Hhk All- N- 02- 013 C3

0." 8 t:0 P YC 8i.Y

Diseases of Cultured Penaeid Shrimp

Ii e i s I

in

Asia and the United States

Edited by
Wendy Fulks
and
Kevan L. Main

The Oceanic Institute

Copyright
! 1992byTheOceanic
institute
Ail RightsReserved

For additional copies, please contact:

The Oceanic Institute

Makapvu Point
P,O. Box 252BO
Honolulu, Hawaii 96825

ISBN %9617016-5-X
 V
Preface ...

Part I: Introduction
Introduction.

PartII: ContributedPapers- CountrySituations

Major
Diseases
ofCultured
Shrimp
inAsia:
AnOverview
37
M. Shariff and R.P.Subasinghe ....

An Overview
of theDisease
Situation,
Diagnostic
Techniques,
Treatments
andPreventives
Usedon ShrimpFarmsin China
OouChen. ,4
Occurrence,
Diagnosis
andTreatment
ofShrimp
Diseases
inThailand
Timothy
Flegel,
Daniel
F.Fegan,
Sumana
Kongsom,
Sompoach
Vuthikornudomkit,
Sin-
porn
Sriurairatana,
Sitdhi
Boonyaratpalin,
Chaiyuth
Chantanachookhin,
JoanE.Vickers
andOliver
D.Macdonald. ... ........,.57
Diseases
ofPenaeas
monody
in Taiwan:
A Review
from1977to1991
113
I-ChiuLiao,Mao-SenSuandCheng-Fang
Chang.

Prevalence
andGeographic
Distribution
ofMBVandOther
Diseases
in
Cultured
GiantTigerPrawnsPenaeus
rnonodon!
in thePhilippines
Josh
M.Natividad
andDonald
V.Lightner. 139
TheStatus
of CultureandDiseases
ofPenaeid
ShrimpIn Korea
161
Myoung
Ae Park.
Baculovirus
Infectionof PenaeidShrimpin Japan
169
TokuoSanoand KazuoMomoyarna.

Part ll: ContributedPapers - Viral Diseases

Infection
Route
andEradication
ofPenaeus
rnonokm
Baculovirus
h48V!
in
LarvalGiantTigerPrawns,
Penaeus
maruxton
Chen,
S.N.,
P.S.
Chang
andG.H,
Kou 1
 canterlts

Viral
Diseases
ofCultured
Penaeid
Shrimp
inJapan
Kazuo Momoyama..

'art!l: Contributed
Papers
- Bacterial
Diseases
Studies
onthe
Epizootiology
andPathogenicity
ofBacterial
Infections
in
Cultured
Giant
Tiger
Prawns,
Penaeus
mortem,
inTaiwan
S.N.
Chen,
S.LHuang
and
G.H.
Kou............. 195
Partli: Contributed
Papers
- Diagnostic
Procedures
Current
Diagnostic
Methods
forAgents
andDiseases
ofFarmed
Marine
Shrimp
James A. Btock...

Neer
Developments
inPenaeid
Virology:
Application
ofBiotechnology
in
Research
andDisease
Diagnosis
forShrimpViruses
of Concern
in the Americas
D.V.Ughtner,
B.T.Poulos, L Bruce,
R.M.Redrnan,
J.Marl,
andJ.R.Swami........... 233
Part II: ContributedPapers- SPFStocks
Selective
Breeding
ofSpecific
Pathogen-Free
SPF!
Shrimp
forHighHealth
and Increased Growth
James
A.Wyban
. 257
Developing
Specific
Pathogen-FreeSPF!
AnimalPopulations
for
Aquaculture:
A CaseStudyforII' VirusofPenaeid
Shrimp
Jelfrey M. Lotz 269

Growth and Survival of Virus-infected and SPFPenaeus

vaniiarneion a
Shrimp Farm in Hawaii
NickCarpenter
andJames
A.Brock 285
ShrimpProduction
in Texas
UsingSpecific
Pathogen-Free
Stocks
Fritz Jaenike,Kieth Gregg and Louis Hamper. ., 295

Part lt: ContributedPapers- Research,Regulations,

and Health Management
ShrimpCultureTechnologies
Inc.: Research
to ImproveShrimpGenetics
and Health
RoiiandLaramore........,........,.............. . ............ ....,... ............... 30
 Contents

DrugsandChernotherapeutants
for ShrimpDiseases:
TheirPresentStatus
in the United States, with an Overview of Researchand Approval Processes
ThomasA. Bell 311

Precautions
for ImportingandCulturingNon-nativeShrimp
Catt J. Slndermann

IssuesRelatedto Regulation
of PenaeidShrimpDiseases
in Texas,U.S.A.
Stetting
K.Johnson 333
Shrimp HealthManagementProcedures
Brad LeaMaster .

Part III: Discussion Group Surnrnaries

Discussion Group Summaries.

Appendices
Appendix 1: WorkshopParticipants 379

Appendix 2: WorkshopAgenda
 Preface

TheAsianInterchange
Program
wasfoundedat TheOceanic
Institutein 1989.
Theprogram's
goalisto facilitate
theexchange
ofapplied
aquaculture
informa-
tionandtechnology
between
theUnitedStates
andAsia.Thisis accomplished,
in largepart,throughtheorganization
ofinternational
workshops
anddistribu-
tionof workshop proceedings
to information
networks
throughout
theUnited
States and Asia.

This is the third workshopproceedings

issuedby the AsianInterchange
Program.Previous
volumes
dealtwiththeculture
of cold-tolerant
shrimp
in
Asia,andthecultureof rotifersandmicroalgae
in AsiaandtheUnitedStates.
Forourthirdyear,wechose tofocusontheinfectious
andnoninfectiousdiseases
ofculturedshrimpin AsiaandtheUnitedStates.Notonlyis thisa timelytopic
in manyAsiancountries
whereshrimpdisease
problems
havebeenmounting
overthepastfiveyears,
butit alsotiesinwithrecent
efforts
bytheGulfCoast
Research
Laboratory
Consortium
todevelop
andmaintain
specific
pathogen-free
stocksfor shrimpfarmersin the UnitedStates.

The Workshop
Thisyear,participants
traveled
fromJapan,
Malaysia,
thePhilippines,
the
People's
Republic
of China,
South
Korea,
Taiwan,
Thailand
andtheUnited
States
mainlandArizona, Horida,Mississippi
andTexas!
toattendtheworkshop
in Honolulu,HawaiifromApril27- 30,1992seephoto!.Everyonepresented
a
paper
during
thefourmorning
sessions.
Afternoons,
bycontrast,
werespent
in
discussion
groups,
whereparticipants
hadtheopportunity
toshare
information
andideaswith their colleagues
on a varietyof disease-related
issues.
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 Preface

The Proceedings
Thevolumeis dividedintothreeparts:theintroduction,
contributed
papersand
discussion
groupsummaries.
Theintroduction
anddiscussion
groupsummaries
werewrittenby WendyFulksandeditedby KevanMain.
Thepapers
aregrouped
intosixsections:
Country
Situations,
ViralDiseases,
Bacterial
Diseases,
Diagnostic
Procedures,
Specific
Pathogen-Free
Stocks,
and
Research,
Regulations
andHealthManagement. The21papers represent
a wide
range
ofperspectives
shrimpfarmers in everycountry
faceuniqueproblems.
Forexample,growingconditionse.g.,temperature,
waterqualityandsoil
conditions!
varybetweenregions;
also,different
speciesmaybegrownand
differentpathogens
maybe encountered.
Importantly,
regulations
governing
culture
practices,
suchastheuseof drugs,andtheirenforcement
arealso
different
in everycountry.
Therangeofviewpoints
presented
in thisvolumeare
thoseof researchers,
farmersand/orextensionagentsfrom eight different
countries.

Evenwith all of thesedifferences,severalcommonthemesrecurredin both the

papers
andthediscussion
groupsessions.
Oneofthose
issues
wasthespread
of pathogens
especially
viruses!
viatheuncontrolled
movement
of shrimp
stocks.Thetransferof stockswhosedisease
statusisunknownmayposea threat
to wild shrimppopulationsand couldalsoharmshrimpcultureventures.
Resolutionof thisproblem
will notbeeasy,andwill mostlikelyinvolvemore
widespread
adoption
ofquarantine
regulations
andincreased
usageofspecific
pathogen-free
SPF!stocks.
Everyone
agreed
thatinternational
cooperation
is
necessary
to defineandimplement
feasible,
effective
quarantine
regulations.
Furthermore,
eachcountrymustdetermine whichpathogens to targetfor
exclusion,
andtherewill probably
bea different
exclusion
listfor everyspecies
grown.

A quarantine
system,
however,
is useless
in theabsence
of standardized
diagnostic
techniques.
Experts
needto agree
aboutwhichtechniques
arebest
fora given
pathogen,
andhealth
inspectors
and,eventually,
hatcheries,
will
needto be certified.Furthermore,better morereliable,moresensitive,easier,
quicker
andlessexpensive!
diagnostic
methods
needto bedeveloped
and
transferred to field diagnosticians.

Anothercommonthemewastheneedfor improvedhusbandry
techniques.
Since
manyserious
diseases
of culturedpenaeids
arecaused
by opportunistic
organ-
isms,disease
losses
canbeprevented
byproviding
optimal
conditions
forgrowth
andbycarefully
monitoring
thehealth
ofanimals
throughout
therearing
cycle.
 Preface

TMsisespecially
trueinsemi-intensive
and
intensive
culture
conditions.
Inthe
areaofdrug
use,most participants
agreed
thata number
ofcompounds,
including
antibiotics
andso-called
"probiotics,"
are
beingused
irresponsibly
in
many
shrimp
farming
areas.
Theprophylactic
useofantibiotics
inhatcheries
can
foster
thespread
ofantibiotic-resistant
bacterial
strains
andmay
also
weaken
larvae
inthelongrun.Also,
extreme
care
must
betaken
when
using
drugs
during
growout
toensure
thatnoresidues
remain
inshrimp
thataresoldfor
consumption.

Environmental
awareness
wasanother
important
issue
thatwasraLaek
several
times
duringtheworkshop.
Almost
nothing
iskern about
howshrimpfarms
affect
their
surroundings.
Inareas
where
there
isa high
concentration
offarms,
ananswer
is needed
to thequestion,
"Howmanyshrimp
farrrN
canthe
ecosystem
support
overthelong
term!"
Inturn,
wealso
need
toknow
how
fluctuations
inthenatural
environment
mayaffect
thehealth
ofcultured
shrimp.
Thedynamics
involved
arequite
complex,
andanswers
willnotbeimmediately
forthcoming.

Internationa0y,
shrimp
culture
isrecognized
asa valuable
industry;
if it isto
prosper,
theproblems
ofdisease
diagnosis,
prevention
andtreatment
mustbe
dealt
withimmediately.
Progress
hasbeen
made
toward
resolving
some
ofthe
vitalissues
mentioned
above,
andsome
of thatprogress
isdetailed
in thisbook.
Forexample,
advances
in disease
diagnosis
aredescribed
herein,
asarerecom-
mendedhusbandry
practices
toprevent
andtreatdiseases
ofcultured
shrimp.
SPFstocks
of Penaeus
vannurnei
arenowbeingusedthroughout
theUnitedStates,
andawareness
oftheneedforquarantine
measures
isincreasing.
In aneffortto
safeguard
thefuture
ofshrimp
farming,
researchers
inAsia
andtheUnited
States
areincreasing
ourknowledge
of knownshrimpdiseases,
andidentifying
and
characterizing
newdiseases
anddisease
agents.
It ishoped
thatthisvolume
will
furtherthe effortsof bothresearchers
and producers by makingavailablethe
latestinformationandtechnology relatedto the diseases
of culturedpenaeid
shrimp.
 Preface

Acknowledgments
TheAsianInterchange
Program
is fundedby theNationalOceanic
andAtmos-
phericAdministration,
UnitedStatesDepartment
of Cominerce
Grant4
NA90AA-D-SG483!.
The editorsthank the Universityof Hawaii SeaGrant
College
Program
forits administrative
supportthroughout
theproject.
A numberof individualscontributedto this work. Most importantly,we would
liketo thankall theauthorswho participated
in theworkshopandprepared
the
papersincluded
in thisvolume.
DonaldLightner
assisted
in theidentification
of workshopparticipants
andprovided
valuable
guidance
in thedevelopment
of the discussion
groupagenda
andthroughout
the workshop.In addition,
Cheng-Sheng
Lee,I-ChiuLiao,RuiyuLiuandByung-Ha
Parkassisted
in the
identification
of Asianworkshopparticipants.
We acknowledge
our capable
interpreters,
StellaGuillory,Hongja
Harrison,
Lynette
Shi,Taeko
Wellington
andMasakoYamatani,for assistingin the implementation
of theworkshop.

Donald
Lightner,
JamesBrockandS.K.Johnson
reviewed
theintroduction,
and
Donald
Lightner
andJamesBrockreviewedthediscussion
groupsummaries.
WethankStephanie
Frank,
Rose MarieNorton,
andDebbieFritzforproofread-
ing portions of the text.

Thefinalproduction
oftheproceedings
wasdone
byPattiKillelea-Almonte,
with
assistance
fromAlcianOmosoandElizabeth
Reynolds.
Thecoverwasdesigned
by ElizabethReynolds.

llfllL l-lMIlill- MR-94- 08

Introduction
 Introduction
Worldwide, almost 700,000MT of cul-
tured shrimp were produced in 1991
28% of all the shrimp sold that year
Rosenberry,
1991!.More than80%of
that record total represents shrimp
grownin Asia,andproductionin Asia
and in the western hemisphere is ex-
againin 1992Rosen- pines,Vietnamand Taiwan.In fact,
pectedto increase
berry, 1991!.
A practicalway to increaseproduction
is to adopt more intensive culture
methods, and this is exactly what is
happeningin manyshrimpproducing China arePenaeus
nations,especiallythosein which suit-
able land is expensive or scarce. In-
creasingthe density at which shrimp
are cultured, however, increases the
amount of stress the animals experi-
ence,makingthemmorelikely to suffer
from disease. Another concern result-
ing from the expansion of penaeid
aquacultureis the transfer of patho-
gens, especiallyviruses, betweencul-
ture locales and species, As the global
shrimp culture grows, so will the im-
portanceof recognizing,treating and
preventingdiseasein culturefacilities.
For the purposesof this paper,disease
is definedas "any departurefrom nor-
mal structure or function" and includes
infectious diseasescausedby microbial
pathogensor parasites,and noninfec-
tious diseases genetic and environ-
mentally induced abnormalities! see
Sinderrnann, 1990!.
Shrimp CUIturein Asia and
the United States
In Asia, the largestproducersof farm-
raisedshrimp in orderof importance!
are the People's Republic of China,
Indonesia, Thailand, India, the Philip-
China, Indonesia and Thailand are the
three largest producersin the world
Rosenberry, 1991!.
Eighty percentof the shrimp grown in
chirrensisalso known
as P. Orientalis!,but P. rrrerguiensis,
P.
penicillatus
andP. morrodon
arecultured
ona largescaleaswell.Relatively
small
numbersof P.japonicrrsand P. semisul-
catus are also grown in China Liu,
1990!.Penaeus rnonodorr,
a fast-growing,
hearty species,dominatesproduction
in the remainingAsian countries,but
P. rriergrriensis,
P. irrdicusand P, japmi-
crrsfigureprominentlyin somelocales
Rosenberry,1991!.Most of the shrimp
culturein Asiawould probablybe clas-
sified as extensive or semi-intensive,
yielding between 500 and 5,000
kg/ha/crop.
The United States has a small, young
shrimpcultureindustry.Almostall of
the farm-raisedshrimpareP. vanrrarrrei,
which are grown either semi-inten-
sivelyor intensivelyin SouthCarolina,
Texas and Hawaii. The United States is
the secondlargestconsumerof shrimp,
 Introduction

however,
importing ofitssupply Viral diseaseoutbreaksin populations
much
fromfarmsin Asia. in whicha virushasremainedlatent
can resultfrom stress;commonstres-
sorsareovercrowding,
abnormal
tem-
Pe@acid
ShrimpDiseases peratures,
andlowdissolved
oxygen
Severaldetailedreviewsof the litera- levels.Preventionof viral diseases
con-
turepertaining ofpenaeid sistsof quarantining
todiseases all introduced
shrimp
have including stockto virus-freefacilities,anddisin-
beenpublished,
thoseby Couch 978!, Ruangpan fectionof infectedfacilities.There are
W8!, Lightner
983,'1985!,
Proven- no knowntreatments for viraldiseases;
zano9&3!,Saticados
988!,Sinder- moreover, diagnosing
someviralinfec-
mannand Lightner988!, Johnson tionsis currently
an expensive,time-
989!,Lightner
andRedman
991! consuming
taskundertaken
onlyby
andSell991!.Nearly 30diseases
and highlytrainedpersons.
diseasesyndromesof cultured
penaeids,
withbothinfectious
and Rickettsia
noninfectious
etiologies,
havebeende-
scribed Sindermannand Lightner,Rickettsiaare rod-shapedmicroorgan-
1988! understood. isms known to cause diseasesin a
butmanyarepoorly
Themostimportant
pathogens
ofcul- variety
of taxa.Rickettsia
orrickettsia-
tured shrimpare viruses,bacteria, likeorganismshavebeenfoundin P,
fungiand protozoa. vannamei
Krolet al., 1991!,P. margi-
rtafusand P. merguiensis.
Penaeus
mono-
dortand P. siylirosfris
have been
Viruses experimentally
infectedBrock,pers.
comm.!,Tetracycline
maybe an effec-
tive treatment Brock, pers. comm.!.
"Viral diseasesand associatedmortali-
tiesareemergingas oneof the most Rickettsiaare not, however, usually
important
problemsin penaeidshrimp consideredseriouspathogensof cul-
culture"Sindermann, 1990!.Viruses turedpenaeids.
in crustaceans
werefirstreported
in the
rnid-1960s,
andto date,nonehavebeen Bacteria
adequately
characterized
Sindermann,
1990!,Thus far, 12 viruseshave been Anotherimportantcategoryof patho-
identifiedfrompenaeid shrimpLight- gensisbacteria. A groupofGram-nega-
ner et al., thisvolume!,and sixfatal tive, rod-shaped bacteria, most of
viral diseases of penaeidshavebeen whichbelongto the genusVibrio,is
reported.Infectionsin larvaeandjuve- associated with serious disease out-
niles are most cornrnon.Some viruses breaks in cultured populations of
arespecifKto oneor onlya fewspecies shrimp mortalitiesof up to 10096
«shrimp,whileothers
appearto be have been reported Lightner, 1983;
capable
ofinfecting
aHpenaeids. Lightneret al., 1984!. One disease
 introduction
causedby thesebacteriais calledvi-
briosis a type of bacterialsepticemia!,
and infections can be chronic, subacute
or acute. There are a number of patho-
genic speciesand strains,and their
virulencecan vary markedly.All spe-
cies of shrimp can be infected. Al-
thoughbacteriahavefrequentlybeen
associated with mortalities, most bacte-
rial infections areof secondarybacterial
etiology and result from extreme
stressesand opporturusticpathogens.
A number of chemicals and antibiotics
have been used to treat shrimp vi-
briosis, including EDTA, furanace,
furazolidone NF-l80!, erythromycin,
terramycinand chloramphenicol.
Chitinolytic bacteriamay invade exist-
ing breaksin the epicuticle,enlarging
wounds through the secretionof ex-
tracellular enzymes bacterial necrosis
and shell disease!, and filamentous
bacteria usually Leucothrixrnucor!may
attach to setae and gill lamellae. The
latter can affect respiration,and their
presenceis consideredindicativeof
stressful culture conditions.
Fungi
Fungal diseasesare also common in
shrimp.In manycases,infectionresults
from opportunistic invasions of shrimp
that have been injured or exposed to
stressful conditions. Fungi can cause
~ mass mortalities, especiallyin hatcher-
ies, where the disease larval mycos5
has proven to be quite deadly. Larval
mycosis'is usually caused by either
Lagenidiumcallinectes,Sirolpidiumsp. or
Haliphthoros sp., and mortalities can
reach 100% within 48 h after the onset
of infection. Treflan~ and malachite
greenarecommonlyusedto treatfun-
gal infectionsin hatcheries.
Another important fungal pathogen is
Fusarium solatti, the most common eti-
ologicalagent of Fusariumdiseaseor
black gill disease.All penaeidscan
contract Fusarium disease, but some,
like P. japotticus,
seemto be particularly
sensitive and suffer a high rate of mor-
tality when infected.
Protozoa
All shrimparesusceptibleto foulingby
epicommensal protozoa.Theseorgan-
isrnsarenaturallypresentin the culture
environment, Protozoa may attach to
the gills, appendages
and bodiesof
culturedpenaeids.A numberof genera
may be encountered,including Zoo-
thamnivrn,Epistylis,Vorticella,Acineta
and Iagenophrys.Late larval through
adult stagescan all be affected,and
high-density cultures are probably
more susceptibleto serious infesta-
tions. Extensive colonization of gills
may interferewith respiration,result-
ing in mortalities.
Microsporida,internal parasiticproto-
zoa,areresponsiblefor the abnormality
known as cotton shrimp or milky
shrixnp.Thus far, rnicrosporidosis
has
not beena major problemin cultured
shrimp, presumablybecausethese
pathogensrequireeitheran intermedi-
ate, or, more likely, a "conditioning"
host to completetheir life cycle.This
diseaseis more apt to occur in extensive
 p culture
situations,
whereinter- P.vsnnamei,
ees,
and
P.stylirostris
andP.setif-
gut-and-nerve
syndrome
ofP.
mediate
hostssuch
asfishmaybe
present
intheculture
envirorunent.
JapotiKks.

Finally,
gregarines
are inhabi- TheStatusof Penaeid
common
tants
ofpenaeid
intestinal
tracts,
Bell Diseasesln Asia
and
Ughtner
991!
reported
thatpopu-
lations
witha high
prevalence
ofgrega- Penaeusmonodon
rineinfestation
sometimes
exhibit
reduced
feeding
andgrowth
rates,
in- Asthemost
important
species
ofcul-
creased
surface
fouling,
and/or to tured
slight shrimp
intheworld,
P,rrirmodori
moderate
increases
in mortality. hasbeen
a frequent
subject
ofdisease
investigations
pathologists
have
NutNional,
Toxicand beenstudying
P. remod0ii
for years.
EnvironmentalDiseases Naturally
distributed
throughout
the
IndoWest
Pacific
regionfrom30'Eto
A fewofthemostcommon in 155'K
diseases longitude
andfrom35'Nto35'S
thiscategory
arevitamin
C deficiency,
latitude,
P.eonodon
is mostabundant
alsoknown asblackdeath;
black
gill in thetropical
watersof Indonesia,
disease,
a resultof exposure
to toxic Malaysia
andthePhilippines.
It has
levels
ofsubstances
suchasheavy
met- become
animportant
culture
species
in
als,ozone,
orarnrnonia nottobecon- countries
withinits range,especially
fusedwiththeblackgilldisease
caused Indonesia,
Thailand,India,thePhilip-
byinfection
withthefungus
FMsariiim
pines,
Vietnam
andTaiwan.
Penaegs
sp.!;
andgas
bubble
disease,
causedmonodon
is normally
considered
to be
by supersaturation
of culture
water exceptionally
hearty,but increasing
withatmospheric
gases.
Allspecies
of culturedensitiesand environmental
penaeids
arepresumably
susceptible
to degradation
havecontributed to dis-
thesediseases,
thoughsomemaybe ease
problems.
Serious
diseases
of vi-
moresensitive
thanothersto subopti- ral, bacterial, fungal, protozoan,
mal environmental conditions or cer- rickettsialandunknownetiologieshave
tain toxins. beenreported.
Diseasesof UnknownEtiology Infectious and Parasitic Diseases,
Fromaneconomic
standpoint,the most
In additionto the previouslymen- important
viralpathogen
ofP,~odour
tioned diseases and disease syn- maybe Pensees
rnoriodori-type
bacu-
dromes,there are a numberof lovirus MBV;Lightnerand Redman,
conditions
reported
fromcultured 1981!.Thisbaculovirus
affectsall life
penaeids
forwhichnocause
hasbeen stages
andhasbeenimplicated
in mass
discovered.
Examples
includeblack mortalities,especiaHy
in shrimp that-
spermatophore
disease,
affecting
male
 Introduction
are cultured at high densities or ex-
posed
to someotherstressor.
Accord-
ing to Saticados
988!,MBVhasbeen
detected in P. monodonfrom Taiwan,
thePhilippines,
Malaysia,
French
Poly-
nesia,Hawaii, Kenya,Mexico, Singa-
poreandIndonesiaTable1, alsosee graphicrangeof thevirusandthatP.
Colorni, 1990!.Ruangpan978, cited
in. Anderson et al., 1987! reported an
instance of 50% mortality in pond-
rearedP. monodonat a farm in Thailand
alsoseeFeganet al,, 1991!,and An-
dersonet al. 987! statedthat MBV is
likely to be normallypresentin all
culturedpond populationsof P. mono-
donin Malaysiaat low endemiclevels.
Infectious hypodermal and hemato-
virus IHHNV!hasalso ity, increased
poieticnecrosis
been found in cultured P. monodon.
Lightner983! reported80- 90%cu-
mulative mortalities within two weeks
of onset of IHI~ disease in 0.05- to
1-gP. mon
odon.Postlarvae
and juve-
niles are considered particularly sus-
ceptible,and P. monodon
infectedwith
IHI&lV have been reported from Ecua-
dor, Guam, Tahiti, the Philippines, Ha-
waii, Singapore,Israel,Panamaand
CostaRica Baticados, 19N; also see
Colorni, 1990!.Furthermore,IHICNV
has been found in southeastAsian cul-
ture facilitiesthat useonly captive,wild
P. monodon broodstock,suggestingthat
this regionis within the naturalgeo-
numodonmaybe a naturalhostspecies
LightnerandRedman,1991!.
Hepatopancreaticparvo-like virus
HPV!is yetanother
viralpathogen
of
P. monodon.
It has beenreportedfrom
MalaysiaLightnerandRedman,
1985!,
Israel Colorni,1990!,the Philippines
and Kenya Baticados, 19N!. HPV-in-
fectedP, rrurnodonexhibit poor growth
rates,anorexia,reducedpreeningactiv-
surface
fouling,
andoc-
casional
opacityof tail musculature.
A
reo-like virus REO! has also been
found in P. monodonin Malaysia An-
derson et al., 1987!.
There areno treatmentsfor the diseases
caused
byMBV,II~K, HPVorREO.
The only recourseis to prevent con-
taminationof virus-freestockandfac6i-
 g P ~ted
States
approval
isreqviref
fordrugs
and
chemicais
used
onshrimp
grown
forhuman
consumption.

tiesbydestroying
infected
animals
and juvenile
P.monodon
cultured
in Malay-
fluarantining
all importedstock. sianbrackishwaterponds. Anderson et
al. 988! isolatedbacteriaof the genus
A numberof bacterialpathogenshave Vibrio from the hemolymph, and also
beenreported
for P. tiumodori
Table2!. found Pseudomonas sp. and other Gram-
«angpan 982! and the Philippine negativebacteria. Theynotedthat vi-
Councilfor Agricultureand Resources briosis in juvenile shrimp has been
ResearchDevelopment PCARRD! treatedsuccessfullyby adding antibiot-
985, both cited in Baticados, 1988! ics to the diet; for the semi-intensive
pondsstudied,however,chemother-
reported that vibriosis affects proto-
~ai stages
andcausesheavymortali- apy was not an economicaloption.
ties up to SNABin P. moriokm Mortalities were reduced after the farm-
»tcheries. Anderson et al. 988! in- ers began to reprove excessdetritus
vestigatedseveralcasesof vibriosis in

from dried pondsfollowedby applica-
tions of CaO at the rate of 0.5 kg/m .
Chitinoclastic bacteriawere responsible
for exoskeletallesionsin pond-cultured
P. rrt0rtodonLio Po and Pitogo, 1990;
Baticados, 1988; Chen, 1990! and a
bacterial disease of larvae and young
postlarvae
termed"necrosis
ofappend-
ages"hasbeenshownto causemortali-
ties in hatcheries in the Philippines,
Indonesia, Malaysia, Singapore, Thai-
land and Taiwan Baticados,1988!.Ba-
ticados 988! discussed a disease
causedby luminous bacteriathat had
causedsignificantproblemsin hatcher-
ies in the Philippines, Indonesia, Ma-
laysia and Thailand. Filamentous
bacteria of the genus Leucothrixsome-
times attach to the cuticles of cultured
P. remodort.Finally, Liu 989! studied
the histopathologyof bacterialinduced
hepatopancreatitis
of culturedP. mono-
don in Taiwan.
With respectto the treatmentof bacte-
rial diseasesin Asia, a numberof drugs
have been tested in the Philippines to
combatlarval necrosis,including eryth-
romycin phosphate, streptomycin-
bipenicillin, tetracyclinechlorhydrate,
sulfarnethazin, furanace and chloram-
phenicol.Rigid sanitarypractices,such
as the chlorination of culture water,
removal of wastes and sediments from
the tank bottom, and increasingthe rate
of water exchangehave effectively re-
duced mortalities from luminous
ria. And finally, filamentous bacterial
diseaseis controlled in the Philippines
with Cutrine4'-Plus, a copper com-
pound, given upon onset of the disease
Introduction
at 0,1 rng Cu/l for 24h or 0.25-0.5 mg
Cu/L for 4 - 8 h Baticados,1988;light-
ner, 1983!.
Anderson et al. 987! reported on a
diseasesyndrome of P. morrodon
in Ma-
laysiain which rickettsiawere believed
to be the primarypathogens.MBVand
a reo-like virus were also detected in
the diseased shrimp, and Gram-nega-
tive bacteriawere implicated as secon-
dary pathogens. Interestingly, this
diseasesyndrome did not affect the P.
merguiensis
or P. indicesinhabiting the
same ponds.
One of the most serious fungal dis-
eases,larval mycosis,affects all cul-
tured penaeids,P. moron being no
exception.Lagenidiurrt
caltinectes
is the
most common agent, causing heavy
mortalities in larvae and postlarvae in
the Philippines,Thailandand Indone-
sia Baticados, 1988!. Simtpidium sp.
and Hatiphthorossp. have also been
reportedto causelarvalmycosisTable
3!. If theincidencerateis low, Baticados
988! reports, lagenidiumspp, infec-
tions can be managed by removing
sediments and dead larvae, increasing
+raterexchange,and reducingstocking
densities. In addition, malachite green,
Treflan~ trifluralin!, Formalinand de-
tergenthavebeenused to combatthe
disease,and potassium permanganate
is considered to be a useful disinfectant
Baticados, 1988!. Water management
bacte- techniquesand Treflan~areusedin the
Philippinesto combatSiroIpidiurrt
sp.,
and Hatiphfhorossp. infections are
treated with furanace, malachite green,
Formalin and potassium permanga-
Formalin and potassium perrnanga- The following epicommensalciliates
nate,HnaHy,detergent,calciumhypo- have been reported to infect P. mcmo-
chloriteand Resiguard+are used to don:Epistylissp.,Vorticetla
sp., Zoothum-
nium sp., Ephelota
disinfedsystemsin which Haliphthoms gemmiparaand
sp. has beena problem Baticados, Aorta sp. Baticados,
1988;Liaoet al.,
19M!. 1977; Gacutan et al,, '1977! Table 4!.
Kpistylissp. is quite corrunonin the
Adult P. monodomare sometimes in- Philippines and Taiwan, whereas
fectedby Fusariam
sp., the agent re- Zoothamnium sp. is prevalentin Thai-
sponsible for black giH disease in land, Indonesia,Malaysia and Taiwan,
penaeidshrimp.Accordingto a report and on juveniles and adults in the
by Ruangpan 982; cited in Baticados, Philippines.Ephetota
gemmipara is less
1988!.about50%of a samplepopula- common, but, nevertheless, caused
tion of cultured P. morudon suffered heavymortalitiesat a SEAFDEChatch-
disease,which resulted in ery in 1976 Gacutan et al., 1977!.
4u'ge
losses.
Liaoetal.977!,however, Ruangpan986! concludedthat the
observed
thatFasariumsp. wasrarein most important pathogenic protozoa
culturedp. m~kpn in Taiwan. Other for P. monody and P. merguiensis
larvae
~gi renownto infect P. moeodonare were the peritrichs Zoothameium sp.
Hyp~yces sp,, $aprotegnia
parasiticaand Epistylis sp. In the Fhilippines,
and4ptokgaia marmaBaticados,1988!. Zoothamniumsp. and Epistytis sp. at-
 Introduction
tackingjuvenileP. monodyarecontrol-
led with chloroquine diphosphate or a
Formalin bath Table 4!.
Gregarines, internal parasitic protozoa
that use bivalves as either intermediate
or "conditioning" hosts,togetherwith
luminous bacteria, were reported to
infect P. morrodonpostlarvae, resulting
in massmortalities within two days. In
Thailand, two speciesof gregarines
were found in the guts of 94% of the P.
monodyfrom a privatefarm Baticados,
1988!. Microsporidians also infect P.
monodon. The disease was observed in
fernale broodstock that manifested
white ovaries and were subsequently
found to be sterile Baticados,1988!.
Nutritional, Toxic and Envimnmental
Diseases.Like all cultured shrimp, P.
monodonwill suffer poor growth and,
perhaps,mortalityif exposedto exces-
sive levels of toxins or if deprived of
essential nutrients. The follovring dis-
eases and disease syndromes were
summarized by Baticados988! Table
5!.
Chronicsoft-shellsyndrome.Affecting
both juveniles and adults, chronic soft-
shell syndrome is a major causeof
decreasedproduction in ponds in the
Philippines.It is causedby nutritional
deficiencies,
pesticidesin thewaterand
poor water and soil conditions.This
syndrome also afflicts pond- and tank-
reared shrimp in Indonesia, Malaysia
and Thailand,
Red disease. This disease has caused
heavy mortalities Liaoet al., 1977;Liao
and Chao, 1983;Liao, 1984!. It affects
juveniles and adults in Taiwan, the
Philippines, Thailand and Malaysia.
Gradual mortalities in one case reached
98%. It may be caused by microbial
toxins in either rancid dietsor in organi-
cally rich pond detritus.
Fatty infiltration of the hepatopan-
creas.This condition has been observed
in P. remodon in Texas, Mexico and
Hawaii. Juvenilesand adults, especially
extensively cultured ones, had exces-
sive depositsof lipid in their hepa-
topancreatictubule epithelium. The
condition may be due to improper die-
tary lipid levels,impropercaloric-lipid
balance, or dietary toxins.
Blue disease. First observed. in brood-
stock in Tahiti where it caused mass
mortalities in 1978-79,blue disease has
also been diagnosedin P. mormfortcul-
tured in Indonesia, Malaysia, Taiwan
and Thailand. Blue P, monody have
low levels of astaxanthin; hence, this
diseaseis thought to be mainly due to
a nutritional deficiency. A viral etiol-
ogy, however, has not been dis-
counted. Blue disease is controlled at
Aquacop in the Philippinesby using
low densities,high-quality feedsand
frequentwaterexchangein broodstock
ponds and tanks.
Cramped tails.Reportedin Taiwan,the
Philippinesand Indonesia,this condi-
tion is seen in juveniles and adults.
Afflicted animals have a flexed, rigid
abdomen. The causeis probably a sud-
den increasein temperature,such as
thatexperienced
by shrimpwhenthey
are handledon warm days also see contamination
with toxicpollutantslike
Liao et al., 1977!. cadmium, copper, potassium perman-
ganate,ozone,ammonia, and nitrate
Hernocytic
enteritis.In the PhiTippines, Lightner,1985!.This conditionhas
bloomsof blue-green algaebelonging been observedin Thailand, the Philip-
caused pines,Indonesia,
to the family Oscillatoriaceae Malaysiaand Tai-
hemocyticenteritisin primarily young wan.

Iuvenilesas weH as subadult prawns

Lightner,1985!. Muscle necrosis. Associated with
stress,musclenecrosisis prevalentin
Heavy trtetai poisoning. Exposure to high-density
cultures.
Thechronicand
excessivelevels of mercury, copper, infected form of the disease when it
cadmium and zinc have caused disease affectsthe distalportionof the abdo-
in cultured P. monody, and cadmium menis termedtail rot Lightner, 1985!.
andcopperpoisoning weresuspected If large portions of the abdomenare
o cause mortalities in hatcheries in affected,mortalitiescan result Light-
Taiwan in 198Q-81 Kouet al., 1984!. ner, 1985!.This is apparentlya corn-
monproblemin Indonesia,Malaysia,
Thismorphological Taiwan, Thailand and the Philippines.
Bl ckgilldisease.
c nditioncanbecaused
by a number
of bioticandabioticagents,
including
v+4 bacterial,
fun~ andprotozoan Gas bubble disease. This occurs as a
~~ "s. vitamin C deficiency,and result of supersaturationof seawater
 Introduction 13

with atmospheric gases Lightner, piration. Penatespenicitlatusand P.

1983!. semisuicatus
presentin the sameponds
did not become sick. After an effort was
Finally,Nashet al. 98S! describedthe made to use only fresh fish as a feed
pathologicalchangesoccurringin P. ingredient, the diseasedid not recur;
monodoncultured in acidic, brackishwa- hence, red discoloration may be a nu-
ter ponds.The gills of the shrimpwere tritional disease,or it may have resulted
brown, and upon doser examination, from a toxinthat was presentin spoiled
were found to be clogged with ferric and rotting fish flesh.
hydroxide precipitate. Hypoxia and
stressresulted, causing low yields. Larval black spot syndrome was re-
ported from Aquastar hatcheries in
Diseasesof Unknown Etiology. Some Thailand Flegelet al., this volume!.
of the previously mentioneddiseases Zoea-1or zoea-2are occasionallyfound
have uncertain etiologies. In addition with a black lump of unidentified ma-
to these, at least six diseases of un- terial at the junction of the stomach,
known etiology have been reported to hepatopancreas
andmidgut.Theeffect
afflict P. monody specifically Table 6!: of this disease on larval survival is
red discoloration Liao et al., 1~ unknown,
larval black spot syndrome, spongy
tissuesyndrome,one-monthmortality The presence
of very spongymuscle,
syndrome, and yellowhead shrimp nerve, and other tissues has been ob-
Flegel et al., this volume!. served in P. monodon postlarvae,
growout shrimp and broodstockin
Red discoloration occurred in Taiwan ThailandHegelet al.,this volume!.No
where pond-rearedshrimp gradually other information is available on
turned yellowish green, red and then spongytissuesyndrome.
Hegelandhis
pale. Heavy mortalities accompanied coworkers also described one-month
the symptoms, which also included xnortalitysyndrome,a growoutdisease
loss of appetite and difficulty with res- characterized
by suddenmortalityfour
 trNroduction
ease prevention. Meanwhile, Taiwan
continues to recover from the 1988 dis-
aster. Some farmers are stocking P.
monodortat lower densities, while oth-
ers have switched to different species.
Economic Impact of Disease. No one
has estimated the extent to which dis-
ease affects the P. monodom culture in-
dustry in Asia. At best, isolated
incidences of mortality have been at-
tributed to certain diseases or disease
syndromes.For example,Andersonet
al. 988! reportedthat vibriosiscaused
70 - 95% reductions in the expected
harvests for three farms in Johore, Ma-
laysia,during 1986and 1987.While the
1988collapseof the Taiwaneseshrimp
industry could not be attributed en-
tirely to disease,the costof the crisisin
its entiretywas approximatelyUS$376
million given that exportsworth US$
470million in 1987decreasedby 80% in
1988[Liao, 1989]!.
Penaevs chinensis
The Chinese have been culturing
shrimp for centuries,and, in the 1980s,
China became an important player in
the global shrimp market.Approxi-
mately80%of theshrimpgrownin the
People's Republicof China are P.
chi~sis. Penaeus chinensis also known
as P. orientalis! is a temperate species
with a small natural distribution. The
main fishing groundsare in the Bohai
Bay in northernChina'sYellowSea,
butthespecies
extends
to themouthof
the Pearl River in southern China and
is also relatively abundant along the
western coast of the Korean peninsula.
15
In addition to the farms and hatcheries
in China, there are several P. chinensis
hatcheries and farms in South Korea,
and the speciesis beinginvestigatedfor
productionin Taiwan.The majority of
production is extensiveor semi-inten-
sive, and high growth rates have been
reportedfor this speciesMain and
Fulks, 1990!.
Infectious and Parasitic Diseases. To
date, the only viral disease agent re-
ported to infect cultured P. chinensis
is
hepatopancreatic parvo-like virus
HPV, seeTable7! Lightner and Red-
man, 1985!. HPV-positive P. chinertsis
have been found from South Korea and
China. Wang and Ma 990! reported
on a diseaseaffecting shrimp in China
whose grosspathologywas similar to
the baculoviral midgut gland necrosis
virus HMNV! that afflicts P. japotticus,
but no viral pathogenswere isolated
from diseased individuals.
The usual bacterial pathogens have
been reported to infect P, chirtensis
in
China and South Korea, and in both
countries, antibiotics are sometimes
added to the feed to combat bacterial
infections Table 7! Main and Fulks,
1990!. Vibrio spp. and Pseudomonas
sp.
can cause serious mortalities in hatch-
eries, as well as during growout Liu,
1990;Wang and Ma, 1990!.Liu 990!
reportedthat infectionsare more seri-
ous in southern China where tempera-
tures are higher, and that copper
sulfate has been successfully used to
control vibriosis in P. chinensis hatcher-
ies. Treatment with 1 - 2 pprn chloram-
phenicolis also effective Wangand
~, 1990!.
Inaddition,
filamentous
bac- Table
7!.According
to WangandMa
t«ia,Leuarthrix
sp.andThiofhrix
sp., 990!,in Chinathesepathogens"do
aresometimes
foundadhering
to the veryserious
damage andcandestroy
surfaces
ofeggs
andlarvae,
butkeepingalltheshrimpfryproducedduring
the
theculture
water
deancanprevent
this breeding
season,fin
some hatcheries]."
WangandMa,1990!. Preven.tive
measures
include
carefully
chsmfechngfemalebroodstock,
and
Asfarasfungalinfections
arecon- regular
treatment
oftherearing
water
cerned,
P. chinensis
eggsandlarvae with6 - 10ppbmalachite
green.In-
maysuffer
fromlarval
mycosis,
caused fected
larvae
aretreated
withnystatin,
~yLugenidiuer
sp.orSirolpidium
sp. a "fairly
effective"
cure.Formalin
0
 Introduction
Table8. Nutritional,toxicandenvironmental
diseases
reportedfromculturedPenance
ppm! is usedto disinfectrearingtanks
in South Korea to prevent both fungal
and protozoan infections Main and
Fulks, '1990!.
Wang and Ma 990! discussed para-
sitic ciliate disease,caused by a new
Strain Of ParanophrySCarCirli,an en-
doparasiticciliate Table 7!. In China,
the disease is found in larvae and over-
wintering adults. Parasitic ciliate dis-
ease often causes 10' mortality in
infected tanks. While no effective treat-
ments have been found, the disease can
be prevented if tanks are disinfected
with 25 ppm Formalin prior to use.
D. Chen this volume! listed three mi-
crosporidians that occasionally infect
cultured P. chiriensisin China, causing
cotton shrimp disease.They are Pteisto-
phorasp., Thetohania sp. and Nosema sp.
Table 7!.
Kpicommensalciliates also causeprob-
lems in China, and to a lesser degree
in South Korea Main and Fulks, 1990!.
In China, 38 species of ciliates have
been identified from cultured P. chinen-
57
chi~is,
sis. Zoothamniumsp., Carchesiurnsp.,
Vorticellasp. and Fpishjtissp. are the
most common Wang and Ma, 1990!.
Serious infestations are prevented by
keeping the culture water clean and
supplying a good diet. In addition to
disinfection with Formalin, the Koreans
treat protozoan infestations by chang-
ing 90% of the water in affected tanks.
Finally, Wang and Ma 990! made a
brief reference to another disease
causedby a protozoanpathogen,"flag-
ellate adhesive disease."
Nutritional, Toxic and Environmental
Diseases.The diseasesin this category
reported for P. chinerisisare bubble
disease,deformitydisease Wang and
Ma, 1990!, body cramp, muscle ne-
crosisand floating head syndrome D.
Chen, this volume! Table8!. The latter
diseaseoccurson hot summer days in
high-densitygrowout pondsthat have
insufficientphytoplanktonbloomsand
poor water quality. Severemortalities
can result; the cause is probably an
insufficient supply of dissolved oxy-
gen.
Diseases
of Unknown Etiology. Al- japonicus,and in Taiwan, that species
thoughtheydid notmentionthemby comprised 30% of production Rosen-
name,Wangand Ma 990! said that berry, 1991!. Although the species is
thereare some diseasesof P. chinensis relatively expensiveto culture due to a
for whichno causeis known. Also,Liu high protein requirement and its need
etal. {1989!
reportedon red leg disease for a dean,sandysubstrate,P.j aponicus
of P,chmensisand P. penicillafus.It was nonethelessfetch a high price, tolerate
reportedlyrampantin the FujianProv- low temperatures,and can survive long
inceof southernChina, and may be journeys out of water.
causedby a bacterium.Other diseases
in this category
are white-blackspot Infectious and Parasitic Diseases. Lar-
disease,
white spot disease,and soft val and postlarvalP. japonicusare sus-
shellsyndromeseeD. Chen, this vol- ceptible to a severe epizootic viral
ume!.Finally,P. chinensis
culturedin disease,baculoviralmidgut gland ne-
China
sometimes
sufferfroma poorly crosisvirus BMNV! disease Table 10!
understood
condition that has been Sano et al., 1981, 1983, 1984, 1985;
caHed
black
gGIdisease
whichmayor Momoyamaand Sano,1989;Lightner,
may not be the sameas either Fusarium 1988!.Mortalitiesof up to 90'%%d
have
disease
orthetoxicdisease
bearing
the beenreportedin somehatcheriesSano
samename MengandYu, 1982! Table et al., 1984!.Known since approxi-
9!. mately 1971, BMNV infections occur
orally or by exposureto water-borne
«o<ornic
Impact
of Disease.
Theeco- pathogens,resultingin drasticallyhigh
nomicimpactof diseaseon cultured p. mortality within the first week of an
inensis is unknown. outbreak.

