Lyme Disease--A Tick-Borne Spirochetosis?

Author(s): Willy Burgdorfer, Alan G. Barbour, Stanley F. Hayes, Jorge L. Benach, Edgar
Grunwaldt and Jeffrey P. Davis
Reviewed work(s):
Source: Science, New Series, Vol. 216, No. 4552 (Jun. 18, 1982), pp. 1317-1319
Published by: American Association for the Advancement of Science
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 A

ant. They are representativeof 11 out of
15 isolates thus far obtained. The other
four isolates showed barophilicgrowth
characteristicsin a pressurerangeof 300
to 500 atm.
The barophilicisolates describedearli-
er (4, 13) were obtainedfrom decompos-
ing deep-sea amphipodsand from inver-
tebrateintestines, that is, from nutrient-
rich niches. Whilewe have also recently
been able to isolate baro- and psychro-
philicstrainsfromthese niches as well as
from decompressed deep-sea water,
most organisms in these samples ap-
peared to be of the same type as those
thus far obtained with the isolation
chamber, namely, psychrophilic and
highlybarotolerant.
An explanationfor the distributionof
more or less temperature-and pressure-
adaptedbacteriain the deep sea mightbe
found in accumulatingdata on the con-
siderableparticle flux from surface wa-
ters to the deep sea and observationsof
bacterialattachmentsto these particles,
such as organicdetritusand fecal pellets
(14). Most of the deep-sea bacteriain the
sediments and water column may thus
represent relatively recent arrivals.
Theirmetabolicrates are affectedby the
decreasing temperature and increasing
pressurebut, as comparativedata from
undecompressedand decompressednat-
ural deep-sea populations show (ll), at
least some bacteriarecover immediately
and exhibit maximumactivity at 1 atm.
While these surface-originatedbacteria
are favored by decompression, some
barophilicorganismsmay be irreversibly
aSected by a decrease of pressure as
manypsychrophilicbacteriaare affected
by an increase of temperature.This ap-
proach of samplingand isolating micro-
organismsin the absence of decompres-
sion may provide answers to questions
concerningthe viability of decompres-
sion-sensitivedeep-sea bacteriaand will
Epidemiologicevidence suggests that
furnish undecompressed pure culturesLyme disease is caused by an infectious
for studies of pressure effects on the
agent transmittedby ticks of the genus
physiologicaland molecularlevel. Ixodes. In the Northeast and Midwest
HOLGERW. JANNASCH Ixodes dammini and, in the West, I.
CARL0. WIRSEN pacificus have been incriminatedas po-
CRAIGD. TAYLOR tential vectors (6, 7). Until recently, all
Biology Department,
WoodsHole OceanographicInstitution,
WoodsHole, Massachusetts 02543
Referencesand Notes
1. H. W. Jannasch,C. O. Wirsen,C. L. Winget
DeepSea Res. 20, 661 (1973)-H. W. Jannasch
C. O. Wirsen, C. D. Taylo;, Appl. Environ.
Microbiol.32, 360 (1976);P. S. Taborand R. R.
Colwell,Proc. MTS/IEEEOCEANS'76, 13D-I
(1976);P. S. Tabor,J. W.-Deming,K. Ohwada
H. Davis, M. Waxman,R. R. Colwell, Microb.
Ecol. 7, 51(1981).
2. H. W. Jannaschand C. O. Wirsen,Appl. Envi-
ron. Microbiol.33, 642 (1977).
3. C. O. WirsenandH. W. Jannasch,Environ.Sci.
Technol. 10, 880 (1976); D. M. Karl, C. O.
Wirsen, H. W. Jannasch, Science 207, 1345 C. D. Taylor, Undersea Biomed. Res. 6 147
(1980). (1979). ,
4. A. A. Yayanos, A. S. Dietz, R. Van Boxtel 13. J. W. Deming, P. S. Tabor, R. R. Colwell
Science 205, 808 (1979);Proc. Natl. Acad. Sci. Microb.Ecol. 7, 85 (1981).
U.S.A. 78, 5212 (1981). 14. S. Honjo,J. Mar.Res. 38, 53 (1980).A compre-
5. P. M. Saundersand N. P. Fofonoff,Deep-Sea hensive listing of papers on particleflux mea-
