
struvite or apatit e, or various combínations, and are often
referred to as triple phosphate stones. Urcasc-p roducing bac-
teria are implicated- in dogs, chiefly coagulase-positive
staphylococci, and to a Iesser extent Proteus mirabilis.
Inhibitio11 of urease activity by urea a11alogues (e.g., aceto -
hydroxamic acid) can suppress iI1fectious ston,e formation .
t Uroliths in ruminants are relatetl to 11utriU011al faclors
rather that infec'tio1 1s onf's .
•
Virulence Factors of Bacteria! Uropathogens
Bacteria capable ot in itiating U 11 require virulence deter-
minants, the most firmly established of "''hich are adhesins
(e.g., t11e pyelonephritis-associated pilus, Pap). Fimbria! at-
tachment of E. coli to surface slime glycoproteü1s by Type J
(rnannose-sensilive) pili is probably of less s.ignificance
than that mediated by several mannose-resistant adhesins,
one of which is Pap, that attach to ccll mcmbranc glycol-
ipids . .Cscherichia coli isolates from different animals appear
to nlaintain the sam.e Pap alleles. Mannose-resistant ad-
hestns occur co1nn1only in u ropathogenic E. coli, but lr.reg-
ularly in other strain s. Other properties associated wit h
E~c,lict íc,ltiu e.olí i5 lhe uropall1oge11ic É. t:oli i11clude rc:;i:;tancc to serun1. bacterici-

Ta ble 74.8 . Leading Bacteria! Causes of Urinary Tract lnfections in Dogs

Preva!ence Typical Colenies On: (Qnfirmatory Tests.

. ' •
:i:
Spedes (o/o) BIOOd Agar MacConkey Agar Gram Staln Oxidasl! Catalase

EsGherichia co/i 42-46 Smooth grey; often hemo~ytic Red discrete, surrounded by red haze Negative rods Negative NA
Entcrococcus 11 14 Very small (<1 mm) Nogrowth Positive cocd NA Negative
Coagulase-pos1tive lL White or off·wllite (otten nemolytic) Nogro.wth Positive coccl NA Positive
Staphyfococ<us
Proteus mirabllis 6- 12 Swarms; no diserete colonies Colorless ttegative rods Ne~ative NA
KfPh5ie//;¡ 8- 12 large, wet mucoid, whitish·grey Pink, slimy coalescing; not surrounded Negative rods Negative NA
by red haze
PSeudomonas <5 Gray to greenish·gray; fruity or ammonia Colorless, surrounded by blue-greeil Negative rods Positive NA
odor; often hemolytk pigment

NA: not applicable.


F 1G U R E 7 4 . 3 . Hyperemic mucosa/ surface of the bladder
with whlte mucfnous material from a sow with Actinobaculum suis
cvstitis.
dal action; hcmolytic activity; and possession of certain 0-
antigens, 1ron-scavengíng protcins, and bacteriocins. ·rhese
properties are rare in random E. coli strains, suggesting that
agents of urlnary rract infections representa select subpop-
11 b1tion within thPir ~pecies
Pili-mediated attachn1ent to urotheliun1 and urea hy-
drolysis are considered critica! virulcncc factors in patho-
gencsis of the Corynebacteriun1 re11ale group infections. Urea
breakdown witl1 production ot a1nmonia initiates an in-
flammatory process, high alkalinity in urine (pH 9.0), and
suppress1on of antibacterial defcnscs, possibly through
complement inactivation by ammonia.
Miscellaneous Urinary Tract-Associated lnfectious Processes
Fungí are rarcly involved in urinary tract infections.
Fungal hyphae can be detcctcd in thc urine of dogs with
dissem1nated Aspergillus infections. Rarely, mycotic cystitis
is identified. 'fhe yeast Candida ci/bitans is sometimes iso-
latctl .frorn tite urine uf tlogs with diabetes mellitus.
Kidncy tumor.~ ;ire associ;:itpcl with inff'ctions with the
avian leukosis/sarcoma complex of viruses. Renal lyn1-
phoma is associated with feline leukemia virus infections
in cats.
The Genital Tract
The pri1riary function of the genital tract is reproductio11.
·rhe genita l tract in mammals includes the ovaries, ovi-
ducts, uterus, cervix, vagina, and externa genitalia in fc-
males and the testes, ductus ctcfcrcns, accessory sex or-
gans, pcnis, and p repuce in males. Tn poultry, the genital
lracl diff1::r:. sul>stautially frorn mammals. Tn males the
testes are interna] and, lacking ;icc<'~~ory c;ell'. glands anda
distinct copulatory organ, the ductus deferens parallels
the ureter into the cloaca. Females typically have only a
single functional ovary (left) and oviduct with the shell
gland (uterus), which empties into the cloaca.
r:hapter 74 TJrogenital System 501
Antimicrobial Defenses
Antimicrobial defenses are in place at all levels of the gen-
ital tract and include:
l. Anatomic Defenses. Stratificd squamous epit helium
in the v agina and vulva provide for resistan.ce to in-
fection. ·rhe cervix provides a physical barrier to in-
fections of the upper genital tract, especially during
pregnancy. The long urethra in males provides a
barrier to retrograde infections.
2. l lorn1011al Dr:fr:11sr:s. l-lo1111u111::~ ¡.Jlay d ¡.Jal l i11 p1ult:cl-
ing the genital tract from disease. Estrogens increase
the blood supply to the vagina and uterus, the num-
ber of polymorphonuclear neutrophil leukocytes in
the cervix and uterus, and the myeloperoxidase ac
tivit y o f phagocytlc cclls In the uact. These activi-
ties are importi'lnt becatisc the vagina and perhaps
the uterus may become con1an1inaled wilh JJulcu-
tially harn1ful agents during coitus.
3 . Immune Definscs. '!'he immune system of the genital
tract appears to be similar in structure and tunction
to other mucosa) surfaces. ·rhere are lymphoid folJi
ele~ ir1 tl1e ~ubmucosa from the cervix caudally.
These follic]p_<; snpply cPll~ th::tt will ultimately se-
crete IgA. IgG a11d lgM will be fou11c.l i11 tlús area as
well, but these isotypes probably arrive through
tr;:insudotion. In thc utcrus, lgG nnd IgM, nnd ~omc
slgA are found. In the prcpuce, mainly slgA is
found; it is p robably secreted by the accessory
glanc.ls or locally frorn cells arlslng from the Jym-
phoid folliclf's in thi~ ;ire;i . The defensive potential
of these antibodies depends upon tl1e lsolype.
Antibodies of the IgA isotype make particles inore
hydrophilic, thcreby negating any surfacc attrac-
tion an organism might have for usually hyctropho-
bic host cell surfaces as well as sterically hindering
attachment. lgG anct IgM, on the other hand, will
opsonize, trigger the complement cascade, and ster-
ically hinde1 allacl1111enl. Ali in1n1uuoglolJuli11::;, ií
specific for epitopes comprising flagella, immohi-
lize bacteria that use motility as a way to ascend the
tract from the n1ore caudal rcgions.
Normal Flora
Lower portions of the genital tract in all ailin1als possess a
normal flora. The flora varíes by animal and within an in-
dividual is dynamic over time. Factors including age, par-
ity, and hormones affcct flora makeup. ·rhe actual micro-
bial flora of the genital tract is likely to be more complex
Lhan p1ese11lly recug1liz1::c.l IJ1::cause opti1nal culture meth-
ods for highly fastidious org;inisms h;ive not always been
employed in past studies on normal flora prevale11ce. Pop-
uiation densi ty of individual flora members is less well un-
derstood, yet it is probably equully or more important than
the particular flora makeup 1n the ovcrall ecology and sta-
bility of the microbial population of the genital tract.
111 ge11eral, the female genital traer possesses a resictent
microhial flor;i c;:iucl;:il to the externa! cervical os. The
uterus is normally sterile or transienllyconlau1i11aleu \vith
small numbers of microorganisms. The vagina contains a
502 l'ART IV Clinical Applications
flora that is mainly composed of species of obligate anaer-
obic bacteria, including both gram-ncgativc a11d gram-
positive species. l 'he aerobic and lacultative anaerobic or-
ganisms, about 011c-tenth the number of obligate
anaerobes, include gram-posttlve and gram-negatlvc
species, as well as Mycoplasma anct Ureaplasrna.
A-s i:; the c<1~t: witl1 ull1t:r 111ucosal surfaces, Lhe flora
shoulrl h<> thought of as protective insofar as other, per-
haps more pathogcnic, strains are excluded through colo-
nization resistancc. Mechanisms tor exclusion include
blocking attachment, efficient use of availablc substrates,
and production of antimicrobial substances. In a more
practica! sense, the normal or transient flora does include
sorne specles that \-vill contarnit1ate tltc uleru::. ::.l!ould it be-
come compromised. Somi> i>x;impl~s include Streptococcus
equi subsp. z:ooepide111icus in mares with endometritis, Arca-
nobacterium pyogenes along with Fusobacterium necro-
phorurn in cows with pyometra, and Escherichia coli in
bitches with pyometra. Ali of the organisms mentioncd
above are part of the normal or transient vaginal flora of
the affected animals. The potential fur tl1es1:: urga11is1ns to
cause disease does not simply ri>~11lt from their presence
uul u:qulres a critica! leve! of replicative dominance over
other flora before disease can occur. lf the normal flora is
<listurbed, as duriI1g antibiotic trcatmcnt of bacteria] en-
dometritis in the mare, the vagina w1ll be repopulated
with othcr, more resistant strains, which may ultimately
infect thc uterus if the under1y1ng compromising condi-
tion goes uncorrected.
TlH.: n.:sil.lt:11 l flo.ra of the externa! genitalia il1cludes
c.01n mensal anaerobes, which are largcly gram-negative,
non-spore-forrners; Mycoplas1na spp.; alpha-hcmolytic
and beta-hemolytic streptococci; Jactobacilli; Haemophilus
spp. (particularly H. hae1noglobinophilus in dogs); coryne-
bactcrla; propionibacteria; and coa&'Ulase-negative staphy-
lococci. Taylorella equigenitalis, a venereally transmittPrl
vatl1oge11 of n1ares, n1ay be carried inapparently in the cli-
toral fossa.
The prepuce a11d the distal urcthra of thc n1alc genit al
tract possess a resident Uora tl1at plays a role similar to that
p layed by the flora in the vagina. 'fhe origin of organisn1s
responsible fo r bacteria! c.lisease ln these areas is alm.ost al-
ways endogenous. Sorne venereally transmitted org;inisms
rt:~iut: i11 Lhe prepuce (e.g., Car11pylobacter fctus subsp. vene-
reali.~ in cattle, 1aylorella equigenitalis in stallions) causing
no advcrsc cffccts at tl1cse sites. The preputial cavity, in
cluding the preputial diverticulum of swine, is home for
agents of pyelonepl1ritis in cattle (C. renale group) and
swine (Actinobaculurn suis) .
Disea~e~
Host and Environmental Factors. For d1sease to occur in the
genital tract frequently sorne predisposing host and envi-
ronmcntal factor(s) must be in place and include:
1. Anatornic Factors. Vulvar conformation of mares is ;i
wcll-k11uw11 predisposing factor LO infection of the
vagina and uterus. 'fhe more horizontally posi-
tione<l the vulva, the grcatcr thc tcndcncy for fecal
contamination ot the vagina to occur. Urine pool-
ing in the vagina predisposes to cervicitis and en-
domctritis. Phimosis in males can lead to nonspe
cific inflammatíon and infection of the prepucc and
pcni.s.
2. Hormonal Factors. Huru1u11e~ µI<1y a role in n1aking
the genit;il tr;ic.t rnorP. susceptihle to disease. In gen-
eral, under the influence of progesterone, the uterus
is more prone to infection. Neutrophil activily in
thc genital tract is suppressed under t he influence
ot progesterone, inc1uaing aecreased m1.grar1on an<.1
phagocytic ability. At least in bitches, d11ring thP
luteal vJ1a~e, recepto1s for E. coli are expressed.
Coloni7.ation hy F.. coli expressing appropriate ad-
hesins allows for establishment and can ultimatcly
lead to developmcnt of pyometra. 'vVhether the
sume occurs in other species is unknow11.
::S. Uther Factors. 'frauma that may occur during partu-
rition can compro1nise integrity of the epithelial
barrler leading to infectiu1i. Dy~Lucias ai1d retained
fetal me1n bTani>s increase chances for post-parturi-
c11t infections. Nutritional factors sometimcs influ-
cnce genital tract disease development. for exam-
plc, posthitis in shcep occurs typically in anjmals
011 rich legume pastures that are high in proteins,
which increase urea excretion, and estrogens. This
leads to preputiaJ swelling and urine reter1tiu11 ir1
the sheath.
•
lnfectious Processes. For most genital tract pathogens, the
routes of exposure are venereal or by ingcstion. Localiza-
tion to the genital tract occurs by ascer1ding or hernatoge-
nous routes. In sorne diseases, although venereally trans-
mitted, pathogens localize to areas in the genital tract by a
hcmatogenous means. Some genital tract pathogens re-
side In the digestive tract a11u 1Je1.:u111e lJlood-borne Lo
ca11si> clisi>;isp in thi> genital tract (e.g., Campylobacter fetus
subsp. fetus in sheep).
Fcm;ife Reproductive Tract. The most common ü1fections of
the female reproductive tract involve the vulva, vagina,
cervix, and uterus. Vulvitis in cattle is associated with
Ureaplasma ar1ú Jvlycuplu:;rnu ~1'1'·• bul a solid eliological
link n~ m ai ns to he shown . Infectious bovine rhinotra-
cheitis virus causes vulvovaginitis in cattlc. Various col-
iforms, especiaJly E. coli, cause vaginal-vuJvar discharge 1n
sows. Such infections are often related to hygiene and
management practlces. Vaginitis in <lug:; i:; 111u~t 1.:u1n-
monly associated with Staphylococcus spp, Streptor.nrr.u~
spp., ur E. c:uli. !J1 prevubt:rlal fe1nales, vagmitis tends to rc-
solve on its own as the dog matures. Tn older bitches, therc
is usually an underlying factor involvcd.
Uterine intections are typically related to breeding,
pregnancy, or parturition, the nonpregnant uterus being
fairly resistant to infection. Durlngbreedll1g or parturitiuu
when the cervix is open, infections of the uter11s ílrP most
often a:;cenc.li11g. I1lieclions of Lhe uterus can involve thc
Pnclometrium (endometritis) or t he entire wall of the
utcrus (metritis). Endomctritis occurs in all animals but is
most common and ot greatest consequence in the horse.
Tnability of sorne horses with endometritis to resolve infec-
tions is in part due to defects in rnlgratlon of neutrupllil~

to the site of infection as well as decreased phagocytic abil-
ily of ll1e neutropllils. Streptococcus equi subsp. zooepiderni-
cus is the patl1ogen most commonly associated vvith en-
dornctritis in 1narcs, although othcr organisrns (cntcrics,
other streptococci, Fseudomonas) can be involved.
Jf p rior to resolution of a uterine infection th e cervix
closes and pus accumulates, a pyometra develops. Pyo-
metras occur in ali animals but are most frequent in dogs,
cats, and cai-tle. Overall, E. coli is tl1e organisn1 n1osl oflen
associated with pyometra. A numher of other organisms,
including Streptococcus sp. and Pseudo1nonas, can also be
involved . In cattle, Arcanobacterium pyogenes and oblig-
ately anaerobic bacteria are frequently found.
Fetus. Abortion, especially in farm animals and horses, is
one of the n1ost C<>111n1011 and eco11on1ically sig11ificanl
genital tract diseases. Abortion is essentially expulsion of
the fetus before full dcvclopmcnt. A numbcr of viral, bac-
teria!,. and fungal agents can trigger this process (see ·tables
74.1- 74. 7). During p regnancy, the placenta and/or fetus
are infected predon1inately by the hematogenous route.
Ma rf's arf' a n f'XrPption; bacteria 1 and fungal placen tal in-
fections most often originate through the cervical os.
A nurnber of viruses can cause abortion in animals. The
herpesviruses are pronlincnt among thc viruscs involvcd
and affect a number ot ditterent animals, most notably
horses (equine herpesvirus 1). Viral induced abortions re
sult subsequent to fetal 1nflammatory lesions With n ecro-
sis, fetal anoxia, ar e11dometrial lesions.
Bacle1ia tespo11sible for abo1Lio11 oflen cause a p lace11 li-
tis or placental edema. lf the fetus becomes in fected, typi-
cally large numbers of organisms are found in the fetal
stomach fluid (Fig 74.4). ln some bacteria! abortions, le-
sions in the fetus rnay suggest the likely agent. As an exam
ple, the focal areas of hepatic necrosis in the fetus are sug-
gestive of- however, not exclusive to- abortions in st1eep
caused by C:ampylobacter fe.tus subsp. fetus (Fig 74.5). Whlle
most bactf'ria 1 :ibortions orC11r in thf' l:ist trimester, early
fetal death ar1d resorption occurs with son1e agents. In
FIGURE 74 . 4 . Gram stain with carbolfuchsin counterstain
of abomasal fluid from an aborted bovine fetus due to Orucella
abortus. Mu/tiple gram-negative rods to coccobaciffi are present.
<:hapter 74 1Jrogenita 1Systf'm 503
F 1G U R E 7 4. 5 . Areas of hepatic necrosis in the fíver of
aborted ovine fetus. Campylobacter f et us subsp. fetus was isolated.
those cases, the infection prese11ts clinically as an infertil-
ity problem (e.g., Camp)'lobacter fetus subsp. venerealis in
calllt:).
Fungal ahortions are most c.ommon in horses and cattle
and typically are the result of a placentitis. The portal of
entry in cattle is either the respiratory or gast rointestinal
tract with subscqucnt hcm:atogcnous spread to the pla-
centa. In horses, an ascenct1ng 1ntection through the
cervix is the most co1nn1on route. Fetal lesions in cattle, if
present, are for the most part limited to focal, hyperkera-
totir lf'sions on thf' skin; in horses fetal lesions are uncorn-
mon. Mortierella wolffi, a zygon1ycete, causes a fuln1inating
p11eumonia with a high mortality rate in about 25ºh of af-
fected cows as a rcsult of a lung-utcrus-lung infcctio.n
cycle. Fungal elements absorbed from tl1e uterus cause a
fulminating embolic pneumonia in the cow that typically
follows the abortion.
Sorne v iral infections o f the fetus result in congenital
abnorn1alities. Facto1s iniluencir1g wl1elher abnonnaliUes
develop are dependent on the stage of fetal development,
immunc cornpctcncc of tbe fetus, and the particular viral
strains responsi bJe (e.g., teline panleukopenia v irus;
bovine viral diarrhea virus; and Akabane, Cache Valley,
Border disease, and bluetongue viruses in sheep).
Details on the pathogenesis and pathology as it relates
lo individual abo rlifacienl age.u ts are found in specific
chapters on those agents.
Mate Reproductive Tract. ln males, the predominant genital
tract infections are orch itis, epididymitis, and infections
o f the accessory sex glands (prostatitis, semin.al vesiculi-
tis). Orch itis can develop by an ascending or hematoge-
nous rouLe. Occasiu11ally periorchitis develops from de-
scending peritonitis, which suhsequf'ntly involves the
testes. Epididymitis affects all animal species but is n1ost
common in rams (Fig 74.6). In younger rams, an ascending
infection by agc-dcpcndcnt prcputial residents ("Actinoba-
cillus seminis", Histophilus somni r"Histophilus ovis" is the
obsolet e name]) is the rule. In adult rams, Brucella avis is
Lile rualu agent tnvolve<.L Tt locallzcs to the epiaiaymal
ductule.s vi;:i t hf" hf"1natogenous route. Inflammation and
504 PART lV Clinical Applications
F1G U RE 7 4. 6. Epididymitis in yf!i:Jr/íny ldfll (rdfl1-ldfl1ú
epídidymitis) caused by Histophilus somni ("Histophilus ovis" is the
obsolete name). Tail of the epididymis is greatly en/arged (A) and
co11tai11s a greenísh purulent n1aterial (8).
cxtravasation of spern1 lead t o development of a spermatic
gran u loma.
J3acteri<1' pro<;t;:ititis most cornmonly occurs in dogs and
is, in facl, the n1ost con1mon cause of prostatic diseasc in
clog<;. ln fPctions are mostly ascending in origin and can be
acute or chro11ic. Consequenccs incl udc prostatic ab-
F 1G U R E 7 4 . 7 • Oophorítis in a chícken with pullorum dísease
(Salmonella Pullorum). (Courtesy Dr. H. ~hivaprasad.)
scesses, which c::in r11pt11re and cause peritonitis. Seminal
veslculitis occurs most commonly in bulls and is thc most
common cause for inflammatory cells dctected during
semen examination. Many potcntial agents have been im-
plicated, \·v ith Arcanobacterium pyoge11es t l1e n1ost fre-
quently rccovered.
lnfections of the penls Qr prepuce can l.Je fru111 t::J 1doge-
nous or cxogenous sources. Vario1 1o; hf'rpPs viruscs cause
tJalauupustl1ilis i11 ani1nals. Men1bcrs of the C7orynebactc-
r i11111 renale group cause a necrotizing inflammation of the
prepuce a11d adjacent tissues in wcth.crs or rams. The dis
ease develops in the presencc ot these urealytic agcnts 1n
an area constantly irrigated with urine. Ammonia is
thought to initiate the inflammatory process. A similar
condition occurs occasionally in goats and bu lis.
Genital Tract of Poultry. Tnfection s of the oviduct (salpingitis)
in poultry can be the result of an asccnding infcction from
thc cloaca o r occur in con junction with colibacillosis
whcn the left abdominal air sac is involved. Escherichia coli
is the most co1nmon parhogen recovercd. Although pullu-
rum discase and fowl typhoid havP hPcome uncommon
tlislo!ases i11 developed countries, oophoritis, salpingitis,
and orchitis are all possible consequences of the sep-
ticemia associated with infcctions by Salrnonel/a serovars
Pullorum and Gallinarum , respectively (Fig. 74.7).
Decreased egg production and/or hatchability can re-
sult from a number of systemlc infectiuns il1 puultry. A
number of viruses (e.g., avían ac1P novin1s, infectious bron-
~J li Lb vh u:s, avían in f1 ucn z:a virus) and bactcrin (c.g.,
Mycoplasrna, Haemophílus paraga llinarurn, Salmonella) can
uffcct ovcrall egg production and hatchability. So1ne poul-
try pathogens (e.g., 1\tJycoplasr11a, Salmonella) are rransmit-
ted vertically in eggs.
 Index

AAF adhesin , 61 structurc, 91 Blastomyces, 293

Abiotrophia, 167 transmission, 93 Bordctclla, 100
abortion, 503 treannent, 94 Burkholderia, 115
Acholeplasrna, see Mycoplasma variability, 93 Catnpylobacter, 134
Absidia, ?97 wooden tongue, 94 Candida, ?70
acitl fa~L ~Lain, 223 Actinobacillus h e1nolysin, 92 C/1/utnydiue, 235
acquired iinmunity, 10 Actinobacillus spp., 92- 94 Clostridiurn diftici/e, 207
activa tion of n1acr.ophages, J ?., 48 Artinnhao1/11rn <1Ji.<, 180 C lostridiurn perfringens, 198
ADCC (antibody depen den t cellu1ar A cti1101nyces pyogenes, see Arca11obacteriu1n Coccidioides, 286
cyt.otox1c1ty), 12 pyogenes Corynebacrerium, 178
antihody dependen t cellular cytotoxic- Actinornyces, 215- 218 Dichelobacter, 195
ity (ADCC), 12 abortion, 216 Erysipelothrix, 181
antibody production, 10 biochenlical characteristics, 215 Esclzerichia coli, 61
• cats, 216 f:lelicobacter, 1.41
antigen presen tation, 1O
detecbon o f, 13 cati:le, 216 l·listophilus, 95
endogenous pathway, 10, 48 c:u11 trol, 218 Hisloplus1nu, 289
exogenous pat hway, 10, 48 disc:ospondylitis, 216 Lawsonia, 139
lgA, 11 dogs, 216 Listeria, 185
¡,,.E 11 epidenliology, 2 17 Mannhcirnia, 84
" ' 10, 11
IgG, goats, 216 Moraxella, ] 19
IgM, 10, 11 gn)\Vth rharartl'r istirs, 21S MSC:RAMM, 1S3, 1S9
second ary response, 10 humans, 216 norn1al flora, 6
Actinobacillus, 91- 94 immunity, 217 T'asteurella, 84
\iltl'le., 94 laboratory diagnosis, 2J 7 Pseudomonas, 122
cc ll products of medica! interest lumpy jaw, 216 Sa.lmonella, 69
adhesins, 91 marsupials, 216 Sporolftrix, 279
Apx toxin, 92 n1astitis, 216 StaphylococaJs, 153
Aqx toxin, 92 n1orphology, 215 Streptococcus, 1S9
cap~ule::,91 Nocardia, con1pared with, 216 l'ersinia enterocolitica, 79
cell wall, 91 pat hogen esis, 215 Yersinia pseudotul1erc11/osis, 78
iron, 93 p lant >1wns, ?.16 Adennviridal'. (;i<l0novin1.~P.s), .~1 7-319
RTX toxin, 92 rcsistancc, 215 adcnovirus, avían, 317, 318
urease, 92 sheep, 216 adenovi rus, bovine, 318, 319
composit ion, 91 staining, 215 aden ovirus. c:hicke.n, 318
control, 94 structurc, 215 adcnovirus, dccr, 318
epidemiology, 94 sulfur granules, 217 adenovirus, equine, 318, 319
horse, 94 sv.rine, 216 adenovirtL~, ovine, 318, 319
¡,'Towt h chara cteristics, 93 transnlission, 215 adenovirus, porcinc, 318
irr1rr1u11ity, 94 lreat111e11t, 218 aden ovirus, turkcy, :·H8
laboratory diagnosis, 94 actin on1yc:osis, 215 adenovirus-1, canine, see infectious
morphology, 91 arti nomycotic my<"Ptom;1, ?.19 canine hepatitis vi.rus
pathogcncsis, 93 acyclovir, 43 adenovirus·2, canine, 319, 3¡8
swine, 94 J\DCC (antihody dependent cellular cyto- adenylyl cyclase toxin (Bordetella), 102
reservoir, 93 toxicity), 9. 11. 12 adjuvant, 48
resistance, 93 adhesin, ·1, 57 J\cgyptianclla, 257
septicernia, 94 Acl inobacillus, 91 aerobactln, 63, 82
staini ng, 91 Aspergil/us, 294 African heart•vater disease. 254