~~ftaeosj
aponicus Preventionconsistsof rinsing and
transferringeggsto a disinfectedtank
PNtaess
jspori~ is a colorfulshrunp immediatelyafterspawning.Accord-
grownprimarilyin Japan,Taiwan,and ing to Momoyarna
and Sano989!,
SouthKorea
forconsumptioninJapan "rinsing eggswith seawater has now
In Japan,
9>o%%d
of the shrimpgive beencarriedout on an industrial scale
weight!producedin 1991were P. in Japansince1985and,to date,BMN
 Introduction 19

Table 10. infectiousdiseasesand diseaseagents of cultured Penaeusjaponicus.

has never occurred in the hatcheries be associatedwith gut-and-nervesyn-

where it has been practised" also see
Sano and Mornoyama, this volume;
Momoyama, this volume!.
A reo-like virus REO! was first discov-
ered by Tsing and Bonami 987! in
juvenile P. japmicusin France.Reo-like
viruses have also been reported in Ha-
waii, among P.j aponicusimported from
Japan lightner et al., 1985; Ughtner
and Redman, 1991!.Tsing et al. 985!
suggested that infection by RKO may
drome GNS!, an idiopathic condition,
which was found in chronically iQ
populationsof P. japmicusculturedin
Hawaii Lightnerand Redman,1991!.
Two serious bacterial diseases have
beenreportedin P.japonicusgrown in
Japan,vibriosis and bacterialgill dis-
easeTable10!,The formerhascaused
remarkablyhigh mortality,especiallyin
the summer and autumn seasons. The
pathogenis apparentlyan unidentified
species
ofthe
Vibrio
genus,
and
affectedium sotani, the etiological agent of
shrimp brownpigmentationFusariumdiseaseor black gill disease
manifest
organandthegillas Table10!.It hasrecentlybeendiscov-
in thelymphoid
wellascloudiness
in the muscular
tis- ered that F. monjtiformecan also cause
sueof the3rdto 6th abdominalseg- Fusarium disease Rhoobunjongdeet
ment
Pakahashi
etaL,1985;Sanoand al., 1991!.No effective chemotherapies
Fukuda,1987!,Duringgrowout,Japa- arein usefor P.japonicusinfectedwith
nesefarmerstreatvibriosisin P. japonj- Fssariumsp. While mortalitiesmay ap-
cssbyincluding
oxytetracyciine
in the proach100%,Shigueno cited in Main
feedat the rateof 50 mg/kg shrimp and Fulks,1990!reportedin 1990that
weight treat- Fusarium diseasewas rare in P. japotri-
perday.Bylaw,however,
ments
mustend25daysbeforeharvest- cusculturedin Japan.Rearingat lower
ing if theshrimpare to be usedfor densities may prevent the disease
human consumption Sano and Lightner, 1983;also see Momoyama,
Fukuda,1987;alsoseeMain and Fulks, 1987and Bian and Egusa, 1981!.
1990;Shigueno,1975;Davy, 1991!.
Finally, epicommensalciliates have
Shigueno97S! reported that an un- beenobservedin P. japonicusraisedin
identifiedbacteriumis responsiblefor Taiwan, South Korea and Japan Main
bacterialgmdisease. This conditionis and Futks, 1990! Table 10!.
prevalent in high densityculture tanks
and is most commonlyseen in the Nutritional, Toxic and Environmental
spring.Lossesfrombacterialgill dis- Diseases.Penatesjaponicus is suscep-
easearenotashigh as from vibriosis, tible to those diseaseswith nutritional,
andtreatment consistsof bathingthe toxic and environmental etiologies,
shrimpin 2- 3 ppmfurazolidone for 2 suchasgasbubblediseaseand muscle
to 4 consecutivenights. Bacterialgill necrosis,that affect other species of
diseasemay be the sameas gill rot shrimp.
disease,
whichwasreported
by Sano
and Fukuda987! to inflict heavy Diseasesof Unknown Etiology. Black
losses
ontheJapanese P. japmicus
in- spot diseaseand gut-and-nervesyn-
dustryseeTable'10!. drome GNS! are two diseasesof P.
japonicus for which etiological agents
»cterial infections have also been have not been discovered Table 11!.
P. japonicus
cultured in South The latter is known from France and
Korea
andTaiwan,
andLcscefhrix
sp. Hawaii where shrimp exhibited ano-
attach
to cultured
p. jupogjcgs
in Japan rexia, lethargy, poor feed conversion,
andSouthKoreaDavy,1991;Main epibioticfouling and muscleopacity
a«Fulks,1990!. Brock,pers. comm.!. Black spot d.is-
easeis poorly described,and affected
According
toLightner
953!, P.japmi- P. japmicuscultured in Taiwan Main
particuiarlysusceptibleto Fmar- and Fulks, 1990!.
 lntroductloh 21

Table 12. Disease-related losses of Penaeusjaponicus in Japan in 1984 Fisheries

Econoxnicimpact of Disease, In 1984, Infectious and Parasitic Diseases.

70.2 MT of P. japmicusworth Y 466.1 There are few references to the viral
mNion were lost in Japandue to dis- diseasesof P. merguiensis in the litera-
ease Sano and Fukuda, 1987!. The ture, althoughthe speciesis known to
greatestlosseswere attributed to vi- be susceptibleto MBV observedin
briosis 0.8 MT! and unknown dis- SingaporeandMalaysia!and HPV ob-
eases 7.3 MT! see Table 12!. served in Singapore and Australia!
Fusariumdisease,BMN, and gill rot Lightner, 1985; Roubal et al., 1989!
diseasewere the other major diseases Table13!.LightnerandRedman985!
impactingJapan'sculturedP.j aponicus stated that in juvenile P. merguiensis,
industryin 1984. HPV has caused cumulative mortalities
of up to 50%. An unidentifiedbacu-
Penaeus rnerguiensis lovirus was also isolated from a wild
specimenin Australia Doubrovskyet
This speciesrangesfrom the Arabian al., 1988!.
Gulf and Pakistanthrough the Malay
Archipelagoand South China Sea to
Australia Dore and Frimodt, 1987!. No referencesto the bacterialox fungal
Penaeus merguiensis
is an importantcul- diseases of P. merguiensis
were foundin
ture speciesin Indonesia, Vietnam, the literature.Presumably,this species
Thailand and the Philippines Rosen- is susceptible to the samebacteriaand
berry, 1991!. pathogenic fungi infectionsthat affect
other shrimps.P. rnerguiensis is, how-
ever, one of four penaeidspeciesto be
diagnosedwith rickettsial infections Diseasesof Unknown Mology. Little
Lightneret al., 1985!. information
was found regardingP.
merguiensisdiseases that have un-
Ruangpan
986!studiedthe patho- known ot uncertain etiologies.Liu
genicprotozoaaffectingculturedP. 990!,however,did report that P. mer-
monlionandP.merguiensis
in Thailand. ', alongwith P. chinensisand P.
Shediscovered
thatin P. merguiensis
penicillaRs,cultured in China suffer
"cotton"
or"milkydisease"
produced fromredlegdisease. Thisdisease
may
bythemicrosporidian,
Thebheria
sp., becaused
by a bacterium.
caused
losses
upto55%.
Furthermore,
two epicommensal
protozoans
were Kconomic
Impactof »sease ~e
identi6ed
asimportant
pathogens,
ns, Ep- '- nomic
impactof diseaseon cultured P.
shjlis
sp.andZoothetmium
sp. Table mcrguiensts
is unknown.
13!.

Nutritional,
ToxicandKnvtro tal Penaeuspenicillatus
lseases.
P.merguiensis
issusceptible
penaeus
prnici7tatus
is a minor ~
tothose
diseases
withnutritional
o, toxic
an environmental
and suchas species.
etiologies, Grownonlyin the south«n
gasbubble
disease
andmuscle
ne- provincesof China and in Taiw~ '
crosis,
thataffect
other specioff
r species
culture
techniques
aresirrular
« tho
s rnp. ofP.monodon
andP.cdnensis.
P.P
latus
reportedly
hasa low
protei«e
 introduction 23

Table 14. Disease agents of cultured Penaeuspenicillatus.

quirementand an unusuallyfavorable Diseases of Unknown Etiology

head to taBratio Liao and Chien, 1990!.
Infectious and Parasitic Diseases
VeryBttlehasbeenpublished
aboutthe
diseasesof cultured P. penicillatus.In
Main and Fulks 990! it was reported
that cultured P. penicillatusin Taiwan
have been diagnosedwith the follow-
ing: MBV, bacterialinfectionsof the
hepatopancreas
andepicommensal cili-
ates and bacteria Table 14!.
Nutritional, Toxic and
Environmental Diseases
P. penicithfusis susceptibleto those
diseaseswith nutritional, toxic and en-
vironmentaletiologiesthat affectother
speciesof shrimp.
The only diseasesof uncertainor un-
known etiologyobservedin P. penicil-
tafusare blackspot diseaseand red leg
disease. The former afflicts pond cul-
tured P. penicitlafusand P. japonicusin
Taiwan Main and Fulks, 1990!.A bac-
terium is suspectedto causered leg
disease,a seriousproblemin P, chinen-
sisand P. penicillatus
grown in China's
Fujian Province Liu et al., 1989!.
Economic Impact of Disease
The economicimpact of disease on
cultured P. penicilhtusis unknown.
 Introduction

TheStatusof Penaeid ing in Texas South Carolina and Ha-

Diseasesin the United waii Rosenberry,1991!.
States Infectiousand ParasiticDiseases.At
least four viruses infect cultured P.
PsosBus
vannamei
vetnamei:
Baculovirgs
penaeiBP!, infec-
tioushypoderinal
and hematopoietic
necrosis
virus IHHNV!, REOand HPV
Pensees
vsnnamei
is the dominant Table15!.BPcauses
high,acutemor-
penaeid
cultured
in the westernhemi- talities Lightner, 198S;Overstreetet
sphere.Seventeen
percentof the al., 1988!,but the effectsof IHHNV on
shrimpproduced
worldwide
in 1991 P. vannamei aremoresubtle,oftenre-
.as P. vennamei
Rosenberry,
1991!. sultingin lowergrowthratesand an
Tlienatural
distribution
ofthisspeciesuneven
sizedistribution.
IHHNVmay
extends
fromthePacificcoastof south- betheetiological
agentofrunt<ef
orm-
ernMexico
to peru;Ecuador
is the
largest
producer P.van- Ity
offarm-raised syndromeRDS!in P. vannamei
Kalagayan
etal.,1991!.
A reo-likevirus
namei.
Because
it hasoutperformed
na- hasonlybeenreported
oncein P. van-
tiveshrimps,
p. vannamei
isgrown
in namei
culturedin theUnitedStates,
and
t"eUnited
States,
where,
in 1991
there then it was found concurrently
with
were22farms
and3 hatcheries
operat- shrimp experimentally
infected
withBP

Krol et al., 1990!. HPV, by contrast,
has only been found in cultured P.
vanriamei from Brazil, Ecuador and
Mexico Lightner et al., 1990,Lightner,
pers.comm!.Viral diseases
areconsid-
ered to be a seriousthreat to the shrimp
culture industry in the United States;
hence, intense efforts are underway to
ensure that P. vunnarnei cultured in the
United States are free of all detectable
viruses seeWyban, this volume!.
Penaeusvannameiare susceptible to all
the common bacterial diseases.
briosis and filamentous bacteria, for
example, are commonly encountered
on farms and in hatcheries. In addition,
infection of cultured P. vannamei with
an acid-fastbacteriumwas describedby
Krol et al 989!. Outbreaks of filamen-
tous bacteria are treated at The Oceanic
Institute hatchery by increasingthe rate
of water exchange and "fine tuning"
fe e ding schedules Wyb an and
Sweeny, 1991! Table 15!.
Larval mycosishas causedseveremor-
talities in P. vannamei hatcheries. At The
OceanicInstitute, the agent responsible
has been identified as Sirvlpiditcrnsp.,
and mortalities can be as high as 100%.
The disease is now controlled by the
addition of 0.1 ppm Trefian~ once per
day. The incidence of larval mycosis
can also be reduced by periodically
disinfecting reservoirs and water lines
Wyban and Sweeny, 1991! Table 15!.
Outbreaks of epicorrunensal protozo-
ans are prevented at The OceanicInsti-
tute's P. vannamei hatchery by 1!
completely drying out the hatchery be-
Introduction 25
tween rearingcydes, and 2! water fil-
tration Table 15!. Formalin was re-
cently approved by the U.S. Food and
Drug Administration for controlling
epicommensal protozoans m cultured
shrimp Code of Federal Regulations,
1991!.
Nutritional, Toxic and Knvlronmental
Diseases
Lightner and Redrnan982! studied
the histopathology of aflatoxicosis in
Vi- juvenile P. vannameiand P. stylirostris,
and found the penaeids to be remark-
ably resistant to aflatoxin. Aflatoxin is
created by fungi and is occasionally
present in the ingredients used to make
fish and shrimp feeds. Periaeus vminaeei
are, however, susceptibleto gasbubble
disease,body cramp and other shrimp
diseaseshaving nutritional, toxic or en-
vironmental etiologies.
Diseasesof Unknown Ktiology.There
are two noteworthy diseasesyndromes
that fan into this category: Z-l syn-
drome and Texas necrotizing hepa-
topancreatitis alias "Texas pond
mortality syndrome"!. The former was
first encountered in 1987 at The Oce-
anic Institute's hatchery where high
mortality ratesat the zoea-1stagewere
coupledwith severedeformities Wy-
ban and Sweeny, 1991!.While the de-
finitive agent is unknown, the disease
was effectively prevented by the addi-
tion of the chelating agentEDTA to the
rearing water, prompting speculation
that heavy metalsmay have beenre-
sponsible.
 tices, instituting quarantine proce.
Texasnecrotizing
hepatopancreatitis
hasthusfarbeen onlyinP. dures,
observed stocking only speciflicpathogen
free SPF!shrimp,and vaccinabon
rsoeumw'
cultured
inTexas.
Thedisease
ischsri~ized inmortal- Preventiveculture practicesare neces.
byin@eases
ityandmorbidity,
poor soft sarytoexclude
growth, or reduce thenumber
sheUs, empty
intestinal thin of pathogens
tracts, presentin the cuitiu
tails,lethargy,
surface andele- environmentand to minimize the stress
fouling
vated
foodconversion in pond- experienced
ratios by the animals.Examples
cultured etal.,submitted!. includedisinfecting
shrimpBell tanks and drying
While ispoorlyunderstoodponds
thedisease between cymes,
optimizing feed-
andprobablyhasan environmental ing regimes,providinghigh-quality
component,
Vibrio bacteria feeds,etc. An example of researchin
orVibrio-like
have been identifiedfrom diseased this area is the work of LeBlanc and
shrimp during epizootics, and re- Overstreet991a and b!. The authors
searchershavefoundseveralotherpo- testedthe meansby which culturefa-
tentiallypathogenicmicrobesin cilitiescouldbe disinfectedto prevent
diseasedindividuals.Texasnecrotizing Baculovirus penaeiBP! infections.They
hepatopancreatitis has been treated found that 48 h desiccation inactivated
with oxytetracydinewith promisingre- BPin hepato pancreatic tissue LeBlanc
sultsBellet al., submitted!. and Overstreet,1991a! and conduded
that desk@ation may be, in many cases,
Economdc Impactof Disease themostpractical meansof preventing
Theeconomic
impactof disease
on P. BP infections
in aquaculturefacilities.
vennenei culture is unknown. BP was also inactivatedby treatment
with chlorine at concentrations of 200
Currentand FutureAvenoes mg/Lfor 1 h or 1,600 mg/L fot 20 s
of Research LeBlancand Overstreet,199lb!.

Shrimppathologists
aroundtheworld Anotherdiseasepreventiontool is
are currentlyengagedin researchto quarantming
importedstocksa
osis, an ingonlystockthat has beencert ed
eatrnentof diseasesin cultured
treat
SPF.p numberof viruseshavealready
penaeids.This sectionwill discuss beenintroducedinto previously
someof themostcurrentadvances in 11cleantt areasvia infected i p
penaeiddiseaseresearch.
these
introductions
aleregarded
as
OiseasePrevention rious
setbacks
totheglobal
shrimp
~
tureindustry.
Therearea numberof ways toprevent
andspreadof disease inally,it is theoretically
p~
Finail
thee occurr
occurrence
prevent the onset of some dis ~
among
cultured
penaeid
shrimp,
in-
dudingadopting
better culture prac- vibriosis
forexample
by "u
tion. Ev
Eventhough
marineinvertebra

have nonspecific immune systems, a
number of bacterial vaccines have been
developed and tested on shrimp e.g..
Giorgetti, 1990; Itami and Takahashi,
1991; Laramore, this volume!,
mixed results. Vaccination is an expen-
sive prospect, however, and one prob-
lem that must be solved is the efficient
delivery of the vaccine to a cultured
population Dunn et al., 1990!.
Diagnostic Techniques
In recentyears, a numberof researchers
have brought attentionto the needfor
betterdiagnosfictechniquesfor shrimp
viruses Lewis, 1986; Lightner et al.,
1983b, 1990; Bell et al., 1990a; Thurman
et al., 1990; Baticados et al., 1991!.
Commonly, health experts employ
light microscopyto detectcharacteristic
signsof viral infection e.g., occlusion
bodies! in stained preparations of tis-
sues. Electron microscopy is important
in someapplicationsas well. In addi-
tion to the problemsof cost,time, and
accessibility,these techniques,practi-
cally employed, are not sensitive
enoughto detectlatent infections,ne-
cessitafingthe useof enhancement and
bioassays. Enhancement is accom-
plished by stressinga suspectpopula-
tion of shrimpto triggeranylatentviral
infectionsinto patency.To conduct a
bioassay,one feedsthe suspectshrimp
to a specificpathogen-freeSPF!labo- Thurman et al. 990! tested the efficacy
ratorypopulationof an extremelysen- of fluorescent microscopy with wet-
the population, mount tissue squashesstained with
sitivespecies,stresses
and subsequentlytestsfor the presence
or absenceof the virus. Bioassaysare
extremely labor- and time-intensive;
furthermore, SPF shrimp are increas-
Introduction 27
ingly difficult to find, and they mustbe
maintained.
Ideally, new diagnostic methods
with should be rapid, simple, inexpensive,
more sensitive than existing tech-
niques,and easilystandardizedLight-
ner et al., 1990!. Lightner et al. 990!
state that "methods using tissue cul-
ture, serologic methods, and gene
probediagnostic
techniquesthat have
becomecommonplace in human and
veterinary medicine are being devel-
oped for penaeid shrimp" also see
Lightner et al., this volume!. An exam-
ple of one of these "high-tech" ap-
proachesta penaeidviral detectionis
the use of the enzyme-linked immu-
nosorbent assayELISA! methodto de-
tect BP in Permeusduorarurrr Lewis,
1986;Lightner et al., this volume!. Fur-
thermore, the first documented pri-
mary cell cultures for shrimp were
describedby Chen et al., 1986.
Improvementshave alsobeenmadein
the standardtechniques.For example,
Bell et al. 990a! recently develapeda
nonlethal biopsy procedure for D.IHNV
that involves the excision of the first
pereiopod,followedby standardhisto-
logical examinationof the appendage
nerve cord. Animals tested by this
method need not be sacrificed and re-
main available for use as broodstock.
phloxineto detectbaculoviralocclusion
bodies.Theyachievedgoodresultsand
concluded that the time required to
diagnosebaculoviralinfectionscould
be reducedusing the new technique
also see Sano et al., 1985 and Mo-
moyama,1988!.
In the meantime, new diseasescon-
tinue to be discovered. Owens et al.
991! recently found evidence for a
new shrimpvirus, lymphoidalparvo-
likevirus LOPV! in farmedP. trronodotr,
P. eerguietrsis
and P. escalates.The
virus appearsto be closelyrelatedto
IHEQW.
Drugs/Chemotherapy
"Chemotherapyshould be consid-
ered as an emergency or last-resort
measure. Although chemicals may
reduce
theincidence
of pathogensor
contxol the abundance of facuhative
organirrms, they alsomayhave nega-
tive effects on desirable pond biota
andon the flora of biologicalfilters.
Somechemicals
maybe hazardousta
the user or leave undesirable or
haxmful residues in the cultured ani-
mals" Meyer, 1991!.
When preventativemeasuresfail, it
may be necessaryto treatdiseases
with
antibiotics or chemicals. A number
treatmentsare currently being usedin
many Asian countries,where the use
of compoundslike saponin,Formalin,
malachite green, Treflan4, chloram-
phenicol, oxytetracydine and furanace
is commonplace. Such therapy is most
practically
applied in hatcheries,where
densegroupsof animalsare presentin
smallvolumes of water. It is simply not
cost-effective to cornbat diseases en-
countered in extensive and semi-mten-
sive pondswith antibiotics
or other
expensivedrugs.
In the United States, Cutrine-Plus~ is
approvedfor use as an algicide,and
Formalin may be used to cornbat epi-
comnensalprotozoans. Sincethese are
the only forrnaHyapproved substances
for the preventionor treatment of dis-
easeson U.S. shrimp farms, efforts are
u.nderwayto developnew drugsand to
obtainregulatory approval for those
which have been shown to be safe. The
following statementby Meyer 991!
representsthe views of many U.S.
aquaculturists also see Sinderrnann,
1986!:
"An urgentneedexistsfor regulatory
approvalof therapeuticdrugsfor use
in combatingdiseases in aquaculture
... increasedfederalsupport and
eatercooperationand involvement
regulatory agenciesare vital if
producersareto beableto controlthe
economiclossespresentlycausedby
diseases.Without such support, the
aquacultureindustry in the United
StateswN fail to achieve its full po-
tential and be unable to compete in
the world market" Meyer, 1991!,
ConclusionS
of
Accordingto Meyer 991!, "Disease
problems constitute the largest single
cause of economic losses in aquacul-
ture." Whether this holds true for
shrimp culture remains to be seen;
since, as this paper has shown, few
estimates of the economic impact of
shrimp diseasesare available. Clearly,
diseasescan causeserious problems in
the culture of all shrimp species,wher-
everthey are are grown. In the future,
it wiH be important to have accurate
estimates of the economic damage
 introduction 29

wroughtby the variousdiseasesin growingshrimp.Too many farmers

orderto effectivelymanagethem.Such had little or no trainingin cultureprac-
estimateswill, in essence,help define ticesthat preventdisease.In addition
the problemso that it can then be to prevention,shrimp farmersshould
solved.Accurateestimateswill depend alsobe educatedabout how to recog-
uponaccurate Identification nizeand quicklyrespondto thosedis-
diagnoses.
of the causesof diseasesfor which no eases for which sophisticated
etiological
agentshavebeenfoundwiD equipment and/or training is not
enhanceeffortsto defineand manage needed. A farmer would probablybe
"the diseaseproblem"aswell. unableto diagnosea viral disease,for
example,but he or shecouldbe trained
In addition to documentingtheir dis- to recognize
andrespondto vitaminC
easesituation,it may in the bestinter- deficiency,
gasbubbledisease, infesta-
est of shrimp producing nations to tions of filamentous bacteria, etc.
institute quarantineregulationswher-
ever they have not already been Literature Cited
adopted.Suchregulations
will slowthe
Anderson,I,G., M. Shariff, G, Nash and M
spreadof virusesandotherpathogens Nash.1987 Mortalities
of juvenileshrimp,
from their natural geographicranges Penaeusmonodon,associated with Penaeus
and hostspecies. nronodon
baculovirus,
cytoplasmic
reo-like
virus, and rickettsialand bacterial infec-
tions,from Malaysianbrackishwaterponds.
There is also an indisputableneedfor Asian Fisheries Science. 1:47-64.
more research into all aspects of Anderson,l.G., M. Shamsudin,M. Shariffand
G. Nash. 1988.Bacterialsepticemiain juve-
penaeiddiseases.Penaeidculturegen- niletigershrimp,
Penaeus
rnonodon,
cultured
eratesbillionsof dollarseveryyearand in Malaysianbrackishwater
ponds.Asian
Fisheries Science.2: 93-'108,
diseasescan seriouslyimpact produc- Baticados,M,C.L. 1988. Chapter6: Diseases.
tion levels.Key researchareasinvolve In:Biology
andCulture
ofPenaens
nronodon,
preventinginfectiousand noninfec- SEAFDEC
Aquaculture
Dept.lloilo,Philip-
p! nes.
tious diseases, developing better Baticados,M.C.L., C,L. Pitogo,M,G, Paner,L.
quicker, less expensive,more sensi- de la Peha and E.A. Tendencia,1991. Oc-
tive! diagnostictechniquesand finding currence
andpathology
ofPenaeus
rnonodon
baculovirus infection in hatcheries and
safe, effective treatments for infectious pondsin thePhilippines.
Bamidgeh.
43!:
diseases. 35-41.
BeV,T.A. 1991.Overviewof diseasesand drug
needs for major aquaculturespecies:
Finally, in someareas,shrimpfarmers Shrimp,
Vet.Hum.Toxkol.
33 Supplement
need to be better educated about mat- 1!: 19-23.
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andJ.A. Brock.1990a,
A biopsy
procedure
forthenondestructive
panelinvestigatingthe 1988culturecri- determination
ofinfectious
hypodermal
and
sis in Taiwan concludedthat rapid hematopoietic
necrosis
virusIHHNV!in-
fectionin Penarus
osnnanta'.
J. AquaticAni-
growth in the P. rttortodort
industry in rnal Health. 2: 151-153.
Taiwan encouragedinexperiencedper- Bell,T.A. andD.V.Lightner.
1991.Chemother-
sonsto try to makequick, easymoney apyin aquaculture
todayCurrent
practices
 IntrorMtiott

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P.J. Invertebr. Pathol. 57: 277-286,
utierrez and G,L. Po. 1977. A suctorean
 Introduction 31

LeBlanc, B. and R.M. Overstreet. 1991b. Effi- shrimp, Perraerrs

rrrorrodorr.
J. Invertebr.
cacyof calciumhypochlorite as a disinfec- Pathol 38 299-302
tant againstthe shrimpvirus Baculovirus Lightner,D.V. and R.M. Redman.1982. His-
penaei.J. AquaticAnimalHealth.3:141-145. topathologyof aflatoxicosts in the marine
Lewis,D. 1986.An enzyme-linked immunosor- shrimpPertaerrs stylirostris
andP. rrartrtarrtei.
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baculovirus,J, Fish. Dis. 9: 519-522. Lightner,D.V., R.M. Redmanand T.A, BeU.
Liao, I,C. '1984.Statusand problemsof grass 1983a. Infectious hypodermal and he-
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Liao, I.C. 1989. Penaeus rrrorrodorrculture in in Perraerrs
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im-
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Contributed Papers

Country Situations
 Shari and Subasi he

and mortality among cultured Asian cultured outside Japan Lightner and
shrimpincludeviruses,b'icteria,
fungi Redman, 1991!. Kuruma shrimp, P.
and protozoa.Although it has been japonicus, have been successfully in-
saidthatvirusesand bacteriaare, by fected with BMNV by Momoyama and
far, the most important causative Sano 988! in Japan. However, BMN-
agents,emerging trends and recent lit- type agents have been reported from
erature
showthat mycosisand parasi- Australia, Indonesia, Japan and the
tosis also play significant roles in Philippines.
mortalityand morbidity of both larval
andgrowoutshrimp {Boonyaratpalin, MBV-type baculoviruses appear to be
1990;Hegel et al., 1992!. extensively distributed and have be-
come established in cultured shrimp
populations in almost every country in
Viruses Asia. Recent surveys by Dana and
Sukenda 990!, M,D. Hassan unpub-
Baculovlruses lished data! and Natividad and Light-
ner 992! suggest that MBV-type
Sixdiseases
ofbaculoviral
etiology
have baculovirusesarewidely spread among
beendescribed
in Asia. Rod-shaped, shrimp culture enterprises in Indone-
enveloped,DNA viruses approxi- sia, Malaysia and the Philippines, re-
mately70 nrnby 300nm in sizearethe spectively.
members of the Peereus monodon bacu-
lovinisgroup. This group consistsof Shrimpbaculoviruses
causehigh mor-
Penaeus irumodonbaculovirus MBV!, talities in cultured penaeids. BMNV
Penaeusmoriodonbaculovirus-type generally infects larvae and early
MBV-type!,baculovirusmidgut gland postlarvae, while MBV-type bacu-
necrosisvirus BlvPPQand baculovirus loviruses cause mortalities in late
midgut gland necrosis-type virus postlarvae and juvenile shrimp Brock
BMNV-type!. MBV is infectious to a and Lightner, 1990!.Baculoviruses in-
widerangeof cultured Asian penaeid fect hepatopancreatic
and midgut epi-
species,including Penaeus
esculentus,
P. thelial cells, which are of endothelial
kerathurus,P. rnerguiensis,
P. nmnodon, origin, and replicatewithin the nuclei.
P,peniciltatus
and P. semisukatusLight- MBVandMBV-typevirusesform single
ner and Redman, 1991!. Plebejus or multiple spherical inclusion bodies,
baculovirus PBV!, a inonodon-type causing hypertrophied nuclei, but
baculovirus,hasbeenreportedfrom P. BMNV and BMNV-type viruses are
pkbejusin Australia by Paynteret al. nonoccluded. Infected cells undergo
985!. Although BMNV is reportedto necrosisand sloughinto the gut lumen.
causeseriousepizooticsamong hatch- I'ree and occluded viruses are shed in
ery-rearedP. japmicus in southern Ja- the fecesChenet al., 1989!and during
pan Sanoand Fukuda, 1987!,this virus spawning Sanoet al., 1985!,causing
"as not beenreportedin P.japonicus transmission through ingestion Over-
 Diseases af Cultured Shri in Asia

street et al., 1988!. The unregulated Parvo-like Viruses

transfer of subclinically infected wild-
caught spawners and larvae appears
to be the primary mode of entry of
shrimp baculoviruses to hatcheries
and culture facilities Brock, 1991!.A
recent transmission experiment con-
ducted by Paynter et al. 992!
showed that exposure of one day-old
postlarvaeto postlarvalhomogenates
with MBV-type virus resulted in de-
velopment of inclusion bodies after
two days. The number of inclusion
bodies reached a peak by eight days,
a second peak in 16 days and disap-
peared in 23 days.
The importance of MBV to Asian
shrimp culture has been well docu-
mented. It hascausedheavy mortalities
of all stagesof P. rnonodonin the Phil-
ippines Baticados,1988;Natividadand
Lightner, 1992! and is considered par-
tially responsiblefor the collapseof the
shrimp culture industry in Taiwan in
late 1980s Lin, 1989!. The prevalence
of MBV among juverule shrimp in Ma-
laysia was about 30% in the late 1980s
Anderson, 1988!,but it is now almost
100% M.D. Hassan,pers. conun.!. The
diseaseis more pronounced under in-
tensive and semi-intensive culture
practices, where heavily stocked
shrimp live under environmental, nu-
tritional, behavioral and other stres-
sors. The occurrence of MBV has little
or no consequenceto shrimp grown
under extensiveconditions Nash et al.,
'l988b!. The diagnosis, control and
management of baculoviral infections
in Asian shrimp culture are well docu-
mented by Brock 991!.
These isometric DNA viruses, includ-
ing hepatopancreatic
parvo-like virus
HPV! and infectious hypodermal and
hematopoieticnecrosisvirus II~/,
are the known parvo-like viruses of
cultured Asian marine shrimp. HPV is
22 - 24 nm in diameter and occurs
within intranuciear inclusion bodies in
hepatopancreaticand epigastric caecal
epithelium Brock, 1991!. The geo-
graphical distribution of HPV is similar
to that of MBV, and HPV is found in
P, esculentus,P. indices, P, chinensis, P.
rnerguiensis,P. monodon,P. penicillatus,
P. semisutcatus and P. vurtnarneiLight-
ner and Redrnan,1991!.The main route
of entry of HPV into shrimp farms is
believed to be via postlarvae.Although
HPV has been documented from nine
speciesof penaeids, its significance in
causing epizooticsand economiclosses
is not fully understood.
IHHNV is 20 22 nm in diameter and
considered highly contagious and in-
fectious to many penaeid species
Brock, 1991!.BIHIM has been recog-
nized from P. japonicus,P. chinensis,P.
rnonodon and P. semisulcatms cultured in
Malaysia, Philippines, Singapore, Sri
Lanka and Taiwan Lightner and Red-
man, 1991!.Anderson 988! observed
a few foci of IHI~V-like nuclear inclu-
sionsin two P. rnonodonjuveniles from
a farm in Sabahin Malaysia. However,
the etiologicaland economicsignifi-
cance of IHHNV in Asia remains un-
clear. The diagnosis, control and
managementof parvo-hkeviruses are
describedby Brock 991!.
R~ike Viruses Luminescent Vibriosis

viruses{REO!of Penaeid Significant

geo.like larvalmortalitiesassociated
shrimp
arenonenveloped, with
icosahedral luminescentvibriosis,causedby
cytoplasmic
agents
withanaverage
size Vibrioharveyiand V. splendidus,
were
ofpgrun.WithinAsia,REOhaveonly reportedfromP. monodoii and P, mer-
beenrecorded
fromP. eonod'on
in Ma- guiensis
hatcheries
in IndonesiaSu-
~ in conjunction
withMBV,a rick- naryanto and Mariam, 1986!, the
ettsiaand Gram-negativebacteria PMippinesBaticados
et al., 1991!and
Anderson
etal.,1987!. their in Thailand Tansutaparutand Ruang-
However,
status
aspathogens
of penaeidshrimp pan,1987!.Lavilla-Pitogo
et al. 992!
reneinstobe established.
Brock 991! found that the main sourceof lumines-
rnenhonedthat the establishmentof cent bacteria in P. eonodonis the
rapid,
sensitive
diagnostic
methods
for midgutcontents
of themother,which
RKOanddetermination of the distribu- are shed into the water almost simulta-
tionof REOin culturedshrimpstocks neouslywith the eggsduringspawn-
is important
in understanding
more ing.Lavilla-Pitogo
etal.{1990!reported
aboutthecontroland managementof that near-shore sea water could be a
thesepathogens. major sourceof V. harveyiand V.
splendidms
for shrimphatcheriesin the
Philippines. Baticadoset al. 991!
Bacteria
tested 24 antibacterials against V.
Perhaps asa consequence of thelarge harveyiandV. splerrdidus
in P. monodon
numberof Vibriospeciesin the normal larvaeand showedthat only chloram-
shrimpmicroflora,opportunisticVt'brio phenicol,sodiumnifurstyrenate
and
spp. arethe mostcommonbacterial the ni trof ur ans cause r easo nable
pathogens of culturedshrimp.Vibrio growthinhibitionof thebacteria.
Based
spp.appearto establish lethal infec- on their results, they concluded that
tionsfollowingprimaryinfections with the most reliableand effectiveway of
otherpathogens,environmental stress, controlling luminous vibriosis in.
nutritionalixnbalanceand/or predis- penaeidshrimphatcheriesis through
posinglesionsLightner,1988!.Ac- rigorous
waterrenewal
andbyexclud-
cordingto Lightneret al. {1992!,some ing luminousvibriosfrom the culture
morerecentlyoccurringdiseasesyn- water.
dromes
of penaeidshrimparecaused
by Vibrio
spp. thatbehavemorelike MiscellaneousVibriosisand Other
truepathogens
than opportunistic
in- Bacteria
vaders. Involvement of other Gram-
~egativebacteriaand Cytophaga-type Takahashiet al. 985a! and Egusaet al.
filamentousbacteriain seriousdisease {1988!reported seriousVibrio sp. epizo-
outbreaksof cultured shrimp in Asia oticsin postlarvalandjuvenileP.japoni-
"asrarelybeendocumented. cusin Japan.This condition,which
 Diseases Ot Cultured StMl
existedsince1981, is characterized
by
cloudinessof the hepatopancreas in
postlarvae,
and cloudiness
of muscle
and brown spotsin the gills and lyrn-
phoid organin juveniles Takahashiet
al., 1984;1985a!.The seriousepizootic
in P.j aponicus
hasbeencontrolledwith
oxytetracycline-medicated
feed at 50
100mg/kg body weight/day for four to
six days Takahashi et al., 1985b!. In
vitro vaccination of juvenile P. japonicus
with Formalin-killed Vibriosp. hasbeen
shown to provide some protection
against subsequent challenge by live
Vibrio sp. Itami et al., 1989!.
In Malaysia, Anderson et al. 988!
experiencedheavy mortalities in almost
market-size5 - 33 g! P, monodonasso-
ciated with multifocal necrosis, herno-
cytic inflammation and nodule
formation in the lymphoid organ,
heart, gills, hepatopancreas,antennal
gland, cuticular epidermis and subcu-
tis, and in other connective tissues.
Some hemocyte nodules contained
Gram-negative bacteria within the
granulomas,or within intracytoplasmic
vacuoles.From the hemolymph of such
shrimp, V. alginolyticus, V. paru-
haemolyticusand Pseudomonas sp, were
isolated. According to Lightner et al.
992!, this condition could be the same
or related to "red disease" in Pe@acus
monodon.Anderson et al. 988! recorn-
mended decreasingshrimp density by
partial harvesting and increasingwater
exchange toward the end of the pro-
duction cycle to prevent mortalities.
They further suggested that proper
draining and drying of ponds and ap-
plication of CaO at 0.5 kg/m to the
in Asia 41
pondbottomcouldbe effectivein con-
trollingthe condition.
Anderson 988!, in addition to Vibrio
spp., isolated Pseudceumas sp., Mor-
axellasp. and AIcaligenese
sp. from the
hemolyrnph of affected shrimp in Ma-
laysia and considered them secondary
invaders.
Filamentous Bacteria
Leucothrix mucor and similar filamen-
tous, bacterial, ectocommensalfouling
organismshave been reported to cause
mortalitiesin all stagesof shrimp under
poor water conditions, Baticados988!
mentioned that L. mucor in larval
shrimp in the Philippines has been
successfully controlled by vigilant
water management, while postlarvae
and adults havebeeneffectively treated
with Cutrine+-Plus,a coppercompound,
at 0.1 mg Cu/L for 24h or 0.25-0.5 mg
Cu/L for 4- 8 h. Anderson 988! found
that L. mucoris a common secondary
invader in Penaeusrrtonodonpostlarvae
affected by MBV in Malaysian shrimp
hatcheries, causing 100% mortality
overnight. He suggestedthat early di-
agnosisis essentialto minimize losses.
Rickettsiasand Chlarnydias
The true taxonomic position of these
small, coccoid to rod-shaped, intracel-
lular Gram-negativemicroorganismsis
not fully understood. The presenceof
these organisms in diseased Asian
penaeidshrimpshas only beenreported
from Singapore Chong and Loh, 1984!
and Malaysia Anderson et al., 1987!.
 SharilfandSChei

Although
switching to pirmtsisandSirolpidiue
fromP.monohm sp. hasbeen
p, merguiensis
culturehasbeensug- reported
fromlarval
shrimpin thePhil-
gested
tocontrol infectionsippinesbyBaticados
rickettsial '1988!.
Baticados
Anderson
etal.,1987!,
detailed
knowl- 988! recommendedusingTreflana,
a
edge
onthedistribution, 5-ppm
pathogenicity bathfor1 hforspawners
before
andthediagnosis
oftheseorganisms
in spawning,
and/ortreatmentof eggs
shrimp
hasyettobeascertained. before
stocking
inhatchery
tanks!
with
20-ppmtidedetergent
for 2 h. Boon-
yaratpalin
990!recommendsdailypro-
Fungi phyl<tietreatment
ofpenaeid
larvaewith
causedby 7agenidiue Treflan+
Larvalmycosis, at0.01- 0.05ppm.A numberof
sp. andHdiphthorvsotherfungicides
sp.,Sirolpidise havealsobeentested
sp.,andsubadultandadultmycosis againstLagerridium
sp.andH. philip-
causedby Fusariamsp.,arethetwo pineesis
Lio-PoandSanvictores,
1986!.
major
funsal
diseases shrimp Althoughthelosses
ofcultured dueto larvalmy-
reported
in Asia. cosis seem to be substantial, data on
economiclossesin Asian shrimp cul-
ture are not available Brock, 1991!.

Boonyaratpalin thatlarval FUsarium Oisease

990!stated
mycosis
commonly
occursin zoeaand
mysis
stages,
causing upto The causativeagent of this disease,
mortalities
100%within two days.We affected Fusariurrt
solani,hasbeenreportedfrom
larvae show extensive, nonseptate, P.japonicus
in japanIshikawa,1968;
highly branchedfungal mycelia Egusa andVeda,1972!andP.iieruxhm
thebodyandappendages.inthePhiTippines
throughout Saticados,
1.988!.
Fusar-
the larvaltissues iurn sotni affectsall stagesof shrimp,
In heavyinfections,
turn paleandyellowish-green. The causing
locornotory
difficulties
asa result
are ofitsmycelial
fungi that causethis condition gnat&.Thefungusalso
Lagenidiute
sp.andSirolpidircm
sp.An- causes
blackening
anddestructionof giR
derson988! notedthatlarvalmycosis tissuehence, thename"blackgill dis-
causes100%mortalityof larvaewithin ease"!,resultingin heavy mortalities
24h in Malaysia.He alsonotedthat SianandEgusa, 1981;Baticados,
1988!.
larvalmycosis
is generally by Chemotherapy
followed andchemoprophyiaxis
of
bacterialnecrosis,and that there is a I'usariumdisease
is notweil documented
closeassociation
betweenantibioticuse and needsfurtherresearch.
andlarvalmycosis.Antibioticsremove
bacterial
epibiont
competitors,
produc- Protozoa
inganincrease
in fungalpathogenicity.
Protozoanparasites,both facultative
Larvalrnycosisinvolving>g~id<~N and obligate,havebeenfrequently re-
calli~teg,L sp., Halipkt4710s
philip portedto causesignificanteconomic
 Diseasesof CulturedShri e Asia

lossesin Asian shrimpculture.Two cados

andEnriques,
1982
[citedby An-
major groups, peritrichus ciliates and dersonet al., 1989]!and from Thailand
other epibionts that colonize cuticular
Donyadol et al. [citedby Flegelet al.,
surfaces,and systemicMicrosporidia 1992]!.In thePhilippinesandThailand,
have been recognized, microsporidians with eightcapsules
werefound in adult P. merguiensis
and
Peritrichus Ciliates and Other were identified as Agama.mrna
- Ttmto-
Epibionts hania!sp. Andersonet al. 989! re-
porteda microsporidianbelongingto
All speciesand life stagesof penaeids the genusAmesoninfectingthe hepa-
are susceptibleto epibioticfouling by topancreas of pond-reared adult P.
one or more protozoa. They include monodori.More recently, F1egelet al.
Zoothamniumsp., Epistytissp., Carche- 992! identified a microsporidian Aga-
siiim sp., Vorticellasp. and, less com- masomaperu''! from pond-reared P.
monly, Acineta sp. and Euphelotasp. momodon in Thailand that caused mor-
Anderson, 1988;Boonyaratpalin,1990; talities up to 24%. The artificial infec-
Nash, 1990;Brock, 1991!.Theyarefound tion of P. monodonby feeding and
on unhatched Artemia cysts, and in injection of tissue homogenateswith
tank and pond-bottom sediments.The spores were carried out with limited
presence of protozoan epibionts on cu- successFlegel et al., 1992!.Future re-
ticular surfaces of various stages of search on the mode of infection and
penaeid shrimp, in small numbers, is a transmissionof microsporidiansis rec-
common phenomenon in shrimp ornmended.
hatcheries and growout facilities. How-
ever, an abundance of these organisms Noninfectious Diseases
indicates heavy bacterial loading, or-
garuc pollution, nutritional and/or en- A few noninfectious diseases and can-
vironrnental stress in shrimp, and ditions of environmental and nutri-
inadequatehygiene.Undersuchcondi- tionaloriginhavebeendocumentedin
tions, heavyinfectionscouldcausedif- Asianshrimpculture.The following
ficulty in moltingandhypoxiain larval sectionbrieflyoutlinesthe mostimpor-
shrimp leadingto death.Formalin,1$ tant of those.
- 20 ppm for 30 min to 1 h, prevents
infection without adverseeffects Boon- Chronic Soft-shell Syndrome
yaratpalin, 1990!.
This has beenrepeatedlyfound in P.
SystemicMicrosporidia monody in thePhilippinesBaticados et
al., 1986;Baticadoset. al., 1987!.The
Microsporidian infections in Asian syndrome,
characterized
bya persist-
l
penaeidshrimpshavebeenreported ently soft exoskeleton
for severa
fromMalaysiaAnderson, Ander- weeks,resultsfroma nutritional
1988; defi-
posure
son et a1.,1989!,the Philippines Bah- ciency, expos
tochemical
pesticides,
 accumulationand associatedgill
~r pond
soiland water
conditions,
dequate practiceschanges,
management whichledtohypoxic damage
in othertissues,were probablyrespon-
Saticados
etal.,2987!.
Histopathologi-
sibleformanyoftheclinical abnormali-
~ and tustochemical
studies
revealedtiesobservedNashet al., 1988a!.
thatthemulti-layered
exoskeleton
of
soft.
shelled
shrimp
issignificantly
thin-
shrimp. OtherDiseasesof Relative
nerthanthatofhard-shelled
purtherrnore,
thecalcium
content
ofthe importance
hepatopancreas
ofsoft-shejied
shrimp Reddisease
of P. monodon
caused
by
lowerthanthatof normal shrimp aflatoxin hasbeendetected in thePhil-
aticadosetal.,2987!,
Saticados
988! ippines by Baticados988! anddela
andSautistaandIaticados
989!men- Cruz989!,andgas bubble disease and
tionedthatthesyndrome canbecon- cramped tailsyndrome in P. rttcttodon
trolled
byadequate nutrition
andby in thePhilippines, whichareof uncer-
maintaining
goodwaterandsoilquality. tainetiologies,
havealsobeen reported.
Detailedinformationon thesediseases
Blue Disease is limited,andtheir economicimpacton
fromMalaysia Asian
Thishasbeenreported shrimp
culture
isnotunderstood.
M.Shariff,
pers.
comm.!,
thePhilip-
pinesBaticados,
1988!andThailand Literature Cited
Nash,
2990!
andis characterized
by Anderson,
I.G.1988,
Disease
problems
faced
bluishdiscoloration
of theexoskeleton. bymarine
prawn
farms
andhatcheries.
Jn:
Affected
shrimpoften
become
soft,thin T.Singh
andK,l.AngEds.!.
Marine
Prawn
Farming
in Malaysia
PresentProblems,
andlethargic
andeventually
die.The Future
Prospects.
Malaysian
Fisheries
Soci-
etiology
ofthecondition
isbelievedto etyOccasional
Publication
No.I. pp,6743.
benutritional
inadequateastaxanthin! Anderson,
I,G.,M,N.Shamsudin,
andG.Nash.1988,Bacterial
M,Shariff
septicemia
in
and reducedstocking density, im- juvenile
tiger
shrimp,
Pe@acus
rnonodon,
cul-
proveddietqualityandfrequent
water turedin Malaysian
brackishwater
ponds.
AsianFisheries
Science.
2: 93-108.
exchange
have
been
recommended
for Anderson,
I.G,,M. Shariff,G. NashandM,
controllingit. Nash.1987.
Mortalities
of juvenileshrimp,
~ monodon, associated
with Penaeus
menodonbaculovirus,cytoplasmic
reo-like
Pathological
Conditions
Related
to virus,and rickettsial
andbacterial
infec-
Acid-sulfate Soils tions,
fromMalaysian
brackish
water
ponds.
AsianFisheries
Science.
1: 47-64.
Anderson,
I.G.,M. Shariff
andG,Nash.'1989.
>rowndiscoloration
of the gills, soft Ahepatopancreatic
microsporidian
inpond-
»e"s and decreasedsurvival were realed
tigershrimp,
Perjaeus
monodon,
from
arrtong
theproblems affecting Malaysia,
P.ttt0rt0- Baticados,J.Invertebr.
Pathol.
53; 278-280.
M.C.I..1988,
Diseases
of prawnsin
~ adults culturedin pondswith po- thePhilippines.
SEAFDECAsianAquacul-
acid-sulfatesoilsin Malaysia. ture, 10: 14.
Baticados,
M.C.L.,R.M. Colosoand R.C.
H tologyandult~d 4 st dy Duremdez 1986.Studieson the chronic
veaiedthatlamellaferrichydroxide
 Diseases of Cultured SIMI
soft-shell syndrome in the tiger prawn,
PenaeusmonodonFabricius, from brackishwa-
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Baticacfos,
M, C.L., R.M, Colosoand R.C.Durem-
dez. 1987.Histopathologyof the chtonic soft-
shell syndromein the tiger prawn, Penaeus
monodon,Dis. Aquat. Org. 3: 13-28.
Baticados, M.C.L,, C,R, Lavilla-Pitogo, E.R.
Cruz-Lacierda, L.D. de la Pelta and N.A.
Suitaz. '1991. Studies on the chemical control
of luminous bacteria Vibrio harvey' and V.
splendidusisolated from diseased Penaeus
monodonlarvae and rearing water. Dis.
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Bautista, M.N. and M.C.L. Baticadcu. 1989. Die-
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SEAFDECAsianAquaculture.11:1-2.
Bian, B,Z. and S. Egusa. 1981.Histopathology
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Boonyaratpalin, S. 1990. Shrimp aquaculture
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Brock, J.A. 1991. An overview of diseases of
cultured crustaceans in the Asia Pacific re-
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Brock,J.A. and D,V. Lightner.1990.Microbial
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Chen,S.N., P.S.Changand G.H. Kou. 1989,
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Chong,K.C. 1990.Asianshrimpaquacultureat
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Chong,Y.C. and H. Loh. 1984,Hepatopan-
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Dana, D. and Sukenda. 1990. The occurrence
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de la Cruz, M.C. 1989. Aflatmin in commercial
prawnfeeds.SEAFDEC
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Egusa, S.Y., Takahashi,T. Itarni and K. Mo-
moyama. 1988.Histopathology of vibriosis
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associated with black gill disease of the
Kuruma prawn, Penaeusjtrporucus Bate. BuU.
Jap. Soc. Sci, Fish. 38: 1253-1260,
Flegel,T.W., S, Boonyaratpalin,D.F. Fegan,
M. GuerinandS. Sriurairatana.1992.High
mortality of black tiger prawns from cotton
shrimp diseasein Thailand. Irc M. Shariff,
R.P. Subasinghe and J.R. Arthur Eds.!,
Diseasesin Asian Aquaculture L Fish Health
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Philippines, pp. 181-197.
Ishikawa,Y. 1968,Preliminaryreport on black
giU diseaseof the Kuruma prawn, Penaeus
japonicus Bate. Fish Pathol, 3: 34-38. In
japanese!,
Itami, T., Y. Takahashi and Y, Nakatnura 1989,
Efficacyof vaccinationagainstvibriosisin
culturedKurumaprawnsPenaeus japonicus,
J. Aquatic Animal Health. 1: 238-242.
Lavilla-Pitogo, C.R., L.J, Albright, M.G. Paner
and N.A Sur1az. 1992 Studies on the sources
of lutninescent
Vibrio~ in Perseus
mono-
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Lavilla-Pitogo, C.R., M.C,L, Baticados, E.R.
Cruz-Lacierda and L.D. de la Pefta. 1990.
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Penaeus
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larvae in the Philippines,
Aquaculture. 91. 1-13.
Liao, I,C. 1990. The world's marine prawn
culture industries: today and tomorrow. In;
R. Hirono and I, Hanyu Eds.!. Second
Asian Fisheries Forum. Asian Fisheries So-
ciety, Manila,Philippines.pp. 11-28
Lightner,D.V.1988.Diseases of cuhuredpenaeid
shrimp and prawns, In: C.J. Sindermann
 and
D.V.Ughtner
Eds.!. DiagnosisCrverstreet,
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W.EHawkins.1988.
Experimentalinfections
andControl
in NorthAmerican
Marine with Bricuiorrirus
penaei
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Aquactdture.
Hsevler,
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tner,
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procedures
penaeid
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ofconcern
to 175-187.
shrimp
culturists
in theArrunim.
IniP. Paynter,
J.L.,D.V.Lightner
andR.j.G.Lester.
1985.Prawn virus from juvenile Penaeus
Deknrch,
W,J.Dougherty
andM A.David-
sonEds.!.
Fronliers Research. escrrfentug.
in Shrimp In: P.C.Rothisberg,
D.j. StaplesEds.!,Second
B.J.Hill and
Australian Na-
Bse~r Science
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pp.173-196. tional PrawnSeminarNPS2. Cleveland,
Lightner,
D.V.,
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Mohney,
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j.L., j.E. Vickers
andR.J.G.Lester.
A.Poernceno.
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A revuiw
ofsome
major
1992.Experimental
transmission
of Penaeus
diseases
of economicsignificance
in
rrionorfon-type
baculovirus
MBV!. In: M.
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prawns/shrimps
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and J,R.Arthur
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Rosenberry,
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Sano, T., T. Nishimura, H. Fukuda, T.
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Bacu-
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larvae,Penaeus japorricus,of differentages.
20j: 19-20. In: A.E.Ellis Ed.!,Fishand ShellfishPa-
Momoyama,K,andT. Sano.1988,A method thology.
AcademicPress,
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ofexperimental
infection
ofkuruma shrimp Sunaryanto,
A, andA. Mariam.1986.
Occur-
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Nash,G.L. 1990.Penaeusmonodongrow out hatcheries.
Bull. Brackishwater
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Tharlochan
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nomic
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FVoceed- 1984.Pathogenicity
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of
ingsof theAQUATECH Conference 990: Vibrio sp,isolated fromdiseased postlarvae
KualaLumpur!. pp. 172-190. of Kuruma prawn, Prnaeus
japonicus,Bate.j.
Nash,G.,LG.Anderson andM. Shariff.1988a, ShimonosekiUniv, Fish. 32: 23-31.
Path~ changesin the tigerprawn, Takahashi,Y., Y. Shimoyamaand K. Mo-
Penaeus rironodonFabricius, associated with moyama. 1985a. Pathogenicity andcharac-
culturein brackishwater pondsdeveloped teristics of Vibriosp.isolatedfromdiseased
frompotentially acid-sulphatesoilsj. Fish postlarvae ofKuruma prawn,Penaeus j aporri-
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Nash,G.,A.Poe or a d Ivf.B.Nash., 1988b. Takahashi,Y., T. Itami, A. Nakagawa,H.
Baculovirus infectionin brackishwater pond Nishirnura and T, Abe, 1985b.Therapeubc
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val stages of blacktiger prawn,Perrarus Tansutapanit, D.S,O.A. andL. Ruangpan. 1987.
monodorr Fabridus,to monodonbaculovirus Vibrio hameyi a causative agent of white
MBV!. Jrr:M. Shariff,R.P.Subasinghe and shrimpnauplii,Penaeus merguicrrsis.
Irr: Ab-
j.R,ArthurEds.!. Disr~ in Asian Aquacul- stract, Tech. Paper No. 6t30,Third Natl
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Society,
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Philippines-
pp.111-125, 1986,NSRC,Bangkok,Thailand.
Jn10years,morethan2Dshrimpdis- rexia and reduced preening activity.
easeswere identified, most of which Mortahty
is higherif shrimphaveboth
had alreadybeendescribed
in the lit- HPV and either Vibrio disease, black
erature Sindermannand Lightner, gill syndromeor epicommensal
foul-
1988!.However,newdiseases
continue
to emerge.
Standard
treatment
andpre-
for shrimpdiseases Intranuclearinclusion bodies in hyper-
ventiontechniques
oannotbe appliedto somenewly dis- trophiednucleiof hepatopancreatic tu-
covered diseases,such as white-black buleepithelialcellsand the presence of
spotdhease,becausetheir etiologyis virusparticlesin the intranuciearinclu-
unhumanXu andLiu, 1988!. sionbodiesof affected hepatopancrea-
tocytes
havebeenobservedin cultured
Diseaseis a seriousproblem,and the penaeidshrimpand in wild shrimp
situationin Chinais rathergrim, This from the HuanghaiSea Wang et al.,
paperwil providean overviewof the 1985!. Viral diseases have not been
shrimpdhole situation,and discuss takenseriouslyin China because
their
treatmentsand preventivesused on impact is subtle and it is difficult to
shrimpfarmsin China. distinguishtheir symptomsfrom other
diseases.Thus far, few works have
Diseases and Disease dealtwith the viral diseasesof penaeid
shrimp in China.
Syndromes
The mostcommonand importantdis- Vibrio Diseaseof Larval Shrimp
easesand diseasesyndromesof cul- Septicemicvibriosisis the most serious
turedpenaeidshrimp in China will be andubiquitousdiseaseof larval shrimp
listedaccording
to theiretiology;infec- in China. Most pathogenic strains ap-
tious diseases, noninfectious diseases pear to be Vibrio parahaemolyHms, V.
anddiarieswhoseetkQp isunlrmmm. Ntginolyticus, other V. spp. and other
Someimportantdiseasesthat remain relatedbacteria. Affected shrimp ap-
to be investigatedare not included. pear turbid, exhibitreduced motility,
reducedphototaxis,
empty guts, in.
Infectious Diseases creasedsurfacefouling and a tendency
to sink Microscopicexamination shows
HepatopancreaticParvo-like Virus, an abundance of bacteria. In serious
HFV cases,mortalitycanreach100%within
Lightner and Redman985! first re- oneor twodays Meng and Yu, 1982a!.
portedthathepatopancreatic
parvo-like
virus had been found in P. chinensis.At Application
of 1 - 1.5 ppm cMoram-
the sametime, Wang et al. 985! con- phenicol,
2 - 3 ppm terramycinor 1
firmedthisobservation.Grosssignsof ppm furacinto the tank water is effec-
HPV infectioninclude atrophyof the tivebothasa treatment
andto prevent
hepatopancreas,poorgrowthrate,ano- thisdisease.
Live animalfeedssuchas
 ahri Diso'ases in China 49