Res. 23, 109( 1976). surementswith the aid of sedimenttraps was
6. J. S. Poindexter, Adv. Microb. Ecol. 5, 67 providedby C. S. Reynolds, S. W. Wiseman,
(1981). and W, D. Gardner[FreshwaterBiol. Assoc.
7. R. Y. Morita Bacteriol.Rev. 39, 144(1975). Occas. Publ. No. 11 (1980)]. Representative
8. C. E. ZoBel; and R. Y. Morita, ibid. 73, 563 paperson bacterialattachmentare J. T. Turner
(1957). [Trans.Am. Microsc.Soc. 98, 131(1979)]andJ.
9. C. E. ZoBell, Bull. Misaki Mar. Biol. Inst. T. TurnerandJ. G. Ferrante[BioScience29 670
Kyoto Univ. 12, 77 (1968). (1979)]. ,
10. R. E. Marquisand P. Matsumura,in Microbial 15. We thankK. W. Dohertyfor engineeringcalcu-
Life in ExtremeEnvironments,D. J. Kushner, lations and design and M. C. Woodwardfor
Ed. (AcademicI?ress,New York, 1978);J. V. constructionand fine mechanicaldetails of the
LandauandD. H. Pope,Adv.Aquat.Microbiol. isolationchamber.Supportedby NationalSci-
2, 49 (1980). ence Foundation grants OCE77-19766and
11. H. W. Jannaschand C. O. Wirsen,Appl. Envi- OCE79-19178.ContributionNo. 5142 of the
ron. Microbiol.43, 1116(1982). WoodsHole OceanographicInstitution.
12. C. 1). Taylor,Arch.Biochem.Biophys. 191, 375
(1978);Appl. Environ.Microbiol.37, 42 (1979); 21 January1982
Lyme Diseasc A Tick-BorneSpirochetosis?
Abstract. A treponema-likespirochete was detected in and isolated from adult
Ixodes dammini,the incriminatedtick vector of Lyme disease. Causally related to
the spirochetes may be long-lasting cutaneous lesions that appeared on New
Zealand Whiterabbits 10 to 12 weeks after infected ticksfed on them. Sampl@sof
serumfrom patients withLyme disease were shown by indirectimmunofluorescence
to contain antibodies to this agent. It is suggested that the newly discovered
spirocheteis involvedin the etiology of Lyme disease.
Lyme disease is an epidemic inflam- attempts to isolate the causative agent
matorydisorderthat usually begins witk either from ticks or from patients were
a skin lesion called erythemachronicum unsuccessful.
migrans(ECM). Weeks to months later Recentlywe isolated from I. dammini
the lesion may be followed by neurologic a spirochetethat binds immunoglobulins
or cardiacabnormalities,migratorypoly- of patientsconvalescingfrom Lyme dis-
arthritis,intermittentattacks of oligoar- ease. We also recordedthe development
ticulararthritis,or chronicarthritisin the of lesions resemblingECM in New Zea-
knees (l). land White rabbits on which ticks har-
Althoughin the United States cases of boringthis spirochetehad fed.
ECMwere firstreportedfrom Wisconsin Adult I. dammini were collected in
(2) and southeastern Connecticut (3), late Septemberand early October 1981
Lyme disease as a new form of inflam- by flagginglower vegetation on Shelter
matory arthritiswas first recognized in Island, New York a known endemic
1975 in Lyme, Connecticut (4). It has focus of Lyme disease (8). Of 126 such
since been reported from other north- ticks thatwere dissected, 77 (61 percent;
eastern, midwestern,and western states 65 males and 12 females) contained spi-
(5) rochetes.The spirocheteswere distribut-
ed mainly in the midgutbut were occa-
sionally also seen in the hindgut and
rectal ampule. No other tissues, includ-
ing the salivaryglands, contained spiro-
chetes. The organismsstainedmoderate-
ly well with Giemsa (Fig. 1); in wet
preparationsexaminedby dark-fieldmi-
Fig. 1. Ixodes dam-
mini spirochetes in
midgut tissues of its
tick vector. (A) Giem-
sa staining (x 1200).
(B) Serum of patient
J.G. examinedby in-
direct immunofluo- t.*
rescence ( x 570).