505
506 lndex

African horscsickness virus, -103-40-1 anacrobcs, obligatc, 193 antibiolic, scc antimicrobial
control, 404 anacrobes, obligatc, non-spore-forming, antibody dependent cellular cytotoxiciry
discasc, 403 193- 197 (/\DCC), 9 , 11, 12
distribution, 404 abscesses, 194 antibody, function, 48
l1ur~e~, 403 cats, 195 anlifungal agents, see antimicrobials
host response, 404 cattle, 195 anti tungal chcmott1erapy, 41- 42
infectivity for other specics, 403 cellular products of medicill intP.rPSt, ilntig<'n pr<'S<'Otill'ion, se.e 1n acro phage
lab()ratory diagnosis, 404 193 antigcn proccssing, cndogcnous puthway,
pathogenesis, 404 capsule, J93 48
propC'rtit>s, 403 cell 1-vall, 193 antigcn processing, exogenous pathway.
rcscrvoir, 404 entero toxin, 193 '18
transmlssion, 404 control, 195 antlmicroblal ct(.tivity, 26
treatment, 404 dogs, 195 antimicrobial drug combinations, 37-38
African s"ine fevcr virus, 312-314 draining tracts, 194 anti microhial drug dosage predictions,
<.:on trol, 314 goab, 195 36-37
discasc, 312 gr(nvth characteristics, 193 antimicrobiai arug, mechanisn1 of act1on,
<listribution, 312- 313 horses, 195 ?.6,28
host rcspousc, 313 i1111nu1lil y, 194 ccll mcmb ra ne function, damagc to, 28,
infectivity for other .species, 312 laboratory djagnosis, 194, 195 ji
laboratory diagnosis, 313 n1orpho lop,y, 193 cell wall synthesis, inhibiU.o n o f, 26, 28
pathogenesis, 313 pathogencsis, 194 nuclcic acid synt hesis, inhib ition of, 28
properties, 312 pericardial effusion, J 94 pro leln synt hesis, inhibitio11 or, 28
rC'~<'rvoir, :~12-31 :~ peritoneal effusion, 194 anti1nicrobial drug rcsistance, 38- 41
rcsistancc, 312 pleural effusion, 19'1 acquired, 38, 46
swine, 313 reservolr, 193, 194 cu11jugdliun, 39, 46
transmission, 312- 313 sheep, 195 constitutivc, 38
treatment, 313 314 staining, l 93 integrons, 39, 46
Agf adhes ln, 69 ~lruclure, 193 niutation, 38, 45
Ail, 78 transmission, 193, 194 normal llora, 4ó
Aino virus, 367 treatment, 195 R plasm icl, :{8, 39
Akabane virus, 367 anaphylotoxins (C3a, CSa), 7 transduclion, 39, 46
•
Alagoas virus, 378 A naplasrna, l:::il-l:::i9 transfcrable, 39
Alcephallne h erpesvin1.~- 1 ilncl -?., c:attle, 2S7, 2S8, 2S9 transfonnation, 46
325-326 control, 258, 259 transposilion, 39, '16
cattlc, 325- 326 dogs, 258 anthnlcroblal reslstance, public health
malignan! catarrhal fever, 325 goats, 258, 259 issues, 40, 45-47
sheep, 326 horses, 258, 259 anti1nicrobial spectrum, 26
wlh.lebecst, 326 iuuuuuity, 257, 258, 259 anli111i~1ubia.l ~usccpl.ibilil)' testing, 35-36
Alcutian mink disease, 309 laboratory diagnosis, 258, 259 antimicrobial therapy, miJestones, 27
Aleulian mink virus, 309 pathogenesis, 257, 258, 259 antimicrohial~, topiral ;intifnngills, 41
Aleutian mink disease, 309 rcserYoir, 257, 258, 259 antitoxin, 52
control, :~09 sheep, l.'.>8, l.'.>9 a n t1v1ral chcmothcrapy, -'!2- 43
clistrih11tinn, 309 tick horne fever, 259 Apfu1110111,vccs, 282
host response, 309 transmission, 2S7, 258, 259 Aplltho1•irus, 339, 3~0
infectivily for other species, 309 treatment, 258, 2 59 aquareovlruses, 406
laboratory diagnosis, 309 A naplasrna bovis, 257 Are, 70
pathogencsis, 309 Anaplasl'na caudat11n1, 257 Arcnnobncteri11111 p)loge11es, 168- 169
propertie~, 309 Aru1µ/u~1nu cen/1ule, 257 abscess, 169
reservoir, 309 Anaplas111a nu1rgi11alc, 257 a rthritis, 169
resistance, 309 Anoplns1na ovis, ?.57 hiorh<'mical characteristics, 168
transmission, 309 A11aplas1na pl1agocytopllilr1111, 258 cntllc, 169
treatment, 309 Anaplas111a platys, 257, 258 cellular products of medica! lnteresr,
Alpllnvir11~, 1."il anaplasmosis, 257-258 168
Alpllarctrovirus, 410 antibody cell wall, 168
Altcn1aria, 283 antlbacterlal, 11, 48 llt!UICllllillÍl..ld~t!, 168
amantaclinc, 43 antiviral, 11, 48 pyotysin o, ló!:>
amikacin, see a n1inoglycosides colonization resisla11re, 4 toxi ns, 168
anünocycl ito ls, 28, 33, 34 IgA, 11 control, 169
an1inogJycosicles, 28, 33, :-;4 lgE, 11 endomctrltls, 169
absorplio11, cl istribntinn, i>xcrPtion , :~4 fgG, 10, 11 epidemiology, 169
adverse effects, 34 lgM, 10, 11 growth charactcristics, 168
antimicrobial activity, 34 normal flora, 6 immu ntty, 169
rC'~i~tanct>, .~4 opsonin, 8, 11, 48 Jaboratory diagnosis, 169
aminopcnicillins, scc pcnicillins Anguillina coli, see Brach)'spirn pilosicoli liver abscess, 169
amoxlclllln, sce pcnictllins anthralysin O, 171 1norphology, 168
amphotcricin. see polyene antimicrobials anthrax, 170 pathogenesis, 169
ampicillin, see pcnicillins anthrolysin O, 171 reservoir, 168
 lndex 5 07

res1stance, 168 Atadenovirus, 318 rrans m 1ss1on, 3 91

staining, 168 atrophic rhinilis, 87, 103, l04 treatment, 392
structure, 168 Aujeszky's d isease, 326 avian influenza virus, 365- 366
summer mastitb, 169 autoclielin, 155 birds, 365
transmission, 169 Australian bat lyssa v irus, 378 control, 366
tr<~»tment, 169 >1vi;in (nnn-pn11ltry) di<P:l <;P, ~6S
Arcobactcr spp., 138 Aspergillus spp., 294- 297 distribution , 365
Arcobacter, 134- 139 avian influenza virus, 365 laboratory diagnosis, 365
abortion, 138 avian paramyxovirus, 370 pathogenesis, 366
cattle, 138 botulism, 209, 210 poultry, 365
d1arrhea, 138 Bru~hyspiru pilushuli, 131 re~ervuir, 365
cpidcmiology, 138 Gandida albicans, 271 transmission, 365
growth characteristics, 138 Chla1nydia spp., 237 treat ment, 366
hnnJunity, 139 C!tlaniydop!tila spp., 237 avían leukosis/sarco1na complex, 417- 419
Jaboratory diagnosis, 1:~9 Clostridium botulinurn, 209, 210 chickens, 41/
111 nrp h o l ngy, 1~4 Clostridium colinum, 209 cnn1Tol, 419
rcscr voir, 138 duck hepatitis virus, 345 discasc, 417
staining, 134 castern cquine cn ccphalitis Virus, distr ibution, 418
structurc. 134 351- 353 host response. 419
sv1ine, 138 Erysipelothrix, 183 infectivity for ot her species, 417
trans111ission, 138 goosc p<1rvovirus, 306 labo1alo1y diagnosis, 4 19
treatrncnt, 139 Helicobactcr canadensis, 145 pathogenesis, 418-419
Arizona, see Saltnonella f·lelicobacter pametensis, 145 properties, 417
aroA mutant vaccine {Saltnonella), 72 llelicobacter pulloru1n, 145 reservoir, 418
Arterivrrus, 384, 393- 397 H1gh lands] virus, 351, 353 resistan ce, 41 l
infPc'tivity fnr othf'r ~pPciPS, ~84 Japane.se f'ncepha litis vi r us, ~S~, ~.'í.'í tr11n~mi.~sion, 4 18
propcrtics, 384 Jvturrny Vnllcy cnccphnlitis virus, 353, trcntmcnt, 419
resistance, 384 355 aVian 1nyeloblastosis Virus, 410
arll1ritis, 471 Mycobacteriu1n avium ssp. aviurn, 227 avian myelocytomatosis virus, 410
Asfarviridao, 312 314 AíJ•copltJsn-JCJ spp., 2 4 1-248 avían r0t iculoendotheliosis virus, 410
Asfiviru.~, 312- 314 Newc,1sllc disease virus, 373 avían sarco1na, sec avían Jeukosis/sarcoiua
aspergillosis, 294-297 Ornithobacterium rhinotracheale, 99 co111plex
Aspergillus, 294-297 •
poxvirus, 334 Avipoxvirus, 334, 336
abor tion, 296 St Louis encephalitis virus, 353 avoparcin, vancornycin resistance, 26, 28,
airsacculitis, 296 thrush, 2/1 41, 165
b irds, ?.96 VEF. virus, 3S1- 353 azithromycin, see n1acrolides
cats, 296 Vcnczuclan cquinc cnccphalitis virus, aztreonam, 30
cattle, 296 351-.353
cellular products of medica! interest, WEE virus, 351-353 B ccll, see lymphoc:y-te
294 West Nile virus, 353, 355 bacillary angiomatosis, ?62
adhesin, 294 western equine encephalitis virus, 1.J¡1cillary dyse::11te::ry, ~ee:: Sltizellu
cell wall, 294 351- 353 bacillary hen1oglobinuria, 204
iron, 295 Yersinia pseudotuberculosis, 78 baci.lle de Caln1ette et Gu~rin (BCG), 2 28
lUe l<tllill, 295 avian adenovi(us, 317, 318 Bacillus anthracis, 170- 174
pigment, 295 avían arizon osis, see Saln1onella biochemical cl1aracteristics, l/l
control, 297 aviiln ch o lPra, 88 cats, 17:~
discospondylitis, 296 avinn cnccphalomyclitis virus, 340, cattlc, 172
dogs, 296 343- 344 cellular proclucts of medical interest,
e.piden1iology, 296 chicken, 344 170
gro~vth characteristics, 295 control, 3·11 anthralysin O, 171
guttura.l pouch, 2 96 disease, 343 ant hrolysin O, 171
horses, 296 laboratory diagnosis, 344 capsule, 170
imrnunity, 296 properties, 344 Dps-like protein, 171
11101phology, 294 treatn'lent, 344 ederna factor {EF), 1.70
nasal cavity, 296 avian erythrohlastosis virus, 410 edema toxin (EdTx), 170
nasa l sinus, ?.96 avi;in in fect io11s hron<h itis v in 1s, ~91-~9?. in1m11ni> inh ih itor p rotf>in (Tnh), 171
pathogencsis, 295- 296 chickcns, 391 lethal factor (LF), 170
pneurnonia, 296 control, 392 lethal toxin (LeTx), 170
poultry, 296 disease, 391 multiple peptide resistance factor
reservoir, 29,S distribution, 391 (MprF), 171
srrucnue, 294 host response, 392 pOX1, 170
transmission, 2 95 infectivity for other species, 391 pOX2, 170
treatn1ent, 297 laboratory diagnosis, 392 protective antigen (PA), 170
Aspergilh1s fu1nigc1tus, 294 pat hogenesis, 39·1 1egulalion o[ virulence deleuninants,
Aspergillus terreus, 296 properties, j91 171
A ~pP.rgillus defl!!r.his, ?96 rf'.~Prvnir,
.391 toxins, 170
aspiration pncumonia, 495 rcsistance, 391 control, 174
508 Indcx

Bacillus anthracis (continued) Bartonella spp. , 260, 261 control, 358

dogs, 173 Basid.iobolus, 297 disease, 351>
epidenüolo8)', 173 1-l(~G (bac:ill P ciP C;ilmPttP Pt Gnc>rin), ?.?.8 clistrihn tinn , 358
gamma phage, l 73 beak and feather diseasc virus, 309- 310 hairy sh¡¡kcr lan1bs, 358
goats, 172 control, 309 host respon se, 358
growth characteristics, 171 disease, 309 infectivity for oth er species, 358
horses, 172 distr ibution, 309 laboratory diagnosis, 358
llun1ans, 173 host response, 309 p<1thugcuesb, 358
imn1unity, 173 infectivity for other specics, 309 properties, 358
laboratory d iagnosis, 173 Jaboratory diagnosis , 309 reservoir, 358
iuurpllulugy, 170 p roperlies, 309 resistance, J58
pathogenesis, 172 psittacine birds, 309- 31 O sheep,3.)8
reservoir, 171, 172 reser,vo ir., 30() transmission, :~58
rcsistancc, 171 resistance, 309 trcutmcnt, 358
sllecp , l/Z transmission, 309 Bordecel/a, 100-104
staining. 170 treatment, 309 atrophic rhinitis, 103, 104
string of pcurls test, 173 bcnign cnzootic paresis, 312 biochemical activities, 102
structure, 170 benzyl penlclllln, see pen icillins u::llul<ir prouucls o f 111edical inlerest, 100
swine, 173 Birnaviridae, 407- 408 adenylyl cyclase toxin, 102
tran ~;mission, 172 beta-lactamases, 29, 30 adhesins, 100
treatmeut, 174 Beturetrovirus , 410 capsule, 100
variability, 171 Bfp adhesin, 61 cell wall, 100
Bacil/11s cere11s, 170, 171, J 74 highParl, 7.03 filamentous haemaggluti.n in (rha),
biochemical characteristics, 171 Bipolaris, 284 100
cattle, 1/4 black dJsease, 203 ft.mbriae, 100
cellular products of medica! interest, 174 blackleg, 206 iron, 102
ccrculidc, 174 bladder stones, 500 outer membrane protein, 101
enreroroxins, 174 Blastonzyces de11nalilidis, 292- 294 ll"'' la<: Un, 100
HBL, 174 cats, 293 pertussis toxin, 100
NHE, 171 ccllular products of 1nedlcal interest, 293 regulation of virul.ence genes, 102
T, 174 adllcsiu, 293 lracheal colonization factor, 100
•
diarrhea, 174 Badl,293 tracheal cytotoxin, 102
food poisoning (human), 174 Wl-1, 293 type III secretion system, 10?.
grvwl!J cl1a1aclelislics, 171 c.o nlrol, 294 con1positio n 1 100
mastitis, 174 dogs, 293 control, 104
morpholo8)', 170 PpidPm inl ngy, ?.CJ3 clng, 104
resistance, 171 growth charactcristics, 293 cp idcmiology, 104
rice, J74 imn1unity, 293 growt h characteristics, lOZ
staining, 170 laboratory diagnos is, 293- 294 imm uníty, 104
structure, 170 morpholo¡:,ry, 292 kennel cough, 104, 319, 375, 388
.
bacitracin 26, 28
bacteria] endocarditis, 262, 263, 440-441
pathogeuesís, 293
reservoir, 293
lal!ur<1lury uiaguu~b, 104
morphology, 1.00
bacteria] overgrowth (sn1all intestine), 17 structure, 292 pathogenesis, 103
baclerial v,1ccine, 53 1-ransnJ iss·ion, 293 p n eurnonia, 104
bactericida! antünicrobials, 26 t reatment, 294 reservoir, 1U::S
bacterir1, 48, 57. Vilriilhili ty, ?.9.3 resistance, 103
bacteriocins and iru1ate immunity, 6 blastotnycosis, 292- 2 94 staining, 100
bacteriostatic antimicrobials, 26 bluecomb disease, 392 structure, 100
Bacteroides spp., 193-197 b luetongue virus, 401- 403 swine, 103
Bartonella, 260 261 cattle, 401 transmission, 103
baci llary angioma tos is, 262 cuutrul, 403 lrea L1nenl, 104
cat scratch disease, 262 deer, 401 turkey coryza, 104
cats, 262 disease, 401 variability, J 03
control, 263 distribution, 4 02 liordetella adcnylyl toxin, 102
dogs, 263 goats, 4Ul Bordetella dern1onecrotic toxin, 102
Pnclncaniitis, ?.6?., 2.63 host response, 402 Bordete//a spp., 101- 104
growth chllrllctcdstics, 261 infcctivity for other species, 101 Borna disease virus, 376
11un1ans, 262 Jaboratory diagnosis, 402- 403 Bornaviritlue, 376
immunity, 263 pathogenesis, 402 Borrelia, 128- 130
laboratory dia6'11osls, 263 properties, 401 avian, 129
n1orphology, 260 rcscrvuir, 402 ca ltle, 129
reservoir, 261 sheep, 401 cellu lar products ot medica! interest,
rodents, 263 transmission, 402 128
slaining, 260 treatmen t, 403 ccll wall, 128
structure, 260 bone, see musculoskelctal system hemolysín, 128
tr:.1nsmissinn , ?.61 horder disease virus, 353, 358- 359 outer surface protein A (OspA), 130
treatme nt, 263 central ncrvous syste1n, 358 control, 130
 lndex 509

growth characteristics. 128 control, 424 cell wall, 131

in1munity, 130 disease, 423 cytotoxin, 131
laboratory <liag11o~is, 129 distribution, 424 flagella, 131
Lyme disease, 129 host response, 424 hemolysin, 131
morphology; 128 infi>ctivity for othvr spPcies, 424 Hly, 131
pathogenesis, 129 laboratory diagnosis, 424 Tly, 131
poultry, 129 pathogenesis, 424 control, 133
reservoir, 128 properties, 423- 424 dogs, 132
staining, 128 reservoir, 424 growth characterist.ics, 131
structure, 128 tra11s111bsiuu, 424 iJUJJlU llÍ Ly, 133
trans1nission, 129 treatrnent, 424 intestinal spirochetosis, 133
treatment, 130 bovine leukernia vin1s, 419-420 laboratory diagnosis, 133
variabilily, 128 Cllttle, 419, 420 morphology, 131 •
Borrelia afielli, 129 control, 420 pathogenesis, ·132
Borreli1111nserina, 1?.8, 1?,9 <iisPase, 419 reservoir, IJ2
Dorrelia burgdorferi, 128, 129, 130 distribution, 420 staining, 131
Borreliagarinii, 129 nost response, 420 strucn1re, 131
Borrelia herrnsii, 128 infectivity for other species. 420 swine dysentery, 131, 132
Borrelia parkeri, 128 laboratory diagnosis, 420 S'A'Íne, 131, 132
Burreliu lheileri, 128, 129 palhogenesis, 420 Lransnlission, 132
Rorrelia turicata, 128 properties, 419-420 treatment, 133
botulinurn toxin, 209 reservoir, 420 varia bül ty, 132
botulism, 209 resistance, 420 Brachy~pira aalborgi, 131
bovine adenovirus, 318, 319 transmission, 420 Brachysp1ra carus, 131
hovinecoronavirus, 387- 388 treatment, 420 Brachyspira hyodysenteriae, lJl, 1J2, 133
cattlc, 387, 388 bovine mammillitis, 325 Brachyspira intermedia, 131, 132
control, 388 bovJne mycobactertal ulceratJve lymphan- Brachyspira rnurdochii, 131
diarrhea. 387 gitis, 233 Brachyspira pilosicoli. 131 , 133
disease, 387 bovine papillomavirus, 315 Brachyspira innocens, 131, 132
dblriUUlÍOIJ, 388 bovine papular slon1alilis virus, 334, 335 bradsot, 205
host respon se, 388 bovine parainfluenza 3 virus, 370, 375 braxy 205
infectivity for other species, 388 bovine parvovirus, 306 brilliant green ag<ir, 59
laborarory diagnosis, 388 • bovine pyelonephritis, 179 Urk, 101
pa thogenesis, 388 bovine respiratory syncyt.ial virus, 370, bronch1tis, 493
properties, 387 37.S Bntcel/a, 105- 112
reservoi r, 388 bovinc rhinovirus, 340, 345 C3ttle, 107, 108
resistance, .387 bovine spong¡form encephalopatl1y (BSE) cellular products ot medtcal lnterest
sh ipping fever, 387 agent,431-433 A antigen. 105
rrnns1nission, 388 bovine, 431 cell waJI, 105
treatment, 388 control, 433 eryll1rilul, 105
>vinter dysentery, 387 disease, 431-432 M antigen, 105
bovine enterovirus, 340, 345 host prion relationship, 432 porins, 105
bovine cphcrneral fevcr viru~, 378, 281 laboralory diagnosis, 432 vir operon, 106
bovine farcy, 219 properties, 432 control, 112
bovine herpesvirus-1, 323-325 trP;itmPnt, 41.~ ciogs, 107, 108
bovine herpesvirus-2 (bovine mam- bovine syncytial virus, 410 cpidcn1iology, 108
1nillitis), 32~ bovine torovirus, 384, 393 growth characteristics, 106
hovi ne herpesvirus-4, 325 bovine virus diarrhea virus, 353, 356- 358 horses, 107, 108
bovine bcrpcsvi.rus-5, 325 cattle, 356 immunity, 109
lJovine herpesviruses, 323-325 control, 358 laboratory diaguusis, 110
conjunctivitis. 323 diarrhea, 356 morphology, 105
control, 325 disease, 356 pathogenesis, 106
tlbea~e, 323 distribulion, 357 (eservoir, 106
distribution, 323 host response, 357 resistance, 106
genital ];; horatory cii;:ign()$i!>, :\S7-:\SR ShPPp, 107, 108
host response, 323 pathogenesis, 357 staining, 105
infectious bov1ne rhinotracheitis (IBR), properties, 356, 357 structure, 105
323 reproductive problems, 356 s1"1ine. 107, 108
lahorntory diagnosis, 321 325 reservoir, 357 trans1nission, 106
meningoencephalltls, 323 resistance, 357 treatment, 111
pathogenesis, 323 transmission, 357 variability, 106
rcscrvoir, 323 treatment, 358 Bruce/la milk ring test, 111
rhinotracheitis, 323 Bn1chyspi ra, 131- 133 8rucella ssp., 105-106
transmission, 32:~ birds, 132 BSE, see bovine spongiform encepha-
tre;itmi>nt, .~?..'> cats, 1::\?. Jopathy
bovine immunodeficiency virus, 423- 424 cellular products of medica] interest, bubo, 76, 77
cattle, 42.3, 424 1.31 bubonicplague, /6, 11
510 Index

bumblefoot, 157 transmission, 135 javelinas, 370

Bunyaviridae, 367- 368 treatrnent, 137 laboratory diagnosis, 371
Bunyavirus, 367 variability, 135 leopards, 370
Hurkholderia, 113-116 Carnpylobacter coli, 134 lions, 370
hiorh1'mira l rha ra\tf'r istics, 113, l lS C:arnpylnhacter 1:nncis1L<;, 1:~4 minks, 370
cattle, 115 Campylobacter fctus, J.34, 135, 136, 1.37 mongoosc, 370
cellular products of n1edical interest, Ca1npylobacter helveticus, 134 neurologic discase, 369
113, 115 Carnpylobacter hyointestinalis, 134 old dog encephalitis, 369
adhesins, 115 Cmnpylobacter jejuni, 134, 135, 136, 137 panda, 370
capsule, 113, 115 Cur11pylolJut te1 lulÍ, 134 palhogenesis, 371
con trol, 114, 116 Carnpylobacter mucosa/is, 134 pneun1on ia, 369
dogs, 115 Campylobacter sputon1111, 134 propertiE's, 369-370
ep iue1 11io logy, 114, 115 Can1pylobac:ter upsaliensis, 134, l3(i reservoir, 371
glanclers, 113, 114 Can adian horse pox, 177 resistance, 310
goats, 115 r;in;irypnx vin1s, 3 .1 4 skunks, 370
growth characteristics, 113, 115 Ca11dida a/bicans, 270-272 tigcrs, 370
horses, 114 bu<ls, 271 transrnission, 371
irnm unity, 114, 11 .S cats, 271 treatn1ent, 371- 372
laboratory diugnosis, 114, 116 cattlc, 271 canine herpesvirus, 328-329
rnelioidosis, 113, 115 cellular p rocluc:ts of mellical iutercst, 270 a1Jor lio11, 328
morphology, 113, 114 adhesins, 270 control, 328-329
pathogencsis, 114, 115 control, 272 disease, 328
reservoir, 114, 115 dogs, 271 distribution, 328
resistan ce, 114, 115 epidemiology, 272 dogs, 328
staining, 113, 114 growth characteri.stics, ?.70 clogs, :~28
sl1uclure, 113, 115 horscs, 271 host response, 328
treatrnenl, 114, 116 immunily, ·¡,;;-¿ infectivlt.y for other specics, 328
BTJrkholderio spp., 113 laboratory diagnosis, 272 laboratory diagnosis, 328
bursae, infection, 471 nio rphology, 270 pathogenesis, 328
bursal disease, see infectious bursal pathogenesis, 271 properties, 328
disease reservoir, 271 rese¡voir, 328
structure, 270 transm ission, 328
Cache Valley virus, 367 s~viue, 271 caJ 1iue leproid g1anulo1na synctron1e, 233
Cafl, 75 thrush,271 can ine 1núnocytic ehrlichiosis, 253
C:ag (cytotoxin associated gene product), transmission, 271 canine parainflucnza Type 2 virus, 370,
142 treatn1ent, 272 375
Caliciviridae, 346- 350 Candi da spp., 270 canine parvovirus 'l'ype 2, 306-.308
¡-;ifi•ivirll!;, fpfinP, SPP fp]i nP c<il icivin 1s canclicliasis, 270- 272 conl'rol, 308
California encephalitis viruses, 367 canine adenovirus 1, 31 7, 318 diarrhea, 306, 307
ca1nelpox vlrus, 334 canine aclenovirus-2, 319, 318 <lisease, 306
CAMP reaction (test), 164, 188 canine coronavirus, 388- 389 distribution, 307
Ca1npylobacter, 134-138 control, 389 dogs , 307
ca Ls, 134 discase, 388 infectivity for other species, :307
cattlc, 134, 136 distribution, 389 laboratory diagnosis, 307- 308
cellular products of n1edical interest, <logs, 388, 389 pathogenesis, 307
134 gastroenteritis, 388 properties, 307
adhesins, 1:14 11ost response, 68Y reservoir, 307
Cilps11IP, 13.S in fectivity for other species, 388-389 transmission, 307
ccll ,,vull, 13 5 laborat ory diagnosis, 389 treatment, 308
Cia, 135 pathogenesis, 389 canine respiratory corouaviru~, 384
cytotoxin, 135 properties, 388 caprine arthritis en cephalitis virus,
enterotoxin, 135 reservoir, 389 421- 422
type nr secretor y systcrn, 135 re~hta11ce, 388 arthritis, 421
dogs, 134, 136 transmission, 389 control, 4¿;¿
epiden1i0Jogy, 136 treatn1en t , 389 clisease, 421
g<.>élt:->, 136 cuninc distcmpcr virus, 369- 372 distribu tion, 421
gro•vtl1 cl1aracterislics, 135 baclgcrs, 370 enccphaliti>, 421
imrnunity, 136 bears, 370 goats, 421
laboratory diagnosis, 137 civets, 370 host response, 422
morpl10Jogy, 134 control, 371-:~72 i11fcctivity fur oll11::r :.pecies, 421
pathogenesis, 135 disease. 369 laboratory dia¡,rnosis, 422
reproductive disorders, 136 distribution, 371 mastitis, 421
reservoir, 135 Llog~, 369 pat hogencsis, 421- 422
sheep, 136 fecrets, 370 pneu n1onia, 421
staining, 134 hard pad di seilsf', 369 p roperties, 421
5tructure, 134 host response, 371 rcscrvoir, 421
s~vine, 1:14 infect1v1ty for other species, 370- 371 resistance, 421