Artemianauplii are believed to be the severe outbreaks. The disc

principal meansof transnussion of
pathogenicVibriospp.; therefore, aH
animal feeds should be thoroughly ster-
ilized before feeding. Certain microal-
gal feeds, however, have an inhibitory
effect on pathogenic Vibrio; hence, the
application of selected microalgae to
the shrimp rearing system not only
serves as supplemental feed, but also
helps to control the Vibrio population.
Therefore, it is possible to prevent Vi-
brio diseasein larvae with ecological
measures Chen et al., 1989; Chen et
al., In press!.
frequently in growout ponds in wh ~
the top layer of bottom mud has not
been removed prior to stocking or
when the densityof shrimpis toohigh.
Application of 2% mashed garlic and
0.2% terramycin or 0. 1% chlora-
phenicol! to the feed for 7 to 14 days,
in conjunction with the addition of 1-
2 ppm calcium hypochlorite in the
pond water, is an effective means of
treatment Menget al., 1989;Hongand
Chen, 1991;Wu et al., 1991c;Xu et al.,
In press!.

Red-leg Disease Eyeball Necrosis Disease

The important pathogenic speciesare Zheng 986! describedeyebaHnecrosis
V. parahaemolyticus,
V. alginolyficus,V. diseaseof penaeidshrimpand deter-
anguillarum,
V.campbellii,
otherV.spp., rnined that it was causedby Vibrio
Proteus riulgaris and related bacteria. cholerae
non-01!.Affectedshrimpfloat
Grosssignsof affected
shrimpinclude motionless,lying prone and roHing
anorexia,lethargyand expansionof red over at the surface of the water from
chromatophores
on the periopods
and timeto time.Theeyesofaffected
shrimp
p eopods,
1 givingtheseappendages a swell, and the cornea turns from black
reddish
coloration.
Affected
shrimp
often
to brown.thenfesters andfallsaway,
swim
inshadow
water,
anddead
shrimp causing mortality within a few da
arefrequently
foundalong
thepondside. usa
riurrr sp. is occasionally
isolated
from the eyesof affectedshrim
Pathogenic
strains
arefrequently
iso- mp.

latedfromthehepatopancreas,
which
Adding 0.2% terramycinor 0.1+
becomes
discolored
andsoft.Hemocytechloramphenicol to the feed and
numbersmaybedrasticallyreduced, treating the pond water with mala-
andhemolymph appears turbidand chite e
clots
slowly
ornotatall.GiHsbecome green and formalin can prevent
yellow shrimp this diseaseZheng,1986!,
orpink,andaffected
usually
haveempty,
reddish
guts. BrownSpot Disease
Red-leg
disease
isthemost
harmfuld Brown
spotdisease
iscaused
bycNtin-
'qitous
shrimp in China, degrading
disease bacteria
anda variety
ofre-
lated
bacteria
thatenter
through
she"
tality
can
beashigh
as95%
during wounds. ~...is is a ubiquitousdisease,
especially
in overwintering
shrimp.
Ibe
 0. Chen

inalachite
greenWuetal.,1988; Meng
~~d partofthebody can
become
~darily
infectedwithVibrio
sp.or and Yu, 1982a!.
spOver titne,
theovaries
may Fusarlum Disease
atrophy
andturnred,
and Fusariurn
sp. Thisdiseaseoccursprincipallyin over-
~ncause
tumors whichinvade
the wintering shrimp.
Theetiologic agents
mu~ tissue.InChina,
brown
spot reportedin China areFusarium salami,
aseistreated
with25ppmformalin
for
24h MengandYu, Wuetal., F.
1980; oxysporum,
ineariim.
F.tricirictum
Conidiospores
andF.gram-
enterthrough
1991c!. wounds
andproduce
hyphae
thatextend
intornusdetissue,creatingtumor-like
Flarnentags
Bacterial
Disease swellings.
Mortality
istheinevitable
re-
Thespecies
offilamentous
bacteria
sult there is no curefor Fusarium
identi6ed
inChinaindude leruvthrix
~Ncor andThiothrix
sp. Wuetal., disease.
1991c!.
'Ibis
disease
isfrequentlyfound Gillsaremostsusceptible
to infection
in larvae
andjuvenile
shrimp. If the bybariumspp.;hyphae andconidia
waterquality
ispoor,bothfilamentous fill thegiQs
ofinfected
shrimp.
Appli-
bacteria
andfouling
ciliatesadhere
to
thegills,
andmortality by cation
is caused of mycostatincanreducethe
difficulty andmolting. mortality
inrespiration ratein theearlystages
of
Application
of10ppmteaseed to infection,
cake but it is also necessary
to
promote
molting,
andahighrateof remove diseased shrimpearly Yu et
water
exchange
areeffective of aL,1989;
means Hong etal.,1988;Mengand
prevention
andtreatmentMengand Yu, 1983!.
Yu, 1983;
Wuet al., 1991c!.
CottonShrimp Disease
Cottonshrimpdisease
is causedby
LarvalMycosis microsporidians,
includingPleistophora
Theetiologic
agents
oflarvalmycosissp.,Thelohariia
sp. andNosema sp.
reported
in ChinaareLagenidiuin
sp. MengandYu,1983; Wuetal.,199lc!,
andSielpidium sp.Lagetiidiumsp.has Nosema sp. was also foundin wild
veside that is missing in
itolpaf<iini
sp.Larvalmycosis is ubiq- peeress
chiiiensis
waters
ofQingdao
takenfromthecoastal
HaoandMou,1984!.
inlarval rearingsystemsthrough- Shrimptissueinfected
withmicrospor-
. In seriouscases,cumulative
idiansturnswhiteandbecomes
soft.In
mortality reaches
100%in oneto two
general,
themortality
rate
islow.
There
d s'ys if treatment
is not provided
in
e-The
following
have reportedare
been notreatments,
example,
onlyprevention.
oneshould remove
For
all dis-
s effective
in theprevention
oflarval eased
shrimpandsterilize
the pond
mycosis:
1G- 40ppbrnethylene
blue,
2 bottomtoprevent
cotton
shrimp disease.
pP galinutatraditional
typeofChi-
n e medicine!
or 0.008- O.O1ppm
 Shri Diseases in China 51

Parasitic Ciliate Disease km' and Cothumia!havebeen observed

Paranophryscarcini Leglise is a marine
facultative parasite that inhabits decay-
ing organic rnatter and occasionallyin-
fects shrimp through wounds. The
optimum temperature for propagation
of this ciliate is 10'C.
In the early stagesof the infection, the
ciliate can only be found in the wounds
of the shrimp. In later stages, it can
infect hemolyrnph and damageother
or pns, even the giRs. Shrimp exhibit
reducedhemocytenumbers, and hemo-
lymph becomesturbid and doesn't clot.
on penaeid shrimp in China Song,
1986!.Zoothamnium,
Vorticellaand Epi-
stylis are most common, and the most
serious fouling organisms affecting
penaeid shrimp. The following treat-
ments can remove ciliatesfrom gill fila-
ments and body surfaces of penaeid
shrimp Meng and Yu, 1983;Zheng et
al., 1987;Wu et al., 1991c!:
~ Exchangea great deal of water;
~ Teaseedcakeat 10 pprn in pond
water;

Parasitic ciliate disease only occurs in ~ Potassiumpermanganateat 5 ppm;

overwinteringshrimp in northern China, and
where mortality due to the diseasecan
exceed 90%. To prevent this disease, ~ Formalin at 25 ppm for 24 h.
damage
to theshrimpshouldbeavoided,
diseasedor dead shrimp should be re-
moved quickly, and live animal feeds MiscellaneousFoolingOrganisms
should be treated with fresh water. This largegroup of surfacefouling or-
Formalin and malachite green are also ganisms includes a variety of bacteria,
effectivepreventivesMenget al., 1988!. algaeand other protozoa.Licmophora
ehrenbery,L. panafoxa,Synedratabulata,
ParasiticNematopsisand Cephalolobtcs Nitzschiasp., Amphomsp., Belanws sp.,
Diseases Entenmorphrasp., Ectocarpussp. and
ParasiticNerrratopsis
sinai~~'sandCepha- Cladophorasp. have all been found at-
lolobusperMeus
havebeenfoundin diges- tached to the gills or body surfacesof
tive tractsof penaeidshrimp in China penaeidshrimp Wu and Zeng, 1988;
Mengand Yu, 1980;Wu et al., 1991b!. Wu et al., 1991a!.Acinetapolymorpha,
A,
tuberosa
andEphelota
sp. alsocausefoul-
Epicomrnensal Ciliate Disease ing diseases Meng and Yu, 1980;Wu
Heavyfouling by epicornmensal proto- et al, 1991a!.
zoaon the surfacesof gills and append-
agesmay causemortalities. Thirty-eight Some treatments used to combat 6la-
species of epicommensal ciliates be- mentousbacterialdiseaseand epicom-
longing to nine genera Zoothamnium, rnensal ciliate disease can also control
Epistylis,
Vor6ceVa,
Raxb~h, Myschis- fouling organismslisted in this section.
ton,Pseudamrchesium,?nt
rustylum,Vagm-
 Muscle
Necrosis
t4oninfectious
Diseases Musde
necrosis
usuaMy
occurs
ingrow
outponds
during
summer
months
and
BlackGIIISyndrome is caused
byovercrowding,
lowo!
Mekspecies
ofpenaeid
shrimp
in gen,
sudden
changes
intemperature
or
China
aresusceptible
toblack
gillsyn- salinity
andother unstableconditions
drome,
which
maybecaused
bytoxinsKeeping pondwaterdeanandusing
orbiotic
agents
inthewater
thatharm moderate stockingdensities
maypr~
the
gills.
Inserious
cases,
most
ofthe ventmusclenecrosisand secondary
giM
lamellae
are
affected
and
necrosis
of bacterial
andfungalinfectionsMeng
the
gNs
may
beapparent.
Itisnotewor-
andYu,1983;
Wu et al., 1991c!.
thythat
black
giM
syndrome
isalways
accompanied
byinfection
byVibrio
spp., FloatingHeadSyndrome
FMssriee
spp.,
Zootharrtnhun
spp.,
lageno-Floatingheadsyndrome of penaeid
phryspp.orHPV,and eventually
re- shrimp always
occurs
in high-density
sultsin highlevelsof mortality. growout
ponds
withpoorphytoplank-
Therefore,
to preventthe disease, tonblooms, poorwaterqualityand
comprehensive
andappropriate
rneas- pollutedbottom
conditions,
duringcahn,
uresmustbeconsideredin advance
MengandYu,1982b!.
swelteringsummerdays. Shrimp
usually
floatat thesurface
of thewaterin the
Gas-bubbleDisease
early
morning
dueto lackof oxygen.
Gas-bubble
diseasemayoccasionaMyFloating headsyndrome resultsin se-
occurin hatcheriesas a result of vere mortalities
at some shrimp farms
of seawaterwithat- if it is nottreatedin time.Thefollowing
supersaturation
mospheric
gases. isaccom- areeffectivemeansof prevention:
Prevention
plished
by controlling
thelevelof
dissolved
gases
ataMtimesMeng
and ~ Raising
the shrimpat moderate
Yu, 1982a;Wu et al., 1991c!. densities;

BodyCrampSyndrome ~ judicious
application
of feed
FuMy
or partiallycramped
shrimpcan
be found in growoutponds during ~ EMangmgwater;and
summer months when both air and
watertemperatures 'n4 es-- ' >>y
arehi~ ~ Using
chemxA
andphysical
when the air is warmer th th uresto increasethe level of ~
voidanceof handling during the hot- solved oxygen.
testhoursandtimelyexchan of
in e summermonths are suggested Diseases of Unknown BiologV
meansof prevention Meng and Yu,
19Q; Wu et al., 1991c!. White-b4ck Spot Disease
Thee cause of this disease is
but itusu occursin warmer sea~~'
 ~ Selecthealthy larvaefor stocking.

~ Raiseshrimpat moderatedenst
Shrimp
growout
ponds
aho
can
be tiesin growoutponds.
considered
ecosystems
inwhich
cul-
tured
shrimp
andastodated
ormrartssms,
e Giveappropriate
quantities
offeeds
including
Bveaturnal
and
rmtmalgal to avoid deteriorationof the bot-
feeds,
related
microorganisms
anda tom environment;
variety
ofpathway.ns,
live
together
and
areconditioned
tooneanother.
Disease ~ Sterilizeor treat animal feedsbe-
sitttations
atcertain
shrimpfarmsare
doeelyassociated patterns. fore use to avoid transmission of
withculture
Noninfectious
diseases
of penaeid pathogenic
Vibriospp.;
shrimp
can
beprevented
bycontrolling~ Give medicatedfeeds only during
some
environmental
factorsorimprov-
ing
nutrition.
Toprevent
infectious
dis- hot seasonsor diseaseoutbreaks;
eases,
ecological
control
measures
must
beinstitutedinsteadof chemical
ones. ~ Controlthe transfer of pathogens
Themostimportant
ecological
meas- at all times; and
ures are:
~ Studythe ecologyof pathogens
~ Dry the bottom of growout and applyecological
controlm-
pondsduringthe winter after stead of chemical control.
harvest,then removethe surface
layer; Literature Cited
~ Sterilizethe ponds with calcium Chen,D., X.Y. Liu, Y,D. Yu, Z.H. Yang,T.
XiaoandQ.X.Wang.1989.Variation of
hypochloriteor teaseedcakebe- vibnopopulation
in larvalpenaeid
shrimp
forestocking; feanng
system
anditscontrollingmeasure,
FifthInternational
Symposium
onIvlicrobial
Ecology.abstracts!
159,
~ Always keep pond water dean, Chen,D., X.Y. Liu, Y.D. Yu, Z.H. Yang,T.
pay dose attentionto water tem- XiaoandQ.X, Wang,.In press.'l1ievibno
perature,watercolor,salinity,dis- aseofpenaeid
shrimp
inthelarvalrear-
ing!ngshms
anditsc rehensive preven-
solvedoxygen,etc.and modify - Ocranol. limnol. inica.
these parameterswhen neces- Hao,B andR.P.Ivlou.1984.Prelimi study
sary; oftheparasite
microsporidian
fromtheChi-
nesePrawnPeriueus
orierttalis.
alar.Sci.2:
4~M, In Chinese
with English abstract!
~ Changepond waterin a'ey
a timel Hong,
X.andX,F.Chen.1991.
Histopathologic
fashion; observation
on red-Iegdisease
of Peiiarus
peiticilstiN.
Xiamen
AquaticScience
and
Techno4gr.
2: 3&39.In Chinese!.
~ Ponds
should
bedeeper
eeperthan
than 1.
1.5 Hong,
X., D.H.WuandH.W Zeng.1968.
-2.0 m; StMdieson Fusarium disease of
shnrnp
andetiology,
Bull.FishDis.Yubing
Iia+cun.A: 91-92.In Chinese!.
 Shri Ditusases
in China

LightnerD.V.andK.M.Redman.
1985.
A parvo-
like virus disease
of penaeidshrimp.J. shrimpdisease
Synedradisease,
Xiamen
Invertebr. Pathol. 45: 47-53. Aquatic
Science
andTechnology.
2:32-35,
Meng, Q.XC.H. Song and K.K Yu. 1989. Wu,D.HX. HongandH.W. Zeng.1988.
Studies on red-legdisease.
I GrossSigns, Studiesonlarvalmycosisofpenaeidshrimp
cause,epizootiologyand pathogenicidenti- and prevention.Bull. FishDis,/Yubing
fication.Bull.FishDis./Yubing jianxun.3-4:78-81.In Chinese!.
jianxun.2-3: Wu,
36-38, In Chinese!, D,H,,H.Huang andH.W,Zeng.1991b,
Meng,Q.X.andK.K. Yu. 1980,Investigations Studies
onparasitic
Nerriatopsis
of cultured
on diseasesand parasitesof the saltwater penaeidshrimp.XiamenAquaticScience
shrimp,Penaeus
orieritalis
Kishinouye.
Fish- andTechnology.
2:4649.In Chinese!.
eriesResearch1: 31-45.In Chinese!. Wu,D.H.andH.W,Zeng.
1988.
Licmophora
Meng, Q.X. and K.K. Yu, 1982a.Diseasesof
disease
ofpenaeid
shrimp.BuII.FishDis./Yu-
penaeidshrimp in larval rearingperiod.
bingjianxun.
34:4542.InChinese!.
Mar. Fish.4; 149-152.In Chinese!. Wu,D,H.,H.W.Zeng,
X.Hong
andH.Huang.
Meng, Q.X. and K.K. Yu. 1982b.Notes on the
1991c.
Investigation
on diseases
of cultured
"black gill disease"of penaeidprawn.J. penaeid
shrimpin southern
partof Fujian
province,
XiamenAquaticScience
andTech-
Shandong Collegeof Oceanology.Vol. 'I2. nology.2: 1-10.In Chinese!.
4: 95-100.In ChinesewithEnglishabstract!. Xu,B.,W.S.Ji,M,Y.Ding,X, Li andH.S.Xu,
Meng, Q,X. and K.K, Yu. 1983.Prevention and ln press.
Comparison ofantibacterial
agents
controlof diseases
in growoutperiodot' for controlof pathogens
in cultureprawn,
penaeidshrimp.Mar, Fish.3: 110-116.In J.OceanUniversityof Qingdao.
Chinese!, Xu,G.J.andM.X.Liu.1988.
Preliminary
obser-
Meng, Q.X., L. Zhou, K,K. Yu, Z.H, Luo and vationonwhite-black
spotdisa' of penaeid
H,Z. Liu. 1988.Studieson the parasitic shrimp.Mariculture.
4: 19-22.In Chinese!.
ciliate diseaseof overwinteredpenaeid Yu,K.K.,W.B.ZhanandQ.X.Meng1989.
shrimp.BuII.FishDis./Yubing
jianxun.3-4: Studieson etiologyof Fusariumdisease
of
17-72.In Chinese!. penaeidshrimp. Bull. Fish Dis./Yubing
Sindermann
C.J,andD.V.Lightner.1988.Dis- jianxun.2-3:19-22.In Chinese!.
easeDiagnosisand Controlin North Ameri- Zheng,G.X. 1986.Identification
and patho-
can Marine Aquaculture. Secondrevised enicity of Vibriocholeraenon4! isolated
edition.Elsevier.333p, rom diseasedpenaeidshrimp. J. Fish
Song,W.B. 1986.Descriptionof sevennew China/Shuichan
Xuebao.
10!: 195-203.In
speciesof peritrichs on Penaeus
orierrtolis, Chinesewith Englishabstract!.
ActaZootaxon,Sinica.11;225-235.
In Chi- Zheng, G.XY.L. Shen and H. Li. 1987.Pre-
nesewith English abstract!, liminary studieson treatmentof Zootham-
Wang,W.X.et al. 1985.Studieson hepatopan- niurn diseaseof penaeidshrimp with
creatic parvo-like virus infection of Perroeus potassium peimanganate,Mar. Fish, 3: 'l02-
chiiiensisunpublished
report!. 105. In Chinesewith Englishabstract!.
Wu,D.H,,X. HongandH.W.Huang.1991a,
Preliminarystudieson a new penaeid
 OccUrrence,
Diagnosis
andTreatment
of
ShrimpDiseasesin Thailand

TitT1tWyW.Fugal'
DarielFFey'' SUmanaKiXK~ifT1'
R
Mlhl ~~'.S~S W P.Std~,CI Wl
~'.J E.Vd~~CI D.M M 'kt'

Diseases
ofcommercially
cultured
Penaeus
monodon
tnThailand
arereviewed
withemphasis
on
recent
research
results.
The major
causes
ofeconomic
loss
ingrowout
ponds
can
beultimately
attributed
toenvironmental
stress
resulting
frompoor
managementpractices
orexternal
factors.
Themost common virus
infection
reported
is&tMeus
trtottodon
baculovirus
MBV!, butthisis
welltolerated
byP.rrtortodott
underunstressful
rearing
conditions,
Twonewviruses
ofunknown
impactaredescribed
innormalbroodstock
specimens
ofP.monodon
captured
from theAndaman
sea Oneisa nonenveloped,
polyhedral
particle
diameter
40ntn!
that
occurred
inthe
cytoplasm
oflymphoidorgancells
athighincidence
90%oFrandomlyselected
stock
examinedbetween
1989
and1991!.
Theother,
alsointhelymphoid
resemblingthe baculoviridae.
organ,
wasa rod-shaped,
enveloped
virus
Bacteria
ofthegenus
Vibrio
V.parahaemolyticus
andV.haroet/i
amounted
for83%
ofisolates!
caused
most
growout
shrimp
mortality
inopportunistic
infections,
andtheincidence
ofantibiotic
resistance
in theisolated
strains
washigh.Fungal
infections
in yuwout
shrimp
arealso
opportunistic
andhavenotcaused
extensive
economic
loss.
However,
Lagenid'rutn
sp.isinfectious
inthehatchery
and
can
sometimes
cause
heavy
losses
ifitisnotcontrolled
withtrifluralin
0
ppb!.
Withrespect
toenvironmental
factors,
preliminary
results
from insecticide
testsshowed
that
thesynthetic
pyrethroid,
cypermethrin,
was
extremely
toxic
toP.mottodon.
Itwas
acutely
toxic,
causing
significant
mortality
ingrowout
shrimp
at1 ng/L,
and
inlarvae
zoea!
at10pg/L,
Inlight
ofthis,
theeffect
ofinsecticides
oncrustaceans
isbriefly
reviewed.
Other
environmental
problems
discussed
include
toxic
algae,
crude
oil,release
ofrearing
pond
wastewater,
and
treatment
ofpost-harvest
pondbottoms,
Finally,
a series
ofmysterydisease
syndromes
of
unknown
etiology
aredescribed.
Twoofthese
show indications
ofviral
etiology.
BPN
Aquaculture
Research
%NO, Thailand
Centre,
577Thanadee
Complex,
Koryor-Songkhla
Road,
Amphur
Muang
Songkhla
Faculty
ofMedicine,
Sirirat
Hospital,
Mahidol
University,
RamaV1Road,
Bangkok
10400,
Thailand
National
Institute
ofCoastal
Aquaculture,
Songkhla
90000,
Thailand
Dept.
Veterinary
Pathology,
University
ofQueensland,
Brisbane,
Australia
Hadda
FarmLtd.,Iaigh-Iaighoch
HElSK,Scotland
 introduction ManagementPracticesand
Environmental Stress
General

Thispaperis an overviewof the impact At recentmeetingsin Kuala Lumpur

of diseaseson the cultivation of Penates New et al., 1990!and Korea Work-
eoedoit in Thailand. However, it shop on Fish Health Managementin
would be pointless to simply repeat the Asia-PacificRegion,8 - 15 October
much of the information that has al- 1990, Pusan, Korea!, there was consen-
readybeenpublishedin recentexcel- susamongdiseaseexpertsthat many
lent reviews by Sindermann and of thediseaseproblemsencounteredin
Lightner 988! and by Brock 991!, aquaculture
can be avoidedby good
andwhereverappropriate,we wN re- managementpractices.We strongly
fer to theseworks. There is also a recent concur with this opinion. The two ma-
publicationin Thai Limsuwan, 1991! jor causes
of problemsencountered
by
thathasa goodsectionon diseases
and shrimp farmersin Thailand are poor
theircontrol. The emphasishere will be manage ment and e nvir onmental
on recent investigationsin Thailand, stress.Managementproblemsare usu-
someof which have not yet been pub- ally solved through assistancefrom
lished. governmentextensionworkers e.g.,
from the Departmentof Fisheries!or
We will also present someabnormal from the technical sales staff of the
phenomena
that we haveobserved
in various feed companies. Environ-
P. mammonbut have, as yet, been un- rnentalproblems,however, often can-
ableto explain. not be solved, especiallywhen they
result from natural phenomena e.g.,
As an overall liinitation, we caution the red tides!. Where they could conceiv-
reader that the context of this article ably be solved,majorchangesin social
applies
totheauthors'experiences
with behavior and legislation would be re-
shrimp farming on the "semi-inten- quired,and it is difficult to imagineany
sive" scalein earthenpondsin southern improvementfor sometime to come.
Thailand. According to our definition, Even so, a movehas been made in that
semi-intensive ponds are piscicide- direction in Thailand. In February
treatedpondsstockedwith hatchery- 1992,legislation was passed requiring
raised larvae at 15 - 80 larvae/m and all shrimpfarmsto be registeredand all
suppliedwith dry commercial feedsas farms over 50 rai approximately 8 ha!
a majorpart of their diet. In the BPN to provide a minimum of 10% of the
Aquastar! system,theaverage pondis farm area the area of the discharge
approximately
1.5-mdeepand1 hain canal can be included! for the treatment
area,with an averagewater exchange of dischargewater. The BOD limit of
overthe whole cultivationcyde! of the outlet water hasbeen set at 10 mg/L
approximately30%. by this legislation.
 Shri Oiseasesin Thaiiand 59

In principle,we agreewith suchlegis- shrimpfariners

discharging
salinewa-
lation since it is desirablefor the stable ters into freshwater canals.In a few
future of the wholeindustrythat the cases,this hasled to physicaldashes
carrying capacity of the environment in with ricefarmersand policeinterven-
each farming area be determined and tion.
thatthewaterresources
bemanaged
in
such a way that conflicts between vari- Themostsignificant
poormanagement
oususersincludingnonaquaculturists! activityin Thailand
is stocking
ponds
areavoided.To formulatesuchlegisla- beyondtheircarrying capacity.
In the
tion, data from sound environmental standardAquastarpond, farmersare
studiesarerequired,and thesestudies stocking 15 - 80 larvae/m. BPN
shouldbe carriedout as quicklyas Aquaculture
recommends
stocking
a
possible.Thehastydraftingof lawsin maximum of 30 larvae/in for such
the absenceof such data can result in pondsystems,
but suggests
that stock-
the settingof arbitraryliinits, suchas ing25larvae/m
wouldbeoptimal. This
the 10-ppmBODlimit for discharge optimalnumber wasarrivedatthrough
water in Thailand. If such limits are not discussions
withexpertsoflongpracti-
necessary or realistic,they canbe daN- cal experience in shrimpfarming.
agingto the growth and competitive- Throughexperience, theyfoundthat
nessof the industry. suchstockinglevelsaHowed for stable,
long-term productivity from these
Seriousenvironmental
problemshave ponds.
Theyfoundthathigherstocking
developed
in someshrimpfarmingar- densitiesincreasedthe risk of failure in
eas in Thailand because of the unre- the long term, althoughshort-term
strainedconstruction
andoperationof gainscouldbe higherunderideal con-
ponds.In themostextremesituations, ditions.
single canalsextendingseveralkm
fromtheseaareusedfor bothpond
supply and discharge.In such situ- Unfortunately,manyThaifarmershave
ations,waterqualitydeteriorates
pro- adoptedthegoalof highershort-term
gressivelyfrom the seasideinwards, gains,and averagestockingdensities
and, consequently,farmersat the end haverisento thecurrentlevelof ap-
of the line can face serious disease proxirnately
50- 60/m,contraryto the
problems
as a directresultof poor adviceof almostaHdisease
experts.
water quality. Sometimes, farms are Thispracticehasbeenadoptedalmost
locatednearindustrialareas,soresid- universally, regardless of the con-
ualwastes
fromfactory
discharges
may straints environmental and infrastruc-
alsobesignificant
underlying
causes
of tural! that vary from pond to pond.
diseaseMenasveta, 1987;
Summonok, Thus,it is entirelypredictable that
1990!.The problemsfacedare not al- manyfarmersreacha pointin the cul-
waysonthesideof theshrimpfarmers. tivation cyclewhere they eventually
There
havebeenextreme
examples
of facediseaseproblemsthat either re-
quire
extremetreatment or, to
measures anextended
periodofstress,
ledtogeneral
weakening
which
anda lower-
moreoften,
areuntreatable. ingof normal
defenses.
Thisapplies
lnfefious
VersusOpportunistic
equaHytosecondary
infections
viaviral
lesions,
whichcanbe exacerbated
by
Diseases stress
to suchanextentthatbacterial
Inthefewyears
wehavebeenworkinginfection
canfo5ow seenextsection!.
Mortality
fromsecondaryinfections
withP. raoirohm,
wehave beenim- usuallycausesthegreatest
lossesin
pressedbythehardiness
oftheanimalshrimprearing.
Insomecases,
thesitu-
and itshigh
level
ofresistance
todis- ationcanbe soseverethat an entire
ease
under
goodcultivation
conditions.crop islostormust beterminated pre-
Undertheseconditions,
thereis a maturely. Oftenin thesesituations,
rather
lowlevel
ofmortality
fromdis- massive mortalitybeginsbeforeassis-
eases
caused
byinfectious
agents i.e., tanceis sought,andit is too lateto
agents
that
cansurmount
thedefenses
devisea treatmentthat will improve
ofa healthy
animal!.
Suchagents
in-
survival.
duce
viruses
and.
parasites,
andthey
usuaHycauserelatively
lowfinancialWith respectto thehatchery,wecur-
loss;
i.e.,survival
forgood
pondshar- rently reservejudgment asfarasthe
vestedshrimp/stocked
larvae
x 100%!
isusually
70- 80%, lossesinfectivity
including ofbacteria
we canconfirm
isconcerned,
thatoomycete
but
diseases
fromcauses
otherthaninfectious are definitely
infectious
for healthy,
agents
e.g.,
physical
injury,
predation,
unstressed larvae.
etc.!.
Thus,
webelieve
thatstudying
theinfectious
agents
andformulating
control
orelimination
strategies
could Viruses
raise
theaverage
survival
by1Ã6.Bet- Lightner
etal.990!andBrock991!
tergrowth
rates
andfeedingefficiency havereviewedinformationon shrimp
couldalsoaccrue, and,altogether, viruses,
andonlyinformationrelevant
thesefactorscouldsignificantly
im- toThailandwill beincluded
here.Only
proveproduction
efficiency. P~ triotrodotibaculovirus
MBV!has
In contrast
beenwidelyreported
in Thailand.
tothevirusesandparasites, Other virusesare either uncorrunonor
weconsider
aHof thebacterial
and donotcau.se
highlosses.
Alternatively,
fungal
diseases
thatwehaveencoun- theymaybe overlookedwhenthey
tered
in growout
ponds
inThailand
to occur.Belowis a summaryof recent
beopportunistic
infections.
Weexclude information
onviruses
wehave
found.
rickettsia,
whichhasnot yet been
foundin Thailand,
fromthisgenerali-
zation.
Ourconclusion
isbased
onthe
experience
that
these
infections
can
al-
ways
bedirectly
orindirectly
attributed
 Shn
MBV
We recently published a summary of
our work with this virus infection in P.
monodon
in southernThailand Feganet
al., 1991!.Although the diseaseis well
tolerated in P. monody as lang as rear-
ing conditions are optimal, its elimina-
tion from the hatchery will improve
production. A recent publication by
Liao et al. 990! shows that the inci-
denceof MBV in the hatcherycan be
substantiaHy reduced by using clean
sea water to wash the eggs or nauplii
beforethey are transferredto rearing
tanks, We are accumulating data to
determine whether washing signifi-
cantly improves the overall, long-term
survival in a hatchery,but initial results
concur with those of Liao et al. 990!.
Our procedure is as fallows: after har-
vest from the hatching tank, several
million nauplii are transferredby net to
a 500-L fiberglass tank filled with dean,
filtered sea water. The tank is fitted
with an overflow filter consisting of a
perforated pvc cylinder approx. 25 cm
dia. x 90 cm long!, dosed at one end,
with an outlet line approx. 7.5 cm dia.!
at the other, and covered with
mesh nylon screening. The larvae are
washed for 3 h at a rateof 500L/h total
of three tank changes!,before transfer
to the rearing tanks.
In an effort to further control and un-
derstand this disease, we have been
trying to develop a DNA dot hybridiza-
tion probe for the rapid, on-site detec-
tion of MBV. Vickers et al. 990!
reported preliminary steps towards
Diseases in Thaiiard 61
preparationof sucha probebut, accord-
ing the most recent issue of the Asian
Shrimp Culture Council ASCC! news-
letter issue t8, fourth quarter 1991!,
Dr. S.N. Chen has already developed
such a kit. As a byproduct of the DNA
probe effort, we now have excellent
electron micrographs of MBV virions
Figs. 1-3!, and these may help in the
comparative characterization of MBV
from various geographicallocations.
Upon examining the negatively stained
viral acdusion VO! polyhedra of MBV
Figs.1-3!,we werestruckby the large
size of the polyhedron subunits about
20 nm! and how they could appear to
be "empty" and "full" Fig. 1a,b; Fig.
2a, b; Fig. 3b, d, f!, rather like nega-
tively stained parvovirus densovirus!
virions Tijssen and Arella, 1991!. The
VO polyhedra appearedto ariseby the
association of these globular polyhe-
dronunits Fig.2d-f;Fig.3b,c, e!rather
than the coalescenceof fibers, as occurs
in the insectbaculoviruses Adams and
McClintock, 1991!.
It is possible that the polyhedra from
theseMBV preparationsare paracrys-
150- taQinearrays af parvovirus partides
with embedded baculovirus virions,
and that what we see is the result of a
dual infection,perhapseveninvolving
interdependentreplication of a par-
vovirus and a baculovirus. Further in-
vestigation of this possibility may be
warranted. For example, the polyhe-
dral subunitsareclosein sizeandshape
to parvovirus virions Adams, 1991!.
There are also reports of mixed or mii-
tiple infectionswith insectpalyhedrosis
62 F el et al.

Figure 1. Negatively stained molybdophosphate! preparations of M8V virions viewed with the electron
microscope. a! Fully enveloped virIons bar = 100 nm!. b! Fully enveloped virions accompanied by
unknown filaments bar = 100 nm!. c! Unenveioped vidon with extruded filament bar = 150 nm!. d!
Emptynucl~pslds showlngcapsafboth ends bar= 150nm!. e! Unenveloped nucleocapsldshowing
extruded filament adjacent to narrvwer filament of unknownorigin bar = 60 nm!. f! Nuciereapsid
showing spiral arrangement of protein subunits bar = 80 nm!.
 Shri Diseases in Thailand

Figure2. Negativelystained molybdophosphate!

preparationsof MBV virionsviewedwiththe electron
microscope.a! Complete andunenvetoped v'rrions.
Arrowindicates
partiallytom envelopebar= 100
nm!. b! Virions~ithtomenvelopesbar= 120nm!. c! Lowmagnificationof manyvirionswithruptured
envelopes. Notealso the bundlesof unknownfilamentsbar= 300nm!. d-f! Transmission electron
micrographsof MBV occlusionbodies.Note thepresenceof insertedfilamentsat the fractureline" in
e! bars = 120, 120and 80nm, respectively!.
 Fi el et al.

Figure3. Negativelystained mol>4ahph~phate!preparationsof MBV vin'onsshowingdetails of the

polyhedroncomponentof the occlusionbodies.Arrowsindicateparticlesthat appear "empty" bars:a
= 100 nm; b = 100 nm; c = 75 nm; d = 100 nm; e = 60 nm!.
66 Fle el et af,

Figure 4. Light lnicroscopephotographsof abnormal lymphoidorgans from healthy broodstockof

Penaeusmonition. a! Lowmagnificationviewof an H&E-stainedtissuesectionshowingnormaltubules
separatedby abnormal,solid clumpsof cells with enlargednuclei.Theseabnormalareashave a more
basophilic
stainingreactionthanthenormaltubuletissuebar= 50@m!,b! Highermagnification
of the
same tissue showing deeply basophiltcinclusions in the abnormalcells bar = 72.5 p,m!, c! High
magnificationof the basophilicinclusionbodiesshowingclearlythat theyare cytoplasmic bar= 5 pm!.
d! Toluidineblue-stained,thin sectionof cells with hypertrophicnucleinext to tubulecells with nuclei
of normal size. Arrows indicate differentiaily stained areas next to "normal" nuclei bar = 12.5 p.m!. e!
Asin d!, exceptthata tubulelumenis visageto the left bar= 12.5pm!. f! Asin d! but a higher
magnNcationclearlyshowingthe hypeitrophiednucleiand inclusionbodies that are cytoplasmicand
have a vanaNeappearance bar= 5 ~!.
 Shri Diseases in Thaihnd 67

Figure
5.Transmission
electron
micrograph
ofanabnormal
lymphoid
organ
fromhealthy
broodstock
of
Penaeusmoredon. Thecells
withsmall
indented nuclei
inthelower
partofthephotograph
arefrom
a
region
oftissue
thatappears
normalwiththelightmicroscope.
Intheupper
partofthepicture
areround,
hypertrophied
nucleitypical
oftheareasthatappearbasophilicin
HBEpreparatI'ons
bar=4 pm!.
 Fle el et al.

Fi'gure6. Transmission
electronmL"rographsof an abnorma/ lymphoidorganfromhealthybroodsfock
of Peraeusmoredon.ThecellsshownwerefromfubulesthatappearednormaiinH&Epreparations,
but hereshowcytoplasmic virogenic
areasadjacentfo the nuclei. a-b! Cellsshowingtwo virogenic
areaseach barsin bofh= 2pm!. c! Highmagnification of thelowestnucleusin b!, clearlyshowing
fwovirogenic areasnextto thenucleus.Notethemuitiiameilar membrane structuresbar= 0.6gm!.
 Shri Diseases in Thailand 69

! t.

Figure
7.Highmagnification
ofa virogenic
areafromthecytoplasm
ofthecellin Figure
Gcclearly
showing
unenveiopecf
virions
approximately
40nmindiameterbar= 300nm!.
70 Fle el ei al.

lg
WI
I!

Figure8. Low'magniflcatfon
of abnormallymphoidorgantissuefromhealthybroodstockof Penaeus
tnoredon.Notethehypertrophied nucleiandthevarietyof differenttypesofcytoplasmicinclusions
that
resemblesecondaryphagosomes bar = 4 p,m!,
 Shri Diseases in Thailand 71

Figure9. Highmagnification
of a portionof Figure8 showing
twohypertrophied
nucleianda varietyof
inclusionsin a singlecell bar = 1.2 pm!.
72 Fle el et al.