SCIENCE,VOL. 216s 18 JUNE 1982 003S8075/82/0618-1317$01.00/0

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 ^ tion (MV)
of spirochetes
of the tick's

Table 1. Serologicevaluation(indirectimmunofluorescence)of serumfrompersonswith Lyme larly subcultured and maintained at

disease. 35°C.
Serum Serumdilution When about 300 I. damminiwere al-
Pa- Disease
tient* contracted collected end point lowed to feed on eight New Zealand
Whiterabbits(12);they appearedto have
B.B. May 1978 September1978 1:1280 no immediatelyadverse effects. Blood
B.Br. July i980 July 1980 1:240
E.D. July 1980 July 1980 1:80 smearsexamineddaily for 14 days after
C.G. June 1979 March1980 1:640 placementof the ticks were negativefor
J.G. June 1979 March1980 1:1280 spirochetes. However, 10 to 12 weeks
L.H. June 1980 September1980 1:640 after the ticks had engorged, up to 15
J.S. July 1979 January1982 1:640
March1980 1:80 small (2 to 3 mm in diameter) macules
A.S. July 1977
C.T. June 1979 March1980 1:320 and papules appearedin the skin of the
Controls:Four samplesfrom New York and ten from Montana _1:20 back and lateral trunk of each rabbit.
Diagnosed by E.G. except for J.S., whose serumwas submittedto the New YorkStateHealthDepartment.
Within 3 to 5 days, these lesions had
All patientscontractedthe disease while visitingShelterIsland,New York. enlarged (up to 5 cm in diameter) to
slightlyelevated annularor oval lesions
withbrightred to reddish-violetmargins.
croscopy they moved sluggishly and ro- eter. The ends appeartaperedwith four Similarlesions on the abdomen,the site
tated slowly. The degree of infection to eight filamentsinserted subterminally of tick attachment, were recorded on
varied;some ticks contained only a few at each end. Insertionpoints of the fila- only one of the eight rabbits. All lesions
spirochetes,others containedlarge num- ments are in a row paralletingthe cell's persistedfor at least 8 weeks.
bers often to the extent that clumps of long axis. Cross sections of the cells Sections of biopsy specimens were
spirocheteswere present throughoutthe show six to eight filamentsinterspersed stained with hematoxylin and eosin.
midgut. between the outer membrane and the These sections showed that the skin le-
Electronmicroscopy (9) of midgutdi- cytoplasmicmembranein the asymmet- sions consisted of a thickened, slightly
verticularevealedspirochetesclosely as- ric region of the section profile(Fig. 2). hyperkeratoticepidermis with the der-
sociated with the microvillar brush The I. damminispirochetewas isolat- mis showingdense mononuclearcell in-
border of the gut epithelium (Fig. 2). ed by inoculating0.1 snl of a suspension filtrationand edema of the superficial
Fine structuralfeatures of the organism prepared from midgut tissues of four layer. Limitedattempts to isolate spiro-
were similarto those reportedfor Trepo- infected ticks into 8.5 ml of modified chetes fromsuspensionsof biopsied skin
nema species (10). Irregularlycoiled, the Kelly's medium (11). After 5 days of lesionsin lRelly'smediumwere negative.
spirochetes range from 10 to 30 ,um in incubationat 35°C, all the culture tubes Even thoughmicroscopicexamination
lengthand from0.18 to 0.25 ,umin diam- containedspirochetesthat could be regu- of repleted I. dammini showed that at
least two ticks harboringspirocheteshad
fed on each rabbit, we are not certain
whether the described skin reaction on
the rabbits is causally related to the
spirochetes or is due to other factors
associated with the ticks' feeding pro-
cess.
When tested by an indirect immuno-
fluorescencemethod (13), antibodies to
the spirochetes in titers of _ 1:1280
were present in the serum of all rabbits
l
on which ticks had fed 30 and 60 days
earlier.The serumof rabbitsthat had not
M ^ 13 Fig. 2. Electron mi-
been exposed to ticks did not react at
a F5;=w v+>
> <
crograph of 1. dam-
mini spirochetes dilutionsof > 1:20.
r + associated with mi- Thatthe I. damminispirocheteis anti-
9sh crovillarbrushborder genicallyrelatedto the etiologic agent of
Lymedisease was suggestedby the posi-
; ^ wp A set shows c;oss)senc tive reactionswe obtainedwhen we ex-
amined serum samples from nine pa-
g^< tf x (x 122,100). tients with clinically diagnosed Lyme
disease by means of indirectimmunoflu-
orescence (Fig. 1) (14). Antibody titers
rangingfrom 1:80 to 1:1280were record-
ed for persons who had Lyme disease
currentlyor as manyas 32 monthsprevi-
ously (Table 1). In contrast, serum sam-
ples from four people from New York
andten fromMontanawith no historyof
the disease did not react with the spiro-
chete in titers higherthan I: 20.
Our observations suggest that the
1318
SCIENCE, VOL. 216