transmission, 421
treatn1ent, 4L.Z
caprine herpesvirus, 326
Capripoxvirus, 334, 336
carbapenems, 30
carbcnicillin, scc pcnicillins
Cardiovirus, 340
<.:dr¡Jet.iluy<.:i ll~, 30
caseous lymphadenitis, 177
CiltS
Actinon1yces spp., 216, 217
anaerobes, obligate, 193, 195
anthrax. 170. 173
l\spergillus, 291 297
Bacillus anthracis, 170, 173
Rartonel/a spp., 260- 264
Blaston1yccs, 293
calit:i viru~, 349-350
Carnpylobacter coli, 134, 136
Ca1npylobacter helveticus, 134
Can1pylobactar jcjuni, 134, 136
Ca111pylobacrer lari, 134
Campylobacter upsaliensis. 134
Candida albicans, ?71
Chlurnyúophilu (elis, 237
circulatory system, infcctious agcnts,
439
Clostridiurn difficile, 208
Clostridiu1n per(ringens, 200
C/ostridium pí/iforrne, 208, 209
Closlridium l'etaní, 211, 213
Coccidioides spp., 285, 287
coccidioidomycosis, 285, 287 •
coronavirus, 381, 389· 390
Cpe,200
Cryptococcus neofi>rmans, 267
d<?rmatophytosis, 273- 278
digestivc sysle1n, infeclious agents, 451
EF-4, 90
Enterococcus durans, 166
Enterococcus hirae, 166
Enterococcus villorurn, 166
eugonic fermen ror-4, 90
cyc, iofcctious agcnts, 484
feline corona virus, 384, 389- 390
fcline herpesvirus-1, 329- 330
feline infectious per.itonitis virus, 384,
389- 390
feline leukemia virus (FeLV), 414-416
feline panleukopenia virus, 305-306
felinc sarco1na virus, 414-416
feline spongiform encephalopathy, 427
feline viral rh inotracheitis virus, 329
r:eLV, 111 116
foxra11, 216, 217
Francisella tularensis, 117- 118
gastn>intestinal system, infectious
agents, 4 51
Haemophilus fe/is, 98
}Jelicobacter bi7.zozeroni, 142
llelicobacter ce1nis, 145
HelicofJacter Cinaecli, 145
fielicohacter marmotae, 145
T-l'elicobacter pylori, 115
herpesvirus-1, 329-330
infectious peritonitis virus, 384,
389-390
integumentary system, infectious
agents, 463
Lagenidiurn, 282
leuke.m ia virus, 111 116
Listeria spp., 187
lung, infectious agents. 489
lympho·id lissues, infectious agenlS, 439
Mulu,,.\/::ziu )/Jf'·• 269
Microsporurn canis, 274, 275
Mnra-xP.lla r.anis, 12.0
rnusculoskeletal system, infectious
agents, 469
Mycohacterium bovis, 227
Mycoplasma spp., 2-11 218
nervous system, 1nfectlous agenrs, 476
Nocardía spp., 219, 220
oblígate anacrobes, 193, 195
o<.:ular ~ysteu1, iHfe<.:liuus ¡1geuts, 484
otitis externa, 269
paoleukopenia virus, 30S-306
l'asteurella rnultocida, 84, 85, 88
plague, 75
plant awn, 216, 217
poxvirus, 334
11ytl 1i.u sb, 283
Pythiurn insidiosurn, 283
rabi<?s virus, 377- 380
reovirus, 398
respiratory system, intectious agents,
489
.rhin<)trachcitis virus, 329
ring\vorm, 273- 278
Salmonel/a, 72
sarcoma virus, ,111 116
skin, infectious agents,, 463
spongifonn encephalopat hy, 427
Streptococc11s canis, 163
tetanus, 211, 213
Trichophyton 1nentagrophytes, 274, 275
lul<1remi<1, 117- 118
Tyzzer's disease, 208, 209
urogenlta l syste1n, il1fectlous agenls,
497
viral rhinotrachcitis virus, 329
Yerstnia pesris, 75
Yersinia pseudotuberculosis, 78- 79
cat scratch disease, 262
Clla11hal
e feve1. ' 401
. Coryr11:buclt:riurn p~eudolubt:rc:ulosis, 178
cattle
Abiotrophio, 167
abscess, 178, 195
Actinobacillus spp., 9l, 94
Actinornyces spp., 216- 217
adcnovirus, bovinc, 318- 319
Afr!can llearrvvater disease, 254
Aino virus, 367
Akabane virus, 367
anaerobes, obllgate, 193, 195
Anaplasrna phagoc.vtophilurn, 259
Anaplc1sma spp., 257
anthrox, 170, 172
Arcanobacteriurn pyogenes, 168- 169
Arcobacter spp., 138
Aspergí/tus, 296
bacillary hen1oglobinuria, 204
Bacillus anthracis, 170, 172
Sacillus cereus, 174
Indcx 511
black disease, 203
blackleg, 206
bluetongue virus, 401
botulis1n, 209
bovine adenovlrus, 318-319
bovine coronavirus, 384, 387-388
bovine enterovirus, 340, 343
l.Juvi ue e(JheJueral fever, 378, 381
bovine h erpesvirus, 323- 325
bovine itn1nu11odeficiency virus,
423- 424
bov1ne leukcm1a vuus, 419-420
bovine mammillitis virus, 32.5
bovine parainflucnza virus, 370
bovine parvovirus, 306
bovine respiratory syncytial virus, 370
bovine rhinovirus, 310, 315
!Juvi11e spongiform encephalopatlly
(BSE), 431-433
bovine torovirus, 384, 393
bovine virus diarrhea virus, 353- 356
Brucella abortus, 106
Bruce/la melitensis. 106
Brucella suis, 106
BSE, 431-4'.~3
Burkholderia pseudornallei, 115
Cc1rnpylobc1cter spp., 134- 1:~6
Ca.n dida albica11s, 271
chlamydiosis, 237
<.hlamyrlophila ahorh1s, ?.:~7
Chlamydophila pecorum, 237
circling disease, 187
circulatory system. infectious agents,
110
Clostridiun1 botulinum, 209- 210
Clostridiu1n chauvoei, 206
Clostridium novyi, Typc B, 203
C/ostridiurn pe1fiingens Type A, 200
Clostridiurn per{ringens Typc B, 200-201
C/ostridium perfringens Type C, 201
Clostridiu1n perfringens Type l), 201
Clostrid1u1n sept1cu111, l04-l0~
C'/ostridiurn sordel/ii, 209
Clostridium Ü!tani, 211, 213
Cocciáioiáes spp., 285, 287
coccidioidomycosis. 285, 287
coronavirus, bovine, 384, 387- 388
Corynebacteriurn renale group, 178-179
Coxiella burnetii, 251- 252
Cryptoc(1cc11s 11eofi1r111a11s, 267
dcrn1atophilosis, 220, 221
Derrnatophilus, ?.20, 221
dcrr.n atophytosis, 273-278
eastern equine encepl1alitis virus,
351-353
Ehrlichia ruminantium, 254
Enterococcus durans, 166
Enterococcus hirae, 166
Enterococcus villoru.m, 166
C::IJ l<::1.VlVJ<.<::111Íd, 200-201
enterovirus, hovine, 340, 343
t>ph<>mf>r>l 1 ff>vf>r, bov\ne, 378, 381
F,scherichia co/i (EAggEC), 64
Escherictlia coli (EPEC), 65
Escherichia coli (ETEC), 63
Eschcrichia coli (invasivc), 64
51 2 Index

\..d l l h:: (Lu11Li1111el/) Pseudo111011<1S aeruginosa, 123 mcchanism of aclion, 28, 30

F.scherichia coli 0157:H7, 65-66 pyelonephritis, 178 rcsistance, .$U
cyc, infectious agents, 484 pythin.~is, 7.R1 cephalothin, see cephalosporins
foot and mouth disease virus, 339-342 Pythiurn insidiosun1, 283 ccphurnyc ins, 30
foul s, 197 Q fever, 251-252 ccrcoplthcclne herpesvtrus-1, 330
gastrointestinal tract, infectious agents, rabies virus, 377-380 cervical lyn1phatlcnitis, 162, 216
452 ra in rot , 220 221 cervicitis, 502
gastrointestinal tract, normal flora, 449 rain scald, 220, 221 celacea11 lvforbillivirus, 370
Granulicatella, 167 red ~vatcr disease, 204 chauveolysin, 206
Hae1nophilus so1nnus, see Histophilus reovirus, 398 chickcn adenovirus, 31R
:.u1111 u respiratory syncytial virus, bovin11, 370 chicken infectious anemia virus, 310-311
hcmorrhagic enteritis, 200 respiratory system, intectious agents, Cl1/a111ydia, see also C/1/arnydiaceae
herpesvirus, bovine, 323- 325 490 Ch/a111ydit1ceae, 235- 239
Histoplrilus so11111i, 98 rhinovirus, bovinc, 340, 345 abortion, 237
IBR, 32:$ rinderpest virus, 370 arthrlrls, 237
imm11no<lrficirncy virus, hovine, ringwor1n, 273-278 avian, 237
423-424 rotavirus, 101, 105 cats, 236
1nfcct1ous bovine rl1inotracheiti~ virus, Salrnonella, 71 callle, 237
323 sh ipping fever, 87 cellular products ot medica! interest,
infcctious pododer matitis, 197 skin, infectious agen ts, 464 /.3.'\
lnfecrlous bovine keratucu11ju11clivil b, ~po1adic bovine encephalomyclitis, 237 adhcsins, 235
121 Staphylococcus aureus, l.'>6 cell wau, 235
intcgumentary system, infectious Stnphylororn1.~ PpidPrn1idis, 1:'i6 chickens, 237
agents, 464 Staphylococcus hyic11s, 156 conjunctivitis, 236
Johnc's disease, 230 Staphylococcus sdurí, 156 control, 239
Leptospirn serovar cnnirola, 14R Staphylococcus xylos11s, 156 ecology, 237
Leptospirc1 serovar. grippotyplrosa, 148 Strcptococcus agalactiae, 163 enteritis, 236
Leptosp1ra se rovar. hardjo, 148, 150 Streptococa1s dysgalactlae, 163 eµic.lt::uiiulo¡;y, 238
1.eptnspira serovar. icterohaernorrlzagiae, Streptococcus uberis. 163 goats, 237
148 sununer 1nastitis, 169 growth characterístícs, 7..3.S
Lepcosplra serovar. pornona, 148, 150 TEME, 98 hu111ans, 236
Jcukcmia virus. bovine, 419-420 tetanus, 2 11, 213 imniunity, l.i8
Listeria spp., 187 thromboe1nbolic mcningocncephalitis, lahon1tory d iagnos is, 238
lu11.1vy jaw, 216- 217 98 life cycle, 235
lung, infectiuus agents, 490 tick-borne tevcr, 259 lyn1phogranuloma venereun1, 236
lymphoid tissues, infectious agent~. 440 torovirns, hovinr, 184, :~9:~ morphology, 235
malignan! catarrhal fever virus, 325 Trichophyton vcrrucos11111, 374, 375 ncrvous sys te1n, 237
mallgnant edema, 205 tuberculosis, 226 ornlrhosls, 237
mammillitis virus, bovine. 325 urogenital system, infcctious agents, pathogcnesis, 237
Man11heilnia glucosida, 85 498 pncurnonia, 236
Mannhei1nia gra11ulun1alis, 85 Vl:'~h.:ular ~tu111alili) vil u~, 378, 380-381 psittacosis, 237
Man11'1ei1nia haen1olytica, 84- 85, 87 virus diarrhea virus, bovine, 353-356 reservoir, 237
/'v1an11heimia n1minalis, 85 wooden tonguc, 94 resi~t;i n r-P, 7.1.'\-216
M111111h11i11úa varig11na, 85 Yersi nia e11terocolitica, 79 roc.lents, 236
mastltis, 219, 241, 267 Yersinia pseur.totuberculos1s, 19 sexually transmitted disease, 236
mastitis, in fpcti ons agPnt.~. 467 (~CFA 111edi um, 208 sheep,237
me lioidosis, 115 ccfudroxil, scc ccphalosporins staining, 235
Moraxella /Jovis, !Zl cefamandole, see cephalosporlns stru<.:lur~. 235
musculoskeletal system, infectious cefeplme, see cephalosporins tracho1na, 236
agents, 169 cefopecazone, see cephalosporins transmission, 237
Myco/Jacrerit11n aviu111 ssp. parat11ben11- ct::fula,'tj111e, ~ee cephalosporins treatmcnt, 239
losis, 230 cefoxitin, see cephalosporins turkcys, l.$1
Mycobacteri11n1 bovis, 226 cefpiromc, scc cephalosporin~ variahility, 236
Mytoplusnia spp., 241- 248 ceftazidime, see cephalosporins C/1/an1ydia 11111ridar11tn, 236
nervous systen1, intectious agcnts, 4/ I cefuroxime, see cephalosporins Chlan1ydta SI/IS, 236
Norardia ~pp., 7.19 cell mediated imrnunity, infectious Chla111ydia traclzo1natis, 236
normal flora, gastroin testinal tract, 449 disease, 118 Chla111ydophila, see also Chlamydiacene
nutritionally variant strepto<X>CCI, 167 cellular 1n1munlty, viral, 302, 303 C/ilur11yiloplriht abort11s, 236
obliga te anaerobes, 193, 195 CEM (contagious equinc metritis), 125 Chlan1ydopl1ila caviae, 2 36
ocular system, infectiou s agents, 484 cep halex in, see ccphalos porins Chlan1ydophila ft'lis, 7.36
paralnHuenza virus, bovine, 370 ct::plialu1 idinc, sce cephalosporins Clrlarnydophila pt·coru111, 236
parvovi rus, bovine, 306 cephalosporins, 28, 30 Cl1/an1ydopt1íla pne11111oniae, 236
l'aste11rella lymphangitis, 85 absorption, 30 C/1/a111ydophila psittaci, 236
P<1st<:u1clla 111ultocida, 84, 85, 87 antimicrobial activity, 30 chlamydospore, 270
l'asteurella trelzalosi, 84-8~ <listribution, 30 chloramphcnicul, 28, 33
po<lo<IPrmatítís, infrctious, 197 excretion, 30 absorption, 33
poxvirus, 334 generations, 30 adverse effects, 34
 Index 513

cHlli1uicrobial aclivity, 33 ducks, 210 red water disease, 204

distribution, 33 epidcmiotogy, 211 reservoir, 204
<->xcrf'tion, ~~ fish, 21 O t·ransrnission, 204
rcsistancc, 33 gccsc, 210 trcatincnt, 204
Chloririáovirus, 314 grass sickness, equine, 211 Clostridium novyi, 202- 204
cholcra. avian, 88 horses, 210, 211 bigheacl, 203
Chordopoxviridae, 333 hun1an, 210 black disease, 203
chor1oretln1tts, 486 la!Joratory diagnosis, 211 cellular products of 1n edtcal lnterest,
chromoblastomycosis, 283, 284 lamzickte, 211 203
ch.ronic wasting disease agent, 433- 434 lin1bern eck, 211 alpha toxin, 2 03
control, 434 rnink, 210 beta toxin, 203
deer, 4~3 pathogenesis, :llU, <'.11 delta toxin (novyilysin), 20:~
rl isease, 4:~3 reservoir, 21 O Pp silon toxin, 20~
clk, 433 rcsi:;tancc, 210 eta toxin, 203
host-prion relationship, 433 sheep, 210 gamma toxin, 203
laboratory diagnosis, 434 trans1nission, 210 novyilysin. 203
properties, 433 treatment, 211 zet a toxin, 203
lr e<1l11 re u L, 434 varial>ili ly, 210 control, 204
Cia, 135 waterfowl, 210 epidemiology, 203
C'.1D(co111bined imrounode.fici.en t) foal, Clostridi//rn chauvoei, 206-207 Fasciola hepatica, 203
Nocardia, 219 hlackleg, 206 gas gangrene, 203
ciprofloxacin, sec fluoroquinolones cattle, 206 goats, 203
circling disease. 187 cellular products of rnedical interest, irn1n11nity, 20~
Circoviridae (ciccoviruses), 309 311 206 J.aboratory diagnosis, 203, 204
c1rculatory system and lymphotd ttssue, alpl1a roxin, 206 liver flukes, 203
437- 445 beta toxin. 206 pathogenesis, 203
antimicrobial properties, 437 chauveolysin, 206 reservoir, 203
Jlotcl, 437- 438 della Loxin (chauveoly sin), 206 )lieep, 203
infections, 438 gam1na toxin, 206 transmission, 203
infectious agents and conditions, neuranünidase, 206 treatn1ent, 204
438- 442 sialidase, 206 water buffalo, 203
cats, 439 control, 207 Clostridium perfringens, 198- 202
C<l ttle, 440 •
epid<->rniology, ?.06 1pacas, 7.00
;:i

dogs, 438 imn1unity, 206 cats, 201

goa rs, 4':1U lanoratory a1agnos1s, zoo, 207 cattle, zoo, 201
horses, 439 pat hogenesis, 206 ccllular products of medica! interest,
poultry, '1A2 reservoir, 206 198
sheep,440 tra11~111b~iu11, 206 atlht:!)Í!l), 198
S\vine, 441 treatrnent, 207 alpha toxin· (Cpa, Ple), J99
CJL) (Creutzfeldt-Jakob disease), 427 Clostridi11rn colin11m, 199, 209 beta toxin (Cpb), 199
Cladophialophora, 283 Clu:;tridiurn difTilile, 207- 208 l>t:!la2 loxi11 (Cpb2), 1.99
Cladosporium, 283 cats, 208 capsule, 199
c larithro1nycin, see macrolides cellular products of n1edical interest, entcrot oxin (Cpe), 200
clc1ssical swine Cever virus (hog cholera 207 epsilon toxin (Etx), 199
virus), :~53, :~59 adhesins, <:O/ fibronectin-binding protein, 198
clavnl;:in i c ;:ici rl, 29, ~O AO P-ri hosyltra n sferase, 207 hemolysin, 200
clindamycin, scc l:incosa111·idcs capsule, 207 i()ta toxin (ltx), 199
cJostridial enterotoxemia, 200, 201 Cwp66, 207 kappa toxin (couagenase), 200
Clostridiutn. 198- 214 toxin A (ToxA). 207 mu toxin (hyaluronidase). 200
biochernical charactcristics, 198 toxin B (ToxB), 207 perfringolysin O, 200
growlh charac.lerislics, 198 toxins, 207 regulation of viru lence genes, 200
morphology, 198 diarrhea, 208 sialidase, 200
resist;:incP, 198 rlogs, ?.08 theta tox.in (perfringolysín <)), 200
staining, 198 horscs, 208 contro l, 202
strucrure, 198 pathogenesis, 208 diarrhea, 201
Clostridiurn argentinense, 209, 210 primates. 208 dogs, 201
Clostridium botulinurn, 209- 211 reservoir, 208 enterotoxemia, 200, 201
hotulism, 209 transm ission, 208 epidemlology, 201
cattle, 210 varia bility, 207, 208 focal symmetrical encephalomalacia.
cellular products of medica! \nterest, Clostridium haemolyticum, 204 201
209 lJacillary lit:!11Juglul>iuuria, 204 gas gangren e, 200
botulinuro tox.in, 209 cattle, 204 goats, 200, 201
( '.2 toxin, ?,JO control, 204 horses, 200, 201
C3 cxc>cnz)'·mc, 210 cpidcn1iology, 204 in\n.1.uni.ty, 202
chickens, 210 tmmunity, 204 laboratory diagnosis, <:U<:
control, 211 Jaboratory diagnosis. 204 lamh dysentery, 200
dogs, 210 pathogenesis, 201 Naglc.r rcaction, 202
514 lndex

Clu~li idiurri per (r in¿;ens (curtlirtut:d) gvaL~, 213 con Lagious caprine pleuropnetu11onía,
overeating disease, 201 gro•Nth characteristics, 212 241
pathogenesis, 200 horscs, 213 cornbined i n1munorlf'ficif'nt (<~TD)foal,
pulpy kidney dísease, 201 immunity, 213 Nocardia, 219
reservoí r, l 98 laboratory d1agnos1s, 213 commcnsal microorganis.m.s, 3
sheep, 200, 201 morphology, 211, 212 co1nn1ensal, 3
!.; truck, 201 pathogenesis, 213 con1n1ensalism, 3
svvine, 201 reservoir, 213 competitive exclusion, 72
transmission, 198 sheep. 213 complement, 7
treatment, 202 sivine, 213 anaphylotoxins (C3a, <.::Sa), 7
yellow larulJ tlbt:ast:, ZOO le laJt u~, 213 innale i.L11111unity, 7
yellows, 200 transmission, 213 c:onidioholus, 297
Clostridium pilifornre, 208-209 treatmC'nt, 214 con junctivitis, 48::1
cats, 208 varíability, 212, 213 contagious bovínc plcuropncumonia, 243
control, 208 clotri.n1azole, see imidazoles contagious caprin e pleuropneumonia,
floes, ?.08 cloxacillin, see penicillins 244
epi<lemiology, 208 CNF (cytotoxic nccrotizing factor), 62 contagious ecthyma of sheep ( orf), 334,
gerbils, zos coah'lllase (stapllylococcal), 15 3, 155 335
hamsters. 208 Coccidioides spp. , 285-289 contagious equine metritis (CEM), 125
hares, 208 cats, 287 Corynebocterium pilns11n1 (Type JTT), 178
horst:s, 208 cattle, 287 Coronaviridae, 383-393
lahoratory diagnosis, 208 cellular prottucts ot rnedical interest, Coronavirus, 383, 384
pathogcnesis, 208 28S- 287 corona virus, bovine, see bovine coron-
rabbits, 208 adhcsin, 285- 286 av1rus
rats, 208 beta i,3 glucanosyltransferase, 287 coronavlrus, canine, see cauine corou-
rf'Sf'íVO ir, 208 bcta-glucosídase-2 (Bgl2), 286 avir us
rhcsus rnonkcys, 208 chitinase 1 (Ctsl), 286 287 coronavírus, feline, see feline enteric
snow leopards, 208 serine protease, 287 coronavirus
transmission, 208 SOVVgp, 286 coronavirus. equine, 384, 391
trcatment, 208 urease, 287 coronavin.ts, human, 384
Tyzzer's dlsease, 208 coccidioidiu, 285 COf O!laVit us, 1abbil, 384
Clostridiurn septicurn, 204- 206 control, 289 coronavirus, rat, 384, 391
bradsot, 205 dogs, 287 coronavirus, resp irator y, canine, 384
IJrax.y, 205 epidenliology, 287, 288 Corynebacteriu111 spp., 175- 180
cattle, 206 growth characteristics, 287 c;orynebacteriu111 cystirlis (l'ype 11), 118
cellular products of 1nedical interest, horses, 287 <~orynP.har.tP.riu1n psr.udntuherculnsis,
204 in1munity, 288 175- 178
a lpha toxin, 20.'.> laboratory diagnosis, 288 biocl1e1nical characreriStics, 175
heta toxin, 205 rnorphology, 285 c: anadian horse pox, 177
delt3 toxin (septicolysín O), 205 pathogenesi.s, 287 caseous lymphadenitis, 177
gan1111a toxin, 205 reservoir, 287 cattle, 178
septicolysin O, 205 sheep, 287 cellular products of 1nedical interest,
toxins, 204, 205 spherulin, 285 175
cunLrvl, 206 slruc Lurc, 285 ce11 wall, 175
cpidcmiology, 205 swíne, 287 exotoxins, 175
goats, 206 transmission, ?.87 iro n acquisition gene, 175
imtnunity, 205 trcat1ncnt, 289 iro n , 175
laboratory diagnosis, 205 valiey fever, 285 phosphollpase D, 175
malignant edema. 204 Coccidioides inirnitis, 285- 289 seríne protease, 175
pathogenesis, 205 Coccidioides posadasii, 285-289 control, 178
prlrnates, 206 C()CCidioidin, 285 t:pidenliology, 178
reservoir. 205 coccidioido1nycosis, 285-289 equine type, 176
sheep,206 Cochlíobolus, 284 goats, 177
Lrcut:>Hti5sion, 205 coi ta) exanthen1a, equine, 322 growth characteristics, 175
trcatment, 206 colibacillosis ot towi, 66 rlaematobius irritans, 177
Clostridi1un sordPllii, 199, ?.09 c:olonizat ion resistance, 4, 446 hockey puck, 175
<::lostridiu111 spiro/orme, 199, 209 antibody, 4 horses, 177
Clostridium tetarli, 211- 214 defensins, 4 horses, 177
cats, 213 innate irnn1unity, 6 humans, 178
catt!C', 213 lactoferrin, 4 immunity; 178
celiular products of n1edlcal interest, lysozyrue, 4 laboralory diagnosis, 178
212 norrnal flora, 4, 6, 47 morphol.ogy, 17S
tetanolysin (tetanospa smin) , 212 org<>nic <>cids, 4 pathogf'nf'sis, 177
tctanus toxi11, 21 2 Sa/Jnonella , 70, 71, 72 pectoral ab scess, 177
control, L:l4 Shigella, 82 plgeon fever, 177
<1ogs, 21 :3 stress, 47. 70 reservoir, 176
cpidcn1iolo¡,,ry, 213 contagious hovine pleuropneumonia, 241 resistance, 175
 lndex 515