I J

Figure10Highmagnification
ofhypertrophied
nucleishowing a! whatmaybeincorqoletely
synthesized
viralmaterialadjacent bar= 1.2pm!. b! High magnificationof the 'viral" materia/in a! bar= 0.3pm!.
 Shri Diseases
inThaiiand
73

phology of our material differs mark-

edly from that reported from Australia.
it Cowdry
A bodies!
in specimens
of
For example, the inclusions in our ma-
diseased
pondshrimp
inthepast
four
terial are cytoplasmic rather than nu-
years.
However,
in onerecently
com-
clearand the virions are muchlarger
pleted
setof vitamin
C feeding
trials
p nm in our specimensasopposedto
proprietary
company
study!,
healthy
shrimpwerefixedandexamined his-
about20 nm in the Australianspeci- tologically
at theendof thetrial,and
mens!. Thus, it appears that different
someoftheanimals5/49!werefound
virusesmay give rise to superficiaHy to havetypicalCowdryA bodiesinthe
similarstainingreactionsin lymphoid antennal gland only.Theincidence
ap-
organ tissue sections, and that a careful pearedto behigherin animalsthat had
examinationmay be necessaryto dis- beengivenvitamin
C polyphosphates,
tinguish between them. but the numberof animalsexamined
histologically
for eachgroupwastoo
As to the nature of the virus in our Thai smallto makestatistical
comparisons.
specimens,we may assumethat it is an We will repeatthis trial with test ani-
RNA virus becauseof its cytoplasmic malsreceiving
vitaminC polyphos-
location, but it is difficult to relate it to phateat 500ppmin feed five timesthe
otherundassifiedvirusesreportedfor maximumdosein the previoustrial!
crustaceans Bonami and Lightner, andcontrolanimals
receiving
no sup-
1991!.If sizeis anyindicator,it maybe plementaryvitamin C. Wewill include
a picornavirus or a reovirus. sufficient numbers of animals to allow
statistical analysis.
Although we could not relate the ab-
normal organs to a diseasecondition in HPV
the broodstock, we are concerned that
thevirusmaybe carriedby thebrood- In a preliniir~y vitamin C trial similar
stockin a chronic
infection,bepassed to that describedin the previous sec-
on to the larvae,and causeproblems tion, five of eleven animals examined
laterin the production
cyclee.g.,see histologically showed distinctive baso-
sectionbelowonyellow-head shrimp!. philic HkE staining!and Feulgen-posi-
Morework is requiredto characterize tive inclusion bodies in the nuclei of
the virus and to determinewhether it cells in the hepatopancreatic
epithelium
hasanyimpacton production. especially E cells!. These resembled
typical inclusions of hepatopancreatic
IHHNV parvo-like virus HPV! Sindermann
and Lightner, 1988;Brock,1991!.As
oughinfecfioushematopoietic
and with the trial described above, these
hyp«~~ necrosis
virus IHI~Q! shrimpwerenormalfor growth,exter-
as been reported from P. mongg~ nal appearance,
and behavior,and
Sindermann
and Lightner,1988; would not have been subjectedto dis-
~" 1991!,
wehavefoundnosignof ease examination. Inclusions were
 Shri Diseasesin Thailand
75

Table
1.Antibiotic
sensitivity
ofbacteria
isolated
from
disease
d /enaeus
rrioirrooron
inThailand.
Oxy. Er!/, Poly.B. Chior. Sulf
30 18 300u 30 1,2
0/1 1/G/G 0/1/0 0/0/1 0/0/1
G/3 8/2/0 2/0/8 0/0/10 0/0/1
1/0 5/0/G 4/G/1 GIG/5 0/0/5
0/1 2/0/0 0/0/2 0/0/2 0/OI2
3/0 1/4/0 5/G/0 0/0/5 0/0/5
2/1 2/2/0 1/1/2 0/0/4 0/0/4
2/3/4 13/5/1 4/'1/14 0/0/19 0/0/1
/9/10 32/13/116/3/27 0/0/46 0/0/

/0 1/1/0 0/0/2 2/0/0 2/0/0

/0 2/1/0 1/2/0 0/0/3 0/G/3
Numbers
indicate
thenumber
ofstrains
Resistant/intermediate/Sensitive,
based
onstandard
tables
Becton
Dickinson,
Antimicrobial
susceptibility
testing:
asystem
forstandardization,
1985!
fordiameter
ofinhibition
zones
inMueller-Hinton
agarNaCl
2.5%!
around
paper
discs
impregnatedwith
thequantities
ofantibiotics
indicated
itg/disk
except
forpolymixin
Bgiven
asunits!.
Alsogivenisthenumber
ofstrains
withmultiple
resistance
i.e.,resistance
tomore
thanone
antibiotk!
y/total!
and thesubtotal
foralltheVibrio
isolates.
Note:
V. Vibrio;
algin.
- aigino/yricus;
angui.
= angxi//orxm;
harv.
= hannah;
mari
=roorinus;
para.-
panr/xrrmolyfims;
vul.
=ou/ni~s;
Aer.
=Arroaronas;
P,shig.
=Pfrisiomonos
s/rigrsoidrs;
oep.
- ampicillin;
strep.= streptomycin;
oxy. = oxytetracycline;
ery, erythromycin;
poly.
B polymyxin
B;chlor.
=
chloramphenicol;
acid.
suf./tri.
= sulfamethoxazole
23.75/trimethoprtrn1.25;
nitro.
nitrofurantoin;
nali.nahdixic

shrimpin Thailand,showingthat the referenCes

given in the introductiOrltO
incidenceof multiple antibiotic resis- thispaper.These
superficial
imperfec-
tancewasratherhigh.A similarpattern tionsareusuallysloughed
off with the
of sensitivity/resistance
was found for moltedexoskeleton
and,again,their
the luminoushatcherypathogens
V. presence
in significantquantityis an
harveyi
andV.splendidus
in thePhilip- indicationof underlyingstressthatis
pines Baticadoset al., 1990!.Dixon et interfering
withtheregular
moltcycle,
al. 990!havealsofounda highinci-
denceof antibiotic resistancein Aero- Onesyndroine
thathasbeenreported
morMsspecies in tropical fish from for shrimpin Thailandby Limsuwan
Singapore. 98S, 1991!is caUed"siendMm"in Thai,
whichtranslates
to "blacksplint"in
In addition to mortalitycaused bybac- English.Thegrossappearance of the
teria, thereare bacterialinfectionsthat lesionsis tough,blackfilaments
up to
cause blackdiscoloration oftheshrimp 2 mmor morein width thatarisejust
carapace
anderosionof the append- under the carapacein the tail muscle
agesandthe telson.Theseimperfec- segmentsand penetrateinwards, usu-
tionscanreducethe salevalue of the ally through the connectivetissuebe-
shrimp, and they are describedin the tweenbodysegments,producingfiner
 branchfilamentsin a rootlikefashion Possiblealternative treatments to anti-
as they progressinwards. These fila- bioticsincludethe prophylacticuseof
rnentsare very ugly, persist after vaccinesItamiet al., 1989;Songand
cooking andmaketheshrimp unmar- Sung,1990; Adams,1991;Sunget al.,
ketable.
1991!,immunostimulants Robertsen et
al., 1990;
Raaetal., 1992;Nikl etal.,In
According toLimsuwan988!, thisdis- press!andprobioficsAsianShrimp
figurationiscaused
by V.mdriificus,
and CultureCouncilNewsletter,issue48,
according to Ruangpanand Sa~ui fourthquarter,
1991!. Anyof these ap-
988!,infection+muredonlyduring proaches wouldbesuperiorto theuse
periods
whenthesalinityin therearing of antibiotics,
but somehavenotyet
pondsdroppedbelow10ppt.Although beentested withshrimpandthosethat
lesions
couldbefoundin shrimpfrom havearestiHin theexperimental
phase.
pondsat highersalinities,the authors
reportedthat pond historiesshowed an Vibriosis
ln theHatchery
earlier
period
ofexposure
to10pptor
less.Theyproposedthat infectionsoc- Althoughwe believe that vibriosisin
curredduring
thelowsalinity
period growout shrimp
arises
asa secondary
butwereoverlooked.
Progression
after ortertiary
phenomenon
through
stress,
thesalinity
wasrestored
tohigherlev- we are stiH uncertainwhether Vibrio
elseventuaHyresulted
in the gross spp.canbeinfectious in thehatchery.
symptoms.According to Limsuwan Merearemanyreports
thattheycan,
991!, this disease
canbe treatedwith butthegreatsensitivity
ofthelarvae
to
oxytetracycline
at 2 - 3 g/kgfeedfor 7 nutritional
andenvironmental
changes
to M days.
andtoxicsubstances,
makesit difficult
to eliminatethemastheunderlying
Another
symptom
wehaveobserved
is causeofinfection.Thespecies causing
black
lymphoidorgans Okiorgans!
in difficulfies
arethesame asthosegiven
somepond-reared
shrimp.Histologicalforgrowout ponds above, butspecial
examination
revealedthattheanimals attention
is oftenpaidto theluminous
haveextensivebacterial septicemiaspeciesV.harpeyiandV.splendidusBa-
caused
byV6bio
species
unpublished!. ticadoset al., 1990!.
TIietypesanddosesof antibiotics
com- Afterreading
thepublication
byAdams
monlyusedin shrimppondsin Thai- 991!on theexposure
ofP. monrxton
to
landhave
been
reviewed
byTonguthaia bathof kiHedVibrioceHs,we won-
and Chanratchakool
992!. Recom- dered
whether
shrimpincluding
lar-
mended
withdrawalperiods
aresome- vae!werecontinuously
invaded
by
esshorterthanthoseusedin more wholebacterial
cellsat a fixedrate.If
temperate
countries
suchasJapan so,hightotalnumbers
ofbacterial
cells
Aoki,1992!,andit wouldbebetterif
in thebathing waterwouldeasily
theperiods
were
given
indegree
days. swamp thelarvalhemocytes,
andeven
 Shri Disimsesin Thaiiand

if only a smallfractionof the totalcount low dosesto "slow downthe bacteria

consisted
of pathogenic
Vibriocells,the but not harmtheweaklarvae."Thisis
resultswould be disastrous.Totestthis a forinulafor rapiddevelopinent
of
possibility,we madedaily bacterial resistant strains.
counts marine TSA plate countsand
marineTCBScounts!in eighthatchery Other Bacteria
tanksover15daysandrecorded per-
centchanges
in larvalsurvival
during AlthoughVibriospp.arethemostcom-
the following24-hperiodafterthe moncause
ofproblems
in thehatchery,
count.
Theresults
areshown
inFigures we havealsohad difficultieswith Aem
11 and 12. We found no correlation novassp. during periodsof concomi-
betweensurvivalandtotalcount,Vibrio tantlyhighsalinityaround3Sppt!and
count or a combined index of the two hightemperature around34'C!.The
countsi.e.,totalcountx Vibrio count!. problemsdisappeared
whenconditions
Fromthispreliminarytest,wefeelthat returnedto optimum0 ppt and
the hypothesisis flawed or that other 30 C!.
factorsin therearingaremorecritical
to larval survival.
At someintervals
duringhatchery
op-
eration,we havefoundzoeawith abun-
Treatmentof Vibrioinfectionsin Thai
dant quantities of sessilebacteria
hatcheries
usually
consists
of adding attached
to theirfinefeedingappend-
antibiotics
directlytotherearing
water. ages.We havenot further isolatedor
Theantibioticsusedaresimilarto those
characterized
thesebacteria.
Theyare
employed
in growout
ponds,
although foulingorganisins
thatcanbeseeneas-
the quantitiesareusuallylower.Even
ilywiththephasecontrast
microscope
chloramphenicolofficiallybannedfor
Fig.13!,andtheyarea goodearly
usebytheThaigovernment!
isreadily indicator
ofa problem
in thehatchery
available.
Because
of uncertainty
with tank.If the underlyingcauseis not
respect to potency, we test all new lots removed,massiveinortalitiescan fol-
of antibioticsfor safetylevelswith lar- lowwithinoneortwodays.
Underly-
vae and for antibacterial
activity.In ing causes can be nutritional
Thai facilitiessuchasours, useis con- deficiencies,
toxicity,lackof waterex-
trolled by qualified personneland is change, etc.
permuttedonly when necessary,
and
onlyafterisolationof a disease
organ- Foulingby filamentousbacteriain the
ismfollowedby sensitivitytesting
and hatchery andin growoutpondsoccurs
determination of the minimum lethal inThailandasreportedelsewhere Sin-
dose. We also prohibit the use of dermannandLightner,1988;Brock,
chloramphenicol.In otherfacilities,an-
1991!.
Theoccurrence of theseorgan-
tibioticsare usedindiscriminately ismson the shrimpis associated
with
oftenfor routine"prophylaxis"!
by poorwaterquality especiaHy high or-
unqualified operators,who use them at ganic nutrient content! concomitant
 ei al.

PercentDropin Survival

Figure
11.
Retatfonshp
"Vfbrto"
count
TCBSbetween
agar!,
to log10
the of
drop
inthe
daily
hrtal
bacterial
survival
of
shrimp count
larvae TSA
dun'ng
the marine
agar!
and
the
succeeding
24-h
period.
The
counts
tnplicate
spread
plates!
were
earn'ed
outdaily
over
a period
of15
days
for
8 production
tanks.
TCBScounts
of0 were
converted
to1 and
are
shown
onthe
graph
as0,

II25
20

X c~5
8
o10

40
5 PercentDropin Survival

Figure
12,
Aelatenshjp
bett
veen
a combined
index
ofTSA
count
andTCBS
count
tothe
drop
inlarva
I
survivtl
during
the
fokwving
24-h
period.
The
data
isthe
same
asinFigure
1 except
1, thatthe
counts
were
combined
toobtain
anindex
calculated
aslog
oftheTSA
count!
x log
oftheTCBS
count
+1!.
TCBS
counts
of0 were
converted
to 1,giving
tog= 0.
 Shri
with reduced molting frequency. The
best treatment is to improve the water
quality water exchange! and this often
induces molting.
Fungi
Oomycetes
We sometimes have difficulties with
Lagenidiumsp. in the hatchery, but not
with other oomycetesreported to infect
shrimp Sindermann and Lightner,
1988; Brock, 1991!. The source of the
outbreaks has not been determined,
but when they occur they can be han-
dled easily by the administrahon of
trifluralin Treflan! at 10 ppb directly to
the rearing water every four hours for
as long as the problem persists. How-
ever, the report by Gil-Turnes et al,
989! suggeststhat it may be possible
to use a probiotic bacterium to solve the
problem of oomycete infections. They
found that embryos of Palaemon macro-
dachjluswere protectedfrom Lagenidiilm
sp. infections by a speciesof Altennonas
commensalon the embryo surface.
Figuret3. Freshmountofzoealarvaeshowingunidentified
of Penaeustnonodon.a! Fouledappendagesbar= 25lim!. b! Normalappendagesbar= 25pm!.!.
Diseases in Thailand 79
We have not yet found an example of
loss causedby these fungi in growout
shrimp.
Other Fungi
Fusarium sp. infections have been re-
ported to afflict pond-reared shrimp in
Thailand Sindermann and Lightner,
1988; Brock, 1991; Limsuwan, 1991!,
but losses have been low and the infec-
tions are clearly precipitated by unfa-
vorable rearing conditions. The
problems resolve when these condi-
tions are improved.
Parasites
External Parasites
The external parasitesmost commonly
encountered in Thailand are stalked
protozoans such as Zoothamniumsp.,
Episfylis sp., Vorticellasp. and Acineta
sp. Sindermann and Lightner, 1988;
Brock, 1991!. As with the filamentous
bacteria, they are fouling organisms
related to poor water quality and re-
foviingbacteriaonthefeedingappendages
 F el
duced molting frequencyand treat-
ments are similar.
Wehavefoundonly malachite
greenat
0.95 ppm to be useful againstthese
organisms for earlylarvalstages.Effec-
tive doses for postlarvaeand older
stagesare harmful to earlier larval
stages;the earlierthe stage,the more
harmful the treatment.Compoundswe
have tested include Formkn, chlora-
mine T, bemalkonium chloride and
pavidoneiodine.
Internal Parasites
The only internal parasite that has
causedsignificant lossesin cultivated
penaeidsin southeastern Thailand is
the inicrosporidian, Appnasomapenaei
Hegelet al., 1992!,The parasitecauses
infections in both Penaemmergiiierisis
and P. monody, but it is especially
prevalent in P. merguiensis,which
seemsto have a high incidenceof in-
fectionat all times,in rearedand cap-
turedanimals.By contrast,high levels
of infection in P. monody seem to be
relatedto highrainfall. Forboth spe-
cies, high levelsof infectionseem to
occur only in farms on the southwest
Gulf of Thailandin theareaof Songk-
hla.Theyhavenotbeenreported from
the southeasternGulf in shrimpfarms
around Chantaburi,even thoughrain-
fall thereis seasonally
veryhigh. The
most likely reasonfor the differenceis
absence of the unknown intermediate
host on the southeastern coast.
We would like to havea chemotherapy thesecarried
for this infection;hence,we have been on thelabel,"absolutelyguaranteedto
et al.
tryingto testa numberof coccidiostatic
drugs that are commonly used with
poultry. The problem with this pro-
gram is that we have not found a
method of artificial infection and so we
are limited by the availability of animals
of suitable infection state from ponds
or from the wild. As a result, the work
is progressingslowly.
In the meantime, one Ph.D. student at
Matudol University in Bangkok hasiso-
lated and purified sporal DNA of Ag-
masomapenaeifrom both P. merguiensis
and P. morwdon, and he has identified
sequencesthat we believe will be useful
in preparing a DNA probe to help trace
the life cycle. He is also comparing the
variableregion of ribosomal DNA from
the two isolates to help determine
whether the infections are caused by a
single speciesof parasite.
Environmental Factors
Pesticides
In May 1990,we were informed that
rice farmers in Thailand sometimes use
compounds to eradicate freshwater
crabs that attack rice seedlings. Since
shrimp farms are often located in rice-
growingareas,we became concerned
that thesecompounds
might possibly
harmfarmedshrimp.
Froma localvendorin Songkhlaprov-
ince,we obtainedtwo retail products
sold to kill crabs in rice fields. One of
the followingstatement
 Shri Diseases in Thailard 8'l

Table
2. Effect
ofa single
addition
ofcyperrnethrin
ligfL!
on
survival
ofP. monodonpostlarvae
ina 24-hour
testin 1-Lbeak-
ers.Control
animals
showednomortality.
Concentration % Mortality Time
pp~ppbrpptr
10,000 10ppm 100 IO mi
5.000 5 ppm 100 0 mi
1,000 I ppm 100 IO ml
500-1 500-1 ppb 100 h
0.5- 0.01 500- 10pptr 100 h
0.005 5 pptr 100 4 h
0,001orless~lptr orless none 24h

kill crabsin rice fields." Bothof these one or more unknown environmental
productscontainedwell known insec- stressors that leads to death from a
ticidesas activeingredients.Onecon- varietyof secondaryinfections.
tained methyl-parathion; the other,
cypermethrin.The methyl-parathion Cypermethrin
wasrecommended for useas a 1-ppt The first testwith cypermethrin
was
activeingredient!mixturewith cooked
conducted using20 postlarvae
in 1-L
ricebait,whilethecyperrnethrin
prepa- beakerswith variousconcentrations
of
ration was recommendedfor use as a
activeingredient,from 10 ppm to
spraysolutioncontaining200ppm of 0.0001 ppb. Survival over 24 h was
activeingredient.We immediately
car- recordedto obtain some idea of the
riedoutaquarium trialswithpostlarvae rangeof toxicity.The resultsareshown
of P. rrionodon
and found that these
in Table2. Cl'nicalsymptoms
for the
compounds
wereextremely
toxic. affectedanimalswere restlessness,
swirlingwith uncontrolled
movement,
We wondered whether such com-
swimming
to the surface
followed
by
poundsmightbecausaUy
relatedto the sinkingto thebottomof thetank,mus-
recent occurrence in Thailand of unex-
clecrampsand death.Thesebehavioral
plainedmortality
thatiscommonly
re- changes
suggested
involvement
of the
ferred to as "one month death
nervous system in the causeof death.
syndrome."This syndromerefersto
thesuddencatastrophic
deathofjuve- Theresults
showed
thatcypermethrin
nileshrimpof approximately
1-3g was extreinelytoxic for larvae of P.
aboutone monthafter stockingin monody.Because of this,a secondtest
growoutponds seesectionbelow!.To
wasconducted to determinetheeffectof
date,no specific
causefor thissyn- sublethalconcentrahons of this insec-
dromehasbeenfound,butmanyThai ticide i.e., activeingredient
concentra-
scientistssuggestthat it resultsfrom
 Table3. Survivalof P. monodorr juvenilesi - 3 g fresh
weight!uponexposureto sublethalconcentrations of cyper-
niethrln ng/L!for 10days in 20-Laquaria,Trialswerecar-
riedoul in triplicatebutthe controlconsistedof onlyonelank.

tions af 1 pptr or less! on juvenile mortality within 10 days at 1 pptr for

shrimp. this insecticide.

In this 10-daytrial, 1- to 3-g juvenile Within the last three months, we have
shrimpthesizereached approximately also tested cypermethrin with zoeal
1 month after stocking in growout stagesand found that it gave significant
ponds!were kept in 20-Laquariacon- toxicity 0% mortality in 12 h! at 10
taining 20 animals each. Concentra- pg/L. Electron micrographs of mori-
tionsof cyperrnethrinused were 1.0, bund animals from these tests are
0.5 and 0.1 pptr of active ingredient, shown in Fig. 14. They show extensive
thesesublethalconcentrations
being cellular damage and appear to have
based on the results of the first trial. distinctivelyenlarged nuclei with tubu-
Waterin the testaquariawaschanged lar inclusions. These are preliminary
everytwo daysfor new watercontain- resultsandthetestsarebeingrepeated.
ing the same concentration of insecti- If the resultsare repeatable,thesetu-
cide that was used at the start of the bular nuclei may serve as a marker for
test. The aquariawere abserved for detectingmortality causedby cyper-
mortalities,andsurviving shrimp at the methrinpoisoning.
endof theexperiment werepreserved
in Davidson'sfixative. Thesespeci- Methyl-parathion
menswere later prepared for standard The first test with methyl-parathion
histological examination Bell and was conductedusing 20 postlarvaein
Lightner, 1988!.The results for mortal- 1-L beakers with various concentrations
ity are shown in Table 3. of activeingredient from 5 ppm to 1
ppb. Survival aver 24 h was recorded
Because
thecontroltankwasnotrepli- to obtain some idea of the range of
cated, we could not test the statistical toxicity. The results are shown in Table
significance of our results. However, 4. Clinical symptoms far the affected
thereis a strongindicationof sil ufieant animalswere the sameas for cyper-
 Shri Diseases in Thaiiand

Figuret4.Transmission
electron
micrographs
ofmoribund zoeaI ofPenaeusmonodon e~ed to10
py'Lofcypermethrin
for24h ina recentpreliminary
test.a!Lowmagnification
showinggeneralized
cei/ular
damage.
Notethecol/apse
ofthemicroviili
in thegut bar= 4p.m!.b!Higher
magnification
of
cells
ina more
advanced
state
ofdegeneration,
Notethattheenlarged
nuclei
aimost
comp/etely
fillthe
cellsbar= 1.2@m!.
c!Highmagnification
ofa nucleus
fromb!,showing
details
ofthetubularinclusions
bar = 0.3 p,m!.
 F el et al.

Table
4.Effect
ofa single
addition
ofmethyl-parathion
onsurvivalof Penaeus
monodon
postlarvae
in a 24-h
testin1-Lbeakers.
Control
animals
showed
nomortality.
lity Time

100
100
100
100 <4
'100 <9
100 4
none 24

rnethrin
above,andalsosuggestedin- resultsfor the various treatments in this
volvementofthenervous
system
in the experiment as with cypermethrin
cause of death.
above!.
However, thereis a strong
in-
dication
of significant
mortalitywithin
Methyl-parathionwas 3,000times less 10 daysat 5 ppb for this insecticide.
toxicthan cypermethrinin this acute This is three times lessthan the concen-
toxicitytest.Basedon theresultsof this trationthatcaused
100%
mortality.
trial, a 10-dayexposuretestwascon-
ducted
using
thesameprotocol
andthe Histological examination of the surviv-
samecontroltank as for the cyper- ing juvenileshrimpfrom both insecti-
methrin test describedabove.The re- cide treatmentsshowed multiple
sults are shown in Table 5.
anomalies,
includingenlarged,
vacuo-
latedventralnerve
ganglia,
andgeneral
Because
thecontrol
tankwasnotrepli- necrosis
of thehepatopancreas
andthe
cated,we could not test the statistical skeletalmuscles.Thesecharacteristics
significanceof the differencesin the were sharedby some of the animals
Table
5.Survival
ofP.rrNsnoaon
juveniles
t - 3g fresh
weight!
exposed
tosublethai
concentrations
ofmethyl-parathion
forten
daysin20-Laquaria.Trials
wereconducted
induplicate
andin
parallel
tothetriplicate
cypermethrin
testsabove;
thecontrol
con-
sisted
ofthesame singletankused
forthecyperinethrin
tests.
 Shri Diseases in Thailand

from the control tank. We found no Hatakeyama and Sugaya,1989!,gave

specific indicators for insecticide or
type of insecticide poisoning at the light
microscope level. No preparations of
the juveniles were made for the elec-
tron microscope. Obviously, more de-
tailed studies with more extensive
histological work are required.
Brief Review of Insecticide Studies
Table 6 gives a summary of pesticide
toxicity data for crustaceanstaken from
the literature.
An early study by Eisler 969! ex-
amined the acute toxicities of seven
organochlorideinsecticides,including
DDT, and five organophosphorus in-
secticides,including methyl-parathion,
against three different decapod crusta-
ceans. DDT was the most toxic of the
organochloride insecticides 4-h LCso
3 - 12 ppb and 96-hLCso0.6 - 6 ppb,
dependingupon species!,
while methyl-
parathion was the most toxic of the
organophosphorous
compounds4-h
LC5011 - 23 ppb and 96-h LC502 - 7
ppb, depending upon species!.He also
showedthat temperature
and salinity
could alter the effect of these insecti-
cides. In general, an increase in tem-
perature resulted in increased
sensitivity.With respectto changesin
salinity,sensitivityto threeorganochlo- to three logs more potent against crus-
ridesdecreased with increasingsalinity taceans than parathion-containing in-
overthe rangeof 12- 36 ppt while it secticides. This would translate into
increasedfor two organophosphorous possible NOEC values in the range of
compoundsover the samerange. pg/L.
48-hLCMvaluesfor threeorganophos-
phorousinsecticides in the rangeof 1-
8 ppb as comparedto generallymuch
higher values up to 60 ppb in some
cases!for the crustaceans usually em-
ployed in toxicity tests e.g., cladocer-
ans suchas Daphniamagnaand Moina
macmcopa!.Together with the earher
study by Eisler 969!, it is dear that
sensitivitiesfor individual speciescan
vary by up to 10 tiines or more. Thus,
specifictests are required wherever in-
forrnation is desired for a particular
species.
In a rather extensivestudy, Kuhn et al.
989! reported on the toxicity and "no
observedeffectconcentration" NOEC!
for 73 potential water pollutants to-
wards Daphnia magna.This included
not only insecticidesbut also a long list
of otherorganiccompoundsand heavy
metals. Among the chemicals tested,
the insecticide, ethyl-parathion, was
the most potent, giving an NOEC 1-
day reproductiontest! of 2 ng/L. This
was followed by bis tri-n-butyltin! ox-
ide at 160ng/L, cadmiumat 600ng/L
and perchloro-cyclopentadiene at 9
kg/L. No synthetic pyrethroid com-
pounds were included in the chemicals
tested, but other reports see below!
indicatethat thesecompoundsare up

A morerecentstudy on the freshwater A report by Armstrong et al. 976! on

shrimp, Parafyacompressa
improvisa the toxicity of the insecticidemethoxy-
Table6. SOmein4eCtldde
toxkNes rtedfOrCeStaceans
Inaerdlci&Type TeatCrrtlanhrn LCso or EDso Reference
IhfL
ORGANO' RIDE
Ahjrilrr CrangonsepternspinosaDecapoda30 4! Eisler, 1969
Palaemonetes
vrdgaris Decapoda ! 2,000 4! Eisler, 1969
Pagurus
bngicarpus Decapoda3004! Eisler, 1969
P,F-DDT CrangrmsepternspinosuDecapoda 3 4! Eisler, 1969
Pabaernonetes
vulgans Decapoda 12 4! Eisler, 1969
Pagurus
longicarpus Decapoda 7 4! Eisler, 1969
Cnrngon
septenmpnosaDecapod a 68 4! Eisler, 1969
Palaemonetes
vulgeris Decapoda! 107 4! Eisler, 1969
Pagurus
longinrrpus Decapoda 70 4! Eisler, 1969
CrangonseptenrspinosaDecapoda 2.8 4! Eisler, 1969
Palaerrronedes
vulgaris Decapoda10,34! Eisler, 1969
Paguruslongicarpus Decapoda 27 4! Eisler, 1969
Crangonseptemspi
nasa Decapoda 1104! Eisler, 1969
Pahemnetes vulgaris Decapoda > 6,5004! Eisler, 1969
Paguruskmgicarpus Decapoda 4704! Eisler, 1969
CrangonseptemspinosaDecapoda 14 4! Eisler, 1969
Palaemonetes
vulgaris Decapoda 62 4! Eisler, 1969
Pagurus
longicarpm Decapoda 38 4! Eisler, 1969
CrangonseptemspinosaDecapoda9 4! Eisler, 1969
Palaemonetes
vulgaris Decapod a 16 4! Eisler, 1969
Pagurus
longicarpus Decapoda 9 4! Eisler, 1969
Cancer
magister Decapoda 0.42- 130 96! Arrnst rong et al.,
1976
ORGhNOPHOSPHORUS

Benthiocarb Mysidopsisbahia Mysidscca330 96! Schimmel et al.,

1983
Carbophenothion Mysidopsisbahia Mysidacea46 96!
Trithlon! Cripe et al., 1989
CMorpyrifosDursban, Mysidopsisbahia Mysidacea0.03596!
Lorsban! Schirnrnel et al.,
1983
DichiorvosDDVP, Crangonseptemspinosa
Decapod
a 184!
Vapona! Eisler, 1969

Palaemonetes
vrdgans Decapoda 3904! Eisler, 1969
Pagurus
longicarpus
Decapoda1504! Eisler, 1969
Diaoxathon
Delrrav! Cnrngon
septemspinosa
Decapod
a 3074! Eisler, 1969
Palae~metes
vulgaris Decapoda5004! Eisler, 1969
P urus ion oda 300 24 Eisler 196I9
 Shri Disease in Thailand 87

Table 6. Continued.
Insectlckte Type Test Organism LCso or EDso Referenos
in~
Dlaxinon Spectracide,Daphniamagrra Cladocera 2 8! Hatakeyamaand
Donx Out! Sugaya,1989
Gadocera 9 8! Hatakeyarnaand
Sugaya,1989
Paratyr compressa Decapoda 6 8! Hatakeyamaand
r nrprovrsa Sugaya,1989
Ethyl-parathion Daphniamagna Clad ocera 2* 4! Kuhn, et al,, 1%9
Fenitrothion Daphniamagna Cladocera >50 8! Hatakeyamaand
Accothion, Folithion, Sugaya,1989
umithion!
Moina improvisavulgaris Gadocera 37.88! Hatakeyarnaand
Sugaya,1989
Paguruslongr'carpus Decapoda 40 4! Eisler, 1969
Paratyacvmpressa Decapoda 1.28! Hatakeyamaand
r nrprovtsa Sugaya,1989
Fenthion Baytex, Daphniamagna Cladocera > 508! Hatakeyarnaand
entex, Tiguvon! Sugaya,1989
Qadocera 35,38! Hatakeyamaand
Sugaya,1989
Paratyacompressa Decapoda 1 8! Hatakeyamaand
r mprovisa Sugaya,1989
Malathion Cythion! Crangonseptemspinosa Decapoda2464! Eisler, 1969
Palaemonetes
vulgaris Decapoda 1314! Eisler, 1969
Paguraslongicarpus Decapoda 1184! Eisler, 1969
Mysidopsisbahia Mysid acea 5.3 96} Cripe et al., l989
Methyl-parathion Crangonseptemspinosa Decapoda 11 4! Eiskr, 1969
Prdaemonetes
vulgaris Decapod
a 15 4! Eisler, 1969
Pagurrrslongicarpus Decapod
a 23 4! Eisler, 1969
Mysidopsisbahia Mysidacea 0.78 96! Schirnrnel et al.,
1983
Perraerrs duorarum Decapoda 1,2 96! Schimmel et al.,
1983
Oziotelphrrsa
senexsener Decapoda 1,0008! Reddy et al., 1986a
Mevinphos Phosdrin! Crangvnseptemspinosa Decapoda 13 4! Eisler, 1969
Palaemorretes
vrdgaris Decapoda 1314! Eisler, 1969
Pagurustongicarpus Decapoda40 4! Eiskr, 1969
PYRE' ROH3
AC 222,705 or Mysidopsisbahia Mysidacea 0,008 96! Schimrnel et al.,
Flucythrinate 1983
Perraeus duoraram Decapoda 0.22 96! Schimmel et al.,
1983
Tftbie 6. Continued.
InaectiddeType LCsoor EDso Reference
Ih
Cypermethrin Mysidacea0.019 96! Cripe et al., 1989
0.005 96! Hill, 1985
0.056 96! Clark et al., 1989
Homaruseneruxna Decapod a G.01 96! Schimmelet al., 1983
Daphnia magna Gadocera 1.0-5.04! Day, 1989
Crangonsepfemspnosa
Decapoda0.01 96! McLeeseet al1980
Pai~mnonetes
pugio Decapoda0.016 96! Clarket al., 1989
Penaeusduorarum Decapoda0.036 96! Clark et al., 1989
Ucapugilator Decapod a 0,2 ! Clark et al., 1989
Fenvalerate Homarusamericana Decapoda0.14 96! McLeese et al., 1980
Daphniamagna Gadocera 0.3 4! Day, 1989
0,8- 2.58! Day, 1989
Daphnia
g deata
mendotaeCiadocera 0.16- 0.29 8! Day, 1989
Ceriod~e'alacusfris Cladorma 0,218! Day, 1989
Diapfomus
oregonensisGadocera 0.12 4! Day, 1989
Atfysidopsis
bahia Mysidacea0.008 96! Schirnmelet al., 1983
Penaeusduorarum Decapoda0.84 96! Schimmelet al., 1983
Nitarraspinipes ? 0.38 96! Clark et al., 1989
Palaemonetes pugio Decapoda <0.003 96! Clark et al., 1989
Crangon septemspinosa
Decapoda0.13 96! McLeese et al., 1980
Mysidopsis bahia Mysidacea0.02 96! Schimmelet al,, 1983
Penaeus duorarum Decapoda0.22 96! Schimmelet al., 1983
Penance aztecus Decapoda0.34 96! Clark et al., 1989
Menippemercenaria Decapoda0.02 96! Clark et al., 1989
Nitocraspinipcs ? 0.15 96! Clarket al., 1989
Ucapugilafor Decapoda2.2 96! Clark et al,, 1989
CARBAMhTE
Carbaryl
Sevin! Daphnia
magna Gadocera128! Hatakeyama
and
Sugaya,1989
Moinama'nor Cladocera2008! Hatakeyamaand
Sugaya,1989
Paratya
comprise Decapoda208! Hatakeyamaand
imprmnsa Sugaya,1989
BMPC
~- Daphnia
mapra Gadocera908! Hatakeyarnaand
hutylphenyl
N- Sugaya,1989
methyl~amate!
Moinammooopa Cladocera 2208! Hatakeyama
and
Sugaya,1989
Decapoda8 8! Hatakeyamaand
Su a a 1989
 Shri Diseases in Thailand
chlor to the Dungenesscrab, Cancer
magister,showed that toxicitywas in-
versely related to age after hatching,
and that it increasedwith length of
exposure.The 24-h LC50values were
.92, 12.0 and ! 920 pg/L for zoea,
juveniles and adults, respectively. The
96-h LC50values were 0.42, 5.1 and 130
p,g/L,respectively,for the samestages.
Since most reports of insecticide toxic-
ity to crustaceans deal with adult
stages, aquaculturists working at the
hatchery stagesor rearing should be
awarethat larval stagesmay be many
timesmoresensitivethan adult stages.
Vogt 987! hasshownthat histological
diagnosisof crustaceanmidgut glands
hepatopancreas!can be used to assess
the effect of low levels of insecticides.
At a concentration of 1 ppm of an
insecticidepreparationcontaining di-
rnethoateasthe activeingredient,there
was no mortality in four days but darn-
age to the midgut gland was evident at
both the light microscopeand electron
microscopelevels.Vogt pointsout that
pesticidebehavior and biologicalim-
pact in the environment are compli-
cated by transfer rates, chemical and
biochemical transformations and health
of an animal. As yet, there is no dear
histologicalindicator to specificallyin-
dicate insecticide toxicity in pond-
rearedanimals,asopposedto toxicity
from other compounds e.g., algaltox-
ins, rancid feed, etc.! Sindermann and
Lightner, 1988;Brock, '1991!.
Animal health is related to nutrition,
and poor nutritional status in crusta-
ceans has been shown to increase the
89
sensitivity of crustaceansto insecti-
cides.For example,Cripe et al. 989!
showedthat low foodavailability
in-
creased
thesensitivity
ofMysidopsis
ba-
hia to various insecticides. This has
implications
notonlyforthosefacing
real situations in hatcheries and
growoutpondsbut alsofor thosecon-
ductingtoxicitytest protocols.
Ware 986! definesorganophosphate
insecticides as chemically unstable
compounds containing phosphorous
and derived from phosphoric acid.
Theyaregenerallythe mosttoxic of all
pesticides to vertebrate animals, and
they are relatedto "nerve gases"by
chemical structure and mode of action
i.e., they inhibit the action of acetyl
choline esteraseor ACH-esterase!.Al-
though they are more acutelytoxic to
vertebratesthan organochlorides,
they
have the advantageof beingless per-
sistent; this is part of the reason for
their popularityin agriculture.One of
this family of compounds,dichlorvos,
or DDVP, is used widely for treatment
of sealice in salmon. From Table 6, it
can be seenthat dichlorvoshas an LCso
of 18- 390lrg/L 8 - 390ppb! and that
theseconcentrations areapproximately
5,000 to 200,000times higher than the
LCsoconcentrationof the most potent
of the synthetic pyrethroid com-
pounds.However,the generallylower
toxicity of the organophosphates
does
not meanthat theycanbedisregarded.
These compounds are used in consid-
erablequantity in agriculture.Methyl-
parathion alone was imported into
Thailand to the amount of 1,452 MT
active ingredient! in 1989 Thus, these
 compounds
alsohavethepotential
to lar organization,
suggestive
of massive
cause
damage
inshrimp
culture
if they calciumlossfrom the reticulum into the
contarrunate
theaquatic
environment. myoplasm" Bradbury,'l973b!.In tests
on actomyosinsuperprecipitationin
Metabolic
andphysiological
studies
of the presence of various mixhires of
parathion
andmethyl-parathion
action ATP, Ca" and parathion, he found
in crustaceans
haveshownthatit is not thatanypreparation
containing para-
limitedto inhibitionof ACHwsterase thion showed a massive increase in
activity.
Reddy
etal. 986a,b; 1988! superprecipitation. Thus, there were
havereported
wide-ranging
effects
in- two quite different effects from the
duding
evidence
oftissue
damage
and insectidde. One was destruction of the
alteration
in enzymeactivities.
Their sarcoplasmicreticulum and mitochon-
mostinteresting
observation
wasthat dria causingreleaseof vesicularcalcium
methyl-parathion
atsubiethal
exposureand the other was a direct action on
00 p.g/L for 48 h! couldleadto the actomyosin.
Theconcentration
of para-
accumulation of lactic acid in musde thion used to obtain maximal contrac-
tissuesthroughinhibitionof aerobic ture tension in these studies was 100
metabolism.aliismayrelateto the fact pm, about25 times lessthan the recom-
thatwe havefoundsporadic
occur- mended
dosage
forgeneral
agricultural
rencesof idiopathicmusdenecrosis use.It ispossible
thatmethyl-parathion
MN! insamples
ofshrimpfromsome or other organophosphorous insecti-
farmers'pondsunpublished!;
this cides have similar methods of action.
syndrome
hasbeenlinkedto lacticacid
accumulation
in crustacean
rnusdeasa In mammals,parathionis metabolized
result of unknown environmental to nontoxiccompoundsvia the inter-
stress
factorsNashet al., 1987!.It is mediate paraoxon, which is an even
possiblethatorganophosphate
insecti- more potent ACH-esterase inhibitor
cides,
althoughnotentirely
responsible thantheparentcompoundElmamlouk
forthesyndrome, may,at verylow, andGiessner, 1976!.However,it has
sublethal
concentrations,
aggravate
it beenshownKlmalouk andGiessner,
in anadditive
orsynergistic
way. 1976!that hepatopancreatic
tissueof
thelobster
cannotconvert
parathion
to
ln detailedstudieson the mechanism paraoxon or p-nitrophenol,suggesting
of parathion action on musde of the thatlobster,
andperhaps othercrusta-
crabCarcass sp.,Bradbury 973a,b! ceans,maynot becapableof transform-
foundthat "parathiondoesnotactivate ingparathionandmethyl-parathion to
musdeby a transientdepolarization,nontoxic compounds. Thiscouldhelp
butit doessignificantly
depress
reticu- to explainwhy they areso muchmore
larcalciumuptake.Jnaddition,
para- toxicto crustaceansthan mammals.
thiongreatlymodifiesactomyosin
superprecipitation, and fme-structural Mostof thestepsin theenvironmental
studiesshowgreatdisruption
of reticu- degradative
pathway
formethyl-para-
 Shri Diseases in Thaihnd

thion are biotic transformations. In a Cypermethrinis a fourth generation

laboratorymodelflow-throughsystem, derivative of the natural insecticide,
Bourquin et al. 979! showed that pyrethrum, a mixture of four related
methyl-parathion was rapidly and irre- compoundsextractedfrom Chrysaiithe-
versibly bound to sediments, and that miim family Compositae, the sun-
it was not chemically or biologicaHy flower family! Ware, 1986!. These
degraded in the absenceof sediments, natural compoundsare good house-
even under various sterile and nonster- hold insecticides becauseof quick
ile conditions. Degradation was fastest knock-downand relatively rapid de-
with nonsterile sediments, and the coinposition e.g., instability to sun-
principal transformation product was light!. The first synthetic pyrethroid
amino methyl-parathion. The calcu- insecticide, allethrin, was manufac-
lated half-life for methyl-parathionin tured in 1949but it, like several others
the system used was 40 50 h. to follow, wasnot successfully
usedas
an agricultural chemical.The first suc-
Using another model ecosystemwith cessful agricultural compounds in the
parathion,Yu and Sanborn975! gave group introduced in 1972 and 1973,
a half-life of 15 to 16 days in water and respectively!were the fourth genera-
showed a gradual transforination from tion chemicalsfenvalerateand per-
the insecticideto unextractablepolar rnethrin. These were applied in the
degradation products 5% of the total field at approximately
100-200g active
addedradioactivityover the 38-dayex- ingredient!/ha and were active against
periment!. They suggestedthat the in- a wide variety of insects. They have
secticide would not present a threat been followed by much more potent
from environmental accumulation. compounds such as cyperxnethrin
Theyquotedsourcesindicatingthat the which areappliedat only 10- 20 g/ha
residualamountof 0.3 ppb .3 g/L! in Ware, 1986!.
waterat the end of their 38-dayexperi-
ment was 10 times less than that rec- Since cypermethrin is one of the
ommendedasan acceptable
residuefor crabicides used by Thai farmers, and
water. However, upon referenceto Ta- since it showed the highest toxicity in
ble 6, it is not certain that such a level our preliininary tests with Penaeus
would be harmlessto crustaceans,if the monodon, we have concentrated our
periodof exposurewere long. search of the literature on this com-
pound. The effects of synthetic pyre-
throids on freshwater zooplankton
In condusion,it is clearthat the aquatic have been reviewed by Day 989!. He
contaminationfrom the organophos- reported the range of acutetoxicitiesfor
phate pesticidesused in agriculture ciadoceransand copepodsas 0.12- 5.0
probably presents a much lower threat pg/L, while reduced reproduction and
to rearedcrustaceans
than do synthetic filtration rates could be observed at
pyrethroid insecticides. concentrations as low as 0.01 pg/L.
Qarketal.989!have the in
reviewed therangeof onemilliontimeshigher
effect
ofpyrethroids inverte-than
onmarine that which can harm crustaceans
Megharaj et al.,1987!.
Growthinhib-
bratesandfish;datafromtheirstudy itingeffects werenotseen untilconcen-
arein Table6.Theyareamongst the trationsexceeded10 rng/L. Thus, jt is
mosttoxicinsecticides
for crustaceans.
Withwhatever species possible
thatnaturalshrimpfeedsor
or testcondi- feed componentsderived from fish,
tions,
LC50
valuesreported
for 24-to algaeor plantsmaycontainsufficient
96-htestsarein therangeof ng/L.For
residuesto cause difficulties.
example,
themosttoxiccompound
listed
is cypermethrin
withan LCso To understand the reason for the ex-
value96-htest!of5 ng/L
forMysidop-
sis.Our testwith P. mouton above!, traordinarypotencyof pyrethroid
in-
however,indicatedthatthetigerprawn secticides towards crustaceans, it is
is evenmoresensitiveto cypermethrin, necessaryto examinetheir mode of
since5 ng gave100%mortalityfor action. Chalmers and Osborne 986!
larvalshrimpin 24h, andsince1iay reported
thatlike DDT,symptoms
of
exposure to 1 ng/L pptr!gaveap- pyrethroidpoisoningin insectsinclude
proximately50%mortality with juve- excitation, ataxia and convulsions that
niles. These lethal concentrations correlatewith repetitive dischargesin
contrastwith all of the other insecti- many areasof the nervoussystem.
cidesin Table6 for which LCsovalues Theyalsonotedthat someDDT-resis-
are in the rangeof pg or mg/L i.e., tant insect strains show cross-resistance
concentrationsseverallogs higher are to pyrethroids,
and they, therefore,
requiredto obtainthe samelevel of suggest
thatthechemicals
maysharea
mortality!. common mode of action. However,
Gammonand Sander 985! reported
To obtain 50% mortality with fish in- that TypeI pyrethroidscauserepetitive
cluding fry!, concentrations
of pyre- firing of neurons while Type II pyre-
throids in the range of hundreds of pg throids rarely do and that the two
oz even in mg/L are sometimes re- classes
of compounds
may therefore
quired, but there are exceptions have different physiological targets.
Schimmef et al., 1983; Gark et al., Their data suggeststhat Type I pyre-
1989!.Oystersappearto be very insen- throids act on sites where calcium con-
sitive, with LC50values in the range of trols sodiuminactivation,while TypeI
1- 2 mg/L.Theyalsoshowedveryhigh compoundsmay involve interaction
bioconcentrationfactors BCF! up to with the GABA gamaaminobutyric
4,700for fenvalerateClarketal., 1989!. acid! receptor complex. Different pyre-
Finally, blue-greenand green algae throidsmayhavedifferentspecificitie-
wereshownin laboratorystudiesto be sThealsostatethat "there is growing
unaffectedby concentrations of fen- evidence
thatTypeIf. syndromein-
valerate
andcypermethrin
upto5mg
m /L volves effects on one or more neuro-
i.e., 5,000pg!, a concentrationthat is transmitter-modulated
channelsaswe"
 ahri D~
as affecting voltage-sensitive axonal!
joxi channels.
Rashatwar and Matsumura 985!
showed direct inhibitory effects of py-
rethroids on calmodulin. Since cal-
modulin plays a vital role in a wide
variety of physiological reactionsde-
pendent upon calcium,it may mean
that the mode of action of pyrethroids
will turn out to be quite coxnplex.I:or
exaxnple,Mulla et al. 982! reported a
rather unusual phenomenon during
cypermethrin application to control
mosquitoes in the field. Even 14 days
after application of the insecticide, the
larval population prevailed at a high
level, but no pupae oa~red. This hints
at a rather specific effect on a target
associatedwith morphogenesis,a proc-
ess that is regulated by neurosecretory
hormones. If calmodulin plays a central
role in this neurohormonal process,
pyrethroids would be very disruptive.
Although pyrethroids are known to be
extremely toxic to crustaceansin labo-
ratory tests, several factors must be
taken into considerationin assessing
their impact in the environment. One
important factor is persistence gov-
erned by rates of degradation and xne-
tabolism! and another is sorption Clark
et al., 1989!.In the study by Schimmel
et al. 983! the sedixnent half-lives for
the pyrethroids AC 222,705 flucythri-
nate!,fenvalerateand perxnethrinwere
16,31and2.5 days,respectively.Those
for the organophosphorousinsecticides
methyl-parathion, benthiocarb and
chlorpyrifoswere 1.2, 6.4 and 24 days,
~espectively.
Agnihorti et al. 986! also
inThaiiarKi 93
report persistence of permethrin,
cypermethrin, fenvalerate and del-
tamethrin in sediments beyond 30
days; although, 75 - 95%was lost from
thewatercolumnwithin24h ofappli-
cation.Perhapsmostalarmingfromthe
point of view of feed formulators is the
report by Joia et al. 985! of cyper-
methrin and fenvalerate residues in
wheat and its milled fractions. Half-
lives in stored grain ranged from 69 to
385 weeks depending upon the mois-
ture content and storagetemperature.
Reduction in residues through bread
baking were low 9 - 84% cyper-
xnethrin and 87 - 88% fenvalerate re-
mained after cooking!. This meansthat
anyresiduesin shrimpfeedingredients
would probably passprocessingcondi-
tions and remain relatively undimin-
ished in finished pellets, where they
would pose a significant threat to
reared shrimp.
In their laboratorystudyon sediment-
sorbed fenvalerate and cypermethrin,
Clark et al. 989! concludedthat "di-
rect contactwith, or ingestionof, con-
taminated sediment did not appear to
enhancethe toxicity of fenvalerateor
cypermethrinto mysids,
grassshrixnp
or pink shrimp"andthat "sediment
sorbedchemicalswere not acutely toxic
... until concentrationsin sediment
wereincreasedto the point wherepar-
titioningintooverlying
waterresulted
in acutelylethalconcentrations."
Thus,
theysuggested
that"chemical
parti-
tioningbetweencontaminated
sedi-
xnentsand overlyingwater may be
modeled
to predictacutelethaleffects
on marinecrustaceans
forhabitatswith
 F el et al.