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treponema-likeorganismisolated fromI.
damminimay be involved in the-etiology
of Lyme disease. It is interesting that
10. K. Hovind-Hougen,Acta Pathol. Microbiol.
organismspresentingthe morphological
characteristicsof spirochetes were said 11. (1971)J
to be associated with ECM in Europe as
early as 1948 (15). Although this was
never confirmed, a recent study (16)
showing that resolution of lesions and 12. Fifteen
concurrent symptoms occurs faster in
patientstreatedwith penicillinsuggests a
penicillin-susceptible bacterium as an 13. shaved
etiologic agent of Lyme disease.
Ourresults establishthe susceptibility
of the domestic rabbitto the I. dammini
. .
9. For electron microscopy,diverticulaof midgut saline with 1 percent bovine serum albumin.
were removedby dissectionandwere processed 14. Fluorescein isothiocyanate-conjugated goat
accordingto S. F. Hayes and W. Burgdorfer[J. antibody to human immunoglobulin (BBL
Bacteriol.137, 605 (1979)]. Cockeysville, Md.) was used at 1:100 dilution in
phosphate-buffered saline with 1 percent bovine
Scand. Sect. B S"ppl. No. 225 (1976). serum albumin
Kelly's medium [R. Kelly, Science 173, 443 15. C. Lennhoff, Acta Derm. Venereol. 28, 295
modified by addition of CMRL medi- (1948).
um 1066 (Gibco No. 330-1540)and Yeastolate 16. A. C. Steere, St E. Malawista, J. H. Newman,
(Difco) for finalconcentrationsof 5 and 0.2 per- Pa N. Spieler, N. H. Bartenhagen, Ann. Intern.
cent, respectively(H. G. Stoenner, in prepara- Med. 93, 1 (1980).
tion). 17. Since submission of this manuscript, microscop-
to twenty I. damminifemales and equai
numbersof males for mating(males may ingest
ic examination by one of us (W.B.) of midgut
smears from Ixodes pacificlls from Oregon and
smallamountsof blood)were placed on each of of I. ricinusfrom Switzerland also revealed, in
eightrabbits.The ticks were containedin metal some instances, the presence of spirochetes.
capsules attached by adhesive tape to the 18. We thank the Nature Conservancy Incorpo-
abdomenof each rabbit.
In accordancewith the data of R. N. Philip, E.
ration for permission to collect ticks ih their
Shelter Island Preserve. We also thank E.
A. Casper,R. A. Ormsbee M. G. Peacock, and Bosler, S. Guirgis, D. Massey, and J. Coleman
W. Burgdorfer [J. Clin. Microbiol.3, 51 (1976)] for their assistance in collecting ticks. Special
midgut smears of infected ticks or cultured thanks also to W. H. Hadlow, Epidemiology
spirocheteswere used as antigen. Fluorescein Branch, Rocky Mountain Laboratories, for the

isothiocyanate-conjugated goat antibodyto rab- histologic characterization of the rabbit lesions.