sheep type, 176 growt h charactcristics, 265 bemagglutination inhibition test, 24

sheep, 177 laboratory diagnosis, 267 immunodiffusion, 24
staining, 175 111a~li u~, 267 itn 1nunofluorescence, 23
structure, 175 meningitis, 267 n10Jecular techni ques, 24
treatment, 178 morphology, 265 nu clei<: <icid hybridization, 7-A
ulceralive ly1nphangitis, 177 pathogenesis, 265 poly1nerase chain reaction (PCR), 24
varia bilily, 1/6 res1stance, zo:i rad101.IDmunoassay, z~
Coryne1Jacteriu1n renale (Type l), 178 t ransmission, 265 serology, 24
Cor}nabact<:riurn renale group, ·178 - 180 trcatment, 267- 268 serum virus neutralization test, 24
bJochemical characteristlcs, 179 Cryplulvl·l·us neu(urrnuns, 265- 268 ~pe<.:i11le11 l 1a11tlli1 1g, 20
cattle, 179 CS31A adhesin, 61 specimens to collect, 18- 20
cellular products of medical interest, culture techniques (bacteria!) Western blot analysis, 25
178 "split plates", 16 dend ritic cells, 10
adhesins, 178 aerobic, 16 deoxy-V-glucose (Z-deoxy-l>-glucose), 43
cell w:il l, 178 ;in;ie rohir., 16 /)ermatophi/us congn/ensis, 220- 222
control, 180 brilliant green agur, 59 cattlc, 220, 22J
epidemiology, 179 differential media, 59-60 con trol, 222
goats, 179 enrichment media, 60 epidemiology, 221
growth characteristics, J 79 gen eral, 16 goats, 220, ?21
i.UHllUHily, 179 g 1a1n nega ti ve (GN) brolh, 60 g1 ease heel, 220, 221
laboratory diagnosis, 179 Hektoen enteric agar, 59 gro\-vth charactcrist ics, 221
morphology, 178 MacConkey agar, 59 horses, 220, 221
pathogenesis, 179 selenite broth, 60 inJn1unity, 221
pizzle rot, 179 tetrathionate broth, 60 laboratory diagnosis, 221-222
pyf>lon f>phriti~ (hovinP), 179 XT.O (xylosP lysin <> c!PoxyrholritP), S9 lnmpy wonl, ?.?.O, ?.?.1
reservoir, 179 xylose lysine deoxycholate (XLD) agar, 1norphology, 220
resistance, 179 59 pathogenesls, 221
sheep, 179 curli, 61 rain rot , 220, 221
staining, 178 Curvularia., 283, 284 rain scald, 220, 221
~l1 uclu1e, 178 cycloserine, 26 reservoir, 221
transmission, 179 cystitis, 499 sheep, 220, 221
treatment, 180 cystitis, infectious agents, 500 staining, 220
CO"'POX virus, 333, 334 • cytolethal-distending toxin (Cdt), 63 strawberry footrot, 220, 221
Coxiel/a burnetii, 251-252 cytolysin A, 63 structure, 220
abortion, 7.:'i7. cytotoxic necrotizing factor (CNF), 62 transmission, 221
amoebae, 251 cytotoxic' f' ccll, scc lymph ocytc treatmen.t, 222
cattle, 251 cytotoxin (Moraxella), 119 dermatophllosls, 220
control, 252 cytotoxin associated gene product (Cag), dermatophyte test medium (DTM), 276
crayfish, 251 142 dermatophytes, 273- 278
goats, 251 <.:ab, 275
irn1nunity, 252 danofloxacin, see fluoroqu inolones cattle, 275
laboratory diagnosis, 252 deer adenovirus, 318 chickens, 27$
rnilk, 251 defen.stDs, 6 control, 278
pathogenesis, 252 Veltaretrovin1s, 410 dogs, 2/':>
pneumonia, 252 ciPmatia<Pons f11ngi, 28'.i, 297 epiciemiology, 274-275
Q fever, 251 de1nonstration of infectious agen.t (bacter- goats, 275
sheep,251 ia!, fungal), l'.> growth charactcnstics, 273
trp;1tm f>nt, 7.S?. culture techniques, 16 horses, 275
vasculitis, 252 cytological t cchniqucs, 16 immunity, 275
Creutzfeldt-Jakob disease (CJD), 427 direct smear, 15 laboratory diagnosis, 275
(~rimean-Congo hemorrhagic fever virus, Giemsa stain, 16 morphology, 273
368 Gram's stain, 16 pathogenesis, 274
C1ossiella equi, 219 n1olecular, in1n1unologic technigues, 17 rese1:voir, 273
cryptococcosis, 265- 268 nucleic acid hybridization, 17 resistance, 273
Cryptococcus, 26$- 268 polymerase chain reaction (PCR), 17 sheep, 275
biochemical characteristics, 265 Romanovsky-type stain, lG swine, 275
cats, 267 serology, 1/ trans1nission, 274
r.attle, 267 specimen handli n g, 1.'i tr<>J1tment, 278
cellular prodi1cts of mcdical intcrcst, Wright's stain, 16 variabilily , 273
265 demonstration of infectious agent (viral), dermatophytosis, 273-278
mannitol. 265 17 dermonecrotic toxin (Bordetella), 102
1n elanin, 265 animal inoculation, 23 dermonecrotic toxin (Pasteure/la), 86
pliuspholipasc, 265 co111ple111e11l fixalio11, 24 tliau1outl 5kü1 tli5<::a~e, 182
sialic acid, 265 cultivation, 20-23 cliarrhea, infectious agents, 451, 452, 453,
rontrol, 267-268 <>IP<tron mi<ros<opy, ?.3 454
dogs, 267 enzyrne linked immunosorbant assay cats, 451
epidemiology, 267 (ELISA), 24, 25 cattle, 452
516 Intlex

diarrhea, infectious agents (co11ti11ued) Brachyspirapilosicoli, 131, 132 lung, infcctious agents, 489
dogs, 451 Bn1cella canis, 106 lymphoid tissues, infer.tio11~ agPnt~, 41R
goats, 453 Cantpylobacter coli, 134, 136 Malcrssezía spp., 269
horscs, 451 Carnpylobacter ftelveticus, ·1,34 Microspor111n canis, 274, 275
pn11l1 ry, 454 Carnpylobacter ¡ejuni, 134, 136 Microsporurn gypseurn, 274, 275
shccp,453 Ccnnpylobacter /ari, 134 musculoskeletal system, infectious
swine, 453 Can1pylobac1er upsallensls, 134 agents, 469
Dichelobacter nodosus, 195-197 Candida albicans. 271 Mycobacteriu111 bovis, 227
catlle, 196 canine adenovirus 1, 3 17-318 Neorickeltsi(/ hel11ti11thoecn, 255
cellular proclucts of meclical intcn:~t, <.a1úue tli~te1uver viru), 369-372 ncr vous syste1u, i.nfecliou5 agents, 476
195 canine herpesvirus, 328-329 i\Jocardia spp., 219-220
adhcsins, 195 canine monocytic chrlichiosis, 254 norm:il f1or;i, g;1'>trointf'_~tinal tracl, 450
ccll 1v,1ll, 195 canine parvovirus Typc 1, 306 obligate anacrobcs, 193, 195
virulence associated locus (Vrl), 195 canine parvovirus ·1ype :l, JUó-308 ocular systcm, infectious agents, 484
virulence associatPrl prntPin (V;ip), canine respiratory coronavirus, 384, otitis externa. 269
195 388-389 parvovi rus Type 1, 306
control, LY/ circulatory system, lnfet:tlous agcnts, parvovtrus Type 2, 306- 308
rp ide111io logy, 196. 197 438 Pasteurella canis, 85, 88
growth charactcristics, 196 C /ostridium botulinurn, 209-210 Pcrsle11rella dagnratis, 85
lmmunlty, 197 Cluslridi urn difll<.ilc:, 208 Pasteurr.:lla 111ultodda, 84, 85, 88
laboratory diagnosis. 197 Clostridiun'I perfrinxens, 200 Pasteurella ston1aNs, 85
n1orphology, 195 Clostridium pilifonne, 208-209 pie'~ Pl!r~. Sa/111onella, 72
(JélllJO¡)CllC~b, 196 ClostTidhun tetani, 211, 213 plague, 77
reservoir, 196 c.;occidioides spp., 285, 28/ plant awns, 216, 217
resistance, 196 corridioic1nmyro.~i~. 2R."i, 287 Pse11do111011as aeruginosa, 123
shecp, 196 Cpe, 200 pythiosis, 283
staining, 195 Cryptococcus neo(onnans, 267 Pythl11111 l11sldtos11111, 283
tr;in~mi~~ion, 19¡:; <lermatophytosis, 273-278 rabies virus, 377-380
trcntincnt, 197 distcmper virus, 369 372 reovirus, 398
Dicnes stain, 240 .EF-4, 90 re~¡.iiratucy 1..u1u1 1avi1 us, 384, 388- 389
difloxacin. see fluoroquinolones Ehrlichia canis, 255 resµiralory system, infectious agents,
digestive systcm, 446-457 Enterococcus durcrns, 166 489
antl m lt:robial propert ies, 446 Enlr:ruLUC.LU~ hirue, 166 Rickcttsia rickct:tsii, 250- 251
flora, 447-450 Enterococcus villon11n, 166 ringworm, 273-278
infcctions, 450 ErJ1Sipelothrix, 183 Rock)' Mo11nt;iin ~pnttf'c1 frvP r, 2S0- 2S1
infecliOU$ agent5 and conditions, Escherichia coli (EPEC), 65 salmon poisoning, 255
451-45:~ Escherichia coli (.E.TJ:,C), óJ Sal111011ella spp., 72
<'lit~. 4."i1 E~c/1erichia coli (i nvasive), 64 skin, infectious agents, 463
cnttlc, 452 cugonic fcrmcntor-1, 90 Staphylococc11s hyicus, 157
dogs, 451 cyc, infcctious agents, 48'1 Srapl1ylococL11s inlt'nnedius, 153, 156
goats, 453 Fle.xispira (Helicobacter). 142 Stapl1ylococa1s schleiferi ssp. coaxula11s,
horses, 451 foxtails, 216, 217 153, 156
puultry,454 ga:.lrui11le~li1 1 al l1a1..t, i11ít:1..Liuu5 agents, Streptococcus canis, 163
shccp, 453 451 teta nus, 211, 213
s1.vine, 453 gastrointestinal tract, 11or1nal flora, 450 ti«k-hornp fPvi>r, ?.S9
d iphtheroid, 175 llacn1opltilu.s ltac111oglobí11ophilus, 98 Trichoph)'ton rncntagrophytcs, 274, 275
l1ircct sn1ear, 15 1-1e/1cobalter bilis, 142 Tyzzer's dlsease, 208- 209
rl isrnspondyl itis, 108, 156, 2 16, 296 Helicobacter bizzozeronii, 142 urinary tract, infeclious agents, 500
disscminnlion ofvirus, 301 Hclicobactcr canis, 1'15 urogcnital system, infectious agents,
dlst emper, canine, see canine disremper Helfcobaaer cinaedl, 142 497
distemper, phocine, 370, 372 Helicobacter fe/is, 142 UTI, 500
DNA vac<.ines, -18, 52 Hclicobacter fennelliae, 145 Yersinia pestis, 77
dogs Helkobuc.ler su/0111011is, 1'12 draining tract, 194
Acti110111yces spp., 216, 217 hcrpcsvirus, 328-329 UT 104, see ::,a/1no11ella fyplumunum DT
adene>virus 1, 317-318 Histoplasrna cnp~11lnh1n1, ?.91 104
anacrobcs, obligatc, 193, 195 histoplasmosis, 289-292 DTM (dcrmatophyte test medium), 276
Anaptas111a przagocytopl11lu111, 2;:, 9 integumentary system, lnfectious duck hepatitis vi rus, 340, 345
A11apla.~n1a platys, 259 agents. 463 control. 345
onthrnx, 170, 173 kennel cough, 10'1, 319, 375, 388 di seas e, 345
Aspr1;<;illus, 296 Lagenidium, 282 du<.k~, 345
Racillus anthracis, 170, 173 Cawsonia, 139 host-virus relationship, 345
/Jartonella spp., 260- 264 Leptospircr serovar. cnnicola, 148-149 laboratory diagnosis, 34.<i
/Jlastu111yces, 293 Leplospira scrovar. gríppotyplrosa, propcrties, 345
Rordetella bronchiseptica, 10-l 148- 149 treatmcnt, 345
botulism, 209 l Rptn~pira ~Prov;ir. ictern/1ae111orr/1agiae,
Bmclrrspira canis, 131 148-149 EAggEC, see enteroaggregative Esc/lericlria
lirac11ysp1ra f1yodyse11tenae, 1J:l Listeria spp., 187 col/
 lndex 517

EASTl, 61- 62 endogenous pathway, antigen processing, enteropathogenic Escherichia coli (EPEC),
eastern equine encephalitis (EEE) , 48 65
351- 353 endo111etTitis, 502 cnterotoxemia, clostridial, 200, 201
birds, 351 endothrix, 274 enterotoxigenic Hscf1erichia coli (ETEC),
cattle, 353 Pndotoxi>mia, 64-6.'i 6'.~-64
•
control, 353 endotoxin, 5, 57 Enterovirus, 340
disease, 3~1 CD14, 5, 57 enterovirus, bovine, 340, 345
distribution, 351 host response, 65 enterovirus, porcine, 340, 342
horscs, 351 Toll-Jike receptors, S, 57 enterovirus, simian, 340
llost response, 351 - 352 euriclunent rnellia, 60 Ent.urnupuxviridue, 333
infectivity for other specíes, 351 enrofloxacin, see fluoroquinolones enzootic bovine leukosis, 419
laboratory diagnosis, 353 enteric redmouth, 80 en2yn1e linked immunosorbant assay
pathogenesis, 351 enterics, see En.terobacteriaceae (E.LISA}, 14
properties, 351 enteroaggregativc E. coli heat-stable toxin eosinophil, AVCC 12
reservoir, 35:1 (F.AST l ), 61 - 62 a nti-p;i rasite rPsponse, 12
resistance, 351 cntcroaggrcgativc Escherichia coli major basic prot.cin, 12
svvine, 353 (EAggEC), 61- 62 EPEC, see enteropathogenic Escherichia
transm·i ssion, 351 Enterobacteriaceae, 57- 60 coli
treatrnent, 353 ceJJ anatomy, 57 Eperythrozoon, 248
El>ola viru~, 376 co111posiLioJ1, 57 e¡.il1e111entl fever virus, l.>oviue, 378, 281
ectothrix, 274 culture characteristics, 59 Epherrnerovirus, 377
ectrome.lia virus, 334 grotvth characteristics, 58 epididyn1itis, 503
edema discasc, 66 Jaboratory diagnosis, 59 epizootic hcmorrhagic disease, 399, 403
EEE, see eastern equine encephalitis morpl101ogy, SI epizootic lympl1angitis, 279-281
EF-4, 90 resistance, .'i8 F.psilonretrnvirus, 410
P.HEC, ~ce cntcrohe1norragic Eschcrichia staining, 57 cquinc adcnovirus, 318, 319
co/i variability, 58 equinc arteritis virus, 394- 395
Ehrlich, Paul, 26 enterobacterial common antigen. 57 control. 395
Ehrlichia, 253-254 enterobactin, 7J disease, 394
Africau lleartwater dbease, 254 Enteruiuu.us, 165- 167 di:;Lrj!Ju liu11, 394
canine monocytic ehrlichiosis, 253 biochernical characteristics, 165 horses, 394, 395
control, 254, 255 cats, 166 host response, 395
dogs, 253 • cattle, 166 Jaboratory diagnosis, 395
immunity, 254, 255 cellular prot1ucts ot medica! intercst, 165 pathogenesis, 395
Jabora tory diagnosis, ?.54, ?..SS aggri>gation s11hst;inr.P, 16.1' reproctuctive, ~94
Ondiri disease, 254 capsule, 165 reservoir, 394
pathogenesis, 253, 254 cell wall, 16) respnatory, 3 94
treatment, ?.54, ?.SS cytolysin, 165 transmission, 394
Ehrlichia canis, 253 cxtraccllular supcroxidc, 165 trcatment, 395
J::hrlicl11a cl1affeensis, 253 gel.atinase, 165 equine coita! exanthema, 322
Ehrlichia equi, see Anaplasma phagocy- iron, 165 equine coronavirus, 384, 391
tophilum control, 167 equine encephalosis virus, 399, 404
ehr/ichia ei.vin3ii, 253 Liogs, 166 cquine grass sickness, 211
Ehrlichia ondíri, 254 cpidemiology, 166 equine llerpesvirus-l, 321 -~~22
Ehrlichia ovis, 253 growth characteristics, 165 eq11inP hPrpP~vin1s-?., 3?.?.
Ehrlichia pha¿;ocy/'oplzila., see Anaplas1na horses, 166 equine herpesvirus·3 (equine coi tal
phagocytophilun1 Iaboratory diagnosis, 166 exanthen1a), 322- 323
F.hrlir.hía platys, see Anaplasma platys morphology, 165 cquine herpesvirus-4 (equine rhinopneu-
Ehrlichia risticii, scc lvcorickcttsia risticii pathogencsis, 165 monitis), 323
Ehrlichia rurninantium, 253 rcscrvoir, 165 equine herpesviruses, 320- 323
ELlSA (cnzyme linked iinmunosorbant resistance, 165 control, 322, 323
assay), 14 rodents, 166 disease, 3?0, 321, 3?2, 323
Eloki1n.iu fluke fever, 255 staining, 165 dislribulion, 321, 322, 323
EMJH medium, 151 swine, 166 equine coita) cxanthema, 322
encephalitis, 479 transmiss ion, 165 equine rhinopneumonitis, 323
cnccphalomalacJa, focal symmetti.cal, 201. treatment, 167 horses, 320, 322- 323
enccphalon1yelitis virus, avtan, see avían Enterococcus durans, 16~, 166 llost response, 3Zl- 322
encephalornyelitis Enterococcus faecalis, 165, 166 infectivity for other species, :~20
cnccphalomyocarditis virus, 340, 343 Enterococcus faccium, 165, 166 laboratory diagnosis, 322, 323
discasc, 343 Enterococcus flirae, 165, 166 pathogenesis, 321, 322, 323
host-virus relationship, 343 Enterococcus villorum, 165, 165 properties, 320
laboratory diagnosis, 343 Enterococcus, avoparcin resistance, 41, 165 reservoir, 321, 322, 323
p1operlies, 343 Enterococcus, vanco1nycin resistance, 26, .resi~tauce, 320
rodents, 343 28,41, 165 transmission, 321, 322, 323
encephalosis virus, equine, 399, 404 enterohemolysin (E. coil), 63 treatment, 322, 323
endocarditis (JJorrelia), 262, 263 enterohemorragic Escherichia coli (El IEC), equine irifectious anen1ia virus, 422-423
endocardit1s, bactenal, 262, 263, 440- 44J 6~-66 control, 423
518 lndex

equine infectious anemia virus (continued) erythritol, 105 F6 adhcsin, 61

diseas1?, 422 ('rythroblastosis virus, avian, 410 factor V, 76
disltibulion, 423 erylhron1ycin, see n1acrolides farcy, 219
horses, 422 Escherichia coli, 61-68 ta.tal insomnia, 4li'
bost rPsponsP, 4?.3 cattl<', 63, 64, 6S felin e calicivirus, :149-350
i·n fectivily for other species, 423 cellular products of mcdical intcrcst, 61 cnts, 349
laborato ry cltagnosis, 423 acid tolerance response, 63 con¡uncttvltls, 349
pathogenesis, 423 adhesins, 61 control, 349-350
propcrtü.•s, 422 capsule, 61 disease, 349
rescrvolr, 423 cell wall, 61 tlistri!Ju liuu, 349
rcsistance, 422-423 enteric toxins, 62-63 hosl response, 349
swamp fever, 422 enterotoxins,61-62 infectivity for other species, 349
l1<111)1JU.>.>ivu, 423 J:PEC 5ignaling protcin.1 (.Wp), vJ laboratory diagnosis, 3 4 ?
lreatment, 42] hemolysins, óJ oral ulcerations, 349
eq11inP infl11pn7;i vin1s, 363-364 intimin, 63 pathogenesis, 349
control, 364 iron, 63 properties, 349
d1sease, 363 pathogenicity island (LEE), 63 reservo! r, 349
horses, 363 serum rcsistance, 64 resistancc, 349
infcctivity for othcr spccics, 363 Tir, 63 rhinitis, 349
laboratory diagnosis, 364 type Ill secrerlon system, 63 lr<::a l11 1 ~ 11 l, 349
pal hogenesis, 363 control, 68 feline enteric corona.virus, 389- 390
properti1?s, 363 <logs, 63, 64, 64 cats, 389
resptratory <.liscase, 363 ~de1na discasc, 66 control, 390
treatment, 364 enteroaggregative diarrheal disease, 64 discase, 3~9
equine Morbillivirus, 370, 372 enterohemorragic diarrhe;il nisp;:isi>, <listrihution, 389
<:quin\.'. papillon1avirus, 316 65- 66 host response, 390
cquine pectoral abscess, 177 enteropathogenlc d1arrheal d1sease, 65 infectivity for other spectes, 389
('quine rhil1ilis A virus, 340, .14.'i enterotoxigenic diarrheal disease, 63- 64 laboratory diagnosis, 390
equine rhinitis B virus, 340, 345 goats, 63, 64, 65 pathogenesis, 390
equ1ne rh1nopneumonitis, 323 horses, 63, 64, 65 propert11:!s, 389
cquinc sarcoid, 316 immunity, 66 rese,voir, 389
eq uine torovirus, 381 , 393 invasivc disease, 64 transmission, 389
Erbovtrus, 340 laboratory diag11u~is, 66-67 l1eallnenl, 390
erysipelas, 181 morphology, 61 feline herpesvirus-1, 329- 330
erysipeloid, 181 poultry, 66 cats, 329
E1 y:;ip1dotl11 úc rl1usiopathiae, 181-184 reservoir, 63 control, 329-330
birds, 182, 183 sheep,63,64,65 disease, .52~
rPll11l;ir prorl11rts of mPrlir;il intPrP~t. ~taining, 61 distribution, 329
181 structurc, 61 fclin(' viral rhinotracheitis, 329
adhesins, 181 transrnission, 63 host response, 329
capsule, 181 treatment, 67-68 infectivity for other spec:ies, 329
cell wall, '181 variability, 63 laboratory diagnosis, 329
c:oagu las«:!, 181 E~c:tu:rithfrt culi 0157:H7, 65-66 pallt11g~n~>b, 329
hyaluronidase, 181 esophagus, in fect ion, 453 propertics, 329
neura1ninidase, 181 ETEC, see enterotoxigenic Escherichia coli reservoir, 3?.9
co n lrol, 184 Eubacteriun1 suis, scc Actinobaculu1n suis resistance, 329
cJogs, 18:5 eu1nycotlc inycetoma, 484 transmission, 329
1'pi<1<'miology, 18:~ European brov.•n ha.re syndrome virus, feJine immunodeficiency virus. 424-425
crysipclas, 181 350 cats, 424, 425
fish handlers' disease, 183 European Iyssa virus, 378 control, 425
growth characteristics, 181 cvaluation of immune responses, 13 discasc, 424
humans, 183 exochelin, 223 distribution, 424-425
lmmunlty, 183 t::JCug~uuu:. palhway, antigen p1occssúig, host response, 425
laboratory diagnosis, 183 48 intectivity tor other species, 424
morphology, 181 Exophiala, 283 lahor;:itory diagnosis, 425
pathogenesis, 182 exotoxin, 4 pathogcncsis, 425
rcscrvoir, 182 f:xseroflilum, 283 propcrtles, 424
n'sist:i ne!', 181 extracellular palhogens, immune rcservoir. 424-425
shccp, 183 response, 10, 1l transmission, 424-425
staining, 181 exudative denna tlrls, 157 tn.·at1111::11t, 425
structure, 181 eye, see ocular feline in fectious anemia, 248
swine, 182 feline infe(."tious peritonitis (Fil>) virus,
transmlssíon, 182 Fl atll 1esi.n, 57 384,389-390
trcatmenl, 183, 184 F17 adhesin, 61 cals, .589
turkeys, 182, 183 F4 adhesin, 61 c:ontrol, 390
variability, 181 f41 adhesin, 61 disease, 389
J;rysipelolhríx spp., 181 F5 adhes1n, 61 disrrlbutlon, 389
 Index 519