well-defmed
physicochemical
andhy- Thailand, and these organisms are con
drodynaauccharacteristics."
This in- sidered to be toxic to some marine
formation indicates that the threat of animals. We have exposed postlarvae
pyrethroid
residues
toaquaculture
may to ¹ scintilans in high-density beaker
be reducedby sorption. trials at our hatchery laboratory and
found no indication of acute toxicity.
A preparationof pyrethrumcalledPy- However, reports concerning the pos-
Sal25hasrecentlybeenintroducedin sible dependence on commensal
Great Britain for the treatment of sea bacteriafor toxin productionin dino-
lice on salmon, by Vetrepharm of flagellates Kodama et al., 1989; Tam-
Hampshire.The productis marketed plin, 1990!may explain the variation in
under licensefrom Norsk Pyrethrumof observedeffect of dinoflageilates.
Norway Fish FarmingIntl. vol. 17 no.
6: 2!. Although the preparationis pur- Within the last few months, Dr. Piam-
ported to cause little environmental sak Menasvata Dept. Marine Science,
damage,it would be useful to deter- Chulalongkorn University, Bangkok!
mine if this product affectsshrimp. informedmethat his grouphasisolated
from shrimp ponds a strain of Atexan-
Toxic Algae drium = Gonyaluax!
that rapidly causes
100%mortality to postlarvae of Penaeus
Limsuwan 991! has reported that wotan at levels of under 100 cells/L.
dinoflagellates,
blue-greenalgaeand
somediatoms can causeproblemsin With respect to other algal species,
shrimpgrowoutponds.He statesthat Limsuwan991! reports that Rhizoso-
someeffectsareindirect e.g.,the ef- leniared tidescan causeproblemsin
fectsof Oscillatoria
sp., Trichodesmiue growout ponds through physical irrita-
sp.andNoctilucasp.! in thatdifficulties tion of thegills, but we have alsofound
arisefromlowoxygen
andhighammo- indications
of toxicity.Duringone"red
nia afteran algalcrash.In othercases, tide" dominatedby this diatom, farm-
suchas the dinoflagellates,
toxinsact ers reporteda drop in feeding activity
directlyon theshrimp. of pond-reared
shrimp,but no signifi-
cant mortality. In preliminary beaker
In southernThailand,we have had trialsatourhatcherysite unpublished!
dif6culties
duringsomeperiodsbut with supernatantliquid from heavy
not others!when dinoflagellates
are suspensionsof the alga, we found
presentin pondsin significantnumbers acute toxicity to mysis larvae '100<
unpublished!,and it is difficult to mortalityin 2 h! at dilutions of 1/1000.
makeconclusionswithoutdetailed test- Thiswarrantsmorerigid tests.
ing with specificisolates.However
octiluca
scmtihms
Noctiluca Suvapepun,
1989! By contrastto RhizosoMiu,we have
andProtogonyaulax
spp.Fukuyo etal., found the cyanobacterium,
Trichrxl~s
1989!
havebeenreported
in theGulfof miiimerythraeum,
is notacutelytoxicin
 Shri Diseasesin Thailand
similar beaker trials; but we concur
with Limsuwan 991! that it can pro-
duce low oxygen tensions. On one oc-
casion during a "red tide" dominated
by T. erythraem,we were called to a
shrimp farm where the inlet canal was
covered with a thick, tan-colored scum
of this alga mixed with copious quanti-
ties of a large up to 500-p.mdia.! for-
aging protozoan of the family
Chilodonellidae. The farmers closed
the inlet gates when the scum ap-
peared, and the water in the canal
becamestationary. As a consequence,
a situation of low dissolved oxygen
occurred beginning in the late after-
noon, resulting in the death of fish and
crustaceansin the canal unpublished! ~
This could probably even occur during
mid-day, if a bloom were in the declin-
ing phasewith large quantities of dead
algal biomass in the water. In addition
to alarm caused by the scum, the alga
produced a noticeable red color
through the releaseof copiousquanti-
ties of phycocyaninand phycoerythrin
when it died, which further alarmed
farmers.
Crvde Oil
With increasing frequency, we have
found small .5 - 1.0 cm dia.! to large
.0 - 6.0cm dia.! lumpsof what appear
to becrude oil washedup on thebeach
in front of our hatchery.Inspectionof
another site 30 km south on the same
coast revealed similar lumps, so the
phenomenon
was spreadovera wide
areaand the quantity of oil that caused
thisdepositionmusthavebeenlarge.
Wehavetestedsamples
ofthismaterial
threetestsin triplicate!
for toxicityto
thelarvaebystirringtheoil g/L!in
normal sea water at 60'C for 1 h fol-
lowedby separation
andmiHipore
fil-
tration .45 ~! of the dear water
"extract."Thiswateris acutelytoxicto
postlarvae00% mortalityat no dilu-
tion and 30%mortalityat 1/1000dilu-
tion in 24 h! when comparedto no
mortality in controls unpublished!. We
could not removethe toxicityby treat-
ment with up to 5 g/L of activated
charcoal unpublished!. We are now
monitoring the beach for oil to deter-
mine whether its presencecorrelates
with production difficulties in the
hatchery.
We do not know the source of the oil.
It could originatefrom passingships or
from undersea well heads approxi-
mately 200-kmoffshore in the Gulf of
Thailand.
Waste Water and Pond Bottoms
In Thailand, it is customaryat the end
of a cultivation to "cheef lane," that is,
to flush out the loose material on the
pond bottom with water jets and/or
pumps.Farmers
believetheresiduein
the pond bottom will jeopardizethe
next crop becauseof the high amount
of organicresidue remainingfrom
shrimpfecesand uneatenfood. The
problemwith this practice
is thatit
harmsthewaterqualityin thereceiving
canal,which maybe the sourceof inlet
water for another farmer. Becauseof
the environmental damage,the govern-
 menthasprohibited
thisactivity,
but Mysteries
enfonwmentis difficult.
LarvalBlackSpotSyndrome
To determine
whetherbottomremoval
is neceseey,
we sought theassistanceAt unpredictable intervals in our hatch-
of Dr. ClaudeE. BoydofAuburnUni- ery, we find that zoea1 orzoea2 larvae
versity
tomeasuretheresidual
organic carrya denseblacklumpof materialat
matter
at theendofharvest
in ponds the junctionof the stomach,hepa-
stocked
with25larvae/m. It wasin the topancreas
and midgut Fig.15a,b!. Ex-
range
of0.5-to1.0.g
organic
carbon/kgaminationof this materialin squash
dryweightofresidue.
Dr.Boydcon- mountswith the light microscopere-
cludedthat mostof the loosematerial veals it to be amorphous in nature.
inthecenter
ofthepond
wasclaythat Sometim.es
the materialloosensand is
hadbeenliftedbythepaddle
wheels, passedin the feces.We have not stud-
or sandand clay that hadsettledout ied this phenomenon
enoughto con-
fromtheinletwaterhighsuspendedclude whether it reduces survival.
solidscontentin the monsoon
season Duringpreparation
of specimens
for
and averagewater exchangeof examination with the electron micro-
30%/day!. scope,theseblackinclusionswere dis-
solvedby the solvents.
Based
onthese results,
Dr.Boydrec-
ommendedtilling
thedampened
pond LymphoidOrganRod-shaped,
bottoms
afterharvest
topromote
maxi- Cytoplasmic,
Enveloped
Virus
mummicrobialdegradationof the re-
maining organic matter. He also
tillingonlyin thecenter Duringinspections
recommended of normalbrood-
stocklymphoidorgansfor viral mate-
of thepondto avoiddisturbingthe rial, we foundonestrange cellin
scouredareas
underthepaddlewheels.
ThiswouM reduce
thequantity abnormal
ofclay The lymphoidtissue Fig. 16!.
lifted
bythepaddlewheels the whatcytoplasm
during looked
of this cell contained
likeenveloped baculovirus
next cultivationcycle.Theserecom- vlrlons.
mendations have been tested and
found tobeworkable, soflushing the Spongy
hluscie
Syndrome
pond at the end of the harvestis not
necessary. However, we have not
testedponds
thatarestockedathigher Histologicalexaminationof normal
densities
andsubjected
to higherfeed broodstock,
pond-reared shrimpand
inputs. postlarvae
PL20andolder!sometimes
reveals
thepresence
of spongymuscle,
nerveandothertissuesFig.17!.Wedo
not knowthecauseor significance
of
thisabnormal
phenomenon.
 Shn Diseases in Thailand 97

Figure15.Twomystery
syndromesfoundinPenaeus
monadonin Thailand.a-b!Freshmount
and
squashmount,
respectively,
ofblackamorphous
material
found
lodged
atthej unction
ofthestomach,
hepatopancreas
andmidgut
ofzoea1 and
zoea
2 larvae
bars
= 125
p.m
and50pm,respectively!.
cN!
H&Epreparation
ofsmallblisters
located
onthefineappendages
ofP.monodon
j uveni!es.
Theanimals
wereremoved
froma "Onemonthmortality
syndrome"
pond seetext!.Theblisterswerefilledwith
hemoiymphbars= 125ijm and50gm,respechveiy!.
98 Fle el et al.

Figure16.Unknown virusfourxfin thecytoplasm

nextto thehypertrophied
nucleusof an abnormal
lymphoid
organcell,Thiswasfoundbychance duringexamination
of theabnormal
tissuesdescribed
in thesectionon lymphoidorganvirus ters: a =2.4pm;b = 0.3pm!.
 Shn Diseases in Thaiiand 99

Figure17.Photomicrographs
of spongymuscleandnervetissuefroma normalbroodstock
specimen
H8Epreparation!.
Theanimalwasfixedfora general
examination
afterit hadbeenusedfor egg
productionin the hatchery.lt showednooutwardsignsof disease,a-b!Lowandhighmagnifeatfons
of spongynervetissue bars= 125pm and25 ijm, respectively!.c-d! Transverseand longitudinal
sectionsof spongymuscletissue bars= 25pm and 12.5iLm,respectively!.
 Fle el et al.
100

BrownMuscleSyndromeidiopathic describedabovein the lymphoid organ.

Muscle Necrosis or IMN! There are cytoplasmicvirogenic stro-
mata and what appear to be unen-
Duringsome
harvests
ofP.monodon,
up veloped
virionsapproximately
45nmin
to approximately
5%of theshrimpare diameter.In contrastto the lymphoid
found to have gross, patchy brown organ, however,there are also
discolorations of the muscle tissue in paracrystalline
arrays
of whatmaybe
after proteinaceous
thetailregion.Thecolorremains materialFig.20!in some
cooking,renderingthe shrimpunmar- of the cells.It remainsto be established
ketable. Since there are no external whetherthe virus is responsiblefor the
it is discov- brownmusclesyndromeandwhether
signsof the abnormality,
atthefrozen thereis anythingmorethana superfi-
eredonlyafterdeheading
storageplant.
cial relationshipbetweenthe virus in
the muscleand in the lymphoid organ.
Histological
examination
oftheanimals
with the light microscopeH&Estain- One Month Mortality Syndrome
ing! revealsmassivehemocyticaggre-
gation.
andmelanization
in areasof One month to six weeks after stocking
musclenecrosis Figs. 18 and 19!. A larvaein growoutponds,moderate
to
general of thesarcoplasmmassivemortality can occur. This is
breakdown
and muscle fibers can be seen. The characterized
by a sudden
reductionof
idiopathic feed intake and the appearancein the
picturecloselyresembles
muscle necrosis IMN! reported for feedingnetsof verylethargicshrimp
Macrobrachiarn
rosenbergii
by Nashet al. thatotherwise
appearnormal.Anecdo-
987!,except ofmelani- tal informationsuggeststhat the phe-
fortheabsence
zation in M. rosenbergii.
nomenon is related to transparenciesin
excessof 40 cm and the presenceof
According
to Nashet al. 987!, this floatingplaquesof benthicblue-green
disease arises from stress, leading to algae"kidat"!thatrisefromthepond
excess lactic acid accumulation, fol- bottom during the day, die on the
lowedbytissuedamage andhemocytic surfaceand fall back to the bottom of
Theyfoundthatreducbon thepondif theycannot
aggregation, beremoved.
in stockingdensitieseliminatedthe
problem.Seeearlier
commentsonthe Forthissyndrome,Dr. ChaloreLimsu-
organophosphate methyl- wan 989! coinedthe phrase"one
insecticide
parathion!.
monthmortalitysyndrome""roaktai
dean"!andthetermisnowwidelyused
ElectronmicrographsFigs.20 - 22! by farmersin anindiscriminate
way,so
haveprovidedevidence
of viral ele- it is difficult to know the real incidence
ments in the cytoplasmof hemocytes of the phenomenon.
Our Technical
betweenthe musclefibers; theseele- ServicesDivision for the month of Janu-
ments are somewhat similar to those
 Shri Diseases in Thailand 101

Figuref8. Photographs
of grosssymptomsof mela@ized
muscletissuefromacblescentPenaeus
monodon.
102 Fle el et al.

Figure
f 9.Photographs
with
thelight
microscope
from
anadolescent
shrimp
specimen
with
melanized
muscletissue,
a!Lowmagnification
ofdisintegrating
muscletissue
with
H8E staining
bar
= 40ljm!.
b!High-dry
view
ofareawherehemocytesareaggregating
between
disintegrating
musclefibersbar
= 10p.m!,c!Toluidine
blue-stained
thinsection
showing
large
numbersofgranulocytes
bar= 10
pm!,
 Shri Diseases in Thaiiand 103

FigureZO.Electron
micrographs
of tissuefromanadolescent
shrimpwithbrownmuscle
syndrome.
a!
Lowmagnification
showingdisintegrating
sarcoplasm
and musclefilamentsbar = 3 pm!. Notethe
lymphocyte
in thelowerlettcomerof themicrograph.
b! HighmagnifI'cation
of thecytoplasm
of the
lynplxxyte notedin theprecedingmicrograph,
showinga virogenic
stroma asterisk!virilis curved
arrows!and paracrystallinearrays openarrow! bar= 0.4 pm!.
 Fle el et al.

Figure21.Highmagnifications
of a virogenic
stromaand vinonsapproximately
45p,min diameter!in
thecytoplasmof a lymphocyte
horntissuesamplesof melanized muscle.Comparetheappearanceof
the viral materialwith that fromthe lymphoidorganshownin Figures8 and T. Barin a = 0,4 pm, and
in b= 0.2@m!.
 Shri Diseases in Thailand 105

Figure22. Electronmicrographs
showinga lymphocyte
frommelanized
muscletissueof anaclolescent
shrimp.a! A lymphocyte withwhatappears to be a paracrystalline
inclusion
in theearlystagesof
formationbar= 1.2ljm!, b! Highermagnification
of theparacrysfalli
nearrayshowing whatappearto
be virions.Ith notclearif theyareenveioperlbar= 0.2pm!.
 F et et at,

aqr1992,
reported
sixoutof83problem Limsuwan
991!proposed
thatOMMS
ponds%! with this syndrome. canbeavoided
bymaintaining
a stable
Histological
examination
algalbloom of green/blue-green algae
of moribund ideal!ordiatomslessideal!sothatthe
shrimpfromonemonth mortality
syn- transparencyof the waterremainsat 20
dromeOIS! pondsaroundThai- - 40 cm. If this cannotbe done, he
landrevealeda varietyof infections, recommends theuseof oxytetracycline
butMBVandbacterial septicemiaare at2 -3 g/kg feedforfivetosevendays
mostoftenreported.Theconsensus of in mildcases, and4 - 5 g/kgfeedfor
participants
at a recent
meeting con- fiveto sevendaysin severecases, as
venedbytheFacultyofVeterinarySci- the only efficacioustreatment.
enceat ChulalongkornUniversity in
Bangkok
gune1989!wasthat the dis- As an alternativetreatment,we have
easeswere a secondaryresult of un- recently
beguntestingthedyeAqua-
knownenvironmental
stressors. shade
fortemporary
reliefwhenalgal
bloomscrashorwhentheyareslowto
In onedetailedstudyof five OMMS buildup. Ourworkinghypothesis is
pondsunpublished!,wehistologicaliythatbenthic
bIue-green
algae
areeither
examined
25randomlyselected
animals directly
orindirectly
toxictotheshrimp
inonedayfromfeeding
netsandfound andthatAquashade will preventor
that92%3/25!hadsmallblisterson limit theirgrowthandreduceor elimi-
smaH appendages
Fig.13c,d! and natetheincidence
of floatingplaques.
whatappeared
tobea genera1
edema. To date,we haveinsufficientdatato
Otherabnormalities
werenecrotican- determine
if thistreatment
is successful.
tennal
glands8/25,or 72%!,bacterial
septicemia
/25, or 20%!andMBV Yellow-head Shrimp
9/25,or76%!.Ina separate
samp1e
of
fiveanimals
takenduringthesame Sinceearly1990,therehavebeenre-
interval
fromChantaburi
ontheoppo- ports m Thailandof a phenoinenon
sitecoast
oftheGulfofThailand,three called"yellow-head"shrimpLimsu-
animalsshowedsimilarblisters,one wan, 1991!.In affectedponds, the
hadanecrotic
antennal
gland
andthree shrimp
beginbygrowing
veryfastand
had MBV. Fromour earlierwork eatingmorethannormal.Theythen
Fegan etal.,1991!,
we didnotbelieve abrupt1y stopeating,
andwithinoneor
thatMBVcouldbethecause of mortal- two days, a few animals are found
ity. However,the blistersandantennal moribund or deadneartheedgeofthe
glandnecrosis werenew,andwe be- pond.Bythefol1owing day,thenuin-
lievethatthesefeatures shouldbefur- berof deadshrimpincreases to 100or
ther investigated to deterininethe more,andwithinthesucceeding day
causes.
all of theshrimpdie.Theaffectedani-
malspresenta swollencephalothorax
in theregionof the hepatopancreas,
 Shli Diseases in Thaiiand
and this has a yellowish appearance
when viewed through the carapace.
Upon removal of the carapace,the
hepatopancreasis abnormallylight-yel-
low in color.
Histological examination HkE stain-
ing! of affectedshrimp with the light
microscoperevealsmassivechangesin
the lymphoid organ LO! with inclu-
sions siinilar to those described above
Figs.23and 24!. However,all regions
of the LO are affected, even tubules still
with open lumens. In addition, other
tissuesare affected,including intersti-
tial tissues of the hepatopancreas,the
antennalglandandhematopoietic
tissue.
This may be an infectious outbreak of
the virus revealedin the chronically
infected broodstock described in the
previous section on the lymphoid or-
gan virus. If this syndrome is indeed
caused by a virus, it will be the first
dangerousviral diseasereportedfor P.
monodorr
in Thailand,and urgentaction
is required to understand the
epidemiology and limit its spread.
Conclusions
Therearemoreanswersthan questions
when it comesto diseaseproblemsof
P. monodon.
Obviously,manymicrobial
diseasesare still inadequatelycharac-
terized and many others remain to be
discovered. Much basic work remains
to be done on mechanismsof patho-
genicity, sourcesof infection, life cy-
cles, etc. At the same time, we lack a
set of simple,nondestructive,quantita-
tive measuresof shrimp health that
107
couldbe usedeasilyona farmto detect
problems early. Perhapsstudies on
crustaceanand shrimpimmunity e.g.,
typesof hemocytes,enzymeactivities,
hemolymphfactors!will give us some
oftheneeded
tools.Workisunderway
on rapid diagnostictechniquesfor vari-
ousdiseasesto help dose the windows
of infection.
Although they will neverreplacegood
managementpractice as the best pre-
ventionfor disease,we shouldurgently
pursue potential benefits from the use
of irnmunostimulants,probiotics and
"vaccines" as desired preventivesto
replace antibiotics.
Environmentaldiseases
causedby pol-
lutants such as insecticides should be a
top priority. Rapid and simple detec-
tion methods are available for some of
thesecompoundse.g., EnzyTechde-
tection tickets! but the levels of sensi-
tivity are too low to be of use for
shrimp.
Since most of the diseases discussed
herein are either induced or exacer-
bated by pond managementactivities,
more attention needs to be focused on
the total dynamicsof pond biota inter-
actions and how they influence the
heAth of theshrimp.Computermodels
may derive some first principles. In-
cludedin thetotal management picture
is the urgent need to determine the
capacityof theenvironmentto cyclethe
waste from shrimp ponds. Once this
capacityis exceeded,the desiredgoal
of long-term production wiQ be un-
achieveableWe have the tools at hand,
Figure
Z3.
Light
ni:ro.~oe
photographs
ofHftE'-stained
sections
oflymphoid
cvyan
tissue
from
a
''yeNow-head"
shrimp.
a!Low
magnification
shoeing
v auolated
cells
and hypertrophic
nuclei
accom-
panied
bydensely
b!Higher
staining,
basopNlic
magnification
cytoplasmic
bar= 5pm!.
inclusions
marked
bycurved
arrows
bar
= 10
pm!.
 Shri Diseases in Thailand

Figure24.Light
microscope
phofographs
ofH&Esfainedsections
fromtheshnrnpin
Figure23.Tissues
otherthanthose
ofthelymphoid
organ
contain
cellswithdensely
sfaining
cytopiasrnicinclusions
DCl!.
a!Outer
rimofthehepatopancreas
showing
norma/
tubule
epithelial
cellsbutshowing
DCi curved
arrows!
in theinterstitial
tissuebar= 10pm!. b! Highmagnification
ofinterstitial
tissue
fromthe
hepatopancreas
showingDCl curved
arrows!bar= 5pm!. c!Section
ofthemidgut showingrema/
columnar
epithelial
cellstotheextreme
leftbutshowing
DClcurved
arrows!i
n the
underlying
connective
tissuebar= 5 pm!. d! Cardiac
tissue
section
showingDCI curved
arrows!bar= 5 pm!. e!
Lyrnphopoietic
tissue
showingDCl curved
arrows!bar= 5pm!.
 Shri
baculovirus in Penaeus tnonrxfo» in southern
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1992.High
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evi-
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proce-
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shrimp.Bull. Environ.Contam,Toxicol.25;
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 Diseases of Penaeus monoclon in Taiwan:
A Review from 1977 to 1991

I-Chiu Liao
Taiwan Fisheries Research institute
199 Hou-IhRoad,Keels@,Taiwan

Mao-SenSuandCher~FangChanig
Tunt:IkangMarineLabcratory
Taiwan Fisheries Research Institute
Tmgkarg, Pingt~, Taiwan

Penueus
monodori,
the grassprawn, is an economically
importantprawnspeciesin Taiwan.
Productionincreasedsteadilyfrom 1968,when artificialpropagation techniqueswere
established,
through1987.ln thelate1980s,Taiwan
becametheworldleaderin cultured
prawn
production,
with a peakproduction,for P.monodoir
alone,of 95,000
MTin 1987.A yearlater,
however,an unexpected
massmortalityoccurred,displacingnot only manyprawnfarmsbut
alsoseveral sub-businesses
in theprawncultureindustry.Thecollapseof theindustrywas
attributedto bothpathogenic
andnonpathogenicfactors.Thisreviewdescribessomegrass
prawndiseases
and diseasesyndromesreportedin Taiwanfrom 1977to 1991,includingthe
pathogenic
factorsthatmostlikelyprecipitated
the1988
crisis,andthetreatments
beingused.

Introduction favorable characteristics. Penueus eumo-

Pe@acus monody, the grass prawn, is
one of the most valuable indigenous
aquatic species in Taiwan. Its culture
history in Taiwan spans only a little
more than two decades,beginningin
196Swhen this specieswas first artifi-
cially propagatedLiaoet al., 1969!and
the first hatchery was established.
Grassprawnculturesoonbecame very
popularbecauseof the species'many
donexhibitsthe highestgrowth rateof
aH cultured penaeids Liao and Chao,
1983!.It is eurythermaland euryhaline.
It is omnivorous rather than carnivo-
rous and, therefore, requires a lower
amount of protein in its feed Liao and
Liu, 1989!. This translates into lower
production costs. Grass prawns also
require simple culture facilities such as
claybottompondsfor growout;hence,
investment costs, especially the con-
 314

Tabfe
1.Prehdionandexport
ofPe~i
asfeedmanufacturers,
harvesters,
and
food processors.

A combination
of pathogenic
andnon-
pathogenic
factorswere attributedto
thecollapse
of theindustry.
Ofthese,
thepathogenic
factors
wereprominent.
No majorproblemswereencountered
underthe traditionalpolyculture
and
extensiveculturesystems,with the ex-
ception of natural disastersand the
presence
ofpredators
andcompetitors.
As theculturesystems
shiftedto semi-
intensive
and
intensive
styles,
stockin~
densities
wereraisedto 40- 60ind./m
andformulatedfeedswereused.Water
qualityandthegeneral
cultureenviron-
mentbecame
harderto manage,
and
the culturespeciesbecamemoresus-
ceptible
todiseases.
Eventually,
a vari-
ety of diseases,
suchas foulingby
protozoanepicommensals
and ectozoic
algae,
black
gilldisease,
gilldecay,
tel-
sondamage,
bodycramp,andreddis-
colorationwerereportedto afflictthe
prawn Liao et al., 1977; Chen and
struction budget and maintenance
costs,
arerelatively
low Liao,1989!. Hwang,
1979;
Liao,198S;
Liaoet al.,
1985!.
These andotherfactorscontributed
to
culture As
therapidgrowthof P.irionodon longasculture
conditions
inal, P. monodort
areopti-
appearsto tolerate
in Taiwan,especially
in thelate1980s light tomoderate
infections
Lightner
whenTaiwanbecame
theworldleader et al.,1987!.
However,poormanage-
in culturedprawnproduction.
Peak ment of thecultureenvironment
by
productionfigures
wereregistered
in farmershas,ininanycases,
most
likely
1987whenproduction
volumefor P.
causedoutbreaks
predisposed
bystres-
monodon
aloneclimbedto 95,000Mf
Liao,
1989!
Table
1!.In1988,
however, sors,
suchaspoorwaterquality,
dete-
rioratingenvironmentalconditionsand
mass mortality struck Taiwanese P. poor nutrition.
iiionodon
farms.
Production
dropped
by
70%,resultingin substantial
losses
to From1977
to1984,
thestudyof prawn
many farms and other businessessuch diseaseswas a minor concernin Tai-
 Shri Oiseases in Taiwan

wan. Since 1985, however, diseases Prawns infected by epicornmensalsare

have become a major issue, especially
after the 1988 crisis. Scientists
prawn farmers have been doing their
bestto restorethe industry by looking
into the factors that caused the crisis.
This review deals with the diseases and
disease syndromes of P. monodorrre-
ported in Taiwan from 1977 to 1991,
with emphasis on developments after
the 1988 crisis, and the various meas-
ures that are being taken or proposed
to recover from these diseases,
Diseases and Disease
Syndromes
Epicommensal Infestations
Pathogensand Symptoms
Kpicommensal
organismsare appar-
entlyubiquitousin prawn culturefacili-
ties Lightner,1985!.All life stages
may
be affected, but the most seriouslosses
are encounteredin juvenile and adult
stages,when the gi's of the host be-
comeheavilyfouledby epicommensal 80'%%d
.Whe n thi sspeciesattaches toP.
organismssuchasfilamentousbacteria,
pexitrichprotozoansand pinnate dia-
toms,resultingin variousformsof gill
diseaseLightner,1985!.Amongthe
moreseriousepicommensal
diseases
of
culturedprawns,thosecausedby pro-
tozoanscausethe mostharmto grass ner, 1985;Cheng and Liu, 1986;Chang
prawnsLiao et al., 1977,1985;Cheng and Su, 1990!. Table 2 lists the corn-
and Liu, 1986!. If there is too much
organic matter in the pond, large num-
bers of protozoareadily attachto the
body surfaces,appendages,
or gills
Hgs. 1-3!.
characterized by roughbody surfaces,
and gill diseases
or both Protozoanepicom-
rnensalshavenot beenobservedgrow-
ing internally in the gills or in other
tissues Fig. 4! but, when abundant on
the body surfaces,appendages
or gills,
can cause difficulties in locomotion,
feeding, molting, and respiration,re-
sulting in mortalities Couch, 1978;
Johnson, 1978; Lightner, l983, 1985;
Chen et al., 1989b!
The mostcommonlyreportedprotozo-
ans include the peritrich ciliates
Zoothamnium
spp. and Epistytisspp.
and the suctorian Acirieta spp.
Shigueno 975!, Johnson 978!, Liao
et al. 977, 1985!,Chen 978!, Tareen
982!, Liaoand Chao983!, Lightner
983, 1985!,and Liao 984! reported
that Epistylisspp. infect prawn culture
ponds, in general.Tung et al. 991!
studied P. rnonodon diseases in south-
ern Taiwan and found that the rate of
epicommensalinfection was 6.9%, with
the infectionrateof Zoofhamrriurrr
sp. at
monodon,the prawn turns blackish-
brown and its body surfacesbecome
fouled, movementis impaired,activity
is reducedand,in seriouscases,heavy
mortalities result Johnsonet al, 1973;
Johnson,1978;Liao et al., 1985;Light-
monly reported epicommensalproto-
zoans in Taiwan and proposed
prevention or control methods.
116 Liao et al.

<z4
gg~
.So
pe~

C
I.Q
q!
g g

g gQ!
gg
W.,g

C4

mg~
Fgf
NNg
km

Ng

-c~ ~i

E'y. -~
,8.u.=~
@8PU
g0

~ ~>
g6
 Shri Diseases in Taiwan $17

Table 2. Epicommensal protozoan diseases of Penaevs monodon in Taiwan and

Treatment
Prawns are first screenedfor infections,
and the culture environment is im-
proved before chemical treatment is
used. If the infection is not serious,the
water is replaced,stimulatingmolting.
After the prawns have molted, the
water is changed again once or twice.
If the infectionis serious,10- 15 ppm
teaseedcakecontaining 10%saponin is
applied to the whole pond to stimulate
molting. After the prawns have molted,
the water is replacedtwo to three times
to eliminate the protozoans Liao et al.,
1977!.
 118
Liao et al,

Formalin can also be used. Adult Treatment

prawnsaretreatedwith a 25- 30ppm
bathforonedaygohnson Strictwaterqualitymanagement
etal.,1973; served, is ob-
Chang andSu,1990!;
larvae
aregiven exchangeincludingmorefrequent
water
a 15-ppmbathforoneday;andjuve- 30- 40 cm.to maintain
transparencyat
nilesreceive
15- 20ppmfor 10- 12h
Liaoet al., 1985!.Prawnsarenot fed NematodeParasiticDisease
during Forinalintreatmentand the
wateris drainedafter 24 h to remove
any tracesof the chemical.After treat- Pathogens
andSymptoms
ingwithFormalinforoneortwodays, Thy'.mrsp.istheetiolagical agentof
benzalkonium chloride IKC!is ap- nematodeparasitic disease.Tissuesin-
of0.5- 1.0ppmforone fected
pliedata rate bythisparasite includethegills.
dayto preventsecondary If Diseased
infection. prawns
havedarkbodysur-
andhas faces
thepondbottomdeteriorates andmelanized
hemocytic
giUle-
a highorganic
content,i.e.,highnutri- sions. The nematodeattachesto the
entloadandheavysiltation,100 120 gillsandbodysurfaces,impairingmolt-
kg/1,000 hydrated ing andfeeding.However,thedisease
m zeolitesilicate
alkalialuminum!
isappliedtoiinprove is not serious;only a few mortalities
thepondconditionand to inhibit the havebeenrecorded.Increases
in the
growth
of protozoans
Chang,
1989!. population
of nematodes
mayoccur
because
of deterioration
of the condi-
Yellow Gill Disease tion of thepand,i.e., highorganic
matter content.

Pathogens
andSymptoms Twonematode
parasites,
Thymascaris
Diseased
prawnsappearnormalexter- sp. Fig,7!andSpirocamallanus
pereirai,
nallyexcept
fora slightly
darker ap- canbe transmitted
to prawnseither
pearance
andthepresenceof light to directly
orindirectly
bycopepod eggs
dark
yeUow
inflamed
lesions or freshdiets frominfectedfishwhich
inthegills may serve as an intermediate host for
Fig,5!.If theinfection
is notserious,
prawnactivityandfeedingis narmal; the parasites! Overstreet,1973;
otherwise, is re- Johnson,
activityandfeeding 1978;
Liaoetal.,1985;
and Su, 1990!.
Chang
duced.Microscopic
observation
of the
gill filaments
of diseasedprawnshas
Treatment
revealed thepresenceof diatomsFig.
6!.Prawns
maybecome by One-half
soinfested to two-thirds
of the pond
diatornsfor anextended periodthat wateris replacedand150kg/1,000m
moltingmaybeseriously impaired or zeoliteisapplied
toimprove thepond
beincomplete,resulting
in mortalities bottom.
A one-day25-to 30-ppmFor-
dueto hypoxia. malinbathor a 12-h0.5-to 1-ppm
copper sulfate bath can also be used
Shri Diseases in Taiwan
 120
Liao et al.

Liao
etal.,1985!.
Afterharvesting,the one-day to stimulate
molting.If the
bottomsiltis purgedand50-ppm so- infestation
isserious,
coppersulfateat
diumhypochloriteis appliedfor two
weekstokillthenematodeeggs. 0.3 0.5
ppm isused,
Also, tokillthealgae. one-daydipping,
certainfishesareexcluded
from the When thecopper sul-
pondsastheymayserveashostsfor fatebeginsto takeeffect,wateris ex-
the parasites. changed
morefrequently.

EctozoicAlgal Growth BodyCramp

Fathogens
andSymptoms Pathogens
andSymptoms
Ectozoic
algalgrowthoccurs
whenfew Bodycrampusuallyoccurs
in thesum-
phytoplankton
arepresent mer,whenbothairandwatertempera-
in the cul-
turepond.Pondtransparency turesare high Liaoet al., 1977!,
isclear. Prawns
Thealgaee.g.,
Enteromorpha exhibita dorsal
sp.!grow abdomen flexureof the
andattach
totheshells
ofprawns thatcannot
Fig. i.e.,thewhole bestraightened,
8!.Serious
infestations
inayresultin bodyor certain
parts
theformation
of grass-like
material
on appear
cramped
andcurved,resulting
thebodysurfaces
of theprawns.In- inmortalities.
Thiscondition
mayoccur
fected
prawns or immo- when
arelethargic thetemperatures
andlowerlayers
of theupper
ofthepondwaterand
bile,oftenobserved
lyingbythesideof
those
oftheairandpondwater vary
thepondLiao
etal.,1977;
Liao,
1984!. significantly,
particularly
duringsuin-
Feeding
isdecreased
orevenabsent.
Darkpondsediments to the merharvests
adhere orwhen
prawns
suddenly
shells
becausemolting af- jump
isimpaired, out of the water.In both in-
stances,
thereis a rapidcontraction
of
fecting
prawnmovement. Large
popu- the prawn muscle. Affectedprawns
lations
ofalgae
alsodecrease
dissolved
oxygen
levelsatnight,whichharmsthe usually
donotrecover
Liaoetal.,1977;
prawns Liaoet al., 1977,1985;Bati- ChengandLiu,1986;
ChenandLee,
cados,
1988;
Chang,
1989!. 1989!Fig.9!. Thisconditionis some-
timesobserved
during h otnights,
Treatment
whena stronglight is aimedat the
Attention
isgiven
towater man- pond,
quahty causing
thus,
cramp.
theprawns
Besides
tojumpand,
temperature,
poor
agement
by maintaining
pondwater nutrition,which affectsneuraltrans-
transparency
at 30- 40cm.If pond rnission,
mayalsocontribute
tobody
wateris clean,it is fertilizedwith am- cramp Chang,1989!.
moniumsulf'ate:calcium
superphos-
phate:urea
= 6:1:0.5,
10kg/1,000
m . Treatment
Whenprawnsareinfectedwith ecto- Itisimportant
toprovidea nutritionally
zoicalgae,10-to 20-ppmteaseed
cake balancedfresh
dietat frequent
inter-
containing
10%saponinis appliedfor vals.Harvesting
andhandling are
Shrim Diseases in Taiwan
 Liao et al.

avoidedduringhotweather.
Also,dur- disease
in penaeidprawnsevenwhen
ing hot periods, mechanicalaerationis presentin such large numbers as to
increased to maintain uniform water occludethemidgutor hindgutlumen.
temperature.
Johnson97S! alsoreportedthat ab-
sorptionof food by the protozoais
Gregarine Disease
perhaps detrimental, but does minor
damage
to thehostprawn.
PathogensandSymptoms
Gregarinesarecommoninhabitantsof Treatment
the guts of wild and pond-reared Gregarinesneed a moHuschost to com-
prawns Oohnson, 197S;Overstreet, pletetheirlifecycle;hence,onewayto
1973;Couch,1983;Lightner,1985!.
In control the disease is to exclude this
an investigation
conductedby the mollusc Oohnson,1978; Baticados,
authorson culturedprawndiseases
in 1988!.
southernTaiwan in 1989,80% of P.
monodon
wereinfected
withgregarines Black Gilt Oisease
Fig. 10!.
Pathogens
andSymptoms
Thegregarine Cephafotobus
sp. is di- Penaeus
monodon
sufferingfrom black
videdintolargeandsmalltypes;the gill disease
havebrownor darkgills
largetypeis furtherdividedintocylin-Fig.13!.Seriously infectedprawnsex-
dricalandcalabash
shapes.
Mostof the
hibit softshells,slowlocomotion,de-
largetypeshavetwoorthreesegments, creased appetiteand heavysurface
eachof which has a nucleus,The last
fouling,resultingin mortalities.Table
segment has a sucker that attaches to
sievesFig.11!oftheposte- 3disease.
thegastric listssomeof thefactors
In Taiwan,
causing this
blackgill disease
is
rior chamber of P. mortodon's
stomach. causedby:
Theminimumbodylengthof infected
prawns was 1.5 1.7 cm, while the
~ Poor pond bottom conditions due
maximum
was9.8- 10.2cm.Seriously to the culturesystempracticed,
infected prawns were 2.5- to 5.~
long;P. monod0tt
over10.5-crn
long i.e., high prawndensityandtoo
muchexcrementin the pond, re-
appearednot to be affectedby this sidualfoodor an overlylongcul-
diseaseChangandSu, 1991!. Statisti-
ture period,whichcauseorganic
cal analysis, investigationof culture
matter to accumulate on the bot-
pondsand histologicalobservationsof
tom Chenand Hwang, 1979;Liao
infectedtissueshowedthatgregarine et al., '1985!;
infectionsdid not affectthegrowthof
P. monodonFig. 12! Changand Su, ~ Extendedexposureto toxic pollut-
1991!.
Lightner
985!reported
thatgre- ants such as cadmium, copper,
garinesappearnot to causesignificant potassium permanganate, zinc,
Shrim Diseases in Taiwan 123
124
Liao et al.

ozone, ammonia and nitrite

Lightner,1985!Baticados,
1988; ing Sindermann,
1977;
Lightner,
ChenandLee,1989!; 1983,1985;Baticados,
1988;Chen
and Lee, 1989!.The samecondi-
tion alsooccurswhen numerous
~ Infestation
of blue-green
algae, epizoaattaches
to the gill Bati-
e.g., Oscillatoriu
sp. Fig. 14!, cados,
1988;
Chang
andSu,1990!.
Spirulina
sp.andSchizothrix
sp.
Lightner,1985!,which hinders Treatment
waterflowthroughthegill,stirnu-
lates
thegillepithelial topro- If thedisease
cells iscaused
bypoorbottom
liferate,
andcauses
soilparticles
to conditions
and algalattachment,
the
in the gills Chang, pondwateris changedand zeoliteat
accumulate
1989!, and 100kg/1,000rn applied.If the disease
is causedby epizoaand filamentous
~ Filamentousbacteria Leumthrix bacteria,
Formalin
at25to30ppm,one-
mucor!
andepizootic
infectionFig. daydipping,isapplied.
AfterFormalin
15!,whichgenerallyoccurin sea treatment,water is replacedonceor
water,in conjunction withheavy twice. Malachite
greenat0,5 0.8ppm
siltation, resulting in mortalities or methylene
blueat 8 10ppm,one-
dueto hypoxiaor impairedmolt- daydipping,
arealsousedLiaoetal.,
1985!.
Malachite
greenandmethylene
 Shrim
blue may remain in prawn tissuefor up
to one month; therefore, prawns
treated with these chemicals are not
harvested until about one month after
treatinent has been halted because the
prawns can be carcinogenic.
Red Gill Disease
Pathogensand Symptoms
In the early stagesof the infection, the
gills appear light red. External body
color, feeding and activity are normal.
As the infection worsens, the gill be-
comes dark red, Fig. 16! and the
prawns stop feeding, choosing instead
to lie on the pond side where they can
be easily caught by hand. The prawns
gradually weaken further and eventu-
ally die. Mortalities occur about two to
three weeks after the onset of disease.
Red gill diseaseis caused by poor cul-
ture conditions, i.e., poor water quality
and low oxygen levels. Microscopic ob-
servation shows that the dark-red gill
filaments are filled with red-radiation
line like capillaries Fig. 17!. Vibriopara-
haernolyticusand V. ariguilfarumwere
sometimes isolated from the gill tissue
and hemolymph of diseasedindividu-
als Liao et al., 1985!,but V. damsela and
V. harveyi have also been reported to
cause this disease Huang, 1989!.
Broodstock are susceptible to red gill
disease.
Treatment
To treat this disease, the culture envi-
ronrnent is improved, prawns are
moved to other ponds, or the water is
Diseases in Taiwan f25
changed frequently. At regular inter-
vals of every 3 to 4 weeks, 50% BKC,
50% hyamine benzethonium chloride!
at 1 - 2 pprn Liao et al., 1985!or 20%
furazolidone at 10 20 ppm, one-day
dipping, is used to prevent the spread
of the bacteria. If the disease is caused
by poor bottom conditions, zeolite at
100kg/1,000m is also applied.
Red Discoloration
Fathogensand Symptoms
In the early stagesof the infection, the
prawn body changesfrom dark green
to yellow green. Prawn behavior and
activity are normal. As the infection
gradually becomesserious,the body
turns light red, red, and finally, dark
red Fig, 18! When the infection is
serious, this diseasesyndrome may be
accompaniedby red giH disease, Dis-
easedprawns have respiratory difficul-
ties and the amount of fluid in the
cephalothorax increasesand becomes
smelly; also, the hepatopancreasbe-
comes pale Liao et al., 1977; Sinder-
mann, 1977;Cheng and Liu, 1986!,The
value of affectedprawns may be low if
marketed at this time, and when trans-
ported, the prawns often die. If red
discolorationand red gill diseaseoccur
at the sametime, heavy mortalities can
result.
According to Liao et al. 977!, red
discoloration is caused by rancid and
spoiled diets or pond detritus that is
rich in organic matter, It is not a bacte-
rial infection. Cheng and Liu 986!
reported that this diseaseoccurs when
l26 Liao et al.
 Shri Diseases in Taiwan
the water quality is poor, when there is
a high organic matter content and
when the reduction layer of the pond
bottom is thick. Significant his-
topathological changes do not accorn-
pany the disease. Lightner and
Redman 985! suggested that when
the hepatopancreasatrophied and ne-
crosis occurred, stored 8-carotene and
other carotenoids were released into
the hemolyrnph, spreading into the
whole body.
Treatment
A fresh diet with a high protein content
is given at increased rates Liao et al.,
1977!. The culture environment is im-
proved and water exchange is more
frequent Cheng and Liu, 1986!, and
50% BKC or 50% hyamine at 1 - 2 ppm
are used regularly.
Shell Oisease
Pathogensand Symptoms
The exoskeletons of diseased individu-
als have black spots Fig. 19! at random
locations. In general, the cephalo-
thorax, abdominal segments and
uropod are easily infected. The black
spots gradually cover the' entire exo-
skeleton, with melanin accumulating
around the spots. Diseasedindividuals
lose their smoothness; this decreases
their market value. Liao et al. 985!
reported that stressors such as exces-
sive handling during transfers, crowd-
ing and injuries sustained from other
prawns after molting result in external
lesions that are secondarily infected
with chitinolytic bacteria.
127
Cheng and Liu 986! reported that
shell diseaseoccursfrom April to Octo-
ber. In addition to environmental
stress, Vibrio spp. can also cause this
disease. According to Sindermann
977!, Lightner 983, 1985!and Chen
et al. 989b!, many kinds of production
of lipoprotein, chitinolytic bacteriaand
Vibrio sp., Aeromonas sp., Spirillium sp.
and Flaeobacterium sp. are associated
with shell disease.
Treatment
At the early stages of infection,
exoskeletalbreaks are not yet evident.
The water is changed two or three
times, or teaseedcake containing 10%
saponin at 20 ppm, one-day dipping,
to stimulate molting! are used to treat
this disease.I'ormalin at 20 ppm and
malachitegreenat 0.3 ppm, at the same
time, dipping for one day Liao et al.,
1985!,are also applied, When infection
is serious, BKC at 0.5 - 1 ppm is ap-
plied, one-day dipping, and reapplied
two or three times once every five to
seven days. If a bacterial infection is
diagnosed, oxytetracycline or tetracy-
cline at 40 - 60 ppm or furacin, nitro-
furans, and furanace at 1 ppm with
dipping is applied Chen et al., 1989b!.
Tait Rot
Pathogensand Symptoms
Tail rot has the same manifestations as
shell disease,i.e., it is causedby envi-
ronmental stress,like high density, ex-
cessive use of drugs or poor water
quality; injuries due to collisions with
other prawns; or tail injuries due to
 128
Liao et al,

incomplete
molting.If secondary
infec- glandularepithelialcells Chenet al.,
tionwithchitinolytic
andothertypesof 1989c,d! Fig. 22!.
bacteriaoccurs,necrosis
of the tail de-
velops,resultingin tail rot Liaoet al., Thistypeofvirusspreads
veryquickly,
1985!.The earlystageof infectionis resulting in high larval mortalities.
characterized
by swelling
of the tail, Damage to adults is less severe. In
especiaQy
nearthe margins; feeding seriousinfections,the virus ruptures
andmovementarestill normal.In seri- hepatopancreatic
cells,allowingocclu-
ous infections, tail extremitieshave ne-
sionbodies
topass
through themidgut
crosiswith some portions tom into andtobeexcreted
in thefecesFigs.23
shredsFig.20!.If theshredding
is and 24!.At this stage,the prawnis
two-thirds
ofthetail,prawn
mobility
is weak,exhibitingreduced feedingand
impaired and mortalities result. activity.
Thebodysurfacesandgillsare
easilyfouledwith diatorns,epicom-
Treatments
mensal protozoa and filamentous bac-
Followingdiagnosis,the cultureenvi- teria,makingthe prawnsusceptible
to
ronment is ameliorated first. Water more serious harm Anderson and
changes aremadeor thepondbottom Shariff,1987;Chengand Liu, 1986;
is improvedto reducestress.BKCat 1 Chang,1989;
ChenandChang,1989;
ppmorfurazolidone at 10- 20ppm, ChenandLee,1989;Chenet al., 1989b;
one-day
dipping,is appliedLiaoet al., LightnerandRedman,1981!.
1985!,
If epicommensals
aredetected,
Formalinat 20 ppm and malachite Chenet al. 989a,d!reportedthat in
greenat 0.3 ppmareappliedconcur- Taiwan,MBVinfectedP. monodon, P.
rently,one-daydipping. penictllatus
and Metapenaeus
ensis;P.
monody contractedthe most serious
infections infectionrate: 1984-1986,
PenaeusManodonBaculovirus 18%;1987-1988,
80%!,According
to
MBV! Oisease Liaoet al.990!, theMBVinfectionrate
in femalebroodstocktaken from the
Pathogens
andSymptoms coastal
waters
of Taiwanwasonly33%
ectedprawnshaveno significant in 1987.Theinfectionratejumpedto
externalsignsof disease;
theymay, 100%in October and Decemberof 1988
however, exhibitdecreased
feeding andOctober1989.Theaverageoverthe
rates,slowgrowth,doubletwo-lay- entireyearwas85%.MBVprevalence
ered!shell and havean atrophied among imported female broodstock
hepatopancreas
Fig.21!.Hispathologi-
was only 40% Fig. 25!. Resultsof a
calobservations
indicate
thatthehepa- number of MBV studies are shown in
topancreatic
tissueis notsignificantlyTable 4.
dainaged,
butthereareeosinophilic
intranuclearocclusionbodiesin the
Shrim Diseases in Taiwan 129
 Liao et aI.