spirocheteand demonstratethe possible bit immunoglobulin (ChappelLaboratories)was
value of the indirect immunofluores- used at a 1:50 dilution in phosphate-buffered 26 February 1982; revised 20 April 1982
cence test as a diagnostictool for Lyme
disease. They also suggest the need for
additional investigations not only into
the epidemiology and ecology of Lyme
disease and related disorders, such as Lack of Correlation Between Hepatic Mitochondrial
ECM of Europe (17), but also into the Membrane Structure and Functions in Ethanol-Fed Rats
relationsbetween the spirochete and its
vector I. dammini. Abstract.A currenthypothesissuggests that alterations in the chemical composi-
WILLYBURGDORFERtion and the subsequentchanges in the structureof the membranecould accountfor
EpidemiologyBranch, Rocky Mountain the functional derangementsobserved in the hepatic mitochondriaof animals fed
Laboratories,National Institute of ethanolfor extendedperiods. An examinationof this hypothesisreveals that the liver
Allergyand Infectious Diseases, mitochondria of ethanol-fed rats show a dissociation between the respiratory
Hamilton, Montana 59840 functions and the lipid compositionand microviscosityof the membranes.
ALANG. BARBOUR
Laboratoryof MicrobialStructureand One of the most characteristicfeatures mals. Conversely, mitochondria from
Function,Rocky MountainLaboratories of alcoholic liver disease is the striking the pair-fed control group and Purina
STANLEY F. HAYES alterationin mitochondrialmorphology Chow-fed animals were functionally
Rocky MountainOperations.Branch, and function. This condition has now similar,in spite of the differences in the
RockyMountainLaboratories been reproducedin an animalmodel (1). lipid compositionand fluid state of their
JORGE L. BENACH Hepaticmitochondrialpreparationsfrom memDranes.
State of New YorkDepartm.entof ethanol-fed-rats,which develop a fatty In this study we measured the fatty
Health and Departmentof Pathology, liver, and ethanol-fed baboons, which acid composition of the phospholipids
State Universityof New York, develop cirrhosis, exhibit a decreased and the microviscosity of mitochondrial
Stony Brook 11794 abilityto oxidize substratesand to form membranes and the membrane-depen-
EDGARGRUNWALDThigh-energy phosphate metabolites (1, dent functions of liver mitochondria
44 South Ferry Road 2).- The mechanisms underlying these from ethanol-fed rats and their respec-
ShelterIsland, New York11964 changesare still being debated. Recently tive pair-fedcontrols and then compared
JEFFREY P. DAVIS it. was proposed that these functional these findings to similar data obtained
Departmentof Health and Social changes may be directly related to alter- fromChow-fedrats. For that purpose, 16
Service+Madison, Wisconsin53701 ations in the structuralproperties.of cel- maie Sprague-Dawleyrats (weighing 125
lular membranesproduced as a conse- to 150g each) were pair-feda liquid diet
References and Notes quence of prolonged ethanol consump- containing 36 percent of its energy as
1. A. C. Steere et al., Ann. Intern. Med. 93, 8 tion (3). Since the functions of the mem- ethanol, or alternatively in the control,
(19803. brane are governed in part by their as carbohydrates. The .constituents of
2. R. J. Scrimenti, Arch. Dermatol. 102, 104
(1970). physical state, the hypothesis has been this diet have been describedin (4). After
3. W. E. Mast and W. M. Burrows, Jr., J. Am.
Med. Assoc. 236, 859 (1976). formulatedthat a correlationexists be- 3 to 5 weeks on this diet, the animals
4. A. C. Steere, S. E. Malawista, J. A. Hardin, S. tween changes in the structuralproper- were treatedin the following manner.At
Ruddy, P. W. Askenase, W. A. Andiman, Ann.
Intern.Med. 86, 685 (1977). ties of the membranephospholipidsand 9:00 a.m. on the day before the experi-
5. A. C. Steere and S. E. Malawista, ibid. 91, 730 the functionalalterationsobserved after ments they were given one-thirdof their
(1979)-
6. A. C. Steere, T. F. Broderick, S. E. Malawista, long-termethanol intake (3). However, daily ration, and all food, except water,
Am. J. Epidemiol.108, 312 (1978). our data indicate that such a simple was removed at 4:00 p.m. The animals
7. R. C. Wallis, S. E. Brown, K. O. Kloter, A. J.
Main, Jr., ibid. 108, 322 (1978). correlationdoes not explain these func- were killedon the next day between 9:00
8. The ticks were first ex?mined by the hemo-
lymphtest [W. Burgdorfer,Am. J. Trop. Med. tional changes. Hepatic mitochondrial and 10:00 a.m. At the same time, ten
Hyg. 19, 1010(1970)].Subsequentlythey were preparations. from ethanol-fed rats dis- male Sprague-Dawleyrats, maintained
dissectedfor the preparationof multiplesmears
from gut, malpighiantubules, salivary glands, played severe functionalimpairment,yet on PurinaChow (the Chow-fedcontrols)
central ganglion, and testes or ovary. Smears the fluidityand lipid composition of the were fasted for a similar period of time
were stainedaccordingto the Gimenezmethod
[D. F. Gimenez, Stain Technol. 39, 135 (1964)] mitochondrialmembraneswere remark- beforebeing killed. Standardprocedures
or with Giemsa. Once spirocheteswere detect- ably similar to those of mitochondrial (5) were used to preparethe liver mito-
ed, wet preparationsof tissues were examined
also underdarkfield. membranes from pair-fed control ani- chondria,to analyze the fatty acid com-
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