llu:;L re:;po11~e, 390 Flaviviru., , 354- 356 gauuua JJll<tge, 173

infectivity for other species, 389 disease, 354 gamma-delta T cells, see lymphocytes
labor;itory diagnosis, 390 distribution, 354 Gamrnaretrovin1s, 410
pathogenesis, 390 properties, 354 ganciclovir, 43
properties, 389 reservoir, :154 gas gangrene, 200, 203
reservoir, -~89 TPSi.StilnCP., 354 gastrointestinal tract, normal flora,
transmission, 389 transmission, 354 449-450
treatment, 390 florfenicol, see chloramphenicol cattle, 449
fe li ne leprosy, 233 Florida horse leeches, 283 chicken, 449
fcline leukcmia/sarcorna virus (FeLV), fluconazole, see imidazoles dogs,150
414-416 flucytusiue (5-flucytsine),42 horses, 449
cat, 414, 415 fluoroquínolones, 28, 31 swine, 450
control, 416 foal pneumonia, 92, 94, 191 gastrointestinal tract, 446-457
discase, 414 foamy virus, simian, 410 ,1nlinücrobia.l properlies, 446
distribution, 41.) toca! synunetrical encephalomalacia, 201 flora, 447-450
host response, 415 Fnnser:aea., 28'.~ infections, 450
infectivity for other species, 415 foot and inouth discasc virus, 339- 342 infectious agents and conditions,
JalJuralury úlaguu:;b, 415 carrle, 3 40 '±51-45.'S
pathog-enesis, 415 control, 341 cats, 451
properties, 414 disease, 339 cattle, 452
reservoir, 415 distribution, 340- 34J dogs, 451
resistance, 414- 41.) host response, 341 goats, 453
transmission, 415 infectivil·y for oth<'r spl'Cil's, 340 horses, 451
trcatmcnt, 415-416 laboratory djagnosis, 341 poultry,454
feline leu.l<einia virus (FeLV), see felinc pathogenesis, 341 sheep, 4.)3
leukemia vLrus/ properties, 339-340 s~vine, 453
sarcoma virus (FeLV) reservoir, 341 genital tract, scc urogenltal systcm
felinc panlcukopenia vi1 us, 305- 306 resislauce, 340 gentanl1c1n, see aininoglycosides
cats, 306 swine,340 germ tube, 270
control, 306 transrnission, 341 Gerstmann-Straussler-Schein ker
disease, 305 treatinent, 341 syndro1ne, 427
distribution, 30.) footrot (cattle), 197 Getah virus, 351, 353
host responsP., :~06 •
footrot (shPPp), 19.'i Ghon complex, 225
infcctivity for othcr spccics, 305 foscan1ct, 43 Ghon-Ranke complex, 225
laboratory diagnosis, 306 fouls, 197 (i1emsa staín, ló
pathogenesis, 306 fowl cholera, 88 gill-associated virus, 384
properties, 305 fowl cholera, 88 glanders, 113, 114
reservoir, 306 fowl spirochetosls, 121 G!asser's disease, 98
resistance. 305 fowl typhoid, 73- 74 glomerulonephritis, 498
transn1ission, 306 fowlpox. vi.rus, 334 GN (gram negative) broth, 60
t nc!all ne11 L, 306 fuxlail (pla11l aw11), 216 gualJJUX virus, 334, 336
fe line sarcoma virus, see feline Francisella, 117- 118 goats, sheep
leukernia/sarcoma virus cellular products of medica! interest, 117 Abi.otrophia, 167
fclinc spongiforrn encephalopathy, 427 acid phosphatase (Acp), 117 Acli11obacillus lisnieresii, 92, 94
feline syncytial virus, 410 capsule, ll/ Actinobaci/lus seminis, 92
feline viral rhinotracheitis, 329 cell wall, 117 Actinn1nyces spp., 216- 217
FcLV, sec fcline lcukemia/sarcoma vin1s intraccllular growth locus, 117 adcnovirus, ovinc, 318- 319
(FeLV) macropl1age growth Jocus, 117 AfTican hearn-vater disease, 254
fibronectin and stress, 47 control, 118 anaerobes, obligate, 193, 195
filan1entous haen1agglutinin (fha), 100 epidemiology, 118 Anaplas1na phagocytophilutn, 259
Filovi1 iúue, 376 growl11 cl1aracter i ~tic~, 117 authrax, 170, 172
fimbria, see adhesin imrnunity, 118 Arcanobacteriurn pyogenes, 169
FIP, 384, 389- 390 laboratory diagnosis, 118 arthritis encephalitis virus, caprine,
fish .m orphology, 117 421 - 422
aquareovíruses, 399, 406 pathogenesis, 118 8acillus anthracis, 170, 172
infectious hematopoietic necrosis virus, reservoir, 117 highp;i(i, ?.03
378 382 staining, 117 b lack disease, 203
infectlous 'pancreatlc necrosis vlrus, 408 structure, 117 bluetongue virus, 40J.
Pasteurella skyensis, 85 transmission, 118 border disease virus, 353, 358
Photobacteri11m darnse/a, 90 treatrnent, 118 botulism, 209
Spring viremia of carp, 378, 382 tulare111ia, 117, 118 bradsot, 205
viral herr1orrhagic septicemia virus, 378, Francisel/a philorniraJ(ia, 117 braxy,205
~82 Francisella tularensis, 117 Bruce/la rnelitensis, 106
fish handlcrs' discasc, 183 Pusobactcriu111 spp., 193- 197 Brurellu ovis, 106
fistulous withers, 108, 216 Burkholderia pseudornallei, J 15
flagella (H) antígen, 57 gal lid herpesvi rus-2, :~:~o ('.ache Valley virus, 367
Plaviviridae, 353 360 gallid hcrpcsvirus-3, 330 Can1pylobacter spp., 136
520 Indcx

goats, sheep (co11ti11ued) Mycobacteli11111 bovis, 226 llae111ophilus and llistophilus, 95- 99
caprine art hritis encephalitis virus, 1\4ycoplasn1a spp., 241-248 at)ortion, 98
421-422 Nairobi sheep diseasc virus, 367, 368 biochernical activity, 96
caseous lymphadenitis, 178 Nairovirus, 367, 368 cat, 97, 98
Cfztamydophila pecoru1n, 237 nervous system, infectious agents, 478 cattle, 97, 98
circling disease, 187 Nocardia spp., 219 cellular products of u1edical interest, 95
circulatory syste1u, infectious agents, nutritionally variant streptococci, 167 adhesins, 95
440 obligate an<terubes, 193, 195 <.:avsule, 95
Clostridiun1 botulinurn, 209-210 ocular systern, infectious agents, 485 immunoglobulin binding protein, 96
Clostridiu1n chauvoei, 206 overeating disease, 201 lipooligosaccharide (L()S), 96
Clost1 hlitufl 11ovyí T y pc A, 20J ovlnc cidcnoviru:s, Jl0 - Jl9 do g, 97, 98
Clostriclium novyi 'l 'ype .B, lU3 Pasteurella 1nultoc:ida 84, 85, 88
1
Glasser's disease, 98
l:lnstridiurn perfringens Type A, 200 Pasteurel/a treha/osi. 84. 85, 88 growth ch aracteristics, 96
Clostridiurn pcrfringcns Typc B, 200- 201 peste des petits ru1ninants virus, 3 70 morphology, 95
Clostrídíurn perfringens Type e;, 20 l Phlebovin1s, 367 pat hogenesís, 97
<:lostridiurn perfringens Type D. 201 pizzle rot. 179 pneun1onia. 98
G'lostridium scpticurn, 205 poxvirus, 331 reservoir, 96
Clostridiurn sordellii, 209 progresslve pn eun1onia virus, 421- 422 resbtacu.:e, 96
Clostridh1111 tetani. 211, 213 pulpy kidney disease, 201 septicenlia, 98
Coccidioídes spp., 285, 287 Q fever, 251 Sh PPp, 97, 98
coccidioido1uycosis, 285, 287 rabies virus, 377- 380 staining, 95
Corynebacteriun1 pseudotubercu/osis, 178 reovirus, 398 swine, 97, 98
Corynebacteriurn rPnale gro11p, 178- 179 respiratory system, infectious agents, TEME, 98
Coxiella bur11etii1 251 - 252 491 thromboe1nbolic meningoencephalitis,
der1natoph1losis 1 220-221 Rift Va\Jey fever virus, 367 98
Dennatophih1s, 220- 221 rinderpest virus, 370 transmission , 96
Dichelobacter nodosus, 195- 197 d .n gworm, 275 variability, 96
Ehrlic/1ia ruminantlun1 1 254 rutaVin1s, 404, 405 fluernophí/us ag11i 1 see H is tophilus so11111í
enterotoxemia, 200- 201 Salrnonella spp., 71 Haemophi/us sornnus, see Histophilus son111i
Erysipelothrix, 182 scrapie agent, 427- 431 Haemophilus spp., 97, 98
Escherichia coli (EPEC), 65 s.l<lrr, iuft::l:liuu~ agen Ls, 465 h ae111oplas1nas, 240
Fscherichia co/i (ETEC), 63 Staphylococcus aureus, 156 hairy shaker 1a1nbs, 358
fo.scherichia coli (invasive), 64 sti.ff lamb <lisease, 2 37 hard pad disease, .369
eye, infeclious agcnls, 485 stra,<Vberry footrot, 220- 221 l lavcrhill fever, 222
Flexispira (Helicobacter) rappini, 142, 145 struck, 201 heart, infection, 440, 441
focal .symn1etücaJ encephalomal;;ci;i, tPtrlrlTTS, 7J l , ?.1~ Hektoen en teric (HE) agar, 59
201 tick pycmin, 15 6 Rc/icobact<'r !;pp., 1'11 117
foot and inout11 dlsease virus, 339- 342 tick-borne feveJ, 2-59 bin.ls, 142, 145
footrot, 195 tularemia, 117- 118 cats, 142, 14·5
Francisella tularensis, 117- 118 urogenital system, infectious agents, cellular products of medica! interest,
gaslroinLeslinal Lrac L, infec lious agents, 498 141
453 visna virus, 4 21- 422 adhesins, 141
Granulicatella, 167 Wesselsbron virus, 353, 355 blood grou p antigen-bindine adh Psin
Helicobactcr rappini, 142, 145 \<VOoden tongue, 94 (DabA), 141
Histophilus sonnli, Y8 ye llo'"' lamb disease, iou Cag pathogen1c1ty island, 141
i ntPgu1nPntary systen1. in fec:tious yello'"'s. 200 cell ~val! . 141
agents, 1165 Yersinia enterucolitica, 79 cyto lethal distending toxin, 142
Johne's disease, 230 Yer:sínía psi-itclulubc:rculo:>i.>, 78- 79 flagella, 142
Jamb dysentery, 200 goose parvovirus, 306 sialic acid-bin dlng adhesin (:>abA),
Listeria spp., 187 gran1 stajn, 16 141
loupi11g ill vir us, 3531 355 Gnu1ulicatella, 167 urease, l.42
Jumpy jaw, 216- 217 granzymes, see lymphocytes vacuo lating toxin, 142
lumpy v.•ool, 220- 221 g rass sickness (equine) , 211 chf'f'tilh.~,
14?.
lung, infectious agents, 491 g rass sickness, equine, 2J l control, 147
lyn1phoid system, intectious agents, 440 grease heel, 220 ctogs, 142, 145
lvtannheimia granulo1natis, 85 greasy pig disease, 157 ferrets, 142, 145
Mannheimia hafünolytica, 8 4, 85, 88 griseofulvin, 4 2 g rowth characteristics, 143
Mannhei1nia n1minalis1 85 Gruuv .E SLTeplutut1-us, :.ee Slteplucuccus inununity, 146
Mannheimia varigena, 85 porcinus 1aboi:atory diagnosis, 146
melioi<losis, 115 Gsr, 75, 76, 78 m<i rinP n1ammals, 142
Mon1xel/u boevrei, 120 guidelines for rational use of antimicro- morphology, 141
Moraxella caprae, 120 bials, 4~ nonhu1nan prin1ares, 14 2, 145
Moraxella ovis, 120 (~11mhoro ctisease, 407 pathogenesis, 144
n1usculoskeletal system, infcctious reservoir, 144
agents, 469 H antigcn, sce flagella an tlge n rodeuts, 142, 145
hrfyr.ohar.teriu1n avium ssp. paratubern1- Hae1natobius irritans, 177 sh eep, 142, 145
losis, 230 Haernobartonella, 218 staining, 141
 Indcx 521

.swine, 142, 145 treatment, 292 circulatory system, infectious agents,

transm ission, 144 variability, 290 439
tre::itmPnt, 147 Histoplas111a capsulatum var. duboisii, 289, C/ostridium botulinurn, 209- 210
variability, 143 290 Clostridium difficile, 208
zoonotic potential, 146 Hísroplasma capsu/aturn var. farctmtnosum, Closrridiurn perfringens Type A, 200
hemagglutinating encephalomyelitis 281-282 Clostridium perfringens Type C. 201
virus, 386 387 control, 282 Clostridium piliforme, 208-209
control, 387 epiueuliology, 282 Clu~lr hliurn .1orrlel/i i, 209
disease, 386 epizootic lymphangitis, 281 C/ostridium tetani, 211, 213
d.istribution, 386 growth characteristics, 281- 282 Clostridium J1P.rfringe.ns TypP. B, ?.00
host response, 387 horses, 281 Coccidioides spp., 285, 287
intectivity for other species, 386 1mmunity, 282 coccidioidomycosis, 285, 287
lahoratory diagnosis, 387 laboratory diagnosis, 282 combined immunodeficient (CID), foal.
pathogcncsis, 386- 387 1norphology, 281 219
properties, 386 pathogenesis, 282 contaglous equlne metrltls (CEM),
reservoir, 386 pseudoglanders. 281 125- 1.26
resistance, 386 reservoir, 282 coronavirus, equine, 384, 391
~wi11e, 386 resislance, 282 Cushing's disease, 219
transnlission, 386 skin, 281 Oennatophilus, 220- 221
treat1nent, 387 staining, ?.81 rlPrm;1tnphyto!>is, ?.73-?.78
vomiting, 386 trans1nission, 282 eastern cquine encephalitis virus,
wasting, 386 treannent, 282 351- 353
hemagglutination inhibition test, 24 histoplasmosis, 289-292 .EEE. 351- 353
11emolysin (Actinobacillus), 92 HIV,110 Ehrlíchia risticii, see Z..leorickettsia risticii
he1nolysln (Esc/-zerichia co/1), 63 Hrns phenotype, 75, 76 Enterococcus durans, 166
hemolytic uremic syndrome (HUS), 66, 83 hockey puck, 175 Enterococcus hirae, 166
Hendra disease virus, 370, 372 hog cholera virus, 353, 359- 360 Enterococcus villorum, 166
Hepatovirus, 340 classical svvine fever, 359 enterotoxen1ia, 200-201
Herpesviridae (herpésviruses), 320-332 conjunctivitis, 359 epizootic lymphangitis, 281
herpPsvin1~ R, simian, 330 c.ontrol, 360 e.quine. (He.n ora) Morhíllívirus, .~70
herpesvirus, bovine, see bovinc hcr- diarrhca, 359 cquinc adcnovirus, 317- 318
pesvirus disease, 359 equine arteritis virus, 384, 394- 395
herpesvirus, canine, see canine her- • distribution. 359 equine coronavirus, 384, 391
pcsvirus host response, 359 equine herpesvirus, 320 323
hei:pesvirus, caprine, 326 lnfecttvlty for other spedes, 359 equine infectious anernia virus, 422-423
herpesvirus, equine, see equine her- Jaboratory diagnosis, 359 equine influenza virus, 363-364
pesvirus pathogenesis, 359 equine rhinitis A virus, 340, 342, 345
herpesvirus-1, feliue, 329- 330 properlies, 359 equine rhinitis 13 virus, 340, 342, 345
herpesvirus-1,2, ovine, 326 reservoir, 359 equine torovirus, 384, 393
high pathogenicity island, 75, 78, 79 skin Jesions, 359 .E.srherir.hia coli (F.PF.C), 6.'i
Highlands J virus, 351, 353 swine, 359 Escherichia coli (ETEC), 63
Histophilus, see Haernophilus, Histophilus transmission, 3~9 Esc/1erichia coli (invasive), 64
Histophilus ovis, see J-listophilus .~omni 1-re.arme.n t, .~60 eye, infectious agents, 484
llistoph ilus so11111i, 96, 97, 98 vomiting, 359 fistulous withers, 216
H"istoplasn1a capsulatum, 28.l- 282, horses Florida horse leech.es, 283
289- 292 Abiotrophia. 167 foal p n eumonia, 191
Histoplas1na capsulatum var. capsulatum, Actinobacillus arthritidis, 92 Francisella tularensis, 117-118
289- 292 Actinobacíllus equuli ssp. equulí, 92, 94 gas gangrene, 200
cellular products of medica! in terest, Actinobacillus equuli ssp. haemofytica., 92, gastrointestinal tract, infectious agents,
289 94 451
atlhe::si11, 289 Actinobacillus, 92, 94 gastrointestinal tract, norn1al flora, 449
calcium-binding protein (Cbp), 2':10. Actinornyces, Z16- Z1/ vetan virus, J~J, J~l
H antige.n, 290 adenovirus, equine, 317- 318 glanders, 113-114
iron, 290 African horscsickncss virus, 403 Granulicatalla, 167
M antigen, 290 anaerobes, obllgate, 193, 195 grease heel, 220- 221
melanin, 290 arteritis virus. 384, 394-395 Haematobius irritans, 177
phagolysosome acidification, 290 Aspergillus, 294- 297 he1norrhagic enteritis, 200
coutrol, 292 Ba~illus unlllfulis, 170, 173 Heudra viru~, et¡uiue, 370
dogs, 291 botulism, 209 herpesvirus, equine, 320-323
epiderniology, 291 Burkholderia mallei, 113-114 Histoplas1na capsulatum var. farcimi-
growth characteristics, 290 C'arnpylobacter lari, 134 nosutn, 281
laboratory diagnosis, 291- 292 Canadian horse pox, ·17/ infectious anemia virus, equine,
rnorphology, 289 Candida a/bicans, 271 422-423
pathogenesis, 290 291 CEM (contagious cquinc mctritis), influenza virus, cquinc, 363-364
reservolr, 290 125-126 integumentary system, tnfectious
structure. 289 cervical lymphadenitis, 216 agents, 464
transmission, 290 CID, foal, 219 kunker, 284
522 Indcx

horses (co11ti1111ed) WEE, 351-353 laboratory diagnosis, 318

Lawso11ia, 139 \oVest Nile virus, 3.)3, 3.).) pathogenes1s, 3J7-3l8
l .P(ltn~pir11 ~Provar. hratislava, 148-149 western equine enc:ephalitis virus, properties, 317
Leptospira scrovat. icterohaemorrhagiac, 351-353 rescrvoir, 317
148-149 host re.sponse to viral tnfectton, 302 reslstance, 317
Lcptospira serovar. pornona, 148-149 host-virus relationships, 301 trans1nission. 317
Listeria spp., 187 human coronavirus, 384 treatmen t, 318
lung, ln (ectious agents, 490 llurr1a11 paraiuflue::uza vi ru~, 370 i11feclious coryza, 98
lymphoid tissues, infectious agents, 439 human respiratory syncytial virus, 370 infectious ente ritis, 392
niain drain virus, 367 human spumavirus, 410 infectiotts hPm;itopoiPtic necrosis virus,
Miuo~po11u11 eq11inur11, 274, 275 human T-lymphotropic virus l, 410 378,382
Microsponu11 gypscurn, 274, 275 human torovirus, 384, .5Y.5 1nfecnous laryngotracheitis Virus,
moon blindness, 150-JSt humoral immunity, viral, 302, 303 331-332
Morbillivirus, equine (Hendra), 370 HU5 (hcmolytic urcmic syndromc), 66 chickens, 331
musculoskeletal sy~tem, infectious hypercalcemia, 293 control, 332
agents, 469 diseasc, 331
A.1ycobactcriun1 aviun1 ssp. aviurn, 226 lbaraki virus, 403 distribution, 331
Mycobacteriu1n bovis, 226 'IBR (infectious l>ovi11e:: rliinulraclt~ilb) hosl response, 332
Mycoplasrna spp., 241- 248 virus, 323 infectivity tor othcr species, 331
na vcl ill, 94 les, see Shigl!lla J;ihoratory diagnosis, :~52
NeuriU<ell~iu d~lilii, 255 ldaD adhesin, Bl pathogcncsis, 332
nervous system, infectious agents, 477 idoxuridine, 43 properrles, 331
J\locardia spp., 219 ilt>itl symhiont intracellularis, see reservoir, 331
normal flora, gastrointestinal tract, 449 La1vsonia resistance, 331
nutritionaily variant streptococc1, 167 1midazoles, 28, 31, 41, 42 rransmlsslon, 331
ohlig;itp an;iprohes, 195, 195 imipenem, 30 infectious pancreatic necrosis virus, 408
ocular systcm, infcctious agcnts, 484 immune response to extracellular infectious peritonitis virus, see feline
Pasceurella caballi, 85, 88 pathogens, 10, 11 inf1:ctious peritonitis virus
periodic ophthalmia, 150~151 immune response to infectlous agents, 6 infectious pododermatitis, 197
pigeon fever, 177 immune response to intracellular influenza virus, ¡ivi:in, sPP ;ivi;in infht<'n7.a
poi 1cvll, 216 palhogens, 10, 11 vlrus
•
Potornac horse fever, 255 immunodiffusion assay, 25 intlucnza vuus, equ1ne, see equine
Powassan virus, 353, 355 inununosuppression, vi r.1i l, 303 influenza virus
pscudoglanders, 281 Indiana virus, 378 influenz¡¡ virus, swinc, scc swinc
l'seucto1no11as aeruginosa, 123 infectious anemia, fellne, 248 influenza virus
pythio~i~. 7.81 infectious hronchitis virus, avian, see influenza, zoonotic significance, 366
l'ythit1111 ;,,sidiosum, 283 avian infectious innate immunity, 6
rabies virus, 377-380 bronchitiS virus bati:erioclns, 6
rain rot, 220-221 infectious bovine keratoconjunctivitis, complement proteins, 7
rain scald, 220-221 J?l defensins, 6
n:curn:ul uvcili~, 151 infeclious bovinc rhinol!acheitis (IBR), núcrocins, ú
reovirus, 398 323 natural killer cetl, 7
respiratory system, infectious agcnts, infectious bursal disease, 407-408 norm:il flora, 6
490 chickens, 407 organic acids, 6
rhinitis A virus, cquine, 340, 34L, 64!1 control, 408 phagocytes, 6
rhinitis R virus, equine, :~40, :~42, :~4.'i disease, 407 polymorphonuclear neutrophil .leuko-
!lhodococcus cqui, 190-191 distribution, 107 cyte (PMN), 6
rlngworm, 273- 278 ducks, 407 i11t~gru11, 39, 46
rotavirus, 404- 405 Gumboro disease, 407 bovine, 40
Sa/111011ella, 71-72 host response, 408 Enterobncterincl'nl', .'\R
Sc111llk.i Fu1c~l vi.tu~. 3.31, 3.33 uúcclivity for othcr $pccic$, 4-07 Sa/111011cl/a Typhimuriurn DT 10, 73,
skin, infeclious agents, 464 laboratory diagnosis, 408 104
sleepy foal disease, 94 pathogenesis, 407-408 Saln1011ella, 40, 73
strangles, 162 propcrtics, 407 integumcntary system, 458-467
Screpcococcus equi ssp. equi, 162 reservolr, 407 auti111 ic.:rul.Jial p1upe1 tics, 458
Strcptococcus equi ssp. zooepidemicus, 162 resistance, 407 flora, 459
'lay/ore/Ja asinigenitalis, 126 transmission, 407 infections, 459
1i1ylore//(I equi8er1iluli~, 125 t1caln1enl, 408 infectious agents and conditions,
tetanus, 211, 213 turkeys, 407 463-467
torovirus, cquine, 384, 393 infectious canine hepatitis virus, 317-318 Cllt~, 4n::\, 466
Ttil'/1upt1ytur1 equinu1n, 274, 275 canine adenovirus-1, 317 cattlc, 464, 467
tularemia, 117- 118 control, 318 dogs, 463, 466
Tyzzer's disease, 208-209 clisPa'I', 117 goats, 465
ulcerativc lymphangitis, 177 distribution, 317 horses, '164
urogenital system, infectJous agcnts, dogs, 317 poultry, 465
497 host response, 318 shccp, 465
vesicular i.1:omatitis Virus, 378, 380-381 infectivity for other species, 317 swine, 465
 lndex 523

integu1nentary system, bacteria! infec- kerion, 274 cattle, 149

tion , ·1 60 integumen tary system, ketocon azole, see irnidazoles cellular products of medica! interest,
fungal infcclion, 461 Kilharn 1a l vil us, 306 148
integumentary system, viral infection, Koch, Rober t, 3 cell wall, 148
459 Koch's p ostulates, 3 hP.rnolys.in, 148
inte1feron, 9, 12, 43, 48, 302, 304 Kunjin virus, 354, 355 con tr<>I, 152
intestinal spirochetosis, tri disease, 3:,4, 3;,;, dogs, 149
intestinP, in fPction, 4SS- 4Só distrihution, 354, 355 epidemiology, 149
intjmin, 63 p roperties, 354, 355 growth c h aracteristics, 1'18
intraceJJular pathogens, imrnune reservoir, 354, 355 h orses, 149
response, 10, 11 resistan ce, 354, 355 immunity, 150, 151
Inv, 78 tran smission , 354, 355 laboratory diagnosis, 151
Ipa, see Shigella kuuker, 283 1.UOOU bli11<lJ lt:~~. 150, 151
lridovirdae, 314 Kupffer cell, 8 morphology, 148
Jridovirus, 314 pathogenesis, 149
.in.in, 4, 57, 63 La Crosse vi (ttS, 367 periodic ophth aln1ia, 150, 151
Actinobacillus, 93 Jabile toxin, J::. coli, 61- 62 primates, 149
A '/U?rgi/111s, ?.9S lacta te dehydrogenase elevating virus, recurrent uveitis, 150, 151
aurochclin, 155 384,396 rcscr voir, 149
Bordetella, 102 Lageniaium, 282 resistancc, 149
Corynebacterium, 175 Lagos bat virus, 378 staining, 148
Enterococcus, 165 Lagovirus, 346 structure, 148
t.'l.U<..htlilJ, 22·3 la1ub dy " :nle1 y, 200 L1d1 l:s 111 i s::.iot1, i49
ferritin, 57 lamziekte, 211 treatment, 152
high p:.ith ogPn ic' ity isl::incl, 7S, 78, 79 l .angPrh;ins rc>ll, 8 v;i riah ility, 149
Histophilus, 96 Lnnsficld grouping, 165 lcukc1nla virus, bovinc, scc bovinc
Histoptas111a, 290 Lansfield, Rebecca, 165 leuken1ia virus
lactoferrin, 4, 57 laryngitis, 4 9 3 leukotoxin (Pasteurel/a, Mannhei1nia), 85
lvlannheimia, 96 laryngotracheitis, 493 limberneck, 211
f>vforaxella, 119 Law:;uniu inlrui·el/uluri:;, 134, 139- 140 liucu111y<.:iu, ~t:t:: liuco~auli<lt:~
Mycobacteriu111, 223 blue fox, 139 li n cosamides, 28, 33, 35
mycobactin, 223 cellular products of medica! interest, lipid A, see endotoxjn
Pusleurella, 86 • 139 lipop o lysaccharide b inding protein, 57
Pseudomonas, 122 adhesins, 139 Listeria, 185- 189
pyoch elin, 122 c:ell 1.v;ill, 139 ;ihortion, 187
Saln1onella, 70-71 Lsa, 139 CAMP rcaction, 188
Shigella, 82, 83 deer, 139 cattle, 187
sic.lPrnphon>s, S7, 6'.~. 70, 71, 7S dogs, 139 cellular products of medica] interest,
Staphylococcus, 155 cmus, 139 185
staphyloferrin, 155 epidemiology, 140 ActA, 185
transferrin, 4, 57 growth characteristics, 139 adhesins, 185
Ycrsínia, 75, 78, 79 h amsters, 139 internalin s, 185
isoxazolyl penlcilllns, see penicillins 139
lJU (St;::;, lecilh.inase, 185
itraconazole, see irrlidazoles immun ity, 140 listeriolys in (), 185
laboratory diagnosis, 140 phospholip;:isp l., 18S
]5 vaccine, 73 morphology, 134 chjnchillas, 187
Ja1nestown <..:anyon vin1s, 367 ostrich, 139 colesla\'\1', 187
Japanese PncPph;i li tis vi r us, .1S4 pathogenesis, 139 conjunctivitis, 187
disease, 354 prolifcrativc enteritis of swinc control, 189
distribut ion, 354 rabbits, 139 encephalltis, 187
properties, 354 reservoir, 139 epidemiolo¡,ry, 187
reservoi1·, 35.'f rodents, 139 goats, 187
resistance, 354 ~lai ni11¡;, 134 growth cha1acterislics, 185
trans1nission, 354 structure, 134 horses, 187
Johne's disease, 229 transmission, 139 hot dogs, 187
job nin, 232 treatrnent, 140 immunity, 187
jowl abscess, 162 wet tail (hamsters), 139 laboratory diagnosis, 18/
J11nopox virus, 1'.~4 1.crV. 75, 76, 78 l.ati n-style cheese, 187
LEE,63 livcr paté, 187
.K88 adhesin, 61 Lenei:virus, 410 milk, 187
K99 adhcsin, 61 Leporípoxvirus, 334 morphology, 185
kanamycin, see arninoglycosides lepr<>id granul<>n1a syndr<>me, canine, 233 pathogenesis, 186
Kauffu 1a11 -Wl lile sche1na, 69 lc:prosy, 233 rt:servoir, 186
kennelcough, 104, 319,375,388 leprosy, feline, 233 sh eep, 187
keratitis, 485 Leptospira, 148-152 staining, 185
ken ttoconjunctiv itis sicca, sulfon amides biochemical charact eristics, 149 structure, 185
and, 32 California sea lions, 149 transmission, 186
524 Index