Treatments
ping,is applied.The treatmentis re-
There isnotreatment
forMBVdisease,peatedfiveto sevendayslater,twoto
butit doesnotaffect
healthy
prawns. threetimes.Oxytetracycline
andother
Thedisease
agentisa virus
thatislatent antibiotics
can alsobe used,50 - 100
eventhroughtheadultstages. If the g/MTprawnweight,mixedin thefeed
prawnis unhealthy,or if a stress
such for1 week.
Thedrugs
areadded
tothe
ascrowding
is present,
disease
may feed,e.g.,oysters
orchicken
eggs,
us-
occur.
Therefore,
goodmanagement
is inga blender
foruniform
mixing,
then
necessary.
A densityof 30 ind./m is themixture
isexposed
toairtodry.
maintained,
feeding
is controlled,
a According
toLiaoetal.990!,when
fresh
dietisprovided
andtheprotein culturing
larvae,
theeggsor nauplii
andvitamin
C content
inartificial
feed should
bewashed
withcleanwaterto
areincreased.
If MBV emm with bac- reduce
theinfection
rateofMBVTable
terial
infection,
BKCat 1 ppm
or20% 5!.

Figure
the 25. Monthly
caasta/
waterschanges
of in
the
Taiwan
or MBV
infection
rate
iepavtert
from inspawners
Southeast
AuanofPenaeus
monodon
countries.
The caught
numbers
inthefrom
figure
i/xticate
sanpiesizes.
 Shrim Diseases in Taiwan t31

Table 4. MBV infection in Penaeus monoaon cultured in Taiwan.

Table5. MBVinfectionrate %! in Penaeusmonodonlarvaerearedfromfertilizedeggsor

gB Treatments

A B C

00 0
5.0
00000 0
0.5 11.0 0,5 0.5
1.5 31.0 0.5 1.0 1,5
3.0 49.O 2.0 1.5 2.0
Fertilizedeggsto Ptk in indoortank;PIA - PL38 in outdoortanks.
2
At FromM BV-freespawner,B: FromspawnerinfectedwithMB V; fertilizedeggsandnaupliiwerenot washed.
C: Fromspawner
infected
withMBV; fertilized
eggsandnaupliiwerewashed.
D: Fromspawner
infected
with
MBV; fertilizedeggswerewashed.E: Fromspawnerinfectedwith MBV; naupliiwerewashed.
 Liao et al.

Table
6.Bacterial
diseases
ofPenaeus
rr

BacterialDiseases
hepatopancreas,
hemolymph, gastric
Many kindsot'bacteria
maycause cavity,
heart,
dis- topancreas muscleand gills;
hepa-
easein P.numodorr.,
especially andhemolymph
in post- are moreserious, infections
larvaeandjuveniles
Oohnson,1978; lomatosis resultingin granu-
Lightner,2983!.Onlya fewkindsof of thewhole bodyCheng
bacteria,
however, cause
primary andLiu,1986;
infec- Lee, Chang.1989;Chenand
tion.Mostincidences
resultfromenvi- 1989;Chen etal.,
1989b;
Huang,
ronmental factorsor stress; then 1989;Liu,1990!.Themostcommonbac-
secondary
bacterial terial diseases
infectioncancause Table in Taiwanarelistedin
6,
heavymortalities.
Bacterial
diseases
of
Perseus
monodon
areeither
body
surface
or internalinfections.Tail rot andsheH Pathogens
andSymptoms
disease
are external
infections.
The Diseasedprawns are coveredwith
main internal infections occur in the mud-like
material
andarelethargic
and
moribund,
lyingonthepondsideand
 Shrirn
occasionally surfacing quickly to the
water surface. After a short period of
time, they die. In serious infections, aH
the prawns in a pond stop eating, re-
sulting in heavy mortalities. Externally,
the diseased prawn has no significant
symptoms, but hasa doubleshell in the
cephalothorax region, and the hepa-
topancreasatrophies Figs. 26 and 27!
or swells Figs. 28 and 29! and granu-
lomas or necrosis occur within hepa-
topancreaticcells. Diseasedprawns are
lethargic and have difficulty molting. If
teaseedcakecontaining 10%saponin or
frequent water changes are applied to
stimulate molting, they may cause in-
complete molting or molting that re-
sults in a soft shell, causing heavy
mortalities.
Treatment
Zeolite 00 kg/1,000 m ! is used to
improve the condition of the pond bot-
torn, but little pond water is replaced.
If water is to be changed, the change
should be gradual. BKC at 1 ppm or
20% furazolidone at 10 ppm, one-day
dipping, may be used; treatments are
repeated every five to seven days, two
or three times, Chen et al. 989b! rec-
omrnended the use of furacin
furanace, 1 ppm; or chloramphenicol,
1 - 10 ppm; oxytetracycline, 60 250
pprn, 1- or 2- days dipping, or addition
of 500 - 1,000 rng of oxytetracycline or
100 - 500 mglkg of the prawn diet
NF-180.Increasingthe amount of fresh
feed in the diet and adding high doses
of vitamin C may increase the resis-
tance of prawns to the disease.
Oiseases in Taiwan
Other Diseases
This section briefly describes some
common diseases, such as associated
isopods Liao et al., 1985!,muscle ne-
crosis Liao et al., 1985;Lightner, 1985;
Baticados, 1988; Chen and Lee, 1989!,
chronic soft-shellsyndrome Baticados,
1988;Chen and Lee, 1989!,blue disease
Chen and Lee, 1989!,siderosis Cheng
and Liu, 1986!and gas-bubbledisease
Chen and Lee, 1989!.The pathogens
and symptoms are as follows:
Associatedisopods.Isopods attach to
the prawn's gills, often on just one
side; the attachmentsite appearsswol-
len.
Muscle necrosis. Either the whole
body, or a partof it turns white; muscle
color is white and translucent. This
diseasemay be causedby chronic stres-
sors or a suddeninjury.
Chronicsoft-shellsyndrome.The dis-
eased prawn's shell is soft and thin;
this makes it weak. Death results from
impaired molting or cannibalism. This
disease may be due to a nutritional
deficiency,suchasinadequateamounts
or of calcium and phosphate.The disease
is controlled through proper formula-
tion of artificial feeds and an improved
culture environment.
Slue-disease.Sick individuals are pale
or light blue. We call affected prawns
"sky prawns." Blue diseaseis caused
by a nutritional deficiency or poor
water quality, The diseasemay be con-
trolled by proper nutrition and man-
ciao et al.

,"a s

'8 p

Syp
jgk
p8
4!
R $$
0 py
$p
ski
g5~
~'6ci.si

Lu !

:i~<

4 li.R rn
t36 Liao et aL
in Penaeus monodon.Unpublished research
report. Tungkang Marine Laboratory, Tai-
wan Fisheries ResearchInstitute, Tungkang,
Pingtung,Taiwan. In Chinesewith English
abstract!.
Chen, H.P. 1978. Diagnosis and control of
disease of Penaeus monodon cultured from
overwintered juveniles. China Fisheries
Monthly.309:3-11.In Chinesewith English
abstract!.
Chen, H.C. and B. Hwang. 1979,Investigation
on the diseasesof pond-rearedshrimps in
lian. China Fisheries Monthly. 317; M. In
Chinesewith English abstract!.
Chen, S.N. and P.S. Chang.1989.Applied and
diagnosticmethod of Penaeus monodonBacu-
lovirus infection in cultured prawns. Fish
World. 4: 42-52. In Chinese!,
Chen, S.N. and W.C. Lee. 1989. Diseases oF
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 Preelence and GeographicDistributionof MBV
and OtherDiseasesinCulturedGiantTiger
Prawns Penaeusmonodon!in the Philippines

Jose M. Natividad
BFAR-IDRCFishHeallh Project
Bureauof Fi!faces andA~ Resources
4th BoorEstuarBuiMing,880 QuezonAvenue
QuezonCity,Philippines

DonaldV.Lightner
Departmentof VeterinaryScieni~
Uti varsityof Artma
Tucson, AZ 8572l, U.SA

Penaeus
morrodon
baculovirusMBV!wasthe mostprevalent disease in hatchery-reared
and
pond-cultured
Prnaeusmonodoii
in the Philippines.The incidence of MBVincreased with
increasing
age.Furtherinore,
MBVwasdiagnosed in P,monodonin allsamplingareasprovinces!
every month throughoutthe samplingperiod,There was a low correlationbetweenthe
occurrence
of MBVandtime month!;therefore,
MBVepizooticsarebasically
hatchery
and/or
pondmanagementproblems
anddonotrelatetocertainplaces
or seasons.

A presumed typeC bacutovirus

andreddiseasewerealsoconsistentlydiagnosedinMBV-infected
P.moexbii.Thispresumed typeC baculovirus is thefirstcaserecordin thePhilippines.
The
followingdiseaseswerealsodiagnosed: foulingwith the protozoans Zaothtimnium
sp.and
Rrticetla
sp.,larvalmycosis,
gill necrosis
andhepatopancreatic vibriosis,
A Fewcases of blue
shrimp syndrome, filamentousbacterialdiseaseand bacterialententiswere also recorded,

Introduction fortunately,limitedinfortnationis avail-

ableon shrimpdiseases
in the Philip-
Maintaininga healthyshrimppopula- pines and Asia as a whole. This has
tion in a culturesystemrequiresa basic frequently
resulted
in misdiagnosis
and
understandingof the etiologyof the subsequent mistreatmentand rampant
diseases
that occurin the system.Un- abuseof antibiotics
andothertherapeu-
 Natividad ard L' htner
tants. The indiscriminate use of an-
tibacterial drugs has resulted in the
developmentof resistantstrainsof bac-
teria, making the problemsevenworse.
The classicalexampleof this problem is
the emergenceof someresistantstrains
of luminousbacteria Vibrioharuey'!in
some parts of the Phihppines where
chloramphemcol,
pem91in,ev~omy-
cin, kanamycin, oxytetracycline, and
sulfadrugsareusedregularly.
Because it is so common in hatcheries
andgrowoutpondsin thePhilippines,
Penaeus
rnonodonbaculovirus MBV! is
of considerableimportance.Histologi-
caliy, the diseaseis characterized
by
prominent, multiple, eosinophilic
HdzE stain! occlusion bodies within
hypertrophiednucleiof the midgut or
hepatopancreatic
tubuleepithelialcells.
MBV can cause 70% cumulative mortal-
ity amongjuvenile and adult popula-
tions; hence,infectionby this virus is
nonselectiveasfar asthe life stagesof
the hosts are concerned Lightner et
al., 1983a;Sinderrnannand Lightner,
198S!.
MBV was originallydiagnosedin 1977
by Lightner and Redman981! in a
populationof laboratory-reared,juve-
nile P. monodon.
Ironically,theseMBV-
infected P. monodon were obtained from
a quarantined population in Mexico,
but the postlarvaeoriginatedin Taiwan
Lightnerand Redman,1981;Lightner
et al., 1983a!,The first caseof MBV in
thePhilippines wasreportedin 1981by
Lightneret al. 983a! froma popula-
tion of postlarval PL5! P. rnonodon.
Someof the most important diseasesof
cultured and wild penaeidshriinp are
discussed in the works of Johnson
97S!, Overstreet 978! and Lightner
983, 1985!.Thorough reviews of im-
portant viral, bacterial,fungal, parasitic
and otherdiseasesof cultured penaeid
shrimparepresentedby Lightner975!,
Lightner et al. 984a, 1984b!, Sinder-
mann and Lightner 988! and Lightner
et al. 989a!. Overstreet 978! has
performedextensive
studieson thepara-
sitic and microbial diseases of wild
shrimp in the Gulf of Mexico, while
Fontaine 985! did similar studies in
the West Galveston Bay, Texas.
LightnerandRedman985a! and Light-
ner et al. 987a! have identified several
important viral and microbial shrimp
diseases in Southeast Asia. Similar stud-
ies were also conducted in Malaysia by
Anderson 988! and Nash et al. 988!.
Research Objectives
Knowing the host and geographic dis-
tributions of diseases of cultured
penaeid shrimp is important in terms
of theinterregional
movementof shrimp
stocks.The Philippinesis composedof
about 7,200 islands, and stocks are
moved betweenislands regularly. Map-
ping the geographic distribution of
penaeid shrimp diseasesin the Philip-
pines may help us predict and/or pre-
vent some shrimp diseases on a
regional scale.
This study was designed to document
the incidence and geographic distribu-
tion of MBV and other diseases of
 MBV in the Phil

hatchery-rearedand pond-culturedP. terial Thurmanet al., 'l990!.The rou-

monodon populationsin thePhilippines, tine examinationby histological Bell
and to generatebaselineinformation and Lihhtee, 1988!and wet mount
for the study of their epizootiology. methodsLightneret al., 1983a;Sinder-
datawill bea valu- mannand Lightner, 1988!were used
Thisepizootiological
abletool in formulatingrationalmeas- throughoutthisstudybecause of their
uresforthe preventionandcontrolof simpliaty and speed.
MBV and other shrimp diseases.
Literature Review
Specifically,
theobjectives
of thisstudy
were: Most diseases of cultured penaeid
shrimpin the Philippinesare associ-
~ To determine the seasonal occur- ated with high-density culture, e.g.,
rence of MBV in P. monodonin the 100,000-450,000
postlarvae/ha.Under
Philippines; these crowded conditions, disease has
becomeone of the major problemsof
~ To assessthe geographicdistribu- the industry.
tion of MBV and other P. monekn
diseasesin the Philippines;
Generally,there are threefactorsthat
~ To determine the relationship of affectpenaeidshrimphealth:physical
ageas a host factor in the preva- factorse,g.,me9mnical injuries!,chemi-
lence of MBV; and cal factors e.g., toxins!and biological
factors e.g., viruses,protozoans,bac-
~ To identify other diseasesof P. teria, and fungi!. Thesethreeelements
moriodonand analyzetheir associa- causetwo majorcategories of disease
tion with MBV-infected popula- noninfectious diseases, which can be
tions. causedby physical,nutritional and
chemicalagents,andinfectiousdiseases,
All data in this study came from diag- which are causedby biological agents.
nostic cases received at the BFAR-IDRC
FishHealthLaboratoryin Quezon City, Of the six penaeid shrimp viruses,
Metro Manila or from samplescollected three are known to infect P. monodonin
during routine field trips. the Philippines MBV, hepatopan-
creatic
parvo-like
virus HPV!andinfec-
There arethree methods currently used tious hypodermal and hematopoietic
to diagnoseMBV infection in P. mono- necrosisvirus IHE~!. MBV is wide-
doti: 1! direct examination, 2! enhance- spreadin P.rnonodon
hatcheries,
where
ment of infection and 3! bioassay.The it causesmoderate to high cumulative
newest technique for diagnosing MBV mortalities in postlarval populations
employs an epifluorescent light micro- and poor growth in pond-cultured
scopeto examine phloxine-stained ma- stocks.
 f42 NatMdad and L htner

Another important penaeidvirus is gen-negative

inclusionbodiesin hyper-
HPV.Itstargetorganisthehepatopan- trophiednucleiwith marginatedchro-
creas.
Thisviruswasfirstdescribed
by matin followingfixationin Davidson's
LightnerandRedman985b! from P. AFAorBouin's!. IFIHIMinfectspost-
chinensis
postlarvae,
P.meqpcietisis
juve- larvaeandjuveniles andmaycause 80
niles,P. semmlcatus
juvenilesand P. to 90%cumulativemortality.
mormfottfromthePeople's
Repubhc of
China,Singapore,Kuwaitandthe Phil- REO, the most recent addition to the
ippines.Microscopically,
pathogno- list of penaeid viruses, was discovered
monicsignsofHPVinfection
aresingle froma population ofjuvenileP.japmi-
basophilic
HIVEstain!,Feulgen-posi-cusin Franceby Tsing and Bonamiin
tiveinclusion
bodies
in hypertrophied 1984 Lightneret aL, 1989a!.Thisvirus
nucleiof epithelial
cellsof hepatopan- is bestdiagnosedby transmissionelec-
creatic tubules.
tron microscopybecause
this procedure
can distinctly demonstrate the eosino-
B6ibPPis probablythe most investi- philicto magenta
staining HkE! cyto-
gated shrimp virus. It was most re- plasmic inclusion bodies, which cannot
centlyreportedin the Philippines
in be seenwell by light microscopySin-
1989from a populatio~of P. rnonodon dermann
andLightner,1988!.A cyto-
fromAsturias,
Cebuby Lightnerun- plasmicreo-likevirus was also described
published diagnosticcase!. IHHN from a populationof P. monodoti
with
disease
was first recognized
as virus- rickettsia-associated
disease,although
causedin a population
ofblueshrimp, histologically,
thevirusdid notappear
P. stylimstris,
and white shrimp,P, to causeanytissuedamageAnderson,
veittameiLightneret al., 1984a!at the 1988!,
University
of Arizona's
shrimpculture
facilityon Oahu,Hawaii. Bacterial
diseases
ofpenaeid
shrimpare
alsoconsidered
to be major problems
Infectivityand pathogenicitystudies in bothhatchery-reared
and imported
BellandLightner,1984;1987!showed stocks.Bacterialinfectionsin penaeid
that the targetorgansfor IHHI'Pl are shrimpcanmanifestthemselvesaspits
tissues
of ectodermal
origin e.g.,epi- in the cuticle, sometimescalled shell
dermis,hypodermal epitheliumof the disease," or as localized infections
foregutandmidgut,nervecordandthe within the body, which may lead to
nerve cord ganglia! and tissues of septicemiaLightner,1983!.In thePhil-
mesodermal
origin e.g.,heinatopoietic ippines, the most serious bacterial dis-
organs,antennalgland tubule epithe- ease is "luminous bacterial disease"
lium, mandibularorgan,connective Pitogoetal., 1990!.Althoughtheetio-
tissueand striatedmuscles!.Micro- logic agents of this diseaseare known
scopically, IRWINvirus can be demon- VibrioharueyiandV.splendicusthe
stratedby the presenceof promiiient exactepizootiologyand pathologyof
eosinophilic H8zEstain!,usuallyFeul- this diseaseremain unknown. These
 MBV in the Phit'
bacteria have been isolated from
shrimp eggsand larvae,and from sea
water and pond sediments,wherethey
characteristically glow in the dark.
Studies on other penaeid Vibrio infec-
tions have been performedby Lewis
973!, LewisandLawrence9N!, Taka-
hashi et al. 985!, Egusa et al. 988!
and Itami et al. 989!. A septicemic
form of bacterial disease was described
by Lightner and Lewis 975! from
hatchery-reared
white shrimp, P. setif-
erus.Most penaeidbacterialinfections
involve motile, Gram-negativeand oxi-
dase-positive bacteria such as V.
at ginoly tie us, V. parahaernolytie us,
Pseudomonas sp., Aeromonas
sp. and Fla-
vobacteriurn
sp. Lewis, 1973;Lightner,
1975; Lightner, 1983; Lightner et al.,
1985!.
Bacterial epicommensals are also very
conunon in the estuarine environment
and have been known to cause prob-
lems in pond-reared penaeids McKee
and Lightner, 1982!. An exampleis
Leucothrixmucor, which normally at-
taches to the gills of the host shrimp,
impairing respiration and resulting in
death Lightner et al., 1975!. Other
bacterialepicommensalshave alsobeen
descried Oohnson,1978;Lightner, 1983,
1985;Sindermann and Lightner, 1988!.
The most important fungal pathogens
causing mortalities in penaeid shrimp
are Fusariurnsotaniand Lagenidiurnsp,
Lightner and Fontaine, 1975;Hose et
al., 1984; Bland et al., 1976!. In the
Philippines, Iagenidiumsp. was isolated
from a larval population of P. monodon
ines
Lio-Poet al., 1982!.Otherfungifound
in the PhilippinesincludeHalipthoros
phitippinensisLio-Poet al., 1985!and
Sirolpidium sp. Most fungaldiseases
affecteggs,larvaeandpostlarvae. The
ability of Lagenidiurn
sp. and Sirtotpidr'urn
sp. to exist as free-livingsaprophytes
and their wide host distribution sug-
gest that they may havea worldwide
distribution Lightner, 1985!.
Noninfectious diseases are also com-
mon problemsin pond-rearedshrimp
and in hatcheries. Examples include
gas-bubbledisease Lightner et al.,
1974;Supleeand Lightner,1976!,and
intoxication with pollutants such as
cadmium Nimmo et al., 1977!and afla-
toxin Wisemanet al., 1982!.Substances
toxic for shrimp are alsoproducedby
somemarineorganismssuch as blue-
greenalgae Lightner,1978;1982!.
Materials and Methods
Sample Sources
All P. rnonodon examined came from
two major sources. One group was
obtained from regular samplingsites
including preselectedhatcheriesand
growoutpondsfrom 11locationsin the
Philippines Fig. 1!. Thesecond.
source
wascomprisedof samplessubmittedto
the BFAR-IDRCFish Health Laboratory
in QuezonCity,MetroManila,by hatch-
ery and growout pond operators.
When shrimpin a particularsampling
area were experiencingmortalitiesor
any form of diseaseat the time of the
 NatMdadand L' htner

1 lb

topancreahc andintramuscular injec-

tionof thefixativein thecaseofjuve-
nilesandadults!.
Forlargershrimp,a slit
wasmadein the cuticle,extending
fromthesixthabdominal segment to
thebase
oftherostrum,
enhancing
the
entryof thefixativeintothespecimen.
Postlarvae
werefixedfor 24h, while
juvenilesand adultswerefixedfor 48
h.Afterfixation,
shrimp werestored
in
50%alcohol untiltheywereprocessed
forroutine
histology
Bell
andLightner,
1988;Humason,1967!and examined
usinglightmicroscopy.
The"gut-gill
panorama"
techniquedeveloped
by
BellandLightner
988!wasadopted
in thepreparation
forembeddingthe
tissuesamples.
Thetissues
werepre-
Figure
1.Map
afthePhilip@'nes the paredfor lightmicroscopy
shoiNrng usingthe
aevaas
of Penaeus
mceocke
sarrpfes.= routineparaffintechnique
Luna,1968;
Pangasinarr;
2 Zamba/ee;3 Bataan;
4 Humason,1967!and stained with
Bvhean;
5 MetroManNa; 6 ~Cade;7 = modifiedMayer's Hematoxylin
and
Batangas;
e -Ovezon;
9 =Imk;10=Cebu;
11
~ Cotsbeto;
12~Other
pmvincee Inc/ude Phloxine/Eosine
which stainHkK! Belland
Carnannea
/Vorte,
Parnpanga,
Mindoro!, Lightner,1988!.In caseswherethe
farmers
wanted
immediate
results,
the
sampling,
selective wasdone samples
sampling wereexamined
for MBVinfec-
to includeabnormalanimalssuchas tionsusing a "wetmount"technique
thoseexhibiting or other called'MBVal.,
discoloration Lightner
et. 1983a!,
which
RapidTest."
islocally
typesof morbidity.
Nodeadshrimp
werecollected
if thetimeof deathcould
notbeascertained. re- Method
Farmhistory ofNlBVDiagnosis
cordswereobtained
usingFarmHis-
torySheets Fig. 2.! Diagnosis
ofMBVinfection wasaccom-
plishedby histological
demonstration
ProcessingSamples of prominently
hypertrophied
nuclei,
with spherical
eosinophilic
occlusion
Allsamples
wereiminediately
fixedin bodies
inthehepatopancreatic
cellsand
fixativeBellandLightner, anterior
Davidson's midgutoftheshrimpLightner
andRedman,
1981;
Lightner
et al.,
1988!
eitherbydirectimmersion
in the 1%8a!.
Insome samples
wherethe"wet
case
of postlarvae!
or bothintrahepa- mount"technique
wasused,h4BVwas
 MBV in the Phil'
Figure2, Farmhistoryrecordsheetusedtocollectbackgroundinformation
Penaeus monodon samples were collected.
detectedby the presenceof intensely
green due to the 0.1% aqueousmala-
chitegreenstain!occlusionbodiesthat
were distinctfrom the secretorygran-
ules and lipid dropletspresentin the
tissuesquash.
Diagnosisof other diseaseswas also
accomplishedby routine histopathol-
ogy. Someprotozoanand fungalinfec-
tions were diagnosedusing the wet
mount method.
fromthehatchery''arm
where
Statistical Analysis
The statistical methods used in this
studywere mainlycorrelationanalyses
suchas the correlationof MBV preva-
lencewith variablessuchas age, geo-
graphic distributionand monthly
distribution,and correlationanalysis
between the occurrence of MBV and
other diseases.
All analyses
were done
usingNCSS Version4.1developed
by
Dr. JerryL. Hintze, Kaysville,UT!.
 NatrAdad
andL' htner

Table
1.incidence
ofPenaeus
monodon
diseases
anddisease-causing
organisms
diagnosed
between
October,
1989andDecember,
1990.

Estimation
ofMBVprevalence
wascom-
putedusingthe formulabelow: Overall
Prevalence
of MBV.Outof a
totalof372
cases
from12major
prawn-
farming
provinces
in thePhilippines,
P ~MB
V 100 249werediagnosed
positivefor MBV.
Overall,the prevalence
of MBV was
where: 66.9%.
However,
there
wasa very
low
Pr = Prevalence correlationr = 0.4!betweenthenum-
berofMBVcases
andprovinces
where
MBVp sam- thecases
- No.ofMBV-positive weredocumented
Table
2!.
ples, and
In termsof the total numberof P,
n = Totalnumber
of samples tnonodorr
samples
examined,
5,085were
MBV-positive
and 4,025wereMBV-
negative;
hence,
theoverall
prevalence
of MBVinfections
in termsof thetotal
Results number
of shrimpexamined
was55.8%.
Eleven There
majortypesof organisms/dis- wasa lowcorrelation
r = 0.3!
easeswere diagnosedbetweenOcto-
between
thenumberof MBV-infected
ber,1989and December, 1990in P. animals
andtheirlocationFig.3!.
monodon.
MBVwasthemostprevalent Prevalence
ofMBVin Different
Post-
disease
agent,accounting
for249cases Iarval
Stages.
There
wasa highcorre-
6.9%! out of the total of 372cases lationr 0.92!
between
hostageand
Table 1!.
occurrence
of MBV. The occurrenceof
 MBV in the Phil t47

Table 2. Overall incidence of MBV in Penaeus mortodort based on the total number of
cases examined from October, t 989 to December, 1990.

~Includes
theprovinces
of Camarines
Norte,Negros,
Pampanga,
Mindoro
andsomeunknown
sources

MBV in the different life stages of P.

mottodott in terms of the total number of
cases received is shown in Table 3. With
the exceptionof one MBV-positive PL3,
all PLl to PL6 samples were negative
for MBV Fig. 4!.

Distributionof MSV. All the diagnos-

tic casescarnefrom 12 major provinces
in the Philippines. Nine of these prov-
inces were from Luzon Islands, two
were from the Visayan Islands and one
from Mindanao Island.
Among the provinceswith lessthan ten
casesof MBV, samples from Bulacan
and Cotabato had 100% MBV preva-
lences;4/4 and 5/5, respectively.Among
the provinceswith more than ten docu-
mented cases of MBV, Quezon had the
highest rate of MBV infection; 80.6%
5/31!. The provinces of Bataan and
Batangaswere second and third, re-
s4 5 s r s p t0 n 'lr
Provlncw
Figure3. Overalllnc/denceof MSVfrom October,
1989 to Oecernber, 1990 based on the total num-
ber of PenaeusttKeadon samplesfrom 12prov-
inces where the samples were collected,
indicating that MBV was distributedthroughout
the country. = Pangaslnan;2= Zambales;3=
Bataan; 4 = Bulacan; 5 = Metro Manila; S =
Cavite; 7 = Batangas;8 = Quezon;9 = lloiio; 10
= Cabu; 11 = Ootabato; 12 Other provinces
whichinciude Camarines Norte, Parrpanga, Min-
doro!.
 NatMdad and L ner

Table3.Prevalence
ofMBVlndifferent
postlarval
stages
ofPanaevs
monodon
based
on
thetotalnumber
ofcasesfromOctober,
1989to Oecember,
1990.
PLStage No.of Cases MBV+ %! MBV-
01
02
12 0
10 0.0
0.0
1

22
100.0
100.0
03
04
36
6 4 0
33.3
0.0
4
66
66.7
100.0
05
06
07
08
11
17
08
2
6 0 0.0
0.0
18.2
47,1
99
100,0
100.0
81.8
52.9

12
86
6
4 5
09 18 33,3 12 66.7
10 26 14 53.8 46.2
11 26 17 65.4 30.8
12
13
23
10
18
6 78.3
60,0
21.7
40.0
14 20 14 70.0 30.0
15 38 30 78.9 15.8
16
17
18
19
32
19
28
14
27
14
24
12
84.4
73.7
85,7
85.7
56
2
4 5 15.6
26.3
14.3
14.3
20
21
22
2 16
27 72.7
100.0
02
27.3
0,0
22
92 77,8 22.2
23
24
25- 35
3

12
77
23
10
100.0
100.0
83.3
01
2 0 0.0
0.0
16.7
42
50- 60
45

62 - 180 4
67
3 85.7
100,0
75.0
01
14.3
0.0
25.0
372 249 66,9 120

spectively, where S0% 2/15! and
67.7% 5/96! of the total number of
caseswere positivefor MBV Fig. 3!.
In termsof thepercentage
MBV-posi-
tiveanimals,theprovinceoflloilohad
the highest prevalence, 84.2%
76/209!,followedby theprovincesof
Bulacan and Cavite where 83.6%
1/61! and78.7% 22/155! of the total
number of animals examined had
MBV. Metro Manila had the lowest
MBV incidencerate; 14.7%6/245!.
Monthly Incidenceof MBV. The cor-
relation between the mcidence of MBV
and tilne month!was very low r =
0.3!. In terms of the total number of
 MBV in the Phil ines 149

Table
4.Monthly
prevalence
ofMBV
inPenaeus
monodon
based
onthetotalnumber
of
casesexaminedfromOctober,1989to December,1 990.
of Cae

1
19
16
34
12
12
22
23
45
24
27 13 48,1
41 34 82.9 7

21 52.4 10

29 13 44,8 16

46 32 69,6 14

372 249

120

I3 6 7 6 11 13 16 17 10 21 23
26 66 16 '12 16 16 1~ 20 22 3I
P6silo
rva!Stages

Figure
4.Prevalence
ofM8Vin
thedifferent
postlarval
stages
ofPenaeus
monodon
based
onthetotal
number
of samples
examined
fromOctober,
1989to December,
f990.There
is a highcorrelation
between
theageofthesamples
andtheprevalence
ofM8V,Furthermore,
notethatthe339%incidence
of M8V in PL3 constituted on y one case.
 Nativktaci
andL htner

Ss JanF<4 i!I kn
INsr Avg 4a Qyy
Malhe

Rgvre
5.Monthly
incidence
ofMBV
based
onthe
total
number
ofPenaeus
remxhm
sample
ex- Figunr
6.Incidence
ofotherPenaeus
mono'
aminedfrom
October,
1989toDa~e; 1990. diseasesd/agnosed
fromOctober,1989toDe-
Tlrere
lsa Iow
correlation
behamrnthe
Incidencecember,
1990based
onthetotalnumber
ofsam-
ofMBV andmonths,
Indicating
thatMBVsets plesexamined,
Notethatthesevalues
do not
prevalentin aNmonths, Include
MBV.BVC
= Type
C becuioÃrus;
Zoo=
Zootharnnium;
Vor
= Vorticeifa,
LM= Lanai
my-
cosis;
BSS=Btveshrimp
syndrome;
GN= giN
necrosis;
FB Filamentous
hactena;
BE 8ac-
tedalenteritis;
HV= Hepatoparrcreatic
vkriosis;
cases
received,
themonth
ofSeptemberRD Reddisease.
hadthehighest
occurrence
ofMBVat
82.9%
4/41!,while
themonth
ofMay OtherDiseases.
Tenmajordiseases
or
wassecond
with 78.3%
8/23!.No-
vember MBVpreva- pathogens
hadthelowest werediagnosed from122
lence,
44.896 samples Figs.
3/29! Table4! and correlation 6,7!.Therewasa high
October
actually
hadthehighest r = 0.99!betweentheoc-
preva-currenceof these diseasesand MBV
lence
00%!;however,
onlyonecase
wasdocumented thisperiod. Table
during 5!.Oneof themostsignificant
findings
wasthediscovery
ofa type
C
baculovirusa nonoccluded
baculo-
Based of animals virus!froma population
onthetotalnumber of postlarval
examined,
theprevalence
of MBVwas P.morrow+. This
virus wasdiagnosed
highin September1.1%;587/826!,asa inixedinfection
withMBVin post-
andJuly1%;431/706!.
MBV appearedlarvae,
lovirus
andis thefirsttypeC bacu-
reportedin P.rriotuxforr
fromthe
to beleastcommon in November;
it Philippines.
wasfoundin only34.7% 67/481!
of
theanimals sampled.However,
there
wasa verylow correlation r 0.2! Discussion
between the numberof MBV-infected
shrimpandtheirmonthly prevalenceP.MBVwasthemostprevalentdisease
of
Fig. 5!. monodorr,
accountingfor249disease
cases
6.9%!.These
figuresarealarm-
 MBV in the Phil'
ingto bothhatcheryandgrowoutfarm-
ers.
Theprevalence
anddistribution
of MBV
in P. monodompopulationsis a function
of a complexinteractionof severalvari-
ables,induding age,feedinghabitsand
the extent of cannibalism. On the other
hand, understandingMBV and its mode
of transmission, virulence, persistence
in the environment and ultrastructure
are necessaryto the understandingof
this host-pathogenrelationship.
Host age is one of the most important
variables in the study of baculoviruses
Martin et al., 1987; Watanabe, 1987!.
In this study, the prevalenceof MBV
was higher amongthe older stagesof
postlarvalP. rrrortodon,
whereasyounger
animals e.g., PL1 to PL7! were not as
susceptible
to MBVinfection.Thisfind-
ing is consistentwith the results of
previous work conducted by the
authors in which a susceptibility study
to MBV was perforined on the different
larval and postlarval stagesof P. mono-
don. However, these findings disagree
with those of Momoyama and Sano
989! who studied BMNV infection in
kuruma shrimp P, japonicus!. The
authors found a strong negative corre-
lation between BMNV infection and
host age, indicating that the infectivity
of BMNV in P.japrmicusdecreaseswith
age.
Another important finding of this study
was the associationof other viral, pro-
tozoan, bacterial and fungal infections
with MBV in P. monodonpostlarvae. It
is possible that these co-infecting mi-
croorganisms
exerted
synergistic
effects
with MBV, acting asbiologicalstressors
and enhancingthe overall susceptibility
of the host to infection. In fact, surface
and gill foulingby epiconunensals
such
as Leucothrix mucor and Zoothamnium
sp. are suspected to compromise the
respiratorysystemof MBV-infectedP.
mmiodori,causing significant mortalities
Lightner et al., 1983a!.This kind of
relationshiphasalsobeendemonstrated
in silkworm larvae. Individuals ex-
posed to Pseudomorius sp. were more
susceptibleto viral infections Watan-
abe, 1987!.
There was a very low correlatio~ be-
tweenthe prevalenceof MBV and the
geographicorigin of the samples.In
addition, there was no significant sta-
tistical correlation between the occur-
rence of MBV and time of year. This is
in contrast to findings that the distribu-
tion of several insect baculoviruses is
influenced by geography and season
due to the migration patterns of insects
Tanadaand Fuxa, 1987;Weiser, 1987!.
Hatchery-reared
or pond-culturedtropi-
calaquaticaninialssuchasPe@acus mono-
don are confined to tanks and ponds
and are subjected to very limited sea-
sonal variation.
In Australia, Lester and Paynter 989!
reportedanMBV-likevirus from popu-
lations of P. plebejus,P. moriodonand P.
mergm'ensis,
Until serologicalstudies
can be made comparing this bacu-
lovirus to MBV, it can not be ascer-
tained if this is a new strain of MBV or
a distinctspeciesLightneret al., 'l989a!.
It is, therefore, imperative to examine
552 Natividad and Li htner
 MBV in the Phil' 'nes

Table5, Otherdiseasesof Penaeus

rnonodon
basedonthetotalnumber
ofcasesrece,v~
from October, 1989 to December, 1990.

the problems underlying the preva- eluded that MBV is enzootic in South-
lence of MBV at the hatchery and pond east Asia and cited as proof the high
levels relative to existing hatchery and prevalence of MBV in the regions
pond management practices and biotic where farms use wild P. monodon
factors. Lightner et al. 983a! con- broodstock in the mass production of

Figure7. Photomicrograph
of the dNerentpathologica/
conditionsanddisease-causing
organisms
in
Penaeusmonodon populations.
f. Penaeus
monodon
bacu/ovirus
MBV!.ThepathognomonicsignsofMBVinfection
arethemultiple,
intranuc/ear
sphenca/
occlusion
bodiesarrows!
within
thehypettrophied
nuc/ei
ofthehe/I/opancr'ea«
tissuesof P. monodon post/arva, H&E staining. Bar= 20 pm!.
2. TypeC bacu/ovirus.
Histologicai
sectionof thehepatapancreas of P.monodon post/arva
showing
thehypertrophied
nucleianow!of thehepatopancraatic tubuleepithe/ia/
cells.Notetheabsence
of
vira/occ/us/ons,
whichis themaincharactenstic
ofthistypeofbacu/ovirus,
H&E stains Bar=20iim!.
~ HPnecros/s.
Histological
section
ofthehepatopancreatic
tissueofP,rnonodon
post/arva
showing
/argemasses
of necrotic
areassurrounded
by hemocytes
arrow!.
Thispatho/ogica/
condition
is
probabpsimilarto vibnosis.H&E staining.Bar= 100iim!.
4 Reddisease.
Histological
section
ofthehepatopancreas
ofjuvenile
p.rnonodon
withadvanced
red
disaase.
Notetheextensive
hemocytic
encapsulation
andme/anized
areas
which
contain
masses
of
necrotict/ssuedebns arrows!.H&Estaining. Bar= 200pm!.
Bacterial
enteritis.
esto/ogica/
section
oftheanteriormidgutofaninfected
P.morx>50n
post/arva
necrot/c
areas
andhemocytic
infiltration
in theaffected
areaarrow!.
This
patho/ogica/
is believedtobecausedbybacteria.
H&E staining.
Bar= 50li.m!
8 F//amentous
bacfena/
disease,
Histo/ogica/
section
of thegil/fi/aments
ofP.monodon
juvenile
w/ngheavyinfection
offilamentous
bactena,
possib/y
Leucothrix
mucorsnows!.
H&E staining.
Bsr
=20pmJ
~ Gi//necrosis,
H/sto/ogica/section
ofanecrotic
andmelanized
gilifroma
p.mc~npcs//anat
arrow!
H&Eshining,Bar=~>m!
8-~oothamnium
sp.This
protozoan
causes
extensive
damage
tothegi//s
andappendagesininfected
md<onpostlarva/,
juvenile
oradult
populations,
H8E staining.
Bar= 20
pm!,
 NatiNkhd
andL htner

postlarvae.
MBVis present
in most
hatcheriesandgrowout pondsin introduced spore-forming bacteriumBa-
Southeast
Asiancountries
suchasMa- ci7lus sphaenacs Lightner,
pers.comin.!.
laysiaLightner
etal.,1985;
Anderson,
1988;
JohnsonandLightner, Sin- At present,
1988!, therearenoknown treat-
gaporeBrocketal.,1983;
Lightner
et ments withwhich to cure
MBV, norare
al.,1985!,
Taiwan Lightner
etal., thereanythatcaneliminate MBVfroin
19Na,1987a;Johnson andLightner, thecultureenvironment. Most farmers
1988;Lightner
andRedrnan, 1991;in theVisayan region have reported
Chen etal.,1989a!,
IndonesiaNashet some degree ofsuccess fromincorpo-
al1988; Lightner
etal.,1992!,
Thai- rating1,000rng vitamin C/kgoffeed
landNatividad,
BFAR data given
diagnostic topond-cultured P.monodonwith
fromimported postlarvae!
andthe chronic MBV infections. Although
PhilippinesLightner,
1983;
Johnson there havebeen nostudies tosubstan-
andLightner,1988;Lightner
etal., tiatethisclaim,it is possible
thatin-
1992!.
MBV-infected
spawners
may creased
dietaryvitaminC increases
continuously
excrete contami-hemocyte
feces count
andactivity,
andim-
nated
withfree
MBV andocclu-proves
virions thecollagen
integrity
of the
sionbodies
during Theseanimals,
spawning. thusimproving
theirgeneral
virions
and
occlusion
bodies eas- resistance
could to diseases
suchasMBV.
ilyremrun
associated
with
theshrimp Support
forthistheory
wasprovided
larvae
and
infect
themwhen beginbyPristavko
they and13ovzhenok
974!
feeding. withlarvae
ofthecodling
mothM-
pevresia
pwnonella!
thatwerefeddiffer-
Inhatchery-reared
P.monodorr
postlar-
entconcentrations
ofvitaminC.When
val
populations,
MBV acts
as
a density-
theamount of vitamin
C wasde-
dependent
disease
thatinfects
more creased,
the hemocytecount
of the
hosts
ashost
density The larvae
isincreased. decreased
and their
susceptibil-
rateofMBVinfection P. itytoBeauveria
inpostlarval bassiana
increased.
irs~dorr
populations
mayberelated
to
crowding
stress etal.,1983a;Among
Lightner theother
P.monokmdiseases
SindermannandLightner, In diagnosed,
1988!. themost important
was
a
high-density
stocking,
MBV is nonoccluded
infection TypeC! baculovirus
easily
enhancedthrough that
cannibalism. was foundinsevencases
.9%!,
Thedesignsofmost inthe ostly
hatcheries froin
theprovinces
ofZambales,
Philippines
should alsobereviewed. Pangasinanand Quezon.Pathologi-
ThefinemistgeneratedbyvigorouscaUy,thepresumed type
C baculovirus
aeration
systemsin mosthatcheries was
diagnosedby thepresence
ofhy-
mayfacilitate
theaerosol of pertrophied
spread nuclei,
withmarginated
MBV.Aerosolcontamination
betweenchromatin,
a laterally
displaced
ordis-
aquaria
several aparthasbeen associated
meters nucleolus
withininfected
demonstrated
using hepatopancreatic
theexperimentally tubule
epithelial
cells,
butwithout
occlusion
bodies
Lightner
 MBV in the Phil
et al., 1989a; Momoyama and Sano,
1989;Sanoet al., 1985!.This presumed
type C baculovirus is the first reported
in P. rrtoriodoriin the Philippines. It is
possible that type C baculovirus may
also be present in P. rrtonodorifrom
Australia and Indonesia Lightner, un-
published data!. The only extensively
studied type C baculovirus in penaeid
shrimp is baculoviral midgut gland ne-
crosisvirus BMNV!, which is common
in P.japonicushatcheriesand ponds in
Japan Lightner, 1985;Sanoet al., 1981;
Sano et al., 1985; Sindermann and
Lightner, 1988; Lightner et al., 1989a;
Momoyama and Sano, 1989!.
Some of the frequently diagnosed dis-
easesin this study include gill and body
surfacefouling causedby two protozo-
ans, Zoothamnt'umsp. and Vorticella
sp., and by an unidentified form of
filamentousbacteria.However, elevated
levels of these organisms result from
water management problems; hence,
they are usually regarded as noninfec-
tious epicommensals.These organisms
are common in hatcheries and ponds
and are apparentlyubiquitous in shrimp
culture facilities Lightner, 1985; An-
derson, 1988!.This study showed that
postlarvae,juveniles and adults are all
potential targets of these epicommen-
sals.Epicommensalorganismscancause
gill obstruction, leading to impaired
respiration and, in severe cases,infec-
tions result in heavy mortalities due to
hypoxia and impaired locomotion and
molting Sinderrnann and Lightner,
1988; Anderson, 1988!, One filamen-
tous bacteria, Leucothrix macor, has
beenknown to causeextensivefouling
nes t55
of host shrimp gills, blocking the diffu-
sion of gasesacross the gill cuticle
Lightneret al., 1975;Couch,1978!.
Larval mycosiswas diagnosedvia wet
mount method! in 18 samples.9%!.
Although the etiologicalagentswere
not identified, diagnosis of mycosisin
infected postlarvae was basedon the
characteristicbranching fungal hyphae
protrudingfrom body surfaces,espe-
cially the appendages,cephalothorax
and abdominal regions.Penaeid dis-
easesof fungal etiologyare very com-
mon in the Philippines Hatai et al.,
1980; Lio-Po et al., 1978; 1982; 1985!.
Although the fungal diseases of penaeids
are believed to be ubiquitous in shrimp
facilities Lightner, 1985!,fungal infec-
tions associatedwith Haliphthorosphilip-
pinensishave been reported to cause
serious mortalities in hatcheriesonly in
the Philippines Hatai et al., 1980!.An-
derson 988! claimed that there is a
close association between antibiotic use
and the occurrenceof larvalmycosisin
Malaysia. He speculatedthat this may
be due to the removal of bacterial
epibiont competitors.Although Ander-
son's theory has not been proven ex-
perimentally, it is supported by the
observations of one hatchery operator
in Batangasprovince Mr. Willy Espejo
of SS Marine ResourcesCorp.! where
the use of oxytetracyciineto treat lutni-
nous bacterial disease in P. moriadort
mysis preceded a high incidence of
serious 1arvalmycosisinfections.
Fourteen cases.8%! of giII necrosis
were diagnosedin the P, iramodonsam-
pled. It is believed that this diseaseis
 Natividad and Li htner

of bacterialetiology.It is grosslychar- hepatopancreatic

atrophy and gross
acterizedby brownish discolorationof signs of red coloration that characterize
the gills and disintegrationof the cara- red disease Lightner and Redman,
pace.Histologically, the diseaseis char- 1989a!. Four cases of red disease were
acterizedby the presenceof multifocal diagnosedin this study; all were also
inelanizedcuticularlesionsin the gill infectedwith MBV. This diseaseis char-
filaments.
acterizedby the presenceof multifocal
necrosis,oftenaccompanied
by massive
8acterlalenteritiswasdiagnosedin six hemocyticinflammation and markedat-
cases .6%!. It is characterized
by sep- rophy of the hepatopancreas.
ticlesionsof themidgutepithelialcells
andis normallyaccompanied by mild Although red disease is common in
to severenecrosisand sloughingof the pond-culturedP. monodon populations
midgutepitheliallining. Thisdiseaseis in Taiwanand the Philippines,its eti-
similar to hemocyticenteritis described ologyremainsunknown. Lightnerand
by Lightner 983! and Sindermannand Redman985! suggestedthat the red
Lightner988!. Because
hemocytic
en- coloration of affected P. moriodon is
teritis is often associated with certain causedby the deposition of beta caro-
typesof filamentous
blue-green
algae, tene and other carotenoids into the
midgut lesionsarebelievedto be caused hernolymph. This author, however, has
by endotoxins
derivedfromthealgae seenreddish
shrimpthatwerepathologi-
Lightner et al., 1987a!. cally negativefor red disease, Interest-
ingly, the aforementionedshrimp
Septic, multifocal necrosisand mas- exhibitednormal hepatopancreatic
tis-
sive hemocyticinflammation of the sue,havingno apparentmorphological
hepatopancreatic tissuessimilar to HP signsof hepatopancreatic
atrophy.
vibriosis was another diseasewe found
that may havea bacterial etiology4 Underthesecircumstances,
it is possi-
cases!.Vibriosp. is the etiologicagent ble that the red discoloration exhibited
of vibriosisandthisdisease syndrome by someshrimp could be attributedto
is one of the most studiedpenaeid severalfactors,includingseasonLight-
shrimpdiseasesLewisand Lawrence, ner and Redman,1985a!,feed type, a
1983;Takahashiet al., l985; Takahashi nutritionaldeficiency,and stressfulen-
et al., 1985;SindermannandLightner, vironmental parameters suchashighly
1988;Itami et al., 1989!.Similartypes acidic water. The latter has been dem-
of lesionswerealsodescribed by Egusa onstrated in samples from farms in
et al. 988! from kuruma prawns P. Binmaley,Pangasinan and Calatagan,
j aponicus!. Batangas, wherethepH of pondwater
in the farmswere 4.5 and 4 9, respec-
The pathological signs described for tively. Although these observations
vibriosis in P. japonicusEgusaet al may have been circumstantial, the rela-
1988! did not include the associated tionship between environmental pa-
 Nalividad and Li htner

sp. nov. isolatedfrom cultivatedlarvaeof theblueshrimp,

Penaeus
sfylirostris,
J.Inver-
the jumbotigerprawnPenaeus
nionodon. tebr, Pathol. 32: 139-150.
Transaction
of theMycological
Society
of Lightner,D.V. 1982.Toxiceffectof certain
Japan.21: 47-55.
marine
blue-green
algae
topenaeid
shrimp.
Hose,J.E.,D.V. Lightner,R.M. Redmanand FinalReport.SeaGrantNo. NA 79AA-D-
D.A. Danald.1984.Observations on the 0024,
pathogenesis
oftheimperfect
fungusFgsar-
tunt
soIani
intheCalifornian
brown shrimp, Lightner,
naeid
D.V. 1983. Diseasesof cultured
shrimp.
In:J.P.McVey Ed.!.
Hand-
Penueas
calijbmiensis.J.
Invertebr,
Pathol.44: of Maricuiture,CRCPress,BocaRa-
292-303.
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Humason,G.L. 1967.AnimahTissueTech- Lightner,D.V. 1985,A reviewof thediseases
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andCo., SanFran- of cultured
penaeid
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cisco.
CA,432.p. with emphasison recentdiscoveries
and
Itami,T., Y. Takahashi
andY. Nakamura.
1989, developments.
In: Y. Taki,J,H. Primavera
Efficacy
of vacdnation
against
Vibriosis
in
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Peseeus J.A LlobreraEds.!.Proc.FirstInterna-
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Johnson,
P.T. andD.V. Lightner
1988.The Southeast Asian Fisheries
Development
Cen-
rod-shaped
nuclear
virusesof crustaceans: ter,Iloilo,Philippines,
pp,79-103.
gut-infecting
species.
Diseases of Aquatic Lightner,D.V., T.A. Belland R.M, Redman.
Animals;Dis.Aquat.Org.5; 123-141. 1989a.
A review oftheknownhosts, geo-
Johnson,
S.K.1978.Handbook ofShrimp Dis- graphicalrange and currentdiagnosticpro-
eases.SeaGrantPublication No. TAMU- ceduresfor the virusdiseases of cultured
SG-75-6N.TexasA/kMUniversity, College penaeid shrimp.AQUACOP IFREMER, pp.
Station,
Texas,23p. 113-126.
Lester,
R.J.G.andJ.L.Paynter.
1989.Diseases Lightner,D.V.,T,A.Bell,R.M.Redman, L.M.
of culturedprawns in Australia.
In: Ad- Mohney, J.J.
Natividad,A. Rukyani
and A.
vancesinTropical
Aquaculture.
AQUACOP Foernomo. 1992.
A reviewof major
diseases
IFREMER. pp.97-10$.
Lewis,
D.H.1973.
Response
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shrimp of economic
significance
in penaeid
prawns/shrimps
of theAmericas
andIndo-
to infection
with Vibriosp. Proc.World Paci6c.
In:M. Shariff,
R.P.Subasinghe
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Lewis,
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AquacultureI, FishHealthSection,Asian
noprophylaxis
to Vibrio
sp,in pondn.ared Fisheries
Society,
Manila,Philippines.
pp,
shrimp,Proceedings
of theFirstInterna- 57-&0,
tionalConfetence
onWarmwater
Aquacul-
ture:Crustacea.
Brigham University, Lightner,
Young D.V. and C,T. Fontaine.1975.A
mycosis of the Americanlobster,Hanlarus
Hawaii.
Liao,I.C. 1985.A briefreviewof thelarval aeericanuscaused byFnsarium
sp.J. Inver-
tebr Pathol 25 239-245
rearing techniquesof penaeid
prawns.In:
Y. Taki,J.H. Frimavera andA. LIolnera Lightner,
1975,
D.V., C.T. Fontaine
Some forms of gill
and K. Hanks.
disease
inpenaeid
Eds.!.Proc.FirstInternational
Conference shrimp. Proc. World Maricul, Soc. 6: 347-
ontheCulture
ofPenaeid
Prawns/Shrimps.
365.
Aquaculture
Dept.,Southeast
AsianFisher-
iesDevelopment
Center, Philippines.Lightner,
Iloilo, D.V.,R.P.Hedrick, J.L. Fryer,S.N.
Chen, I.C. Liao and G.H. Kou. 1987a.A
pp 65-78.
Lightner,
D.V.1975. Some serious survey
potentially of cultured penaeidshrimpin Tai-
wanforviralandotherimportantdiseases.
disease
problemsintheculture
ofpenaeid FishPathol. 22!: 127-140.
shrimpin NorthAmerica.
In:FroceedingsLightner,
D.V.andD.H.Lewis. 1975.A sep-
oftheThirdUS-Japan
MeetingonAquacul- ticemic
bacterial
disease
syndrome
ofpenaeid
ture,US-Japan
Cooperative
Program
inNatu- shrimp.Mar, Fish,Rev.37:25-28.
ral Resources.
SpecialPub8cations
of the Lightner,
D.V., andR. Redman.1981.A bacu-
FisheryAgency,Japanese
Governmentand
JapanSeaRegional Laboratory, lovirus-caused
Fisheries Penaeus
disease
monodon.
ofthepenaeid
shrimp,
J. Invertebr.Pathol.38:
Nigata,Japan.pp. 95-97. 299-302,
Lightner,
D.V.1978.
Passible
toxic
effects
ofthe Lightner,
D.V., andR.M, Redman.1985a.Ne-
marineblue-green
alga,Spirulina
subsaisa
in crosis
ofthehepatopancreas
in Penuete
rnono-
 MBV in the Phil' ines

donandPenaeus Arthropoda: Martin, S.W., A.H. Meek andP. Willeberg.

stylirostris
Decapoda! withreddisease. J.FishDis.8: 1987.Veterinary Epidemiology. IowaState
181-188. University Press, Ames. Iowa. 343p.
Lightner, D.V. andR.M. Redman. 1985b.A McKee, C. andD.V.Lightner, 1982.Effects
of
parvo-likevirusdisease ofpenaeid shrimp. several algicidesand surfacbmts onfilamen-
J. Invertebr.Pathol.45:47-53. tous bacteriumIeucothrixinsicorOersted.
Lightner,D.V.andR.M.Redman. 1991.
Hosts, AppLEnviron. Microbiol.
43:715-718.
eographicrange anddiagnostic procedures Momoyama, K. and T Sano. 1989.Develop-
orthepenaeid virusdiseasesofconcern to mentalstages of Kuruma shrimp, Penaeas
shrimpculturists in the Americas.In: P. japonicitsSate, susceptible to baculoviral
DeLoach, W.J.Dougherty andM.A.David- mid-gut gland necrosisBMN!virus. J.Fish
son Eds.!,Frontiers in ShrimpResearch. Dis. 12: 585-5N,
pp. 173-196. Nash,G.I.,I.A. AndersonandM, Shariff.1988,
Lightner,
D,V.,R.M.Redman
andT.A.Bell. Pathological changesin the tiger prawn
1983a.Observations
on the geographic
dis- Penaeus rnonodonFab6ius,meciatedwith
tribution,
pathogenesis
andmorphology
of culturein bradcishwater
pondsdeveloped
the baculovirusfrom Penaeus
rnonodon
Fab- frompotentially
acidsulphate
soils.
J.Fish.
ricius,Aquaculture,
32:209-233. Dis. 11; 113-123,
Lightner,
D.VR.M,Redman,
D,A.Danald, Nimmo,D,R.,D.V. Lightner
andL.H. Bahner.
R,R. Williamsand L.A. Perex.1984a.Major 1977. Effectsof cadmiumon the shrimp,
diseasesencounteredin controlledenviron- Penaeus
duoramrn,
Paleomonetes
pugioandPa-
mentcultureof penaeidshrimpat Puerto leoinonetes
vulgaris.In: J,F.Vernberg,
A,
Penasco,
Sonora,Mexico.In: C.J.Sinder- Calabrese,
F.D. Thurbergand W.B, Vern-
mann Ed.!. Proc.9th and 10thUS-Japan bergEds.!.
Physiological
Responses
ofMa-
Meetings
onAquaculture,
NOAATechnical rine Biota to Pollutants,AcademicPress,
ReportNMFS.16;25-33. New York.pp. 131-183.
Lightner,
D.V.,R,M.Redman,T.A.BellandJ. Overstreet,
R.M.1978.Marinemaladies'
Worms,
Brock.
1984b.Anidiopathic
proliferative
dis- germs
andother
syrnbionts
fromtheNorth-
easeofthemidgutandventral
nervein the ern Gulf of Mexico.Mississippi-Alabama
Kuruma prawn,Penaeusjaponicus
Bate,cul- SeaGrantConsortium,MASGP-78-021-1978
turedin Hawaii.J, Fish.Dis.7: 183-192. Pitogo,
C.L.,L.J.Albright,
M.G.Patter
and
Lightner,
D.V.,R.M.Redman, R.R,Williams, N.A. Suiiaz.1990.Studieson the sourcesof
L.L.Mohney,J,P.M.Clerx,T,A. Belland luminescentVibrioharveyiin Penaeus mono-
J.A.Brock,1985.Recentadvances
in penaeid donhatcheries.In: M. Shariff,R P. Subas-
virusdiseaseinvestigations,
J, WorldMarie. ingheandJ.R.Arthur Eds.!,Liiseases
in
Soc. 16: 267-274. AsianAquaculture
I. FishHealthSection,
Lightner,
D.V.,B.R.Salser
andR,S.Wheeler. AsianFisheries
Society,Manila,Philip-
1974.Gasbubbledisease
inthebrownshrimp, pines,pp.'157-164,
Penaeas
aztecus.
Aquaculture.
4:8WN. Pristavko,V.P, and N.V. Dovzhenok.
1974.
Lio-Po, G.D, C.R. Lavilla and A,T. Llobrera. Ascorbicacid influenceon larvalbloodceII
1978,Toxicityof malachitegreento the number andsusceptibility
to bacterial
and
larvae of Penaeusmonodon.KaJikasan, Phil- fungalinfection
in thecodlingmothIas-
ippineJournal
of Biology.7;338-346. peyresia
pomonella.
J. Invertebr,
Pathol.24:
Lio-Po,G.D., M.E.G. Sanvictores,M.C.L. Ba- 165.
ticadosand C.R. Lavilla. 1982, In vitro effects Sano,T., T. Nishimura,K. Oguma,K. Mo-
of fungicides
on hyphalgrowthandsporo- moyama
andN. Takeno,
1981.
Baculovirus
genesis
ofLagenidiian
sp.isolated
fromPerseus infection of cultured Kuruma shrimp
rsonodon
larvaeand Scyllasernitaeggs.J. Fish Penaeus
japonicus
in Japan.
FishPathol.
15:
Dis. 5: 97-112. 185-191.
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and M E.G. Sanvictores, 1985. In vitro ef- Hayashida
andK, Mornoyama.
1985.Bacu-
fectsoffungicidesonthefungusHafipthoros loviralinfectivity
triaLson kurumashrimp
philippinensis.
J. FishDis. 8: 359-365. larvae,Penaeusjaponicus,of differentages.
Luna, L.G. Ed.!. 1968. Manual of histologic In: A.E, Ellis Ed.!. Fish and ShellfishPa-
stainmg
methodsoftheArmedForces
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Academic
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pp.397-
ofPathology.
McGrawHill,NewYork,259p. 403.
 Nativkhd eed L hlner

Slndernrrmn,
C.J.andD.V.Lightner Eds.!.
1988.
Disa' Dtagrsssls inNorth Thurman,
andControl R,B.,T.A.BellandD,V.Lightner.
1990.
Unique
physico-chemical
properties
of
Arnerkan
Marine
Aquaculture.
Haec~,New theocduded
penaeid
shrimpbaculoviruses
York.431p.
Suplee,
V.C. andD.V. Lightner.1976.Gas- andtheirusein diagnosis
of infections.
J.
Aquat. Animal Health. 2: 'l28-131.
bubbie disease duetooxygensupersatura-Watanabe,
H. 1987.
Thehostpopulation.
Irr:
tioninrrrceway-reared brown
shrimp.
Frog. J.R.
FuxaandY. Tanada
Eds.!.
Epizootiol-
Fish Cult. 38: 158-159.
Takahashi, Y., Y. Shomoyama and K. Mo ogyofInsectDiseases.JohnWileyandSons,
NewYork.555p.
moyama. 1985.Pathogenicity andcharac-
«nisticsof Vibir'rr fromcultured Weiser,
sp.isolated In:
J.1987.
J.R.Fuxa
Patterns
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overplaceandtime.
Tanada Eds.!.Epizooti-
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Bate.Bull. ology of InsectDiseases,
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Y.andJ.F.Fuxa. 1987.Thepathogen
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D.V.Lightner
Toxicity
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and
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John Wiley andSons, NewYork.555p. 44: 1479-1481.
 The Status of Culture and Diseases of
PenaeidShrimp In Korea

MIpmngAe Park
tM IF4 ' tl M~AI I'
6&3 Shikari, IQj;m~, Ymg~pe
Kyong-Nam
626-900,
ftgublicofKorea

Thispaperreviews
thestatusof culturedRrtaeus
japonicus
andP.chinensis,
anddescribes
the
diseasesknown to affectcultured shrimp in Korea.

Introduction prawn, P.japorricus,

Public institutes and private companies
first attempted to culture penaeid
shrimp in the late 1970s,but commer-
cial-scale culture was not successful in
Korea until the early 1980s.Sincethat
time, the development of semi-inten-
sive shrimp culture in Korea has been
accompaniedby diseaseoutbreaks.Un-
fortunately,few studies have beencon-
ducted on shrimp diseasesand there is
an urgent need for more research.
The Status of Penaeid
Shrimp Culture In Korea
Culture Areas
The primary speciesof shrimp grown
in Koreaare the fleshy prawn, Penaeas
chineesis, on the western coast of the
Korean Peninsula, and the kuruma
alongthe southern
coast. There are approximately 30
shrimpfarmsin Korea.Fifteenarecon-
centrated near Chungnam province on
the Yellow Sea; these are responsible
for more than 80% of total production
of cultured shrimp.
Penaeidshrimp seedare producedat
three governmentinstitutesunder the
purview of the National FisheriesRe-
search and Developxnent Agency
NFRDA!. Penaeus
j aponicuspostlarvae
havebeenproducedandreleasedatthe
Koje hatcheryon the southerncoast,
and P. chirtensisseed are cultured and
releasedat Puanand Poryonghatcher-
ies on the western coast Fig. 1.!.
Artificial Seed Production
From 1983to the present,33,565,000
P.
japonicirsand 23,790,000P. chinensis
postlarvaewere producedby NFRDA
 Tggef. Production
ofPenaeus
japonA~andP.chinensis
seedi

forstock
enhancement
andtosellTa-
ble1!. Privateshrimpcultureenter- Production ofShrimp
prises,
by comparison, produced 160
millionP. japonicus
and P. chinensis Thetotalamount ofshrimpharvested
postlarvae
in 1991alonegable2!. in 1990was3,775MT; 312MT from the
culturefisheryand3,463MT fromthe
capturefishery. Productionfrom Ko-
rea'sculture
fishery
wasstillverylow
compared
tothecapture fisheryless
than10%ofthetotalproductionTable
3!.

Diseasesof Cultured
PenaeidShrimpIn Korea
Several
diseases
afQict
cultured
penaeid
shrimp
inKorea;
sometimes
theycause
seriousdamage.In general,little is
known about
penaeidshrimp
diseases
inKorea;
therefore,
fewdetails
regard-
0 25 SO ingtheoverall
status
ofdiseases,
diag-
glI ~
t ~ nosticproceduresand treatmentsare
available
at thistime.ThefoUowing
RYMre
1,Penaeid
shrinycu/lure
areasin
Korea. diseases
havebeenfound in Korea.
 Diseases of Penaeid Shri
Table2. Productionof PenaeusjaponicusandP. chinensisseedin privatecompan/es
Viral Diseases
HPV. Hepatopancreatic parvo-like vi-
rus HPV! is known to infect P. chinensis
Lightner and Redman, 1985!. Symp-
toms of HPV infection are poor growth
rate, decreasedfeed consumption, slow
movement and a propensity to aggre-
gate in areas near the tank edge. Af-
fected shrimp can also exhibit white
patcheson their abdomens.The means
to prevent and treat HPV diseaseare
unknown.
Histopathological observations funded
by The OceanicInstitute's Asian Inter-
changeProgram in 1991confumed the
presenceof HPV-infected P. chinensis
postiarvae at a government hatchery.
This researchwill continue this year.
BMN. Baculoviral midgut gland ne-
crosis BMN! affects P.j aporricus
larvae,
causing mass mortalities as a result of
in Korea
thedestruction
of the epidermai
cellsof
the midgut and hepatopancreasSano
et al., 1984;Lightner et al., 1989!.
BMN-infected mysis and postlarvae
stop feedingan.dswim with an abnor-
mal posture near the surfaceof the
water. Using histopathologicalmeth-
ods, BMN was diagnosedin P.japomicus
larvae in Korea in 1991.
Vibrio Disease
Vibrio disease has been observed in
penaeidshrimprangingin sizefrom 1
g to adult. The mostseriouslossesare
incurred during growout.
Table 4 contains information on six
strains of Vibriosp. isolatedfrom cul-
tured P. japonicusthat exhibitedsigns
of disease Kirn and Chun, 1990!. A
bacterium was isolated from the heart,
lymphoid organand muscleof shrimp
 Park

T~ 4 S~~ ~ zx strainsof ~ sp. isolatedfromculturedkurumaprawns,PenasNIs

from farms in Namhaeand Anhung culture

waterclean
andbyprovidinga
betvreenAugust and October,1989. high-quahty
diet.Formalin
is report-
The strains were then submitted for
edlyeffectivein controllingtheseor-
morphological,
biochemical
andphysi- ganisms on shrimp farms.
ological
characterization
Rg. 2, Tables
5-7!.

Several
workershavereported
effective
therapyofbacterial
diseasesusingan-
tibiotics.
OxytetracycBne
is frequently
usedtotreatVibrio
disease
of penaeid
shrimpin Korea.

Leocothrix Disease

Lmcothnxmoorattaches
tolivingand
nonliving
solidsubstrates,
andit rap-
idly adheres
to thebodysurfaces
of
penaeidshrimp.Affectedindividuals
are duHin appearanceandcovered
withmuddy
debris;
if mortality
occurs,
it isusuaHy
dueto hypoxia.
Lmcothtix
mscor maybemanaged
with
antibiotics
or coppercompounds
Lightner
andSupplee,
1976!.
Ciliate Disease

Zoothamnium
sp.andEpistylis
sp.also
infest
cultured
penaeid
shrimp.
Serious
outbreaks
are
prevented
bykeeping
the Fyore2. S ~
J8poNcus.
r~ mme VI no . ~
 Diseases of Pertaeld Shri in Korea

Note +!: weak or delayedpositive,

the diagnosticcapabilitiesfor shrimp

Problems and
quarantine
andresearch
facilitiesneed
Countermeasures to be upgraded,particularlytechniques
for virus detection and isolation.
Coastal Pollution
Other Needs
Pollution of coastal water with effluent
from factories and with domestic sew- The foHowingmeasures
would help
agehave caused a decline in the popu- control the incidenceof diseaseamong
lations of plankton that were once culturedpenaeid
shrimpin Korea:
availableas feed for shrimp. Red tides
are also more frequent as a result of ~ Trainprofessionals
to conduct
in-
agricultural runoff, especiaHyduring tensive researchon shrimp dis-
the rainy season.These factors have eases;

triggered shrimp diseases.

~ Preventinfectiousdiseases
through
Govemfppnt$upppp an early warning systemfor
shrimpfarms;and
~~onal center for shrimp disease
g osis and control, with field units ~ Increasedinternational
exchange
severalprovinces,is needed.Also of informationon topicssuchas
, 1~; Muroga
etal.,1979;fatahashi
etal.,1985;
4Kvsuda
et al.,1979;Uekiet
 Diseases of Penaeid Shri in Korea 167

shrimpcultureandtheprevention Muroga, K., S. Takahashi,H. Yamanoiand M.

Nishibushi. 1979. NonMolera Vibrio iso-
and treatment of disease. latedfromdiseased
Ayu.BuU.
Jap.Soc.Sci.
Fish. 45P!: 829-834.
Sano, T., T, Nishimura, H. Fukuda, T.
LiteratUre Cited HayashidaandK, Momoyama.
1984.Bacu-
loviral mid-gut gland necrosis BMN! of
Kim, J.H. andS.K. Chun. 1990.A vibriosis kururnashrimp Penaeus j~cns! iarv~ in
occurring in cultured kururna prawn, Japaneseintensiveculture systems.Hel-
Penseusjaponicus.
J. FishPathol.3!: 1-9. golander Meeresunters,37: 255-264.
Kusuda,R.,H. SakoandK. Kawa.1979.Clas- Takahashi, Y., Y. Shimoyama and K. Mo-
sification of Vibrio isolated from diseased moyama. 1985. Pathogenicityand charac-
fish 1. Fish Pathol.'13!: 123-137. tenstics
of Vibriosp. isolatedfromcultured
Lightner,
D.,T BeUandR. Redman.
1989.
A kururna prawn. Penaens japonicusBATE,
review of the known hosts, geographical Bulk Jap. Soc.Sci. Fish. 51!: 721-730.
range
andcurrentdiagnostic
procedures
for Ueki, N,, T. Sugiyainaand K. Mugora. 1988.
the virus diseases of cultured penaeid Vibrio infection in the heshwater shrimp
shrimp.Advances
in TropicalAquaculture, Pakienion
paucidene!
predisposed
by a para-
Tahiti, Feb. 20 - March 4, 1989, Aquacop. sitic isopod Tacheucbinensis!infestation.
lFREMER,Actes de Colloque.9: 113-126. Fish Pathok 23!: 175-178,
Lightner,D.V. and R.M.Redman. 1985.A Yasunaga, N. and N. Yamamoto. 1977.Char-
parvo-like
virusdisease
of penaeid
shrimp. acteristics of bacterial strains isolated from
J. Invertebr,Pathol,45: 47-53. socaUed Vibriosis of cultured red sea bream
Lightner,D.V. and V.C. Supplee.1976.A in the winter of 1977, Fish. Pathol. 12!;
possible
chemicalcontrolmethodforfilamen- 209-2l4.
tousgUl.Proc.WorldMaricul,Soc.7;473.
BaculovirusInfectionof PenaeidShrimp in Japan

Tokuo Sano
L;kior<ttory
afPquabc
Pathology,
Ci8p:vtmient
ofAquatic
Bicisiiences
TokyoUniversity
afFisheries,
4-5-7Hhnan,
MinatoKU
Tokyo108,Japan

Kazuo Momzprna
Vhrnaguchi
Prehctural
NaikaiFisherim
Experiment
Staff
Yanaguchi754,Japan

Baculoviral
midgutglandnecrosisBMN!isa superacute viraldisease,
causing severemortalities
in kurumashrimplarvae,&rureusjaponicus,
Withthegoalof implementingrealistic
prophylactic
measures,
wedeveloped diagnostic
techniquesforBMN,investigated
thehostrangeof BMNV,
andtestedthe efficacyof a varietyaf chemical
andphysical agentsasBMNvirucides. The
methodswedeveloped canfacilitate
diagnosis
in hatcheries.
Washing
fertilizedeggsisthemost
effective
prophylactic
technique.
ln addition
to P,japonicus,
BMNVcaninfectP.rnanodort,
P.
chinertsis
and P.semisutcatus
but not Metapenaeus
ensisor Brrtunustrituberculatus.

Introduction all will be placed on List B. List B

The OIE Office Internationale desEpi-
zooties! has dealt with fish diseasemat-
ters since 1960 through the Fish
Diseases Commission FDC!, which
producesan annualreport on the main
developments regarding current dis-
eases,new pathogens, and diagnostic
and. control methods usecl worldwide.
The FDC has created a separatesection
for fish diseases in the Animal Health
Code. This is currently being revised
and will contain eight pathogens six
viral agents and two bacterial agents!;
contains 89 corrununicable arumal dis-
easesconsidered to be of socioeconomic
and/orpublichealthimportance
within
host countries, and are significant in
the international trade of animals and
animalproducts.Recently,
theFDChas
expanded to encompassdiseasesof
molluscs and crustacea De Kinkeline et
al., 1990!.Consequently,elevenpatho-
genswereplacedon List B, including
Penaeusmonody baculovirus MBV!,
Bacui~rusperrrrei
BP!and baculoviral
midgut gland necrosis BMN! virus.
 170

Thisindicates
theimportance
ofbacu-
lm~estopenaeid
shrimp Sanoetal.,1981!.
culture. factor Themostimportant
allowingfortheprompt
imple-
ListB agents
cause
noserious
socio- mentationof prophylacticmeasures
for
economicproblemsfor terrestrial BMNis rapid,simpleandaccurate
di-
noraretheya publicagnosis,
homeotherms, seed
applicable
production
m situon shrimp
farms,
if possible.
The
health
con~sequence.
However,
thebacu-
lm~ particles fromvictims following
released twodiagnostic
havedeveloped
techniques we
meetthesecriteria.
of BMN,both
moribund
aninmls
and
latently
infected
adults,
undoubtedly
Fluorescent
AntibodyDiagnosis-
The
constitute
an "aquatic
biohazard."
"Aquatic
biohazard"
may bederedas principle
ofthistechniquedepends
on
a hazard
foraquatic
animalssuchas visualizing
thespecific
fluorescence
to
fishandshellflsh
caused theantigenof the baculovirus
byaquaticaffected in the
pathogens
inListB Sano,
1992!
as midgutglandsmearor in the
opposed inListA that intestinalepithelium.The fluorescent
tothepathogens
aredangerous
forhumansandtorter- antibodyFA!stainingof thesmearor
restrial
homeothermal
animals. the organrevealsthat the numberof
juveniles
showing
ubiquitously
visib/e
Examples
ofaquatic include fluorescence
biohazards tendstoincrease
withtime
IPN infectious
pancreatic afterinoculation
necrosis!, withthevirus.Visible
VHSviralhemorrhagic fluorescence
sepflcemia!, in thenucleiof theepi-
andEHNepizootic thelialcellswasrecognized
hematopoietic
ne- at 18h
crosis!.
Aquatic ingeneral,postinoculation
pathogens, Sanoetal.,1985!.
havethepotential
tospread
widely
and DarkFieldMicroscopic Diagnosis.
rapidly,andfurthermore,
tobetrans-
mitted TheresultingThisdiagnosis
intergenericaHy. confirms
nuclear
hyper-
diseases
prevail
asenzootic trophyofthemidgut
orpan- cells, glandepithelial
zootic
andcause
serious
socioeconomic whichis pathognornic
for BMN
consequences.
Thegoalof ourstudies virusinfection,
infresh
squash
prepa-
on BMNis the extermination
of this rationsunderdarkfield illuinination
shrimp
disease. thataim,we equipped
Toward witha wet-type
condenser-
have
studied
three diagnosisTwo
aspects: to fourdayspostinoculation
- 3G'Cincubation
at25
temperature
were
ofBMN,thehostrange
ofBMNV,and
forBMN.Thispaper considered
countermeasures tobesatisfactory
during
the
describesour results. infectivity
trialMomoyama
andSano,
1988!.

Diagnostic
Techniques HostRangeof 8MNV
for 8MN
BMN is a superacutecommunicable Todetermine
thehost
range
ofBMNV,
disease
of kurumashrimpin Japan susceptibility
trialswereperforined
on

the larval stagesof five speciesof crus-
taceans:giant tiger prawn Penueus
mono-
don!,fleshyprawn P. chinensis!,
green
tiger prawn P. semisukatus!,greasy-
back shrimp Metapenaeus ensis!,and
blue crab Portunus trituberculates!.
BMNV was inoculated in accordance
with the water-borne method Momo-
yama and Sano, 1988!. Giant tiger
prawns developedsevere
infectionsmor-
tality!,boththefleshyprawnsandgreen
tigerprawnshad temporaryinfections
no mortality!,and both the greasyback
shrimp and blue crabs were not in-
fected with BMNV. Hence, the host
range of BMN includes Penaeusmono-
don, P. chinensis and P. semisulcattcs.
Countermeasures for BMN
In order to establish realistic
measuresfor BMN, the virucidal effects
of chemical and physical agents were
studied. The results obtained
follows.
Virucidal Effects of Chemicals
BMNV. BMNV is inactivated by expo-
sure for 10 min at 25'C with the follow-
ing chemicals and concentrations:
5-pprn active principle concentrations
of chlorine; 25-ppm active principle
concentration of iodine; 100-ppm ben-
zalkonium chloride; 100-ppm ben-
zethonium chloride; 0.5% Formalin;
and 30% ethanol. Also, the virus is
inactivated with the following treat-
ments: ethyl ether for 18h, at 4'C; 25%
NaCl solution within 10 h and
NaCl solution within 24h Momoyama,
1989a;1989c!.
BMNH1 J 171
Virucidal Effectof PhysicalFactorson
SMNV. BMNV is inactivated with ul-
traviolet irradiation of 4.1 x 10 pW x
stem; summer sunlightexposurefor 3
h atabout30'C; heatingat 45'C for 120
min, at 50 and SS'C for 30 min, and at
60'C for 5 min; drying within 1.5 h at
about 30'C Momoyama, 1989b!.
Sterilizing Effects of Egg-washingon
BMNV. A prophylactic effectcan be
achievedby pouringenoughseawater
over the fertilized eggsof the shrimp or
by soakingthe eggsseveraltimesin an
egg-washingtank.Asa resultofapplymg
this measure iversitu at a hatchery in
YamaguchiPrefecture,the incidence
of BMN has dwindled year by year
since 1985 Table1!. Figure 1 illustrates
the collectingprocedurefor the fertil-
counter- ized eggs of shrimp.
Discussion and ConClusions
are as
The two diagnosticmethodswe devel-
oped are simple, rapid and inexpen-
on sive, facilitatingthe diagnosisof BMN
at hatcheriesor in the field. Using these
techniques, the virucidal effects of
chemicalandphysicalagentsfor BMNV
were elucidated, and the host range of
BMNV was determined. Furthermore,
a preventive method for BMN, egg-
washing,was developedand success-
fully prevented BMN in hatcheries
where it has been implemented. As
shown in Table 1, BMN has not oc-
curred since 1987. Consequently, we
12.5% determined that it was unnecessary to
develop highly sensitive diagnostics
such as a baculoviral DNA probe.
Figure
1.General
procedure
forcollecting
andwashing
shrimp
eggs
prior
toincubation.
! Broodstock
spawn
atnight
inspawning
tankThe
nerrf
day thespawner
isremoved,
aerafionis
halted,
andtheeggs
sink
while
fheupper
portion
ofseawater
isdrained.
! Theeggs
arecollected
ina collection
tankby
means
ofa bottom
drain
prpewith
gentle
ffowingsas
ester,
! Theeggs
areooilected
withanouter100
pmmesh
netalterthey
arefirstfitered
though
theinner
360gmmesh
net.! Theeggs
arewashed
with
stenle
seawater
orbychanging
thewaterin
theegg
washing
tank
several
times.
The temperature
oftherinsing
water
andthewater
intheeggwashing
tank
shoukf
bethesame
asthatin
thespawning
tank! Theeggs
areincubated
r'na rearing
tankwith40- 50cm-deep
seawater
thathasbeen
thermo-control/edand ferti ized.
 BMNin J 173

Table 1. Incidenceof BMN In a kurumashrimp hatcheryin Yarna-

OfficeInternationale
desEpizooties!
in im-
Baculoviral infection posesa danger to proving
awareness
andcontrol
of interna-
natural resources of penaeid shrimp. tional transfersof fish and shellfishdiseases.
The most effective way to prevent the Bull. Eur. Ass. Fish Pathol. 10; 4-6.
Momoyama,
K, andT. Sano.1988.A method
threat of BMNV is to exterminate the ofexperimental
infectionofkurumashrimp,
virus at the spot of a preliminary out- Penaeur
japmicusBate,withbaculoviral
mid-
breakby virucidal measures.The rec- gutglandnecrosis
BMN!virus.
J.FishDis.
11: 105-111.
ommended chemicals are chlorine and Momoyama,
K. 1989a.
Virucidat
effect
ofsome
iodine,appliedin themanneranddoses disinfectants
on baculoviralrnid-gutgland
indicated in this paper. Recently, Batts necrosis
BMN!virus.FishPathol.24;4749.
In Japanese
withEnglishabstract!.
et al. 99'l! reported that a 7.5-sexpo- Momoyama,
K. 'I989b.
Inactivation
ofbaculovi-
sure to a free iodine concentration of raI rnid-gutglandnecrosisBMN!virusby
ultraviolet irradiation, sunlightexposure,
0.14 mg/L inactivated99.9%of infec- heating
anddrying.FishPathol.24;115-118.
tious hematopoieticnecrosisvirus, sug- In Japanese
withEnglishabstract.!.
gestingthat the water-borneroute of Momoyama,
K. 1989c.
Tolerance
ofbaculoviral
rnid-gutglandnecrosisvirus BMNV! to
virus transmission can be blocked by ether, NaCI concentrationand pH. Fish
addinglow iodineconcentrations
to the Pathol.24:175-177.In Japanese
with Eng-
water supplies of hatcheries.However, lish abstract.!.
Sano, T., T, Nishimura, K, Ogurna, K, Mo-
whether these shorter exposure times moyamaandN. Takeno.1981Baculovirus
and lower doses can also inactivate infection
of kurumashrimp,Penaeusjapom'-
BMNV remains to be determined cusin Japan.FishPathoi,15:185-191,
Sano,TT, Nishimura,H Fukuda, T. Haya-
shidaandK. Momoyama. 1984.Baculoviral
Literature Cited mid-gutglandnecrosisBMN! of kuruma
shrimpPenaeusjaprrticus!larvaeinJapanese
Batts, W., M. Landolt and J.R. Winton, 1991.
intensive
culture
systems.Helgotander Meer-
esunters. 37: 255-264,
Inactivationof infectioushematopoieticne-
crosisvirusby low levelsof iodine.Appi. Sano,T., T. Nishimura.H. Fukuda,T. Haya-
Environ. Microbiol. 57: 1379-13&5,
shidaandK. Momoyama. 1985.Baculoviral
De Kinkelin, P., T. Hastein, J, Krecek, S.N, infectivity
trialson kurumashrimplarvae,
Chen and B.J, Hill, 1990. The role of the OIE
Penaeus
japorticus,of differentages.Itt; A.K.
174

Ellis Ed.!.Fishand She15shPatholagr. Sano,T. 1992.Manualfor FishVirusExamina

Academic Press,London.pp.397-4N. tion. FAO,Rome.ln Press!,
ContributedPapers-

Viral Diseases
 Infection Route and Eradication of R.naeus
monodonBaculovirusMBV! in LarvalGiant
TigerPrawns,Penaeusmonodon
Chen,S.N.,P.S.ChangandG.H.Kou
Liep'udgment
of Ztxiogy,Na5snalTaiwanUrimrsity
Taipei,Taivre

Ourexperiments
showed
thaturaeusmonodon
baculovirus
MBV!wastransmitted
orally.MSV
maybe eliminatedfrom hatcheriesby usingMBV-negative broodstock
or by washingnauplii
or fertilizedeggsseveraltimesin cleanseawater,Formalinandiodophore,

Introduction Chen et al., 1989 acerb;Lester et al.,

Of the six pathogenic viruses identi-
fied from penaeid shrimp, Penaeus
monodortbaculovirus MBV! Fig. 1!, a
nuclearpolyhedrosisvirus, is consid-
ered one of the most potentially seri-
ous pathogensto the larval stagesof
shrimp.
MBV has been found in shrimp from
the Indo-Pacific and Pacific Coasts of
Asia, Australia, Africa, and southern
Europe and in North and South Amer-
ica Anderson et al., 1987,Brock et al.,
1983;Chen et al., 1989a;Lightner, 1983,
'1988;Lightner et al., 1983, 1987!.This
virus infects a number of species of
shrimp, including P. varrnarnei,
P. rrgmo-
don, P. esculerrtus, P. semi'setcatls, P.
peniciltu4s,P. kerathurus,P. plebejusand
Metapenaemcrisis Srock et al., 1983;
1987; Lightner and Redman, 1981;
Lightner, 1983,1988;Lightner et al.,
l983, 1985,1987,1988!.The most seri-
ous infections were found in cultured
gianttiger shrimp,P. monodonChenet
al., 1989c;Lightner, 1988!.
Kpizootiologicalstudieson MBV in P.
monodon revealed an incidence rate
higher than 50% in postlarvae,juve-
niles and broodstock obtained from Tai-
wan Chen et al., 1989c! and
SoutheasternAsia Chen et al, 1990!.
Results obtained from a pathogenicity
study showed that environmental
stressors may significantly increase
mortality in MBV- infected larvae in
hatcheries Chen et al., 1989c!.A hatch-
ery experimentalsoshowedthat MBV
mayinitiatemortalityandgrowthretar-
dation in MBV-positive postlarvae
$78 Chen
Chen et al., 1989c;Chang and Chen,
1992!. For this reason, MBV may result
in variable larval production. To obtain
a better quality of postlarval P. rnotio-
don, MBV should be eradicated from
hatcheries.
The present paper attempts to describe
the pathway of MBV infection, In addi-
tion, procedures for the eradication of
MBV are also suggested.
Materials ancl Methods
To investigate the infection route of
Penaeus monodon-type baculovirus
MBV! in P. monody, nauplii and fer-
tilized eggs derived from either MBV-
positive or MBV-negative broodstock
were used Table 1!. All experimental
broodstock were collected from
Figure1a.Sectionof hepatopancreasof Penaeusmonodoninfectedby Penaeusmorodon baculovirus
MHV!. Note. Round occlusion bodies arrows!.
Figure 1b. MBV particles.
Chan and Kau
open sea in southeasternAsia and im-
ported into Taiwan.
For the broodstock, MHV infection was
confirmed by the presence of occlusion
bodies in shrimp feces. These were
detectedwith the aid of 0.1% aqueous
malachite green, 1% Gram's or
Giemsa's staining solution and Olym-
pus IM inverted and BH-2 light micro-
scopes. Experimental larvae were
maintained in hatchery ponds, with
the exception of those used in Experi-
ment 6. Each pond contained approxi-
mately 30MT of 28-to 30-ppt seawater.
Rearing temperatures ranged from 28
to 33'C,
Experiment 6 was conducted in plastic
tanks. Each tank contained 5 - 10 MT
the of seawater and salinitiesand tempera-
 MBV Transmission in Hatcheries 179

o0 D

CL OP
D
o Gl
0 0
O g! 0
o

D PV I Irl I H 0 ev
DO o D ooo

IPP 0
CD' II II II I 0
EPP
PC
O' UJ
cd II II II II 0
CP
X!
c,
IPP
oo I
o II I 5
IPI
I
0
rl
cD IPP
Cd 00

O0
C
Cl
PII O 0
ICI O
IIP oo 0.
CC IO
CP
0
O m
D PPPIPP 4I
IPP
C
O
D IIPrg rll

CCP o o R' 0
CD
0~%
C
O gp o
r 0~o 0C

'U
CI 0
O
~ IIP Ill
O. IPP
Ih
cg 0 rP

-" UO.~~
2 R ~E"
IIP

Cog
Oa ~.u~
C 'g
I >+rll 0
cd C 'cP O '
>E'~z 0.
cd
P CPG.O
0 e
C: 0 IPI0
Q! '0 D
8%
~o IIP Oe IU
CD
O IPI ~ ~ IIPW
Q.~ IIP
C7 !v !
Peg ra 0- 4 Q
CD 2+ XP 2+8 IPP
2 rpl g PIC'
C> 3 IP'
c~
C CD OaC>rp I
bP
CCi
CP
Exp CP~
I-
O

IIP~ a 0 t
D
IOPPP2 CP
~ ~ I eV
 Chen Cha and Kou

tures similar to those described
Approximately 7,000- l0,000 nauplii or
fertiii rwdeggsper ton were placedinto
each tank.
MBV infections in the hepatopancreata
of developing stagesof P. monody lar-
vae from zoea 1 to PL 40 were studied
using tissue squashes and his-
topathologicalstaining with 0.1%aque-
ous malachite green and hematoxylin
and eosin HRE!, respectively, as de-
scnbed by Lightner 983!. For each
stage,5 - 32 randomly selectedlarvae
were examined, and their MBV status
was recorded.
above. sea water with salinity of 28 30 ppt,
200- 300ppm Formalin and 20- 30 pp m
iodophore, as described in Figure 2.
During the course of larval rearing,
aeration was continuous and the alga
Sketettmema costatumwas provided, as
were artificial feeds shrimp flake,
plankton powder and micro-encapsu-
lated feed, rotifers or brine shrimp lar-
vae!, to ensure a sufficient feed supply
and completegrowth of the experimen-
tal larvae. Feed debris and shrimp ex-
crement were removed daily, and
one-third of the sea water in each tank
was exchangedat two-day intervals.

To detect the mode of MBV transrnis-

sion, nauplii derived from MBV-free
broodstock were reared in a similar
tank} with the excrement from MBV-
positive shrimp. The MBV status of
these shrimp in the later stages was
determinedusing the techniquede-
scribed above.
The eradication of MBV infection
achievedby the short-term washing of
nauplii and fertilized eggsusing filtered
ResUIts and Discussion
Pathway of MBV Infection
Penaeus mottodortlarvae produced from
either MBV-positive or MBV-negative
broodstock were examined his-
topathologically. The results in Table 1
was and Figure 3 Experiments 1 and 3!
show that larvae produced from unin-
fectedbroodstockhad no signs of infec-

A! NauplH
Running fodophore Runnirg
in plankton not so pprn ~ 588 wntnr ~
X-2min 30 s 30 5 1-2min poAds

B! FerNtzed Eggs
Running ladoprere
CO/Act
tlrtHired Sol ~ ~ 2OOpprn 2O~ ~ HetCherr
ding 1 -2m' 30 s 1 - 2 min ponds

Fig+a2. Procedures
for theeradfcatfan
of MBVinP.mone~ hatcheries.
Nate:ln a hatchery,na~lii
are much easier to collect than fertilizedeggs, Ftritihermare,fertilizedeggs are much more sensitiveto
Formalinthan rMqo/iiare.
MBV Transmhske in Hatcheries
 Chen Cha

tionwhile rearedin the hatcheryat 28 MBV infection. If this is true, eradica-

- 33'C. However, when these larvae tion of MBV at the hatchery level may
weremovedto a nurserypond,MBV- be feasible; hence, experiments were
positive
individuals
werefound Table conducted to determine the best means
1 and Rg. 3, Experiments1 and 3!. of eliminatingMBV from P. monokm
Whennaupliior fertilizedeggsderived hatcheries.
fram MBV-infected broodstock were
blaredin the hatchery,
MBV-positive Eradication of MBV Infection
shrimpwerediscovered
at the mysisor
postlarvalPL! stage,respectively
ga- To eradicate MBV infection in larval
ble 1 andFig. 3, Experiment2!. shrimp, various washing procedures
were tested.When nauplii and fertil-
MBV-infected PL2 were also found ized eggs obtainedfrom MBV-infected
when nauplii producedfram MBV- broodstock were treated as described in
positivebroodstockwere reared in Figure2, no MBV-positivelarvae were
plastictanksTable1, Experiment 6c!. found in the hatchery pond Table 1
However, larvaeproduced from MBV- and Fig. 3, Experiment4!. One day
freebroodstock rearedin plastictanks after the larvae were moved from the
containingfilteredwater revealedna hatcheryto the nursery pond, how-
signof infection
up to thePL40stage ever, MBV was diagnosedin some of
Experiments
6aandb!. the larvae. In contrast, MBV was not
detectedin the nauplii derived from
Whennauplliproduced
fromMBV-free MBV-free broodstock.
broodstock
wereexposed
toMBV-posi-
tive feces,MBV was detectedat theTheseresults
alsosupport
thehypothe-
mysisstageTable
1 andFig.3,Experi- sisthat MBVwastransmittedby oral
ment 5!. ingestionofMBVvirions.Furthermore,
theyrevealthat in a controlledenviron-
Although no one has found evidence ment,MBVcanbeeliminated
bywash-
of vertical transmissionof MBV in ingnauplii
orfertilized
eggs
thoroughly
shrimplarvae,
thepresent
study
shows withfiltered
seawater,200- 300ppm
thatMBVmaybetransmitted
byoral Formalin
and20- 50ppmiodophore.
ingestion of occludedor free MBV
virions. Baculoviruses
were alsofound Ourresults
alsoshowed
thatwashing
tobetransmitted
orallyin P. daorsrmm withfiltered
seawater
alonemayonl
Couch,1974!andP. japonicus Mo- reduce therateofMBVinfection
ratein
moyama,
1981;Momoyama
andSano, larval
shrimpExperiment
7!. Since
it is
1989!.
easierto collect
naupliithan fertiii'~md
eggs,and because
the latter are much
Theabove
experiments
alsosuggestmoresensitiveto the chemicalsem-
that oral ingestionof fecesfrom MBV- ployed,
commercial
hatcheries
should
positive shrimp is the main source of
treat
nauplii
instead
offertilized
eggs.
 MBV Transmission irt Hatcheries

In conclusion,there are two ways to Chang, 1992!. These results, when

eliminate MBV from hatcheries, consideredin light of the high inci-
thereby significantly improving the dencerate of MBV amongcultured
quality of P. rrtortodort
larvae: shrimp in the worM Chen et al., 1989c,
1990;Lightner et al., 1985!suggestthat
~ Use only MBV-free broodstock, the eradication
of MBV in hatchery-
and rearedlarvaeand the production
of
MBV-free larvae are important. We
~ Wash fertilized eggs or nauplii have also reached the conclusion that
with filtered sea water, Formalin to produce shrimp larvae of superior
and iodophore. quality, a broodstock quarantine sys-
tem for MBV infection shouM be estab-
Studies on baculoviral midgut gland lished, and the development of
necrosis BMN! virusin P.japotticusalso eradication measures for MBV should
showed that viral infection may be be emphasized.
eradicated by eliminating broodstock
excrement followed by washing fertil- Acknowtedgments
ized eggswith cleansea water Mo-
moyama, 1991!. This work was supported by a grant
from the U.S. Departmentof Agricul-
Pathogenicstudies showedthat in P. ture Grant No. FG-TA-111!and the
rrroru/don,MBV infection may initiate Councilof AgricultureandtheNational
damageor loss of hepatopancreatictu- ScienceCouncil Grant No. NSC-81-
bule and midgut tissueleadingto dys- 0209-B002-05!
of the Republicof China.
function of these organs or tissues
Chen et al., 1989b;Lightneret al., Literature Cited
1983!.Consequently,
moltingwasde-
postlar- Anderson,
layedsothattheMBV-infected l.G., M. Shariff,G. NashandM.
Nash, 1987. Mortalitiesof juvenileshrimp
vae were irregularin body size Chang Penaeus
monodon,
associatedwith Psnseus
et al., 1992!.
It wasalsonotedthatlarval monodon baculovirus,cytoplasmic reo-like
shrimpinfectedwith MBV contain virusand rickettsial
and bacterial
infections,
from Malaysianbrackishwater ponds.Asian
fewerhepatopancreatocytes thanjuve- FisheriesScience,1:47-64.
nile or adult shrimp; destructionof Brock,
j.A.,D.V.Lightner andT.A.Bell. 19S3.
A review offourvirusBP,MBV,BMNand
thesecellsmaycause a serious
disease IHHN!diseases of penaeid shrimp with
or mortality.In comparison withcon- particular
referencestoclinical
signiticance,
trol shrimp, MBV-infected larvae diagnosis andcontrol in shrimp aquacul-
ture,Proc. 71st
Intl.Council fortheExplo-
showedrelativelyhigh mortality rates rationoftheSea,C.M.19'/Gen:10/1-'lS.
Chang Chenetal.,1989c!. Chang, P.S.1992.
etal.,1992; Studies
ontheBiological
and
Biochemical
Properties
of Penseus
mono
type
baculovirus,
Ph.D.Thesis,Nadonal
Our recentstudy alsoconfirmedthat it Tai i, Taiwan.113p,
juvenile aremore Chang,
andadultP.monokm . Cten.
P.S.andS.N, . 11992.
Effectof
Penaeus
monodon-type
bacuiovirus
MBV!on
resistant to MBV than larval shrimp
 survhral
andgrowth ofIarvalFlmsrus rerrtxkur. Lightner,D.V. 1988.Diseasesof cultured
Submitted forpublicatkrn to Aquaculture. penaeid shrimpandprawns.I»: C.J.Sinder-
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Investigiatlon
of vtral infectionin cultured Dlagnasis and Control in North American
Jianl
iglgrawn
Pcwws
namcdon!
inAsia.
»: G.. ou, H. Wakabayashi, LC. Liao,
MarineAquaculture.Elsevier,Amsterdam
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Japan Symp.onFishDiseuses. Nadonat Sd- Chen, I.C. Liao and G.H. Kou. 1987. A
enoeCouncil,Taipei,Taiwan.pp. 1W170. surveyof cultured penaeidshrimp in Tai-
Chen,S.N., P.S. Chang,G.H. ka» and D.V. wan for viral and other important diseases.
Ughtner.
1989b,
Studies
onvtrogeneshr
and Fish Pathol. 22!: 127-140.
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hvtnN MBV!in gianttigerprawn Prrums baculoviruswauseddiseaseof the penaeid
srrrrrsdsrr!
and the red taII prawn Perseus shrimp, Pe»rreusrrur»0do».
J. Invertebr.
Psrrfc8lrrtus!.
Rsh Pathol,24:89100. Pathol. 38: 299-302.
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andG.H. Kou.1989c. Lightner, D.V., R.M. Redmanand T.A, Bell
Observationonpath nicityandepizooti- 1983.Observations on the geographicdistri-
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in cultured
shrhnpsin Taiwan.FishPathol. baculovirus from Peered monde» Fabricius.
24: 189-195. Aquaculture, 32/4!; 209-233.
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1989a.The first identification of Prrursus L.L. Mohney, J.P.M. Oerx, T.A. Bell and
r»srurds»
baculovirusMBV!inculturedsand J.A. Brock. 19&5. Recent advances in
shriinp, heaps»mesrrsr's.
Bulletinof the penaeidvirus diseaseinvestigations.J.
European
~tion of FishPathologists. World Marie. Soc. 16: 267-274,
9!: 624<. Momoyarna,K. 1981. Studies on infectious
Couch,
J.A.1974.
Anenzootk
nuclear
polyhe- mid-gutglandnecrosis of kurumashrimp
droais
virusofpinkshrimp:
ultrastructure, Perureus
japrr»tcus
Bate!-Artificial
infection.
prevalenceandenhancr»nent.
J. Invertebr. Bull,YarnaguchiPref.
Naikai Fish.Exp.St.
PathoL24: 31M31.
8: 1-11. In Japanese!.
Laster,
ILJ.G.,
A. Doubrovsky,
J.L.Paynter, Momoyama,K, 1991.Studieson baculoviral
S.K.
Sambhl
andJ.G.Atherton.
1987,
Light mid-gutglandnecrosisBMN! of kuruma
andelectron
microscope
evidence
of bacu- shrimp,Perraeus
jeporricus.
BulkYamaguchi
lovirus
infection
in theprawnPrrrssus
ptbs- Pref.NaikaiFish.Exper.St. 20: 1-91. In
jus.Dls.Aquat.
Org.3!: 217-219. Japanese!.
Lightner,D.V. 1983.Diseasesof cultured
naeld
shrimp.I»:J,P.McVeyEd.!.CRC Momoyama,
mental
K. andT. Sano.
stagesof kuruma
1989.Develop-
shrimp, Perureus
andbaakaf Mariculture.
Vol.1, Crusta- jape»r'cus
Bate,susceptible
to baculoviral
ceanAquaculture.CRCPress.BocaRaton, mid-gutglandnecrosis
BMNjvirus.J.Fish
FL.pp, 289-320. Dis. 12: 5&5-589.
 Viral Diseases of Cultured
PenaeidShrimpinJapan
Y~ hhmvyama
Yamaguchi
prefecturaj
NaikaiRsheries
Experieet Station
Vamagudi 754,Japan

About900miHion
juveniies
ofapproximately
20crustacean
species
areproduced
forseafarming
and pond culture every year in Japan.Pnme jspoaicws, the kuruma shrimp,is the most
important species,coinprising apprmimately90% of aHjuvenilesand nearly1N% of pond
culturedcrustaceansproducedannually.Two viraldiseases,
baculoviralmidgutglandnecrosis
SMN! of P.japmicusand Prnaeus motiodoa
baculovirusMBV!infectionof P.moraxfos,
the grass
shrimp, havebeenrecordedfrom thesespeciesin Japan.BMNis a severe infectious
disease
causinghigh mortalitiesin hatcheries.MBV,by contrast,hasbeendetected onlyoncein
postlarvaethatwereproduced for experimental
purposesfromspawners imported
fromTaiwan.
The researchconducted in JapanonBMNandMBVis reviewed in thispaper.