Listeria (continued) dendritic cell, 10 reservoir, 330

treatment, 189 Histoplasrna, 281, 282 resistance, 330
variability, 185, l86 innate irnmunity, 6 turkcys, 330
listeriolysin O, 185 Kupffer cell, 8 mastitis, 156, 157, 163, 466, 467
liver abscess, 169, 194 Langerhans cell, 8, 10 Mbx, 119
louping ill virus, 351, 355 Listcria, 185, 186 Mbx, 119
dlsease, 354, 355 mesangeal cell, 8 M cel!, 71
distribu tion, 354, 355 Mycobacteriun1, 223- 233 Mycobacl'erium, 229, 230
proper ties, 354, 355 phagocyte, 6 Salmone/la., 71
rt::~t::rvui (1 354, 355 Rhuúucc1tcc1~, 190, 191 Sltigellu, 81, 82
resistance, 354, 355 Saln1onella, 71 Yersinia, 78, 79
transmission, 354, 355 Sporothri.x, 279, 280 lneasles vírns, 170
Lo'"cnstcin-Jcnscn mcdium, 224 Ycrsinia, 76 meleagrid herpcsvirus-1, 330
Lpf adl1esin, 69 maedi vtrus, see visna/maedi/progressive melioidosis, 113, 115
J.T enterotoxin, 61 - 62 pneumonia viruses meningitis, 479-481
lumpy jaw, 216 Main Drain vin1s, 367 mesangial ce11, 8
lumpy sl<in disease virus, 334, 337 Malassez.ia pachyderrnarts, 268- 270 meta.m oviruses, 375
lump y wool, 220 bioche1nical characteristics, 268 Mel'apneumovirus, 370
lung, see respiratory systcm cats, 269 methicillin, see penicil lins
Ly1ne disease, 129 control, 270 metllisazone, 43
lyn1ph nodes, infection, 444 dermatitis, 26~ metnt1s, 502
Lymphocystisvirus, 314 dogs, 269 metronidazole, see nitroimidazole
ly1nphocyte cpidcrniology, 269 Mgts, 70
Al1CC (antibody ctepenctent cellular growth charactenst1cs, 268 miconazole, see imidazoles
cytotoxirity), 9- 1?. immunity, 269 xnicrocins and innate immun\ty, 6
antibody dependent cellular cytotoxic- luboratory diagnosis, 269 Microsporum canis, 271, 275
1ty (ADCC), 9- 12 morphology, 268 Mtcrosporurn gallinae, 274, 275
B lyxnphocyte (cell), 10 otitis externa, 269 Microsporurn gypseum, 274 , 275
cytotoxicT lylnphocyte (cell), 9, 12, 48 pathogcncsis, 269 Microsporum nanum, 274, 275
gamma delta T lymphocyte (cell), 9 reservoir, 269 Middleb1ook 7H10 1nedlurn, 224
granzymes, 12 resistance, 268 milker:s nodules, 334, 335
innate immunity, 7,9 structure, 268 mínima! bactericida! concentration
inLerferon, 9, 12, 43, 48, 302, 304 transnlission, 269 (MDC), 36
natural killer cell (NK cell), 9, 10, 12 treatment, 270 mínima! inhibitory concentration (MlC),
NK re ll (nat111"al k illerrell) , 9, 10, 17. v;.i ri ahility, 7.68 .16
pcrforin, 9 Malassczia .spp., 268 mink enteritis virus, 306
T helper lymphocyte (cell), 9, 10, 12, 48 malignant catarrhal fever, 325 minute virus of canlnes, 306
T lymphocyte (cell), 9, 10 malignantedema,204.205 minute virus of m ice. 306
TNi; (tun1or necrosis factor), 9, 12, 1$ mamn1ary tumor virus, mouse, 110 Mokola virus, 378
nunor necrosis factor (TNF), 12 rnarnmillitis, bovine, 325 rnollicu tes, 241- 249
lymphogranuloma venereum, 236 Mannheimia leukotoxin, 85 Molluscipoxvin1s, 334
lyn1phoid organ virus, 384 Mannheirnia, see Pasteurella, Mannheirnia molluscun1 contagiosum virus, 334
ly111phoid tissuc, see circulatory syste111 111arine n1an1n1<lls n1onensin, 31
and lymphoid tissue Actinobacil/11s delphinicola, 92 monkey ll virus, 330
lysosomf', 7 At:tinohaf'illus .w :ntiaP., 9?. monkPypox virus, 334
lysozyme, 7 Actinomyces spp., 217 n1onobacta1n s, 30
Lyssavirus, 37 7 Bruce/la cetaceae, 106 monocyte, see macrophage
Br11cel/a pinnipediae, 106 monocytic ehrlichiosis, canin e, 253
MacConkey agar, 59 Helicobacter ceton1m, 142 rnoonblindness, 150, 151
n1aci:olides, 28, 33, 35 Leptospira scrovar. por11011a, 148, 149 Moraxe/la bovis, 119-121
absorption, 35 Nocardia spp., 220 biochemical characteristics, 119
antimicrobial activity, 35 phocine diste111per virus, 370, 372 cattle 121
' products of medica! interest,
cellular
dislcibu Lion, 35 Phocoenobacter uteri, 85
excretion, 35 Photobaderium da111sela, 90 l l9
norm;1 l flor;i, ~.'i Streptococcus phocae, J 63 adhesins, 119
rcsistancc., 35 Mar locus (Staphylococcus), 39, 46 capsule, 1.19
macrophage, 6, 8 Marburg virus, 376 cell wall, 119
Af)CC, 12 Marek's disease virus, 330- 331 iron, 119
antibody dependent cellular cytotox- chickens, 330 RTX toxin, 119
icity, 12 coutrol, 331 conlrol, 121
antigen presenting cell, 6, 10 disease, 330 epidenliology, 121
antigen processing cell, 6 , 10 distribµtion , 330 growth c.haracteristics, 119
Bacillus, 170, 171 host response, 331 infectious bovine keratoconjunctiviti:;
cell mediated immunity, 48 intectivity for other species, 330 (IEK), 12.1
r:nr.ddínides, 28.'\- 287 laboratory diagnosis, 331 laboratory diagnosis, 121
Corynebacterium pseudotubcrculosis, 175, pathogcncsis, 330- 331 morphology, 119
176 properties, 330 pathogenesis, 121
 Iuuex 525

reservoir, 121 horses, 226 bursltis, 241

resistance. 11.9 imn1unity to 1nycobacterial infections, cats, 241, 244, 245
staining, 119 223- 224 cattle, 241, 243
~l l U LlUtt:, 119 i.tl1.lllll11.ily, 227 cellular producls of 111cdic,1l inLercsL,
transrnission, 121 laboratory diagnosis, 228 240
tn»ltmi>nt, 1?.1 morphology, 22'.i pt>roxidt>, 240
variability, 119 mycobactcriaJ ulccrallvc ly1nphangitis supcroxidc, 240
Moraxella cytotoxin, U9 (hovine), 234 urease, 240
Moraxella spp., 119-121 pathogcncsis, 224-226 conjunctivitis, 241
Morbillivirus, equine, 370, 372 pri.n1ates, 226 contagious bovine pleuropneumonia,
n1orb11l1vlruses, 369 reservoir, 224 241
Mortierella, 297 resistance, 224 contagious caprine pleuropneun1onia,
mouse hepatitis virus, 384 sheep,226 241
!11.0USC 111.ilf)'l.lllllTY tu111()1. viT'Lt:S, 4 10 staining, 223 conttol, 243
nlouse pox Virus, 334 structure, 223 cysti tis, 241
moxalactam, see cephalosporins swine, 226 dogs, 241, 244
mucoid enteritis (rabbits)., 209 transmission, 224 cpi.dcrniology, 245
Mucor, 297 treannent, 228- 229 feline infectious anemia, 248
mud fever, 392 tuberculosis, 223. 224- 226 FeLV, 248
mtunps virus, 370 u lcerative dermatitis (rapid growers), FIV, 248
n1urine leuken1ia virus, 410 234 goals, 241, 244, 245
murine pneumonia vi.rus, 370 variahility, 224 growth characteristics, 241
m11 rini> sarc.oma virus, 410 ssp. avium, 2?.4
Myr.nhat:fPri111n avi11n1 horses, 241, 244
Murray Vallcy cnccphalitis virus, 353 Mycobacteriurn aviu111 ssp. hon1inissuis, 226 immunity, 245
musculosl<eletal system, 468- 473 Mycobacterium Ctvium ssp. parahiberculosis, Jaboratory diagnosis, 246
antin1ic:robial properties, 468 229- 233 mastitis, 241
infections, 468 cattle, 230 morphology, 240
iufectious ageuts a11tl co11tlitious, control, 232, 233 pathogenesls, 242
469-470 epidemiology, 230 pleuropneumonia, 241
cats, 469 goats, 230 pneumonia, 241
cattle, 469 growth characteristics, 229 poulLi y, 241, 243
ctogs, 469 hnmunily, 223-234, 231 reservoir, 242
goats, 469 • Johne's disease, 229 resistance, 242
horses, 469 laboratory diagnosis, 231 rodents, 241, 245
poultry, 4/0 morphology, 223 se1ninal vesi.culitis, 241
sheep,469 pathogenesis, 229- 230 sheep,241,244,245
:;winc, 470 rescrvoir, 229 staining, 240
mutattonal reststance, 38 sheep,230 swine, 241, 245
mutualism, 3 staining, 223 synovitis, 241
mycetonia, 21.9, 281 structure, 223 transmission, 242
lnycobacterial ulcerative lymphangitis, transrnissiou , 229 treatrnent, 248
bovinc, 233 treatment, 232 varia bility, 242
Mycobacterium. (not 1\lf. avium ssp. paratu- }.lfycobacterium a1d11rn ssp. silvaticum, 229 1nyeloblastosis virus, avian, 410
berculosis). 223-234 Mycobacteriun1 bovis, 224 n1yelocytomatosis virus, avian, 410
birds, 226 Mycobacteriurn chelonae-abscessus, 234 rnyeloperoxidase, 7
c;ininP lPproid g ran11loma syndroml', Myr.ohar.tPriurn flavP.w:ms, 2'.-\4 myoc.a rditis, 441
233 Mycobactcrium fortuiturn , 234 myxo1na virus, 334
cats, 226 Mycobacteriurn genavense, 227
cattle, 226 Mycobacteriurn haernophilurn, 227 nafciJUn, see penicillins
cellular products of medica! intercst, Mycobacterium leprae, 233 Nagler reaction, 202
223 lvfyc;obacterium leprae, 233 Nairobi sheep üisease virus, 367, 368
alkyl hydroperoxidase reductase Mycobacterium lepraemuriurn, 233 Nairovirus, 367, 368
(Ahp), 223 ,\lfycobacteriurn marin111n, 227 nalidixic acid, 31
dimycolyltrehalose (cord factor), 223 °A1ycobacteri11111 ph lei, 234 nasal cavily, Aspergillus, 296
exochelin, 223 1\ltycobacterium simiae, 227 nasal sinus, Aspergillus, 296
iron, 22'.i Myr.ohar.teríum smP3mati.~. ?.::\4 n<i tu ra 1 killer cell (NK cell), see lympho-
mycobactin, 223 Mycobactcrium thcnnoresistiblc, 234 cyte~
phosphatidyl inositol n1annoside, 223 Mycobacteriurn tuberculosis, 224 navel ill, 94
sulfolipids, 223 Mycobacteriurn ulcerans, 234 necrotic Jaryngitis, 493
surface 1nycosides, 223 Mycobact:eriu,.n xenopi, 234 necrotizing fasciitis, 163
waxes, 223 l11ycobacti11, 223 Neethl ng virus, 334, 337
control, 229 Mycoplas111a spp. (haemotrophic), 248 neomycin, see aminoglycosides
dogs, 226 Mycoplasma spp. (nonhaemotrophic), 241 neoplasia o f lymphatic tissue, 445
epidemiology, 227 Mycoplasrna, Ureaplas111a, 240-249 Neorickcttsia., 253, 255- 256
feline leprosy, 233 abscesses, 241 control, 255, 256
goats, 226 airsaccn litis, ?.41 dogs, 256
growth charactcristics, 224 arthritis, 241 Elokimin fluke fever, 255
526 Index

Neu1 itkt:ll~iu (cunlinut'd) dolphins, 220 shecp,485

horses, 255 epidemiology, 220 swine, 485
immuni ty, 255, 256 goats, 219 o lñ ñog Pn<"Pph;i l iti~, ~lí9
laboratory diagnosis, 255, 256 gro,v-th characteristics, 219 OmpA adhcsin, 61
path.ogeoesis, Z.'.l.'.l, Z.'.l6 horses, 219 Ondiri disease, 254
Potomac horse fever, 25.'i immunity, 220 001nycosis, 282-283
rcscrvoir, 255, 256 laboratory diagnosis, 220 oophoritis, 504
Salmon poisoni ng, 2 55 1nastltls, 219 upsonins, 7, 8, 11
transmission, 255, 256 morphology. 218 oral cavity, infeclion, 450-451
lreatment, 255, 256 mycetoma, 219 orbifloxacin, see fluoroquinoloncs
f\leoril*ettsia /1e/111inlhul:'cu, 255 11uca1uiufuu11 placcnlilis, 219 orbit, infection, 466
Neorickettsia risticii, 255 pathogenesis, 219 orbiviruses, 400-404
NeorickC!ltSia SC!lllLC!tsu, 255 rescrvoir, 219 orchiti~, .'íO~, .'í04
ncphritis, 498 resistance, 219 orf (contagious ccthyma of sheep), 331,
nervous syste m, 474-481 sheep, Z19 335
antimicrobi;il propi>rtiPs, 474 staining, 218 organ ic acids, 4, 6
infcclio ns, 474 structurc, 219 Orientia ts11tsugarnushi, 251
lnfectious agcnts and conditions, swine, 220 Or11ilhubulleriur11 rhinvlruch~ule, 99
476-478 tran smission. 219 ornithosis, 237
cats, 176 trea t1nent, 220 Orthon1)'xOviridr1e, 361-366
cattlc, 477 va ria\Jilil y, 219 m orphology, 361
dogs, 476 whales, 220 propert 1es, 36 t -363
goats, 478 nocardioforrn placenlitis, ?.19 ()rtJ1opoxvirus, 333, 334
liu1 ~t::., 477 nocardiosis, 218 Orthorcovirus (rcoviruses), 398 100
poultry, 478 normal tlora cats, 398
sheep, 478 rirr11latory system, 437 cattle, 398
swinc, 476 colonization rcsistuncc, 4, 6, 47 control, '100
ncutrophil, see polymorphonuclear digestive system, 446-450 dlsease, 398, 400
neutrophil leukocyte establishment, 6 d istribution, 399, 400
Newcastle diseasc virus, 373- 375 gastrointestinal tra ct, 449-450 dogs, 398
ch1<.:ke11s, 373 CdLLle, 449 gastrointestinal disease, 398, 400
•
control. 375 chicken, 449 horses, 3\:18
diarrhea, 373 dogs, 450 ho~t r<'~ponse, :199, 400
disease, 373 horses, 449 infectivity for othcr spccics, 398, 400
dislribution, 374 swine, 450 Jaboratory diagnos is, 399, 400
host response, 374 innate immunity, 6 p;ithogenesis, :{99, 400
infcctivity for other species, 374 inte¡,TUmentary systcm, 458 poullry, 398
JabOratory diagnosis, 3/4-J/.'.l lympho1d tissues, 437 primates, 398
neurological signs, 374 musculoskeletal system, 468 propertics,398. 400
pathogenesis, 371 nervous system, 474 reservoir, 399, ..JOO
propertles, 374 olular sy~-rem,482 re:.bla111..e, 398, 400
rcscrvoir. 374 respiratory system, 487-489 respiratory disease, 398, 400
resistan ce, 374 urogenital system, 496 rodents, 398
1 t:~¡ 1i1 al\11 y ubl1 "'"• 373 / \1ovi1'/1abdovi1·us, 377 jhecp, 3?0
transmission, j74 Nori.valk-like vi rus, 346 swine, 398
trl':i tm l'n t, .~7.'i novyilysin, 203 transmission, 399, 400
lurkcys, ~{7:{ n ucleic acid hybrídi zation , 17, 2'1 treatment, 400
new tluck tllscasc, 89 nurrl t lonally varlant Streptoc:uc:cu:;, 167 us1eu111yelilb, 468
New Jersey virus, 378 nystatin, see polycncs osteopetrosis (avian), 417
•
nitric oxide (NO), 9 otitis externa, 466
nitrofurans, 28, 31 O antigen, see so111alil.: <u1li~e11 ove1caling disease, 201
nitroimidazoles, 28, 31 0157:H7, 65- 66 ovinc adcnovirus, 318, 319
'.'!K cell (natural killer cell}, see lympho- obligate anaerobes, 193 ovine herpe~vir11~-l,?., ~26
1..yle~ obligate anaerobes, clucs to prcsence, 45 ovine progrcssivc pncumonia virus, scc
Nocardia, 218- 220 obligate anacrobes, see anaerobes maedi virus
Actinornyces, r.omp;i red wi th, ?.llí ocula r infections, 482-486 ovine pulmonary aclcnocarcinorna virus,
actinomycotic mycetoma, 219 antimicrobial propcrties, 182 410
l11ochcrn1ca1 characteristics, 219 flora, 483 oxat:illiu, ~ee ve11 ít:ill i11~
hovinc farcy, 219 infections. 483 oxidase test, 58
cuts, 219, 220 infectious agcnts and co nd itio ns,
cattle, 219 484- 485 P987 adhesin, (il
combincd imn1unodeficient (CID) foal. cats, 484 Paecilo111yces, Z':J'I
219 cattJe, 484 P" li~an<'~. 17.S
1..011llol, 220 dogs, 464 Palyum virus, 399, 403
Crossiella cqui, 219 goats, 48!'> panleukopenla, fcllne, see feline pan-
Cushing's disease, 7.19 horses, 484 lcukopenis virus
dogs, 219, 220 poultry, 485 papillomavirus, bovine, 315
 Index 527

papillornavirus, 315- 316 modes of dissen1ination, 301-302 Pie, see Shi.gella

papillomavirus, bovine, 315 persistence, 303-304 J>icornavirictae, 339-34~
papillom::ivin1s, 0q11in0, 316 v i rus-host rela tionsh ips, :~01 pi geon fever, 177
papular stomatitis virus, bovine, 334, 335 pathogenicity, 3 pig's ears, Sa/1nonella, 72
parainfluenza 3 virus, bovine, 3?0, 3?5 pathogenicity island, 62 pigeonpox virus, 334
pa rainfl11en7.a 'fype 2 virus, canine, 370, Escherichia co/i, 62, 6~ pilus, see adhesin
375 Hclicoba~tcr (Cag), 111 pinkcyc, 121
paratnfluenza virus, hurnan, 370 RhudulVC:lus, 190 pipl:!racilliu, Sl:!t: .[Jl:!Uicillins
Para1nyxoviridae, 369-375 Salmonella, 69 p izzle rot, 179
Parapoxvirus, 334 Shisella, 81 plague bacillus, 75
parasite, 3 Staphylococcus, 153, 155 plant awns (foxtails), 216
paratyphoid (poultry), /3 Yersinia, 75, 78, 7 9 plasmid, 46
Parvoviridae., 30.5- .309 PCR (polymi>rasP ch::iin reattion), 1.7, ?.4 plasminoger.i ac:-tiva tor, 75, 76
parvovirus Type 2, canine, see canine pectoral abscess, equine, 177 pleural effusion, 194
parvovírus Pef adhesin, 69 PMN, see polymorphonuclear neutrophil
parvovirus, bovine. see bovine parvovirus penicillin, 28-30 leukocyte
parvovirus, porcine, see porcine par- a bsorption, 2 9 P1nt, 86
V(JVi l US adve1 se cffects, 30 )JMTl, 75
Pasteurella dermonecrotic toxin, 86 antimicrobial activity, 29 Pneu1nocystis, 297
PastP.11rP.lla 1P11kotox in, 8.'i clistrib11tinn, ?.9 pneumonia, 489, 490, 491, 492, 493
Pasteurclla, Mannheimia, 84- 8'> cxcretion, 29 cats, 489
atrophic rhinitis, s'"'ine, 87 mechanism of action, 28 cattle, 490
avían cholera, 88 resistance, 29 dogs, 489
av;an, 88 penicillin binding proteins, 28, 155 goat s, 491
cattle, 87, 88 penlclllin G, see penlctlllns horses, 490
dog, cat, 88 penicillin V, see penicillins poultry, 492
cell products of medical interest Peptostrcptococcus spp., 193-197 sheep, 491
adhesins, 84 perfingolysin <), 200 swine, 491
capsule, 84 pertorin, see lymphocyte pneumonia, aspirat ion, 495
fihronectin hind ing p rotein, 84 perica rdia 1 eff11sion, 194 pn P11monic pl;ii'11P, 75, 76
hyaluronidasc, 86 pcricardiu111, infcction, 440, 441 P11cu1novíri11ae, 3 75
iron, 86 periodic ophthalmia, 150, 151 pneumoviruses, 375
Ieukotoxin, 85 •
periodontal disease (Shigella), 82, 83 poliomyelitis suum. 342
ncuraminidase, 86 perltoneal effusion, 191 poll evil, 108, 216
P1nt, 86 Perslan car, myceroma, 274 polyenes, 28, 31, 4 l - 42
Rho activating toxin. 86 pertactin, 100 polymerase chain reaction (PCR). 17
RTX toxin, SS pertussis toxin, 100 polymorphonuclear neutrophil leukocytc
tuxius, 85 peste des petilis fL11ninanLs virus, 370 (PMN), 6, 7
transferrin binding protein, 86 Pestivirus, 353, 356-360 antibody dependent cellular cytotoxic-
co1nposition, 84 pesticin, 75, 76 ity (ADCC), 12
control, 89 Pet (plasmid encoded toxin), 62 complement, 7
horse, 88 pgrn Jocus, 76 innat.e im1nunity, ó
goats, 88 phaeohyphomycosis, 28:~. 284 phagocyte, 6
growth charactcristics, 86 phagocytc, 6 polyomavirus, 316
human, 88 phagocytosis, 7 porcine aden ovirus, 318
immunology, 88 catalase, 7 porcine enteroviruses, 340. 342
laboratory d iagnosis, 89 compiement receptor (CR), 7, 11 control, 342
rnorpholugy, 84 Fe r.;cl:!pl.ur, 7, 8 <.lisei:l~I:!, 342
sheep, 88 halide ions, 7 host response, 342
pathogenes.is, 86 hydrogen peroxide, 7 laboratory diagnosis, 342
pueu111onia, 87, 88 inna Le ilnn1unily, 6 swine, 342
rabbit, 88 lysosome, 7 porcine epidemic diarrhea virus, 387
reservoir, 86 myeloperoxid<ise, 7 porcine parvovirus, 308- 309
resistance, 86 opsonin, 7, 11 control, 309
roaent, 88 pnago1ysosome, 7, s d1sease, 308
staining, 84 respiratory burst, 7 distribution, 308
structure, 84 superoxide dismutase, 7 host response, 308
tri:lu~u.1.b~iuu, 86 ~U.[Jl:!fUXÜ.11:! fi:IUicals, 7 infectivity for other :;pecies, 308
treatment, 89 phagolysosome, 7, 8 laboratory diagnosis, 308
variability, 86 pharrnacodynamic activity (antimicro- pathogenesis, 308
capsule serotype, 86 bials), 26 properties, 308
somatic serotype, 86 pharyngitis, 491 reproductive disorders (SMEDI),308
pathogenesis of viral diseases, 301-304 Phialophora, 283 rf'Sf'rvoi r, 308
host response, 302 Phlcboviru.$, 367 rcsistancc, 308
11nmunlty, 302- 303 phocine distemper, 370, 372 swine, 308
immunosuppression. 303 Photobacteriun1 damsela, 90 transmission, 308
interferon, 302 Phytophthora, 282 treatment, 309
528 Index

porcinc postwcaning multisystcmic wast- Clzlmnydophila, 237 propertics,354,355

ing syn<trome, 311 circulatory system, 1nfecttous agents, reservo1r, 354, 355
porcine reproductive and respiralory 442 rcsistancc, 354, 355
syndrome virus, 395 Clostridium botulinurn, 209, 210 transmission, 354, 355
con crol, 396 Clu:;tridium per(rir18ert:; Tyye A, ZOO l'u.xvi1idue, 333-338
tliscasc, 395 coronavirus, 384, 392-393 poxviruses, 333-338
distribution, 396 digestive system, infectious agents, 454 birds, 333
husL 11:sponse, 396 encephalomyelitis virus, 343-344 cats, 333
Jaboratory diagnosis, 396 i:,rysipelothrix spp., 182, 18.:S cattle, 333
myocarditis, 396 PYP, inffftious ag<'nts, 48.'\ chicken, 333
pa thogenesis, 396 fowl cholcra, 84, 85, 88 control, 336, 337, 338
pneumon1a, 396 fowlpox virus, 334 fowl, 333
rP~Prvoir, 39n Gallibacteri11111 anatis, 85 goats, 333
res pi ratory discasc, 396 gastrointestinal tract, iniectious agents, ho~"t-virus relationship, 333, 335, 336,
SMEDI, 396 454 337
swinc, 395, 396 gastrointestinal tract. norn1al flora, 449 laboratory diagnos is, 333, 335, 336, 337,
trQnsmission, 396 Haen1ophilus paragallinnrum, 98 338
trcaunent, 396 Helic:ubuc:ler pullu1111n, 142 n1onkeys, 333
porcine respiiatory corona virus, 383-386 infectious anemia v irus, 310-311 peacocl<s, 3;~3
contro l, 386 infectious bronchitis vlrtis, 384, prop&r ties, :1:1:1, :136
IJ' 1eu111onia, 383 391- 392 quail, 333
discasc, 383 infectious bursal d1 sease virus, 407- 408 rabbitS, 333
d istribution, 385 infectious laryngotrachcitis virus, 331 racoons, 333
host response, 385 influenza virus, 365 rodcnts, 333
intcctiv1ty for other spec1es, 385 integumenrary sysrem, Jnfectlous seals, 333
l;ihoratory diagnosis, 386 agents. 466 shecp, 333
pat hogenesis, 385 Jaryngotracheitis virus, 331 squirrel, 333
propertles, 384 ly111phctliL :.yslc1n, i1úeclious agents, swinc, 333
reservoir, 385 442 treatment, 3.:Só, 337, 338
rcsistance, 385 Marck's diseilse viri 1~, ~30-.~ 11 tu rkey, ::¡::¡::¡
swine, 383, 385 lvlicrosponun gallinae, 274, 275 pPCPl, 75, 76
transrni ssion, 385 musculos.keletal system, intectious J>l'lJ (punf1ed protein derivative), 228
lreal'1nent, 386 ¡igents, 470 PP!.(), see moll icutes
vo111itin¡;, 383 Mycobacteriu1n aviu111 ssp. avium, 227 primate lcn tiviruscs, 410
porcinc J<ubulc1vin1s, 3l ü Mycoplasn1a spp., 241-248 primates
porcine tcschovirus, 340, 342 nervous system, infcctious agents, 479 anthrax, 170, 173
cont rol, 3'13 Newcastle disease, 373 Bacil/11s nnlhracis, 170, 173
dtsease, 3~2 uunual flora, ga:.lrui11l1:::.li11ctl L1ctc.:l, 449 Bu1to11ella spp., 260-264
host response, 343 ocular system, infectious agents, 485 botulism, 209
laboratory diagnosis, 343 Ornithobncteriu111 rfiinotracl1eale, 99 (_'nn1pylobncter spp., 134-138
:.wiue, 342 Pasteurel/a anatis, 85 Clostridi11111 botulinum, 209
treatment, 343 Pasteare/la avi11tn, 85 C.:lostridi11"1 per(nngens, 199- 201
porcinc torovints, 384, 393 Pasteurella dagrnati~. RS í.lnstridiun1 píliforn1e, 208, 209
I'orphyro111011as spp., 193- 197 f>astcurclla sallinan11n, 85 C/ostridium s<·pticum, 205
postwcaning 1nultisystemic wasling syn- Pasteurel/a langaa, 85 Clostrldtum tetani, 211, 213
dro1ne, porcine, 311 Pasteurella rnultocída, 84, 85. 88 Corynebucleriutn pseudotuberculosis, 178
l'otomac horse fcver, 255 Pasteurella volantium, 85 Helicohncter bizzozeronii, 142
µoult ry 1~ovi1us, 398 I l elicobacter fteil111anii, 142
Arti11obacillus salpingitidis, 92 respiratory system, infectious agents, Helicobacter pylori, 142, 145
adenovirus, chicken , turkey, 318 492 Listprin (pp , 1R7
AnuúettLC!I "!JI'· 138 dlinotracheitis virus, 370 Mycobacteriu111 spp., 227
acizonosis, 74 Rien1erella anatipestifer, 89 obllgate anaerobes, 193, 195
Aspergil/11s spp., 294-297 rinewo rm , ?.73-?.7R reovirus, 398
avian cholcra, 84, 85, 88 Saltnonella spp., 73-74 Sal1nonella spp., 69 7'1
avían encephalomyeht1s VIrus, 343-344 s.ki.n, infecrious agenrs, 466 Shtgella spp., 81-83
avían leukosis virus, 417- 419 Staphylococcus aureus, 157 simian hcmorrhagic fever virus, 384,
avion influenza virus, 365 thrush, 271 397
avJan para1nyxovlrus, 370 Trithuphylun :.irnii, 274, 275 5jn1ian immunodcfi1..iency virus (STV),
Bordctclla avi111n, 104 turkey coronavirus, 384, 392-393 42~-426
Bonle~elia bronchiseptica, 104 turkey hepatitis virus, 340, 345 si mi;in typ<' n rí'trovirus, 416-417
1.Jut u li:.111, 209, 21 O turkey rhinotracheitis virus, 370 Staphylococcus aurcus, 154- 158
bu n1ble foot, 157 turkeypox virus, 334 Srrepcococcus pne11n1oniae, 163
c:nndida a/bicnns, 271 urog&nital system, infectious agents, Streptococcus pyogerzes, 160
chickcn adenovirus, 318 499 tetanus, 211, 213
c111c1'en 1nrecnous anemia virus, 3J0- Powassan vtrus, 354, 355 Tyu.c1 ':. \.li~t:Cl~t:, 208, 209
3 ll disease, 354, 355 Yersinía spp., 75-80
Clilnmydia, 237 distribution, 354, 355 prion, 477
 lndex 529

procine lype C: oncov in 1s, 41 O purified protein derivative (PPD), 228 red water d iscase, 204
progressive pneumonia virus, scc purpura hemorrhagica, 162 redmouth, 75, 80
visna/maedi/progressive pyelonephrltls, 499 Reoviritlue, 398-405
pneumonia vjruses pyelonephritis, bovine, 179 reovirus, see Orthoreovirus
p rophylactic use of antimicrobials, 47 pyochelin, 122 reproduct:ive and respiratory syndrome
prostatitis, 503 pyocln, 122 vjru~, ~ee po1ci11e

Prototheca, 297, 298 pyocyanin, 122 reproductive and respiratory syn-

1'$-C<)mpounds, 30 pyolysin O, 168 drome virus
Pseucluilc::schl-riu, 284, 297 pyoJnetra, 503 reptiles, Sailnonella, 72
pseudocO\'Vpox virus ot cattle, :~34, ::ss::. pytn1os1s, 28.:S resistance (arug), sce anttmicroblal arug
ps<>11<loglanclers, 279- 281 Pythium, 282-283 resistance
Pseudornonas, 122- 124 pYV, 75, 78 resistance plasmids (R p lasmids), 38, 39,
cattle, 123 46
cellular products of medica! interest, Q fever, 251 Actinobacillus, 93
122 quailpox virus, 334 Enterobacteriaceae, 58
a<.lhesins, 122 quinolones, see Jluoroquinolones Escherichia coli, 40
capsule, 122 Mannheirnia, 86
cell wall, 122 r;ihi<"S virus, :'177-380 norn1al flora, 47
iron, 1.22 cuts, 378 Past'eurella, 86
p1gments, 122 cattle, 378 Pseudo111011as, 123
pyochelin, 122 control, 380 Salmonella , 40, 73
pyocin, 122 disease, 377 Staphylococcus, 155
quoru1n sensing, 123 c.listril>utiuH, 378 Yer~iniu, 80
regulation of virulence determinants, dogs, 378 respiratory burst, see phagocytosis
122-123 foxes, 378 respira tory coronavirus, canine, 384
toxlns, 122 goats, 378 respicatory syncytia) virus, bovine, 370,
control, 123, 124 horses, 3/8 3'75
dogs, 17.:S host response, 379 respiratory syncytial virus, human, 370
epiden1iology, 123 infcctivity for othcr species, 378 respir:itory system, 187 195
gro~vth characteristics, J.23 laboratory diagn osis, 380 antJmtcrobtal properttes, 487
horscs, 123 neurological disease, 377 flora, 487
imn1unity, 123 pathogenesis, 379 infec;tions, 489
laboratory diagnosis, 123 • pruperties, 377 i11fecliou~ agenls and condilions,
n1orphology, 122 rabies, 377 489- 492
pathogenesis, 123 reservoir, 378 dogs, 489
reservoir, 123 resistance, 378 cat.s, 489
staining, 122 sheep,378 horses, 490
structure, 122 s kun ks, :'178 cattle, 490
lfans1nission, 123 swine, 378 shccp, 491
trcat1nent, 123, 124 transmission, 378 goats, 491
Pseudornonas aerugi nosa, 1?.::>. treatrnent, 380 swine, 491
pseudorabies virus, 326-328 R plasrnids, 38, 39, 46, 58, 73, 71 poultry, 492
al)ortion, :'126, 327 rabbit coronavirus, 384 respiruviruses, 375
Aujeszky's disease, 326 rabbits reticuloendotheliosis virus, avían, 410
cats, 326 Actinobacillus capsulatus, 92 Retroviridae, 409- 426
cattle, 326 Bo11letel/u. bronchisepth:a, 104 classification, 409
control, 327 Clostridium piliforme, 208- 209 components, 409- 412
diseasc, 326 ClostriditJrn spiroforrne., ?.0() ft>at11rf'$, 409
disltibulion, 326 coronavirus, 384 im1nunologic charactcrization, 413
dogs, 326 Franccsella tularensis, 117, 118 oncogenesis, 413-414
bost respon.~f', :S?.7 Lawsonia, 139 repllcation, 412- 413
infectivity for o thcr spccics, 327 Lcporipoxvirus (myxom:i virsu), 331 Rhabdoviridae, 377 382
laboratory diagnosis, 327 Mora.xella cunlculi, 120 rhln!tis A virus, equine, 340, 345
rneningoencephalitis, 326, 327 l'asteurella 1nultocida, 84. 85 rhinitis B virus, equine, 340, 345
patho genesis, 327 tularen1ia, 117, 118 rhinitis, 490
properties, 327 334
l'iiCO O llf)OX V it US, rhinopharyngilis, 491
pruritis, 326 raclioimmunoassay, 25 rhinopneumonitis, equine, 323
reservoir, 326 rain rot, 220 R11inosporidi11rn, 297
resistance, 327 Ranavirus, 314 rhinotracheiti.s virus, turkey, 370
sheep,326 rapid sporulatJon medium (T{SM), 276 rh1notraclleitis, 493
swine, :~26 rapidly growing mycobacteria, 234 rhinotracheitis, virus, feline, 329
transmission, 326 rat coronavirus, 381, 391 Rhinovirus, 310
psittacosis, 237 rational use of antlrnicrobia.ls, guidellnes r111novirus, bov.tne, 340, 345
pullorum disease, 73 for. 45 Rhizopus, 297
pulmonary adenocarcinoma vi.rus, ovine, fecurrent uveitis, 150, 151 Rho activating toxins, 62, 85, 86, 102
410 reu l>luuu ceUs, lufe1.:liuu, 443 Burcl1:LellcJ, 102
pulpy kidney disease, 201 red nose (IBR), 323 CNF, (Escherichia coli), 62
530 111dex

Rho activating toxins (continued) treatment, 373 RTX toxin, 63, 85, 92, 102, 119
Mannheirnia, 85 ulcerative sto1natitis, 372 Actinobacillus, 92
f'ct,\{CLll t:l/u 1 8(> 1 i 1lgV\1 \Jl 1t1, ~te dt:.1 11 1a LO~'Jl 1y l t::j Boalel~llu, l 02
l'rnt, 86 IZMSJ:'' 250
.
J:'.scherichia coli, 63
Rhodor.or.ciLs equi, 190- 192 Rocky Moun tain .s ported fever, 250 Mannheirnia, 8S
American nlligntor, 191 rodcnts lvforaxclla, 119
bioche1nical characteristics, 190 Acrinobacillus rnuris, 92 Pasteurella, 85
cellu lar producl's of1nedical interest, Bartonel/a spp., 260- 264 Rubulavirus, 370
1.90 Clostridiu1n piliformc, 208, 209 Rubulavirus, porcine, 370
capsule, 190 Enrerococcus durans, 166 rubulavlruses, 373- 375
tell wall, 190 Enterococcus hirae, 166 run1en, infection, 454
cholesterol oxidase, 190 Enterococcus 1•illorum, 166
phospholipctS<:: C, 190 Flc:xispiru (Hc:/iL(1/:JuGler), 142 Suc:c/11.uornyces /Joulardii, 83, 208
virulence associated proteins (Vaps), Francise/la tularensis, 117, 118 Shigella, 83
190 He/icobacter bilis, 145 Clostridi111n d iffir:ilP., ?.08
c.onlrol, 192 Ilelicobacter bizzozeronii, 142 salmolysin, 70
crocodiles, 191 Helico/Jacter c/10/ecystus, 14:l salmon poisoning, 255
epidenüolo¡;,y, 191 Helir.ohar.ter cinaedi, 142 Salmonella, 69- 74
growth characteristics, 190 Hclicobactcr ganrnani, 142 abortioo, 71
horses, 191 flelico/Jacter hepaticus, 145 artzonosi$ (avian}, 74
human, 191 Helicobac/er mesocricetorum, 142 aro A rnutant vaccine, 72, 73
iinmunity, 191 He/icobacter muridarun1, 142 cats, 72
laboratory diagnosis, 191, 192 Hel i<:ubucler rriuslc:luc:, 145 callle, 71
n1orphology, 190 Helicobacter rodentiurn, 142 cellular products ot medica! interest, 69
pathogenesis, 191 Helicobader trogontum, 142 Are, 70
pueu11 1v11ia (fual}, 191 Helicobacter typlilo11icus, 142 capsule (Vi), 69
reservoir, 1.91 lacta te dehydrogenase elevating virus, cell >vall, 69
resistance, 190 384, .396 effector proteins, 69- 70
staining, 190 Lawsonia, 139 cn tcrobactin, 71
structu re, 190 Leptospira serovar. grippotypllosa, 148, 149 en terotoxin, 70
sivine, 191 Leptospira serovar. icterohaemorrhagiae, iron, 70
transni.ission, 191 118, 119 M (:ell, 71
treatment, 192 1Vforaxella caviae, 120 neuropenia, 71
variability, 190 mouse hepatitis virus, 384, 390 pathogenicity islan ds (SPI), 69
Rickett.sia rickettsii, 250- 251 murine pneu1nonia virus, 370 PhoP/Ph oQ, 70
cvutrul, 251 J\tfyl<JjJ/u~rnu .\ f'f'·, 241-248 RpoS iegulaLion, 71
/)errnacentor, 251 Pasleurel7a pneumolropica, 85, 88 seru1n resistance, 71
dogs, 251 rat coronavirus, 384, 391 ShdA, 70
immunity, 251 reovirus, 398 SlyA, 70
laboratory d1a¡,rnos1s, 251. Senda1 virus, 370 stress proteins, 70
morphology 251 Streptococcus equi ssp. zooepidemicus, 163 type III secretion systen1, 69- 70
path<>gcnc!;i~, 251 tularcmia, 117, 118 virulence plasmids, 70
Rocky Mouutaiu spotted fever, 250 Ty;:zer's dbease, 208, 209 cvutrul, 73
staining, 251 Yersinia pestis, 76 diarrhea, 71
ticks, 251 Yersinia pseudotuberculosis, 78 dogs, 72
treatn1ent, 251 Rornanovsky-typc stain, 16 DT 104, 72, 73
J<ickettsia spp. , ;¿:,o, 2!>1 Xon1virus, Jl'.$4 En tentidis phage type 4, 72
Riernerello anatipestifer, 89 rotavi n JSPS ( Rotavi rus), 404-406 fowl typhoi d, 73- 74
rifan1pin, 28, 32 cattle, 404, 405 horscs, 71- 72
Rift Valley fever virus, 367 control, 406 immunity, 72
rinderpest virus, 370, 372- 373 diarrhea, 404 J5, 73
buffalo, 372 disease, 101 laboratory diagnosis, 72
catt le, 372 distrlbutJon, 405 uornt:uclature, 69
control, 373 enteritis, 404 paratyphoid, 73
diarrhea, 372 horses, 404, 405 pathogenesis, 71
Llisease, 372 host response, 405 pig's ears, 72
distribution, 372- 373 intectivity tor other species, 40!> pneumon1a, 71
host response, 37.3 lahor;itory di agnosis, 40S- 406 poultry, 73
infectivily for other species, 372 pathogcncsis, 405 pullorum disease, 73
laboratory diagnosis, 373 properties, 405 reptiles, 72
pathogenesis, 373 reservoir, 405 rcservoir, 70
propcrtics, 372 sheep,101,105 septicemia, 71
reservoir, 372-373 swine, 404, 405 ~heep, 71
resístance, 372 transnlission, 405 structuce, 69
sheep, 372 treat1nent, 406 swine, 71
:,w i11e, 372 Rous sarcon1a virus, 4 10 t.ransmission, 70
transrnission, 372- 373 RSM (rapid sporulation n1edilun), 2/6 treatment, 73

Salrnone/la aroA mut ant vaccine, 72
Salmonel/a culture characteristics, 59- 60
Sulrno11clla .En teritidis p h age type 4, 72
Salmoneila genomic island 1, 40, 13
Saltnonella pathogPnicity i.~lanrls (SPI), 69
Sal!none/la Typhimu rium DT 104, 40, 72,
73
salpingitis, 504
sa1nple collection, 15
sarnple tra n sport, 15
Sapporo-li ke v irus, 346
saprophyte, 3
sarafloxacin, see fluoroquinolones
sarcoid, equine, 316
SA RS (sPVPrP ac:11l'P respiratory syndrom e)
coronavirus, 384
SgE (sporadic bovtnc encephalomyelltis),
237
Scedosporiun1, 284, 297
sc1apie cigent, 427- 431
control, 431
disease, 47.7-47.8
gciats, 427
host genct1c polymorphisms, 429
laboratory diagnosis, 430- 431
pathogcncsis, 429- 430
prion strains, 429
properties, 428 - 429
sheep,427
llt:dlUlt:lll, 431
sealpox virus, 334
sccretory IgA, see antibody
selenite broth , 60
seminal vesiculitis, ~03
SPm li ki Fo rPst vi rus, :i.S-1, :ts3
sen, 81
sepsis, 438
septicemia, 438
scpticolysin (), 205
Serpullna, see Brachyspira
sertun resistance
Escherichia coli, 64
Salrnunellu, 71
Yersinia, 76, 78, 80
seru1n virus neutralization test, 24
severe acute respiratory syndrome coron-
a vir us ()Al{)), 384
" ' xn;i lly t ransm itt ed disease (chlamydio-
sis, h uman), 236
ShdA, 70
sheep, goats
Abiotrophia , 167
Actinobacillus lignieresii, 9 2, 94
Actinohaciilus seminis, 92
Ar.tinomyr.P.~ .~pp. ,
216-217
adenovirus, ovinc, 318- 319
Atrican lleartwater disease, 2~4
anaerohes, ohligate, 193, 195
Anaplasrna phagoGytophilum, 259
ant hrax, 170, 172
Arcano/Jacterium pyoxenes, 169
arthritis encephalitis vints, caprine,
421- 422
Hacillus anthracis, 1/0, 1/2
highParl, 20:~
black discasc, 203
bluetongue Virus, 401
Index 531
horder disease virus, 353, 358 Mannfleimia varigena, 85
botulism, 209 melioi cio.~i.~, ns
bradsot, 205 Moraxe//r¡ boevrei, 120
braxy, 20~ Moraxel/a caprae, 120
Bruce/la melitensis, 106 Moraxella ovis, 120
Bruce/la ovis, 106 muscu loskeletal system, infectious
Burkholderia pseudomallet, 115 agenrs, 469
Cache Vallcy virus, 367 Mycobacterium avium ssp. paratuber-
Campylobacter spp., 136 culosis, 230
capt ine a1th1ilis encephcililis vir us, 1vfycobaLT.eriurn bovis, 226
421- 422 Mycoplasrna spp., 241-248
caseous lymphadenitis, 178 Nairohi .~hPPp cliS<'ilS<' vints, 367, 368
Chlarnydophila pecoru1n, 237 Nairovirus, 367, 368
circling d1sease, 187 nervous system, infectious agents, 478
circulato ry system, in fectious agents, Nocardia spp., 219
440 nutritionally variant. streptococci, 1.67
ClosrrldJun1 /Jorul/nurn, 209- 210 obllgate anaerobes, 193, 195
Clostridium chauvoei, 206 ocular system, infectious agents, 485
Clostridium novyi Type A, 203 overeating disease, 201
Clostridiurn novyi Type B, 203 ovine adenovirus, 318- 319
Clostridiun1 perfi'ingens Type A, 200 l'asteureila multocida, 8 4, 85, 88
<:Jnslridiunz pP.r(iingP.ns TypP R, 200-?.01 PastP.urP.lla trP.halosi, 84, RS, 88
Clostridium perfringens Typc C, 201 peste des petits ruminants virus, 370
Closrriáiurn per(ringens Type r>, 201 Phlebovirus, 367
Clostridiun1 septicurn, 205 pizzle rot, 179
C/ostridiun1 sordcllii, 209 poxvirus, 334
C/osrridium cetani, 211, 213 progrcssive pneumonia virus, 421-422
Coccidioides spp.. 285. 287 pulpy kid ney disease. 201
coccid ioidon1ycosis, 285, 287 Q fever, 251
C(11 yri~}JL-4.(..lt.:.t ittr11 fJ~')tud,Jli1l1c1 c...ul«.1~i.>, 178 1abi.es v i1 u~, 377- 380
Corynebacterium renale group, 178- 179 reovirus, 398
COJfi!?.lla hurnP.tii, ?..Sl - ?.S?. rPspir;ito ry sys tt>rn, infe.c:t io11s agents,
· dermat ophilosis, 220-221 491
Dermatophi/us, 220- 221 Rift Valley fever virus, 367
Dichelobacter nodosus, 195- 197 rinderpest virus, 370
Ehrlichia ruminantiurn, 251 ringworn1, 275
enreroroxemla, 200- 201 rotavirus, 404, 405
Erysipelothrix, J 82 Salrnon.e/la spp., 71
Escherichia coli (EPEC), 65 scrapie agent, 427- 431
Esclu:ric:hiu luli (ETEC), 63 skin, infeclious agen ts, 465
Escherichia coli (invasive), 64 Staphylococcus aureus, 156
eye, infectious agents, 485 stiff la1n b disease, 237
Flexispira (llelicobacter) rappini, 142, 145 strawberry foot.rot, 220- 221
focal symmetrica l encephalomalacia, struck, 201
201 tPt!lDlJS, ?.11, ?.l .~
foot and mouth disease virus, 339- 342 tick pycmia, 156
footrot, 195 ticl<-borne fever, 259
Francíse/la tularensis. 117-118 tularemia, 117- 118
gastroin testinal tract, infectious agents, urogenital system, in.fectious agents,
453 498
G ranuJicatella, 167 visna virus, 421-422
l·Telicohn cter rnppini, 142, 145 Wessclsbron virus, 353, 355
1Iistophilus so1n11i, 98 '"'ooden tonguc, 94
integu1nen ta ry system, inJ:ectious yellow lamb disease, ZOO
agen ts, 46.S yellows, 200
Johnc's discasc, 230 Ycrsinia cntcrocolitica, 79
lan1b dysentery, 200 Yersinia pseudotu/Jerculosis, 78- 79
Listeria spp., 187 sh eeppox virus, 334, 336
louping ill virus, 353, 355 ShE1' 1, 8 1
lumpy jaw, 216- 217 shiga (and shiga -lll<e) roxin, 62, 81
lumpy W()OI, 220- 221 Shigella, 81- 83
lung, in fectious agents, 491 cellular ptoducts of medica! jnterest, 81
lymphoid system, infectious agents, adh esins, 81
440 cell wall, 81
fvfannhP.imia granuln1natis, 8.S PntPro toxins, 81-8?.
Mannhcimia haemolytica, 84, 85, 88 invasion plas1nid, 81
Mannheimia ruminalis, 85 invasion proteins (Ipa), 81
532 lndex

Shigella (continued) laboratory diagnosis, 416 biochemical characteristics, 155

path og<'nicity islands (Shi-0, Shi- 1)), p;ithogenesi.s, 416 bumblefoot, 157
81 primates, 416 cattle, 15 6- 157
regulatory genes, 82 properties, 416 cats, 1S6
shiga toxin, 81 reservoir, 416 cellular products of 1nedical interest, 153
type !11 secretion system, 81 transmission, 116 adhesins, 153
colonization resbtance, 82 treatment, 417 alpha toxJn, 154
control, 83 simian virus 5, 370 aurochel in, l55
diarrhea, 82 Sindbis virus, 353 beta toxin, 154
dysentery, 82 sinusllis, 490 capsule, 153
epiderniology, 83 skin, see integumentary system cell wall, 153
immun ity, 81 sleepy fo;i 1clise;ise, 94 cielt;i toxin, l 'i.'i
laboratory diagnosis, 83 SLT, 62,81 ente rotoxin, 153
morphology, 81 Sly, 70 exfoliative toxin, 153
pathogenesis, 82- 82 SMEDI, 306, 308, 396 ga1n1na toxin, 154
primates, 81, 82 snowshoe hare virus, 367 hemolytic toxin, 151
reservoir, 82 so1natlc (O ) antlgen, 57 tron, 155
staining, 81 Sop. 70 leukocidin , 155
structure, 81 soremuzzle, 401 MSCRAMMs, 153
lla11~ 1 11i)siun, 82 spanowpox virus, 334 111ultiple peptide resistance factor, 155
treatment, 83 specünen handling, 15, 20 Pan ton-Vatentine toxin, i:i:i
variability, 82 spec:rinon1yc:in, see ;iminocyclitols pyrog<'nic toxin, ts :~
Shigella boydii, 81 spherulin, 285 regulation ofvirulcncc dctcrm inants,
Shigella culture characteristics, 59- 60 spHan1yc1n, see macrolides 155
Sh(<?el7a dysenteriae, 81 sporadic bovine enceph alomyelitis (SBE), SCCcap, 153
Shigclla flcxncri, 81 237 SCC1nec, 155
Shigella sonnei, 81 sporotrichosis, 279- 281 staphyloferrin, 155
Shi-0, 81 Sporothrix schenckii, 279- 281 toxic shock syndron1e toxi n, 153
shipping fcvcr, 87, 97, 243, 323, 375, 387 cats, 281 coagulase, 75, 153, 1.55
Siucienuviru:;, 318 celluli:lr vrouucL~ uf 1uecJici:ll iuleres t, cuulrvl, 158
sialodacryade11itis virus, 384, 391 279 derruati tis, 156
siderophores, 57, 63, 70, 71, 75, 79, 82, adhesins, 279 dogs, 156
122, 155, 223 cell wall, 279 epideroiology, 157
aerobactin, 63, 82 n1elanin, 27') exudative dern1atitis, 157
a11rnchC'lin, l 'i'i pi>ptidP-rh;imnnmann;in, 780 go;its, 1.56- 1.57
cntcrobactin, 71 sialic acid, 280 greasy pig disease, 157
exocheli n, 223 control, 281 gro~vth cha racteristics, 155
mycobactin, 223 dogs, 281 horses, 157
pyochelin, 155 epidemiology, 281 imn1unity, 157
~laphylofeniu, J55 growtll cllaracteristics, 279 laboratory diagn osis, 158
yersiniabactin, 75, 78, 79 horses, 281 mastitis, 156, 157
SigA, see Shigella immunity, 281 morphology, 153
simian AIDS-related virus, 416-417 laboratory diagnosis, 281 osteon1yeliris, 156, 15 7
simian enterovirus, 340 morphology, 27') otitis externa, 1S6, 157
sirn ian foa1ny virus, 410 path oge nesis, 280 pa t hogt>nf'sis, 1.'i 6
sin1ian hcrnorrhagic fcvcr virus, 384, 397 prima tes, 2 81 rcscrvoir, 156
simian hcrpcsvirus B, 330 reservoir, 280 resista n ce 155
sirnian immunodeficiency virus, 425- 426 staining, 279 ruminants, ' 156- 157
pritnates, 425, 4 2 6 structure, 279 sheep, 156- 157
disease, 425 transnlission, 280 staining, 153
properties, 425 treatment, 281 structure, 153
infec:tivity for o ther species, 426 variability, 7.79- 780 s'.vinf>, 1'i7
distribution, 426 Spring viremia of carp vi rus, 378, 382 transmission, 156
reservoir, 426 Spu1navirus, 410 treatment, l58
t ra nsmission, 426 spu1navirus, hu1nan, 410 variability, 155
pathogcncsis, 426 Spv plasmid, 70, 71 Staphy/ococcus aurcus, 153, 156
host response, 426 squirrel monl<ey retrovlrus, 410 Stapflylococcus hyicus, 15 3, 157
laboratory diagnosis, 426 squirrel parapoxvirus, 334 Staphylococcus inl'ermedius, 153, 156
treat1nent, 426 Ssa, 70 Staphylococcus schleiferi ssp. coagulans, 153,
control, 426 s~e, 70 156
simian T-lymphotropic virus, 410 Ssp, 70 staph yloferrin, 155
sin1ian type D retrovirus, 416- 417 ST enterotoxin, 61- 62 starlingpox virus, 334
control, 417 St, Louis encephalitis virus, 353 stiff Jamb disease, 237
d1sease, 4lo stat)te tox1 n, c. cot1, hl - hL. stomach, infecnon, 454
distribu tion, 416 Staphylococcus, 153- 158 stones, bladder, 500
host response, 116 abscess, 156, 157 strangles, 162
infectivity for other species, 416 avían, 157 strawllerry fuv ln.>l, 220
 lndex 533

Srrepcnbacillus tnonllifonnis, 222 streptomycin, sce aminoglycosides Francisella tularensis, 117, 118
Stteptococcus, 159-165 stress, 47, 70 g¡¡strointestinal tract, infectious agents,
alpha hemolysis, 159 string of pcarls test, 173 453
l.Jeta heu1vlysb, 159 SlTUCk, 201 gastromtestina l tract, normal tlora, 450
biochemical characteristics, 161 suilysin, 162 Getah virus, 351, 353
birds, 160 .'>uipoxvirus, 334, 337 Hclicobactcr suis, 142
CAMP test, 164 sulbat:Lau1, 29 hemaggluttnatl ng encephalomyeht1s
cats, 160 sulfonamides, 28, 32 virus. 384, 386
cattle, 160 ;ibsorplion, 32 hog cholera vi rus, 353, 359
ccllular products of medica! interest, ad verse effects, 32 iuflueu:i:a, 364-365
l59 antimicrobial activity, 32 integumentary system, in fectious
adhesins, 159 distrihution, ~2 agents, 465
capsule, 159 cxcrction, 32 intestinal spirochetosis, 133
cell wall, 159 resistan ce, 3 2 Japanese encephalitis virus, 353
M protein, 159 sulfur granule, 217 La1vsonia, 139-140
VIS<:'.RAMMs, 159 summer mastitis, 169 teptospira scrovar. bratislava, 148-14?
pyrogenic toxins, 159- 160 superoxide radicals, sce pl1a¡;vt:ylv~i~ Leptospira serovar. canicola, 148-149
streptolysins, 160 swamp cancer, 28:~ Leptospira serovar. grippotypllosa,
suilysin, 162 swa1n p fev~r, 4?.?. 148-149
control, 164 swinc Leptospira se1ova1. icte1ohuernurrhugiue,
uugs, 160 Acttnobacillus indoticus, 92 148- 149
epiden1iolO,l,'Y, 163 Actínobacillus "1i11or, 92 T.eptnspira ~Prnvar. pomona, 148-149
gamma hemolysis, 159 Actínobacill11s ple11ropneun1oniac, 91- 94 lung, infectious agents, infectious
goats, 160 ALliriubacillus porcinus, 92 agents, 491
gro,vth charactcristics, 160 Actinobacillus rossii, 92 Jymphalic tissuc, infec:tious agents, 441
h orses, 160 Actinobaci/lus suis, 92 Mannhei111ia varigena, 85
.1mn11.1n1ty,
. 16?) .., Acti110111yces spp., 216, 217 Mic.Tuspuru111 nanum, 274, 275
¡owl absccss, 162 aden ovirus, jl8, :~lY m usculoskclctal system , infectious
laboratory diagnosis, 163 A frican swi n e fever virus, 312-314 agents, 470
mastitis, 163 anthrax, 170, 173 MycobuLie1 iurn avhtnt ssp. avium, 226
morphology, 159 Arcobacter spp., 138, 139 Mycobacteri11n1 bovis, 226
necrotizlng fasciitis, 163 Asfivin1s, 312-314 Mycoplasrna spp., 241- 248
pathogenesis, 162 • atrophic rhinitis, 87, 88, 103, 104 nervous systcm, infectious agenls, 478
pueurnonia, 162 Bacillus anchracls, 170, 173 t\locardia spp., 220
poultry, 160 benign enzootic paresis, 342 normal flora, ga.~troint<>sti n;il tritCt, 4.50
prlm<'ltP.S, 160 f?ordatalla bronchi.~optica, 103 o c ular oyatcm, i11fcctioua ngcnt3, 485
purpura hen1orrhagica, 162 B111clzyspira /1yodysente1 iue, 131-133 parvovirus, 306, 308, 309
reservou, 162 Hrachyspira innocens, 131, 132 Pasteurella aerogenes, 85
resistance, ·161 Tlrarhyspira pilosicoli, 131, 132 Pastcurclla nu1iri, 85
rodcnts, 160 Bn,cella suis, 106 l'asteurclla r1111ltociua, 84, 85, 88
ruminants, 160 Can1pylobactcr coli, 134 poliomyelitis suum, 342
sheep, 160 r.andida albicnns, 271 porcine adenovi rus, 318, 319
staining, 159 circulatory system, infectious agents, porcine epiden1ic dia11hea vil u~, 384,
strangles, 162 441 387
structure. 159 c:lassical swine fever virus. 353, 359 porcinP parvovirn~ ..~Ol'i , :~OR, '{()()
swine, 160 C /ostridi1nn perfringens Type C, 201 porcine rcproductivc and rcspiratory
transmission, 162 Clostriuiurn teturti, 211, 213 syndrome vtrus, 384, 395-396
treatment, 163 Coccidioides spp., 285, 287 porcine respiratory coronavirus, 384,
variahility, 161 coronavirus, rcspiratory, 384, 383-386 383-386
Str,·ptococcus agalactiac, 160, 162 dermatophytosis, 273- 278 po1cine Ruúuluvirus, 370
Srreprococcus ca11is, 160, 163 d1amond skln discase, 182 poxvirus, 334
Streptococcus didelpllis, 163 digestive system, inf~c.r-ious agi>nts, 45~ pseudorabics virus, 326- 328
Streptococcus dysgalactiae ssp. dysgalactiae, eastern cquinc cnccphalitis virus, rabies virus, 377-380
160, 162 351-353 reovtrus, 398
Streptococcus dysxalactiae ssp. equisimi/is, edem a disease, 66 respiratory c:oro navir11s, ~84, 38~-.:lR6
160, 162 EEE,351-35 3 rcspiratory systcm, infectiou~ agents,
Strcptococcus cqui ssp. equi, 160, 162 Et1terocOLLL1:> cluran:,, 163 491
StTeptococcus equ1 ssp. zooepiden1icus, 160, Enterococcus /1irae, 163 Rhodococa1s equi, 190- 191
162 Enterococc11s villon1m, 163 ringworm, 273 278
Strcptococc11s iniac, 163 enterotoxemia, 201 rvlaviru~, 404, 405
Srreprococcus pl1ocae, 163 cpidemic diarrhea virus, 384, 387 Rubulavirus, 370
Streplococcus pne11111oniae, 160, 163 F.rysipelnthrix, 1R/. skin, infectious agents, 465
Streptococcus porcinus, 160, 162 Escherichia coli (EAggEC), 64 SMEDI, 306, 308, 396
SLreplucuc:t11s pyu¿;enes, 160 Escherichia coli (EPEC), 6.'.> Streptococcus porcinus, 162
Streptococcus suis, 160, 162 Escherichia coli (ETEC). 63 .'\trpptnrnrr11c c11ic, 16?
StrPptococcus 1Jberis, 160, 163 eye, infectious agcnt s, 485 swincdyscntcry, 131 - 133
streptolysin O, 160 fool and 111vull1 uiscasc virus, 339-342 swine influenza, 364- 365
534 lndex

swlne (continued) -ieschen disease, 342 transmissible spongiform encephalo-

swine vesicular disease, :~4:~ Tescfznvirus, :~40 pat h ies, 427-434
Talfan di:;ca!;c, 342 tcschovirus, porcinc, scc porcinc transport media, 1S
Teschen clisease, 342 teschovirus transport of samples, 15
tetanus, 211, 213 tetanolysin, 212 transposons. 39
TGE, 384, 383 tetanus, 211 Trichophyton equinum, 274, 275
tra11s111is~i1Jle gastrueuteritb viru:. tetauu~ tuxiu, 212 7 rkhuphylufl rflenlug1o¡;hytes, 274, 275
(TGE), 384, 383 tetracycline, 28, 33 T1ichophyl'on sin1ii, 274, 275
tularem ia, 117, 118 adverse effects, 33 Trichophyton vern1cos11m, 274, 275
urogel1ita l systen1, infectious agents, 498 antimicrobial activity, 33 trimethoprim-sulfonalnides, 28, 32
vesicular disease, j 4 j Clostric/iu1n diftlcile, 33 absorptíon, d1stnbution, elimination,
vc>sir11lar stomatiti.~ vi rus, ::\78, ::\80-381 normal flora, :~3 33
Yersinia e'11terocolitica, 79, 80 rcsistancc, 33 advcrse effects, 33
Yersinia pseudotu/Jerculosis, 78 Salmonella, 33 antimlcroblal acrtvJty, 32
swine dysentery, 131. 132 TG.E vi rus, see t ransmissible gastroenteri- normal flora. 33
s,.,íne influenza virus, 36'1 365 tis virus resistance, 3 3
cuutrul, 365 Theilovi1us, 3 40 tubercle, 225
disease, 364 thiena1nycin, 30 tuberculin test, 228
laboratory diagnosis, 365 t hromboen1bolic: m en ingoPncPp ha litis t11he.rc:ulosis, 223- 229
pathogenesis, 364 (fLME), 98 tularcm.ia, 117, 118
resp iratory tra ct disease, 364 thrush, 271 tumor necrosis factor, see ly1nphocytes
s~"i'ine, :~64 tiamulin, see n1acrolictes turkey adenovirus, 318
trcatmcnt, 365 tick pyemia, 156 turkey coronavirus, 392-393
swinepox virus, 334, 337 tick-borne fever, 259 1Jluecu1111J dbt:ase, 392
S\.vine vesicular disease, 343 tinea, see dermatophytosis control, 393
con trol, 3'13 Tir, 63 disease, 392
dtsease, 343 Tly, 131 disLribution, 392
host-virus relationship, 343 1'-lymphotropic virus 1, human, 410 enteritis, 392
la boratory diagnosis, 343 T-lymphotropic virus, sirnian, 410 11ost response>, 393
swine, 343 tobramycin, see aminoglycosides infectious enteritis, 392
treatment, 343 Togaviridae, 351- 353 intes:tivity tor other species, 392
symbiosis, 3 Toll-like receptors, 9 J.;ibor!t tnry oiagnosis, :~9:{
syncytial virus, bovine, 410 bacteria! DNA, 9 mud fever, 392
syncytial virus, telinc, 410 bacterial flagella, ':J pathogenes1s, 393
synovif'i.~. 471 inflammation, 10 properties, 392
innatc immunity, 9 rcservoir, 392- 393
T cell, see lymphocytes virus recognition, 10 transmlsslble enteritis, 392
Talfan diseasc, 342 Torovirus , 393 transmission, 392- 393
Taylorella, 125-127 torovirus, bovine, 384, 393 treatment, 393
cell ula1 p1 oducts of 111edical in Le1 es L, LOH'lvi1us, equine, 384, 393 Lu1keys, 392
125 torovirus, h uman, 384, 393 turkey coryza, 104
capsule, 125 torovirus, porcine, 384, 393 turkey hepatitis virus, 340, 34.S
cell wa ll, 125 toxic shock syndrome (dog), 163 control, 345
contagious equtne metritis (CEM), lZ~ toxoid, 48, ~z disease, 345
control, 126 t racheal colonization factor, 100 host-virus relationship, 345
cpidemiology, 126 tracheal cytotoxin, 102 laboratory diagnosis, 3'15
grov\Tth cllaracteristlcs, 125 trachetcls, 49:3 properties, 345
horses, J26, 127 tracheobronchitis, 493 treatment, 345
i1nn1un ity, 126 trachoma, 236 turkeys , 345
labrn alt11 y d iagnos is, 12<) Lransnüsslblc enteritis, 392 turkcy rhinotracheiti3 virus, J70
n1orpholoh'Y· 125 transnlissible gastroenteritis virus (TGE), turkeypox virus, 334
m11l1>~, 1?.6, 17.7 :{8:~-:~86 type 1 adhesin, 57
pathogencsis, 126 control, 386 type 111 secretion system
reservoir, 126 diarrhea, 383 Bordetella, 102
resistance, 125 ctisease. 383 Campylobacter, 135
staining, 125 distributi<>n , 385 Escherichia coli, 63
~ t r uclure, 1 25 hosL response, 385 Saln1011ella, 69-70
transn1ission, l 26 infectivity for other species, 385 Shigella, 81, 8 2
treatn1ent, 126 J;iboratory di ;ignosis, 386 Yersinia , 7S, 76, 78, 79
variability, 125 pathogenesis, 385 Tyzzcr's discasc, 208
Taylore/la equi.~enitalis, 125- 127 properties, :~84
Taylorella osinigenitalis, 126- 127 reservoir, 385 ulcerative lymphangitis, 177
Tbilisi phage, 111 resistance, 385 Urcaplasma, sce Mycoplasma
·1·t.ME(thron1boen1bol!c men1ngoen- s1Nine, 383, 385 urinary traer lnfectlons (UTI), dogs, 500
cephalitis), 98 transm ission, 385 urinary tract, see urogen ital system
tcmocillin, see penicillins treatment, 386 u.rine
tendonltls, 471 vorniting, 383 anlirnlc1oblal pi:operties, 500
 Index 535

direct smear, 16 rcsistance, 346 hircls, 3Sl

culture Lechniques, 16 swine, 347, 348 control, 353
urogenital system, 496-504 trans1niss.i on, 347 disease, 351
urinary tract, 496 treatment, 348 distribution, 351
anti1nicrobia] propert ies, 496 vesicular stomatitis virus, 380 381 horses, 351
llora, 496 cattle, 380 J IU:>l lt:>¡JU11>C 1 351- 352
infections, 496 control, 381 infectívity for other species, 351
infcctious agen.ts and conditions, 496 d isease, 380 labor<itory diagnosis, 3.'i3
<.logs, 496 dbtribution, 381 pat hogenesis, 3S1
genital tract, 501 h o rses, 380 properties, 351
antimicrobial propf>rtif>s, SOl infectivity for oth er species, 380 reservoir, 351
flora, 501 lab<>ratory diagnosis, 381 resistance, 351
infectio n s pathogenesis, 381 transn1ission, 351
infectious agents and conditions, properties, 380 treatment, 353
407 500 rcservoir, 381 v\Test N ile virus, 354, 355
ctogs, 497, 500 resbt<iuce, 380 di5ease, 354, 355
cats, 497 ston1atitis, 380 distribution, 354, 355
horses, 497 s~vinc, 380 propPrtiPs, 3.'i4, .1.'i.'i
cattle, 498 transmissi.on, 381 rescrvoir, 3 54, 3 55
sheep,498 treatmen t, 381 resistance, 354, 355
goa1s, 498 Vesiculovirus, 377 trans1nission, 354, 355
swinc, 498 Vesivirus, 316 wet tail, 139
poultry, 499 Vl antigen, 69 vvhlte blood cells, infection, 443
urolit.h, 500 vidarabine. 43 \.Yinter dysentery, 387
lJTT (urinary tract infections), dogs, 500 viral hem<>rrhagic septicemic virus of Wood's light, 275
uveitis, 486 salrnonids, 378, 382 wooden tongue, 94
viral im1nunosuppression, 303 wooly monkey sarcoma virus, 410
vaccin e, hacterial, 53 viral vaccine, 49- 52 wound, infection, 472
vacc ine, definilion, 48 virulence, J Wright's stain, 16
vaccine, viral, 49- 52 virus disseminatio n , 301
vaccin ia virus, 333, 334 vir11.~ nf'11 t ra li 7.ation test, 24 xylose lysine deoxycholate (XLD) aga r, 59
va¡,>in itis, 502 virus, dcmon stration, scc Dcn1onstration
vaJJey tever, z~::i • of infectious agent Yaba monkey rumor v1ru~. 334
vancornycin resistan ce. avoparci.n, 26, 28, virus-h ost relationsh ips, 301 Yad, 78
41, 165 visna/maedi/progress.ive pneumonia Yatapoxvin1s, 334
vancomycin, 26, 28 vi(u~e~, 421-422 yello>v fever virus, 353
vasculitis. 443 con trol, 422 yello\v head virus, 384
vector, 4 disease, 421 yellow lamh diSf>'1S<', ?.00
VEE, see Veueztu::l<1n eyuine encephalilis distrihution, 421 yellows, 200
Venezuelan equine enceph alitis (VEE), goats, 421, 422 Yersinia, 75- 80
351- 353 host respon~<>, 4??. morphology, 75
bilds, 351 infectivity for oth e·r spccics, 421 staining, 75
control, 35:~ laboratory diagnosis, 422 yersiniabactin, 75, 78, 79
ni'<>;JSP, .1.'il pathogenesis, 421 - 422 Yersinia enterocolitica, 79-80
distribution, 351 ptoperties, 121 cellular products of rnedical intcrest, 79
horses, 351 reservoir, 421 adlleslns 79
host response, 351- 352 resistance, 421 Ail, 79 '
infectivity for other species, 351 sheep,421, 422 ccll wall, 79
laboratory diaguosb, 353 transnüssion, 421 Gsr, 79
pathogenesis, 351 treatment, 422 high pathogenicity island, 79
properties, 351 visn~ virus, Sf>f> visn;i/mai>cli/progressive lnv, 79
reservoir, 351 pneumonia viruses LcrV, 79
resistance, 3~1 vulvar infection (vulvitis), 502 toxins, 79
trans1nission, 3S1 vulvovaginitis, 502 type III secretion system, 79
trcatn1cnt, 3S3 Yad,79
vero toxin, see shiga (shlga-lll<e) toxtn walleye dermal sarcoma virus, 410 Yops, 79
vesicular exanfhema of swine virus. vVEE, see western equine encephalitis Yst, 79
346- 349 Wesselsbron virus, 354, 355 control, 80
co11trol, 348 disease, 354, 355 epiden1iology, 80
disease, 346 distribut ion, 354, 355 gastroenteritis, 79
distribu tion, 347 properties, 354, 355 immunity, 80
host response, 348 reservoir, 354, 355 laboratory diagnosis, 80
infectivity tor otl1er species, 346 resistance, 3~4 1 3~~ mesenteric lymphadenitis, 79
J;ihnratory diagnosis. :~48 transn1ission , .1S4, 3.'iS pathogenesis. 80
pathogcnesis, 317 318 Western blot assay, 25 reservoir, 79
properties, 346 western equ!ne encephalitis (WEE), septicemia, 79
reservoir, 347 351 - 353 structure, 79
536 lndex

Yersinia enterocolitica (continued) Yops, 76 LcrV, 78

transmission, 79- 80 control, 77 toxins, 78
trc<1tu1e11t, 80 cpídculiology, 77 typc lll sccn:tiou systcu1, 78
variability, 79 ilnmuníty, 77 Yad, 78
Yersinia pestis, 75- 77 laboratory diagnosis, 77 Yops, 78
cellular proc\ucts of n~ec\ical interest, pathogenesis, 76- 77 control, 79
75-76 plague, 75 epi<temiology, 19
c<ipsnlC' (C:<ifl), 7'> rC'SC'rvoir, 76 immunity, 79
ccll wa ll, 75 structurc, 75 la boratory diagnosis, 79
coagulase, 76 rransmission, 76 pathogenesis, 78- 79
Gsr, 76 treatment, 77 reservoir. 78
high pathogenicity island, 75 variability, 76 structure, 78
T·Ims phenotype, 75 Yersinia pseudutuberculusis, 77- 79 Lraus1ui s~iv11, 78
iron, 75- 76 cellular products of 1nedical in terest, 78 treatment, 79
LcrV, 76 adhesíns, 78 variability, 78
p eslicin, 76 Ail, 78 Ymt, 75, 76
p lasminogen activator, 76 cell wall, 78 Yops, /5, /6, 18
toxins, 76 c:;sr., 78 Yst, 79
type III secretíon syste1n, 76 high pathogcnicíty island, 78
yersiniabact1n, / :;, Inv, 78 zidovudine, 43
Y1nt, 76 iron, 78 Ziehl-Neelsen stain, 223

•
 Second Edition
The most recent revision of this comprehensive text
covers the bacteria!, fungal , and viral pathogenic
agents that are signiflcant cau::;es uf anin1al disease.
The focus includes pathogenic mechanisms and
processcs in infcctious diseases; methods of diagnosis;
and principies of resistance, prevention, and therapy.
In addition to serving as a resource for veterinary students,
Veterinary Microbiology, Second Edition also serves as a
convenient reterence tor vetennarians and veterinary scientists
whose expertise is outside the area of microbiology.
Veterinary Microbiology, Second Edition is now organized in tour
parts according to the most appropriate methods of instruction:
ABOUT THE EDITORS: --ft-----~~~----r-----------.~
Dwlght C. Hish, DVM, PhD N. JalrleS
• Part 1 deals with the QP.nP.rt!I r:h;:¡r;:¡r:teristics ot the host-parasite
relationship, laboratory uiay11usis uf conditions invoiving an
infectious etiology, antimicrobial trcatmcnt, and prevention of
infectious disease.
• Part 11 (Bacteria and Fungi) and Part 111 (Viruses} µresent the
infectious agents that affect the veterinary species. The chaptcrs
deaiing with the bacteria! agents are grouped mainly by morphol-
oOY <lnct their gram-staining characteristics. The fungai agents
are yruuµe<.J 111ai11ly by n1orQhologic characteristics (yeast, mold}.
The viruses are groupcd along taxonomic grounds.
• Part IV, an enhancement nP.w to this ectition. deals with the
infectious aQents in the contexl uf ll 1e llusl. Tl1is section is
organized by organ system. Each organ system is discusscd first
as a microbial habitat, followed by discussion of those infectious
agents that mainly affect that particular system.
, DVM, PhD RiChard L \Valker, DVM, PhD, MPVM
UETEtlfiAIY KICID810lOCY / OMl&HT C. lll~H, 9. "
SF110 .l HS lOD~ ;
UilCRCIOR: t. ICIP r C
1n111u11,1n111u ~~
I~BN0-8138-0379- 9
11 111111 1 111 111111
9 780813 803791