Introduction fleshy shrimp and P. lett'sulcatus,

the
westernking shrimp,arealsoproduced
Since the adoptionof the 200-mileeco- for seafarming in certain regions.
nomic zone system in many nations,
sea farming is now strongly encour- Althoughjuvenilesof manycrustacean
agedto increaseproductionof coastal species areproducedfor seafarming,
fisheries resourcesin Japan. Everyyear P.
j apmicus
is theonlyspecies
rearedto
about 700 million juvenile crustaceans edible size in pondsin Japan.Since
belonging to approximately20 species 1988,the numberof seedP.j~icus
areproducedatpublicseafarmingcen- importedfromTaiwanforpondculture
ters for stocking into coastalwaters has increased substantially. Other
gapanSeaFarrrung 1990!. penaeid
Association, shrimp,including
non-native
The main speciesproducedarePenaeus species,
have
never
beencultured
nor
japirra~, the kurumashrimp 84%!, havetheybeenimported,
exceptfor
Portents trifubereuhrtus,the blue crab veryrareexperimental
cases.
%!, and Metapenaeus
ensis,thegreasy-
backshrimpP%! Table1!.Threeother Theannualproduction
of pond~-
semisulcatus,turedP.japorricss
penaeidshrimp,Peruieus from19Nto1990
was
thegreentigershrimp,
P.chirtensis,
the almost 3,000
h4T,and about
200
million
Table1.Crustacean
speciesandnumberof juvenilesproducedfor sea farmingand poncl

P JApl&KlL%
]Uvenijes
areproduced
an two penaeid shrimp viruses have been
nuaHyat publicandprivatehatcheries foundinJapan
maybethatjapanrarely
for pond culture. importslive shrimpfromforeignna-
tions. Another factor is the lack of
Because
it isthemajor
crustacean
spe- comprehensive
viral diseasesurveys.
ciesusedfor seafarmingandpond Thecurrentpractice
of importinglarge
culturein Japan,research
oncrustacean numbersof seedP. japmicuswithout
diseaseshasfocused onP.jsptmicus.
To screeningfor viruses, however, will
date,two viruseshavebeenrecorded allow foreign viruses tospread easilyin
in crustaceans
in Japan.Oneis bacu- Japan. IHHNvirusisthemostdanger-
loviral midgut gland necrosisvirus ous of the potentialviral introductions
BMNV!in P. jsponicus
Sanoet al.,
andalsothemostlikelytobeimported.
1981!;theotherisPenseetttonodorr
bacu-
Fukuda ItLightner,
lovirusMBV!in P.monocle is highlypathogenicto P.japonicus
1985!andit existsin Taiwan
et al1988!.Formanyyears,BMNV Lightneret al1987!, from which
causedserious losses duringthepro- manyseedP.japmricus
areimported.
ductionof juvenileP.japorricus.

MBV,firstdetected
inJapan, Research
isthought Japan onpenaeid
shrimp
virusesin
tohaveoriginated
ina foreign hasdealt
almost
country. BMNMomoyama, entirely
with
Aftertheinitialdiscovery,
MBVwas 1991!
only one
never
foundagain
in Japan, paperwaspublished
probablyet onMBV Fukuda
because
it isnotinfectious al.,1988!.
%his
topostlarval resultspaper
summarizes
the
P.japeiacs,andveryfewP.mortar'4ott of thestudieson BMNof P.
arecultured
in Japan. only jspmicrrs
Onereason andMBVinfection
km in Japan.
ofP.mono-
in fresh squash preparations using a then cultured at a farm showed a high
dark Beld microscopewith a wet-type rate 1.4%! of BMNV infection Mo-
condenser.Infected nuclei are dearly moyama,1988!. Regular or frequent
seen in white agametthe dark back- BMN outbreaks on farms where BMN
ground due to the Increasedreflected survivors are cultured suggest that
and diffracted rays produced by the theseshrimp are a sourceof infection.
numerousvirus particlesin the nuclei to larvae.
PvhrMyama,1983!.Because this methyl
hastheadvantages
of simplicity,rapid- A Method of Experimental Infection
ity, precisionand low cost, it is the only
diagrxebc
methodusedin shrimphatch- Since there is no cell line avaHable far
eries. penaeidshrimp virus culture, a rehable
method to experimentally infect larval
Fluoreieent
antibodydiagnosis
is used J'. japmicuswith BMNV has been de-
to detectBMN-specifM.
virusantigenin veloped to faciTitatestudies on BMN.
smearsor sectionedpreparationsof the Water-borneinoculationusing the 61-
midgut. Sanoet aL 985! demonstrated trate of diseasedpostlarvaestored at
BMNVinfectionin postlarvae 18h after -80'C is one means of infection, The
inoculationby detectingQuorescence
in virulence of material frozen at -80'C
thenucleiof themidgutepithelial
cells. persists at almost the same level for at
The method was also used to demon- leastsevenyears Momoyama,1989a!.
strat» the presenceof BMNV in the Demonstration of infection in test ani-
midgutsof spawners
latentlyinfected rnalsis accomplished
by dark 6eld rni-
with the virus Momoyama,1988!. croscopy four days postinoculation
Table2! Momoyama
andSano,1988!.
Source of Infection
SusceptibleStages
Epizootiological
investigations
havein-
dicatedthatspawmers with latentBMNV Therelationship
between
ageandsus-
infectionsmay be the main sourceof ceptility toBMNVwasstudiedusing
infectionin hatcheryepizootics.Histo- the infection method described above.
logical examinationsrevealed nuclear
hypertrophy
of themidgutepithelial Fertilized
eggsandnaupliiwererefrac-
cellsin threeoutof 70spawners
exam- tory to thevirus,showingno evidence
ined.Fluorescent
antibody
techniques of infection
on the 6naldayof the
revealed
the presence of BM¹pea6e
infection
challenge.
Thezoea,mysis
virusantigenin thehypertrophied
nu-
larvae,PL2 two david postlarvae!
cleiofthespawners
Moirnqrama,
1988!. andPL4were"highlysusceptible"
to
infection
withBMNV,
exhibiting
higher
Furthermore,
histological
examination mortalityandlowergrowthratescom-
of themidgutsof youngP.japonicus paredtocontrolshrimp.PL6wereclas-
thathadrecoveredfromBMNandwere si&ed
as"susceptible;"
theygrewonly
 Viral Dseasee in

Table2. A method
for experimentally
Infecting
larvalP.~onlcus
with BMNV.

slightlyslowerthan controls.PL8and highenough,virus-ladenfoodmaynot

PL10, by contrast,were refractory,ex- be necessaryto establishbaculoviral
hibiting no mortalityand no lossof infections.
growth, althoughsomeanimalsdevel-
opedslightinfectionsSanoet al., 1985; infection Cycleof BMNV
Momayama and Sano, 1989!.
Based on the results obtained in the
The route of infection with baculo- epizootiological
and water-bornesus-
virusesin shrimpisconsideredtobeby ceptibility
studies,
thefoiowinginfection
oral ingestionof virus-contaminated cycle
was proposed Fig.
1! Momoyama,
seclliments or by cannibalismof dis- 1991!.Somesurvivorsrecoverfrom
easedshrimp Couch,1978;Lightneret BMNV diseaseand reach maturity
etal.988!estab- withoutentirelyeliminating
al., 1983!.Overstreet the virus
lishedexperimental in larval fromthebodyA!.BMNVgrows
infections inthe
thewhiteleg nucleiof themidgutepithelial
andpostlarvalP. vematrw', cellsof
shrimp,
withBaadovirus
penaei
BP!by thesebroodstockB!. Then the virus
feedingvirus-laden rotifersor brine particles
areexcreted
intotheenviron-
shrimp.In the infection
trialsusingP. mentalwater with fecesand collapsing
jape6rrs
andBMNV, wasnotadded cellsafterbeing
food released
intothelumen
to the water thus, the animalscouldnot ofthemidguttubulesfromthenecrotic
feedduring
theinoculation
period.
How- epithelial
cellsC!.Shrimp
olderthan
ever,peristaltic
movements,
whichare nauplius
become
infected
withthevi-
frequently
observedin theesophagealrusby orallyingesting
it D!.If the
region
ofshrimp,
suggest theintake
of shrimparebetweenthe zoeaandPL6
seawatercontainingBMNVparticles stagesE!,evenif a fewshrimp live
through
themouth. is normally
Thishypothesis bydefeating
thevirusattack
supported
bytheobservation
thatazo- F!,mostshrimp
become
diseased
G!
carmine in thestomach andsomewill die H!. If theshrimpare
G accumulated
lumenof shrimpplaced olderthanPL6 I!, mostshrimplive
andintestinal
in seawatercontainingthis dye Mo- normally
witha slightinfection
0!.
moyama andSano, 1989!. Someof therecovered
If thecon- well shrimpK!as
centration
of the virusin thewateris astheslightly
infected
shrixnp
I',
J! growup to be the sourceof the next treatments:
ethyletherfor 18 h at 4'C;
infectionL!, completingthecycle M!. NaCl, 25% solution within 10 h and
Some instancesof BMNoutbreks were 12.5% within 24 h; pH 1.0 within 10
causedby contamination &om other min, pH 1.5 and 2.0 within 30 min, pH
rearingtankein the hatcheryN!. 2.5 within 60 min, and pH 3.0 and 4.0
within180min Momoyama,1989c!.
inactivatian and Survival of BMNV
Withregards
tophysical
factors,BMI<f
Inactivation
of BMNVbychemical
and was inactivatedby: ultraviolet irradia-
physicalfactors,and survivaltime of tionof 4.1x 1' pW x s/cm;summer
the virus in seawater at differenttem- sunlightexposurefor 3 h; heatingat
peratures
wereexaminedby meansof 45'C within 120 min, 50 and 55'C
water-borne
infectivityexperiments. within 30 min, and 60'C within 5 min
BMNVwasinactivatedby1@min expo- Momoyama,1989d!.In sea water,
sureat 25'Cto anyof thefollowing BMNVcouldnotsurvive
longerthan4
dhhhxtants:
S-ppm
chlorine;
25-ppm d at 30 C, 7 d at 25 C, 12 d at 20'C,
iodine;100.ppmbenzalkonium
chlo- and20d at15'C Momoyama,
1989a!.
rideandbenzethonium
chloride;
30%
ethylakoho4
andO.S%
farmajinMo- BMNV appearsto be much more sen-
moyama,1989b!.The virus was also sitivetochemicals
andphysical
stresses
inactivated
withthefollowing
chemicalthaninsect
baculm~s Aruga,
1979!,
 p T ~ D V Ughtner,1988.Rod- Momoyama.K. 1989c.Toleranceof baculoviral
~ hapednudesrviruses muI-guk
glandnecrosis
virus BMNV! to
~. Dk. ~Oe 5:125-~41 ether, NaCI concentrationand pH. Fish
Laster,
IL ., A. Doubrrrvsky,
J.L.Faynter,
S.K. Pathol.24:175-177.In Japanese
withEnglish
Ssmbhiand J.G. Atherton.1981.Ught and abstract!.
electrcin
micrI apeevidenaeofbsculovirusMomoyama, K. 1989d.Inactivationof baculovi-
Infscfkin
intheprawn,Pesari»
pkhjur. Dis. ral mid-gutglandnecrosisBMN! vtrusby
Aqua@ Org,3:217-219. ultravioletirradiation,sunlight exposure,
Llghtnar,
D.V.1985.
A reviewaf thedheases heatinganddrying. FishPathol.24: 115-118.
af cdturedpenaeldshrhnpsand prawns In Japanese with Englishabstract!.
with emphasison recentdiscoveries and Momoyama,K. 1991. Studies on Baculovirai
ts. Irc Y. Taki, J.H. Prima+ra mid-gutgland necrosisBMN! of kururna
andJ.. Llokirera
Eds.!.Prearedings
af ihe shrimp, Penaei»jspariicus.BuII. Yamaguchi
First International Conference on the Cid- Fref. NaikaiFish. Exper.St. 20: 1-91. In
tureofPenaeid
Prawns/Shrimps.Ilokla
City, Japanese!.
Phlpplnss,4-lDecember,
1984.pp.79-103. Mamoyama, K. andT. Sano.1988.A method
Llghtner,
D.V.,R.P.Hedrick,
J.L. fryer,S.N. ofexperimental
infection
ofkurumashrimp
Chen, LC. Uao and G.H. Kou. 1987. A huvae,Peru»i»japoriicus
Bate,with baculovr-
survey
ofcultured
penaeid
shrimp
in Tai- ral rnid~t gland necrosisBMN! virus.J.
wanforviralandotherimportant
diseases. Fish Dis. 11: 105-111.
FishPakhol.22:127-140. Marnoyama,
K. andT. Sano.1%8.Develop-
Ughtner,D.V., R.M. Redrnan
andT.A. Bell. mentalstagesof kurumashrimp, Peiureus
1%3.Observatians
anthegeogra
hicdistri- japorrici»
Bate,susceptiHe
to bacdoviralmid-
butiorl
pslhogenesisand
morph ofthe gutglandnecrosisBMN!virus. J. Fish Dis.
bsculovtrus
framPresa»srosofori
F us. 12: 585-589.
Aituaculture.
32:209-233. Overstreet,R.M., K.C. Stuck, R.A. Krol and
M K. 1981.Studies
on infectious W.E. Hawkins.1988.Experimental
infec-
~k glandriecrasis
ofkuruma
shrimp tions with Baculopirus
pcnseiin the white
Perks'
japariici»!-1,
OCCurrenCea
andsyinn- shrimpPriuieus
aarrrurruei
CrustaiwaiDe-
toms.BulL Yamaguchl
Fmf. NaikaiFish. capoda!
asa bioassay.
J.WorldArluaculkure
Exper.
St.8:1-'B.In Japanese!. Soc. 19: 175-187.
Momoyama, K. 1983.Stiuliesonbaculoviral Sano,T.,T. Nishirnura, H. Fukuda,T. Haya-
mki-gukgland necnxks ofkururna
shrimp shida and K. Momoyama. 1984.
Baculoviral
Prnarua
japrxricr»!-III,
Presumptivediasnos- mid-gutglandnecrosisBMN!of kuruma
tictechniques.
FishPathol.1F:263-26I.
In shrimpPrnarus japrnicur!
larvae
inJapanese
JapanesewithEnglishabstract!. intensiveculturesyskems.Helgolander
Meer-
Momoyarna,K. 1988.
Infection
source
afbacu- sunters. 37: 255-264.
loviral
mid-gut
gland
necrrisLi
inmass
pro
ductionofkurums shrimp
larvae,Psnarus Sano, T.,T.Nishimura,
shidaand K.
H.Fukuda,
Momoyama. 1985.
T. Haya-
Baculoviral
jqpsrici».
FishFakhol.
23:1%-110. InJapa- infectivity trial on kurumashrimplarvae,
nese withEnglish
abstract!.
Momayama,
K,"3989a.
Survival
ofbaculoviral Perrser»japorucus,
EllisEd.!. Fish
of different
and
ages.
Shellfish
Iu: A.E.
Pathology.
mkk-gut glandnecrosis
virusBMNV! in Academic Press, London. pp.3974.
~ tissues andinseawater. FishFathol. Sano,T., T. Nishimura, K. Oguma, K. Mo-
24:17%.MI,InJ with Enghh ~!. moyama and N. Takeno. 1981.Baculoviral
Momoyams,K.1 9b.Viruadaleffect
ofsome infection
ofkuruma shrimp,Periaeus
j aponi-
disinfectants
onbaculoviral
mid-guk
eland cusin Japan.
FishPathol.15:185-191.
nectrsiis
BMN!virus,
FishFathal.
24:549.
inJapanese
withEnglish
abstract!.
Contributed Papers-

Bacterial Diseases
 Bacterial Infectkes af P. moncWNi 197

Table 1. Isolation of bacteria from hepatopancreata of mor-

in Taiwan. Out of 274 isolates, 201 were For the inoculationexperiments,0.02

V.dam- ml of V. hathi or V. nereisconcentra-
vibrios Table'I!. Vibrioharveyi,
V. tubiashii, tion 2.8 x 10' or 1.3 x 10 C.F.U./ml,
sel, V. nereis,V. znclnificus,
V. anglillarum respectively!
andV. purahaemolytims wasinjected
with a G26
were most abundant in the hepatopan- needleintramuscularlyvia thejunction
creata of culturedP. irxm0don.
betweenthe first and secondabdomi-
nalsegments.
Similarly,
0.01rnlofeach
bacterial
solutionwasinjectedinto the
Pathogenicity
Study hernocoel
ofexperimental
shrimp
atthe
abdominalsurfaceof the fifth segment.
Meth04s
Forty
shrimp
were
used
in thisstudy;
therewereten shrimpin eachgroup.
BecauseV. harveyiand V. riereiswere
Additionally,
twogroups
oftenshrimp
among themostabundant
bacterial inoculated
spe- via with0.02ml of0.85%
NaCl
muscleor hemocoel,
wereusedas
cies,theyweresuspected
ofbeingasso-
controls.
ciatedwith disease
outbreaks.
Iherefore,
thesetwobacteriawereusedin a patho-
genicity
studyusingartificial
inocula-Results
tion techniques.Immersionand Significant
mortality
wasobtained
in
insulationtechniques
wereemployed
to theexperimental
groupsgable
3!.Vi-
inducediseasein experimental
shrimp. brioharveyi
or V.nereis
injected
at a
concentration
of1.3x 10C.F.U.lml
or
Fortheimmersion
experiment,
shrimp moreinitiated
significant
mortality
in
weighing
10g each
were
immersed
in experimental
shrimp
within
fourdays
2.8 x 10 colony forming units afterintramuscular
orhemocoelinjec-
C.F.U.!/ml
ofV.haroeyi
solution
for3 tion.Cumulative
mortality
after
hemo-
min.Tensimilar-sized
shrimp
wereim- coelandintramuscular
injection
of V.
mersed
in 0.85%NaClfor 3 minasa harveyi
ispresented
inTables
4 and
5.
control.
 Chen k and Kotr

Table
2.lsola5m
ofYibrfo
sp.fromhepatopancreata
ofmor-
bidPaelevsmomonduringi 988-t
990.

Table
3.Mortality
inexperimental
andcontrol
groups
aflerhem
t' f Yb6o
ha ' S ' N 770713
Asb VS

at the concentration indicated. rompswere inoculated with 0.02 ml of bacteria

shrimpwere fixed in Davidson'sfixa- Large numbers of densely stained
tive as describedby Bell and Lightner hemocytesand bacteriawere found in
988!. Vibrio tiareeyi was recovered the spongy connectivetissue of the
from all experimentalshrimp; the his- stomach Figs. 7-8!.
topathological changes were as fol-
laws. Heart:Histopathological
changesin the
heart tissue of infected shrimp were
Gills: Histopathological changes in only observedin a few cases. Densely
gills were observed seven days after stained hemocytes, macrohernocytes
intramuscular or hemocoel inoculation and bacteria were found in the abnor-
of V. haraeyi.
However,significantgiU mal hearts Figs. 9-'11!.
abnormalitieswere observedamong
the shriinpimmersedin V. harveyisus- Muscle: Muscle necrosisand hemocyte
pensionfor four days. encapsulated nodules were found at
the injectionsites Fig. 12!.
The main pathologicalchangesin the
gills wereswellingof secondary
gill
Heyatopancreata:The hepatopancreas
filamentFig.1!with an increase
in the
was affectedmost severelyafter either
numberof denselystainedgranules immersionor injection of V. harveyi.
Pig. 2!. The invasion of bacteriawas Four daysafter infection,hepatopan-
observed
insidethesecondary
gill fila- creata became reddish and sinuses ex-
rnent, which inay initiate necrosisof panded significantly Fig. 13!.
thetissueRg.3!.Abnormal hemocytes Multiplicationof bacteria
in hepatopan-
andeosingranulocytes anddamaged creatocytes was also observed in the
lymphoid tissuein the baseof secon- histopathological sectionsFig. 14!.
darygill filamentsarecommon syn- Bacteriawere also found in the basal
drornes resulting from bacterial membrane,sinus, and lumen; this
invasionof shrimpgill tissue. couldleadto degeneration
or lysisof
hepatopancreatocytes
Figs. 16-18!.
Lymphoidorgan:Seven
daysafterbac- Denselystainedhemocytes
and bacte-
terialinfection,
eithertheOkaorgan ria were also found in the infected
Fig.4!orlymphoidtissues
in thegills hepatopancreata
7 to10daysafterin-
Fii. 5!,submuscuiar
layerofthestom- fectionFigs.'15-18!.
Hemocytes or bac-
ach Fig.6!, or subcuticuiar
structures teriaaggregated
in the necroticareasof
wereseriously
affected.
Necrotic
lym-hepatopancreatocytesor in thehepa-
phoidnodules
withmass
aggregations
topancreatic lumen, where fibrella
of bacteria
werefoundin theinfected structuresor granuloma-likestructures
tissues
ofmorbid shrimpFigs.46!. wereformedFigs.18-21!.Fourteento
Stomach andmidgut:Fourdaysafter 21 daysafterinfection
withbacteria,
the
inoculationof bacteria,the stomach number ofnecrotic areas
orgranuloma-
andmidgutexhibited signsofdisease. likestructures in hepatopancreata
in-
creased
significantly.
 Bacterial Infections of P. monodon 201

Figures 1-3. Histopathoiogicalchangesin the gills of Penaeusmonodonseven days after artificial

infection with Vibrio harveyi. Figure t. Swelling of secondarygill filament. Note: dilation of basal
membranefrom epithelial layer arrows!. Figures2-3. Densely stained granulocytes arrows! and
bactem Fig. 3; white arrow! in secondary gill filaments. G: Gill.
Figure4. Invasionof V. harveyiinto the lymphoidorganof P. monodon.Arrowsindicatenecroticlumen,
non-tailed arrows indicate normal lumen.
Figures5-6.Lymphoidtissuesin gill Fig. 5! and stomach Fig. 6! of P, monodoninvaded by V. harveyi.
202 Chen, Huan and Kou

FiguresT-8.HemocytesFig.7!andbacteriaFig,8!aggregatein
thelooseconnective
tissueofintestine
Fig. 7! andintestinalcavity Fig. 8!.
Figures9-11.Histopathoiogical
observations
of heait of P. morodoninfectedby V. harveyi.Note:
presenceof hemocyteencapsulated
noduleFig.9,arrow!,andnecroticareawithaggregated bacteria
Fig 11!. Figure 10showsphagocytosisin hemocytes.
Figure12,Hemocyte encapsulatednodulein muscleof bacteriaatinfectionsite arrow!.
Figures13-14.Histopatholoyical
changesof P. monodonhepatopancreata afterartificialinfectionwith
V. harveyi,Note:&pensionof sinus Fig. 13,arrows!andinvasionof bacteriavia hepatopancreatic
lumensinto hepatopancreatocyteFig. 14,arrows!.
Figure15.Denseiystainedhemocytes
in hepatopancreata
of V. harveyi-infected
P. monodon.
 Bacterial Infections of P. monodon

Figures
16-21.
Various
pathological
changesin
hepatopancreata
ofP.rnoredon
infected
byV.harveyi.
Note:aggregationof denselystainedhemocyfes.
Figures16-18,Obtained
froma morbidshrimp;mosthepatopancreatocytes
havebeendamaged.
Figures19-21.Obtainedfromlive,activeshrImp.Note:Formation
of granuioma-like
structure,
 Chen, Huan
Discussion
After a survey of the diseasesof cul-
tured P. monodonin Taiwan, Lightner
988! reported that the major bacteria
isolated from morbid shrimp were Vi-
brio spp., Pseudomonas sp. and Flavo-
bacterium sp. Our resultsalsoshowed
that Vibrio spp. are the dominant
pathogenic bacteria in cultured giant
tiger prawns in Taiwan. The results
further suggest that Vibrio spp. play a
major role in the initiation of mortality
in cultured giant tiger prawns in Tai-
wan.
Lightner et al. 988! also reported that
V. alginolyticus,V. angaillarumand V.
parahmrmlyticuswere the main species
isolated from morbid shrimp collected
in 1986. However, our results showed
that V. harveyi,V. damsela,V. nereis,V.
fubiashii, V. anguillarum and V, para-
haemolyficuswere the major species
found in morbid shrimp collectedfrom
1988 to 1990.
Although several factors may be in-
volved in a massmortality of cultured
shrimp, our results,the epizootiologi-
cal study incorporatedwith a patho-
genicity test and histopathological
observations,suggestthat V. nereisand
V. harveyiare the important Vibriospe-
cies contributing to the occurrenceof
mass mortality of cultured giant tiger
prawns in Taiwan. Vibrioharveyiwas
also reported to significantly affect the
survival of giant tiger prawn larvae in
a hatchery in the Philippines Pitogo,
1988!.
and Kou
In a pathogenicity study performed on
Penaeus japonicus, Egusa et al. 988!
reportedthat 72 h after inoculationof
Vibriosp., the lymphoid tissuesin vari-
ous organswere seriouslyaffected,but
very little necrosiswas found in lym-
phoid tissue. However, in our study of
P. monodon,hemocyte encapsulated
nodules as well as necrosis of internal
organs were frequently found in in-
fected shrimp. More hemocyte encap-
sulated nodules were found over time.
Serious hepatopancreatic necrosis may
extend from the proximal area to the
distal area of the organ. This suggests
that the route of invasion may be from
the stomach to the primary duct and
the secondary duct, extending to the
hepatopancreatic lumen. We also
found that bacteria could multiply in
the cells of various tissues.
This is the first report of the initiation
of pathogenicityin P. mormonartifi-
ciallyexposedto Vibriospp. Our results
may have important implications for
further studies of the bacterial diseases
of cultured P. monodon,especially in
areassuch as immunology and chemo-
therapy. Work is in progresson the
development of bacterial vaccines for
cultured giant tiger prawns; those re-
sults will be presentedelsewhere.
Acknowledgments
This work was supported by a grant
from the U.S. Department of Agricul-
ture Grant No. FG-TA-111! and the
Council of Agriculture and National Sci-
enceCouncil of the Republic of China.
 Bacterial Infections of P. morxxhn 205

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Baumann P., L. Baumann and M. Mandel.
1971.Taxonomyof marinebacteria:the ge-
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Baumann, L., P. Baumann, M. Medel and R.D.
Allen. 1972. Taxonomyof aerobicmarine
eubacteria.J. Bacteriol.110: 402-429.
Baumann, P., L. Baumann, S.S. Bang and M.J.
Woolkalis. 1980. Reevaluation of the taxon-
omy of Vibno,Beneckea
and Photobacferiurn:
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Baumann, P., L. Baumann, S.S. Bang and M.J.
Woolkalis. 1981. In list No. 6, validation of
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Baumann, P. and R,H.W, Schubert.
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Volume I!. Williams k Wilkins. Balti-
more/London,pp. 516-550.
BeU,T.A. and D.V. Lightner.1988.A Hand- Lightner,D.V. 1988.Vibriodiseaseof penaeid
bookof NormalPenaeidShrimpHistology.
SpecialPubhcationNo. 1, World Aquacul-
ture Society.Baton Rouge, LA. 114 p.
Brenner,D.J. 1984.Bergy'sManualof System-
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Wilkins.Baltimore/London. pp. 408-516.
Cheng,W. 1989.Isolationand characterization
of Vibrio damsela infections for cultured
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Egusa,S., Y. Takahashi,T. Itami and K. Mo-
moyama.1988.Histopathology of vibriosis
in thekurumaprawn,Penaeus japonicus Bate.
Fish Pathol. 23: 59-65,
Glen, R., R.S. Wheeler, and R,K, Sizemore,
1980.Characterization
of brownspotdisease
of gulfcoastshrimp.J,Invertebr.Pathol.36:
255-263.
Huang,S.L. 1989.Bacterial
infectionin cultured
black tiger prawn Penaeus monodon! M.S.
Thesis. National Taiwan University, Taipei,
TBlw an.
H.M. Sommers. 1983. Colour Atlas and
TextbookDiagnosticMicrobiology.2nd ed.
J.B.Lippincott,New York.
Krieg, N.R., J.G. Holt, R.E.G. Murray, D.J.
Brenner,M.P. Bryant, J.W. Moulder, N.
Pfennig, P.H.A. Sneath and J.T. Staley
Eds.!. 1984.Bergy'sManual of Systematic
Bacteriology.Vol. ! WilliamsdxWilkins.
Baltimore/Landon. pp. 408-53$.
Lee, J.V., P. Shrend, A.L. Furniss and T.N.
Bryant.1981.Taxonomyand descriptionof
Vibnofluvialissp. nov., synonymgroupF
vibrios,groupEF6!.J. Appl. Bacteriol.50:
73-94,
Liao, I.C., C.E. Shyu, C,Y. Liu, S.C. Chou,
Y.W. Liu and C.F. Chang Eds.!, 1989.
Extension Handbook on Prawn Disease Pre-
vention.Keelung,Taiwan. In Chinese!.
Lightner,D.V, 1983,Diseases
ofculturedpenaeid
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book of Mariculture. Vol. 1, Crustacean
Aquaculture.CRC Press,BocaRaton,Flor-
ida. pp. 289-320.
shrimp. In: C.J. Sindermann and D.V.
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2nd ed. Elsevier,New York. pp. 42-47.
aticBactetiology.
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Baltimore/London.pp. 550-575.
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wan. pp. 172-179.
 Contributed Papers

Diagnostic
Prcxedures
230 Brack

Theconstraint
of diseases
on thepro- ment of live shrimp wittun
ductivityand economicviability of between shrimp farming regions
shrimpfarmingis one negativeout- has resulted in the transfer of pu-
comeof intensi6cation
Lightner,
1983, tative shrimp pathogens, rn~
1988;
Nash,1990; Overstreet,
1990;
Wy- theshrimpviruses.Thus. cultured
ban and Sweeney, 1991!, Where shrimp populabons have been ex-
shrimp
arecultured
semi-intensively
to posedto "new" pathogens, some
intensively,
epidemicsndendemicdis- of which may be capable of caus-
eases
that resultin mortalitythat mg sigzuficant diseases rn these
appreW or exceed50%are common- new hosts.
place,
In oneAsiancountry
withinthe
pastfiveyears,
theshrimp
farming o Althoughevaluationof stoclcq~
industry,
withan annual
production jty is routine in many region .
valuedat several
hundredmilliondol- there are no uniform standards
lars,virtually
collapsed
duetodiseases for assessing
animalquality i-e-
Un,1989!
ofapparently
complexand, for pathogenand nonpathoge'n-
possibly,
unclear
cause.
Furthermore, related health! when stocks
in mostfarmingregionsthereis little transferredbetween the different
indication
thatin theinunediate
future
culturephases maturation-hatch-
onecanexpect
to seea signkBcant
re-
docthnin disease-related
losses
onin- ery, hatchery-nursery,
nursery-
growout!.
tensively
managedshrimpfarms,
Why
isthe
shrimp
farming
industry
in ~ Thelackof uniform standards and
manyareas
faced
withsignificant
dis- routinemonitoringprotocolsto as-
ease
problems?
Several
contributing surenutritionalqualityof shrimp
factorsare: dietsoncethe feedsare in the
farmer's hands.
' The
widespread
use
ofwildmught
~p. oroffspnng from ~ Failureto applyroutinemonitor-
derived
wild~ught forstocking ingprotocols
shrimp, toassesspondwater
hatcheries
andfarms.
Thenatural qualityconditions
in shrimp
ponds.
suite
ofpathogens
present
in the
wildstocks
arecontinually
intro-
duced
intohatcheries
andfarms. ~Overstocking
of shrimpponds
relative
totheability
ofthesystem
~The
lack
ofuniform
standards
for tosuccessfully
support
growthof
p between a high
biomass
ofshrimp.
widely
divergent
geograpNcal
re-
gionsseeSindermann,
tNs Thevirtual
indiscriminate
useof
urne!.
AsLightner
990!h as drugadditives
in shrimp
ciiiture
ocumented,
theextensjv
reive move- setbngs
withifisome
regions
Lin,
1989!.
 Current Shri
While it is quite apparentthatdiagnos-
tic xnethodsby themselveswill not
solve the disease problems facin.g
shrimpfarming,it is alsoobviousthat
methodsof detectingbiotic and abi-
otic agentswill contributeto the proc-
essof implementing practical
control
strategiesfor many diseases
facedby
the shrimp farmingindustry today.
Historically,interest
in thediseases
of
marineshrimpand pathogendetection
xnethodsprecededthe developxnentof
shrixnpaquaculture Overstreet,1973;
Couch, 1978!.At least in part, this was
due to a considerableconcern over the
potentialimpactof disease oncommer-
cialshrimpfisheries. The development
of shrimp farxning hasservedto boost
theinterestin the diagnosis
andcontrol
of shrixnpdiseases with economiccon-
siderations.
The purposeof this paper is to review
the presentmethodsusedto detectthe
known biotic and abiotic agents associ-
atedwith shrimp diseases,
and to dis-
cussselectedissuesconcerningdisease
diagnosisas this pertains to shrimp
farming.
Table 1. Causal factors associated with dis-
eases of cultured penaeid shrimp.
Dia nostic Methods 211
Disease Causation and
General IssuesConcerning
Diagnostics
A varietyof biotic and abioticagentsare
associatedascausalfactorswith shrimp
diseasesTable 1!. Interactions involv-
ing multiple factors Overstreet,1990!,
as illustrated in Table 2, are postu-
lated, but studies to quantify these
relationships
haveyet to be conducted
orpublished.
Nevertheless,
knowledge
gainedthrough studiesof diseasesin
terrestrial animal farming systems
confirm that interactions are com-
monplace between causal factors
Martin et al., 1987!,and that under-
standing contributing factors may
leadtoimproved,morepractical
meth-
ods for disease control. Thus, it be-
hoovesthe shrimpfarmingindustry to
recognizeand understandcomplex
etiologicassociations
for thediseases
of
farmed shrimp.
One interesting feature of penaeid
shrimp is the rather common occur-
renceof multiplebioticagentsputative
pathogens! within a given host or
populationunit.Someexamples ofthis
phenomenon arepresentedin Table3.
An impact of multiple pathogens
on
shrimpdiisease
diagnosisis to compli-
caterecognitionof the most important
causalagentfor the disease.Dluminat-
ing theserelationships
in thecontextof
the diseaseworkings is importantbe-
causewithout this knowledge,a patho-
genofminorsignificance
xnaybefalsely
attributed to be the causeof a major
dinical disease outbreak.
NotetRF Saadednoptsaf,HFY ~hepatopanaeNcparvo-likevirus,h4BV
prrraeasmonafonbaculovirus,
BIO - m4lhe virus,andGNS - But andnervesyndrome.

Hate: MWV-
vauo0aNon virus
us,hypodermal
andhematopo<etic
necrosis
virus,LOVV lymphoid
org&

An approach usedto clarifyassocia- rialcolonies,

etc.!considered asdiag-
tionsbetween bioticagents
andclinical nosticindicators
for theagent,maybe
diseasesis determinationof infection countedto derivea severityestimate.
prevalence
in population
samples,
or Several
examplesof theapplication
of
quantitativeestimatesof theabundance infection
prevalence
or lesionseverity
of putative
pathogens
severity
index estimatesare listed in Table 4.
estimates!within target tissuesof
shrimpwithdisease
signs.Often,the Thedetection
of specific
bioticagents
numbersofa particular
pathogencan- in samples
of shrimptissuescanbean
not be determineddirectly,and intermediate
steporanendpointin a
structural
changes lesions!,
usually diagnosticprocess,depending
onthe
microscopic
i.e.,viralinclusion
bodies, purpose of theex~ation seeTable
hernocytic
nodules surroundingbacte- 5 fora list
ofsome reasons
toperform
 Current Shri
diagnostictestsfor shrimpdiseases!.It
may alsobe illustrahveto note that the
agentrecognized by a diagnostician is
often the one he/she has been trained
to see.
Current Detection Methods
for Biotic Agents
Viruses
It is well documented that penaeid
shrimp are hosts for a variety of intra-
cellular prokaryotic agents, of which
the most varied, widespreadand sig-
nificantas pathogensare the viruses.
Lightner gn press! lists 'll types of
viruses that have been demonstrated
from tissuesof penaeidshrimp. Some
of these viruses are reportedto be the
direct etiologicalagentsof economically
significant diseasesin specific shrimp
species.Although the detection meth-
Oia 213
ods for viral agentsof shrimp have
recently been reviewed comprehen-
sivelyby Lightner and Redman991!,
a fewpointsconcerning
thesemethods
are discussed here.
Techniquescurrently used to demon-
strateviral infections of penaeidshrimp
includeepidemiological
featuresand
grosslydiscerniblestructuralchanges,
wet-mountmicroscopywith or without
staining, histopathology,eledronmi-
croscopy,
specificantibody
methods, a
DNAprobeandi' vibocultureona fish
Table5. Someapplicationsfor diagnostic
proceduresdevelopedfor agents/diseases
of farmed shrimp.
 21'

Table6. Somelight microscopymethods Forthe majorityof the hepatopancrea-

for rapktdetectionof penaeidbaculovirai tic baculoviruses,patent infection can
infectionsin freshtissuemounts hepa- be demonstratedrapidly in wet-mount
topancreasor midgutfeces!.
impressionsmearsof hepatopancreas
tissues or fecal strands. The use of
selectedstaining procedures has been
reported to improve the visibility of the
diagnostic viral occlusion inclusion!
bodiesin hepatopancreastissue prepa-
rations Table6!. For baculoviral midgut
gland necrosisvirus BMNV! infection
of Penaeus j apcrricrrs,rapid demonstra-
tion of the pathognomonichypertrophic
nuclei of virus-infected hepatopancreas
epitheliuin is reported using dark field
optics Momoyarna and Sano, 1988!.
Additionally, direct demonstration of
baculoviral antigens by a fluorescent
antibody method is applied in shrimp
hatcheriesin Japan for the definitive
ceilline. The majorityof the penaeid diagnosisof BMNV Sano et al., 1984!.
virusinfections arecurrentlydetected
by light microscopydemonstration of An enzyme-linked irnmunosorbent as-
structuralchangesi.e., uniqueinclu- say ELISA! technique was also re-
sion bodies within cells of specific ported for detection of penaeid
target organs! characteristicfor infec- baculoviruses Lewis,1986!.Theauthor
tion by the viral agent. While, for the indicatedthat detectionof 10-ngviral
rriost part, these detection methods protein/100ii.Lwas possibleusing this
allowfor therecognition
of the pres- EUSA method. However, further re-
ence of viral disease because the ports on the application of this conven-
abundance
of the agentis probably ient and useful technique have not
quite high in the specifictargettis- beenforthcoming.
sue s!,thesetestmethodsmaylackthe
sensitivityto detectlatentor pre-patent Histopathology
diagnosisis presently
carrier state infections. Furthermore, the mostreliable measure for IHHNV
available
methods
for detecting
shrimp and HPV infections Lightner, 1988;
virusesdo not allow the diagnostician Brock
andLightner,
1990;
Lightner
and
to discriminate
betweenantigenicaHy Redman,1991!.Bell et al. 990! de-
distinct strains, or discern virulence scribedthe applicationof a nonlethal
differencesbetweengeographically biopsy/histopathology techniquefor
diversepopulationsof a particular detection
of subclinicalHQ&4V infec-
virus.
tionofadultP.vrirrnrrrrrrei.
Morerecently,
 Current Shri
TableT. Examplesof chemicalenhancement
of the prevalenoe
DNA probeshavebeendevelopedfor
IHHN and BP viruses see Lightner et
al., this volume!.
While molecularand cell culture proce-
duresareunderdevelopinent,methods
that rely on conventionaltechnology
have been devised by researchers to
improvethe sensitivityof existingmeth-
ods for detection of shrimp viruses.
Theseprocedures,applied for the dem-
onstration of specific viral agents, in-
vade virus infection enhancement and
amplification,conspecificshrimp chal-
lengebioassay,indicatorshrimp useof
a different shrimp species! challenge
bioassayand iri vitro growth in fish cell
culture systems.
For shrimp viruses, enhancement and
amplification methods are applied to
detect latent or carrier state infections.
Characteristically, this has been done
in groups of shrimp introduced from
distant geographiclocations or targeted
for transfer onto farins using specific
pathogen-free SPF! shrimp stocks.
Enhancement strategies generally in-
volve prolonged holding of the test
Dia nostic Methods 215
of BPinfectionin the
population in an isolation or quarantine
facility with or without intentional
crowding of the shrimp Lightnet and
Redman, 1991!. Also available, as an
experimentalapproach,is infectionen-
hancementthrough sublethal exposure
to selectedpesticidesor heavymetals.
An exampleof the effect of chemical
enhancementon the prevalenceof pat-
ent BP infection in Penaeusdtiorarum is
given in Table 7.
Lightner et al. 985! reportedthe ap-
plication of indicator shrimp bioassay
with the highly sensitive speciesP.
sfylirostris to detect IHHN virus in
carrier state-infected P. vannarriei
populations.An exampleof a field
application of an indicator shrimp
IHHNV bioassayis given in Table8.
Resultswerenegativefor IHHNV infec-
tion by direct histologicalexamination
of tissues from P, mmrurmeithat were
stockedas SPFjuveniles into ponds
thathad previouslysupportedIHH&W-
infectedpopulations;but histological
examinationyielded IIiHNV-positive
results when P. stylirostriswere exam-
ined aftergrowoutin similarpondson
this site.
 8.Anexample
ofttteuse
ofP.styNestds
indioator
shrimp
asa bioassay
applied
for

Y
saatplad
for hlstapathologieal
exaadnatton
whenthepondswereharvested.

Conspectficshrimp bioassaysystems live BMNVin tissuesuspensions.This

for virus detectionwere developedto procedurediffered from that of Over-
detectHvevirus in samplesof shrimp street et al. 988! in that susceptible
tissueor othersubstrates,
Forexample, larval stageswere subjected to bacu-
Overstreetet al. 988! describedan lovirus infection by the water-borne
experimentaldetection procedurefor route rather than through bioencapsu-
live BPin tissuesamples.Artemisnau- lation in live feeds.
pliiwereincubated
withtissue
suspen-
diona from samples of unknown Lewiset al. 992! applieda novel tech-
infectiveBPstatus.Thenaupliiwere niqueto estimate theefficacyof shrimp
subsequently
fedtomysisstageP.eee- bacuiovirus decontamination methods
Ilsrari,
andaftervariouS
inCubationpe- in shrimpfarmsby measuring,after
riods,samples
ofhepatopancreas
tissue exposure to various disinfection treat-
wereexmdned microscoplcaHy
forpat- ments, the viability in an insect ceH
ent BP infection,1%eseresearchers culture asSaysyaternof an oCCIuded
laterappliedthistestmethod
to assess insect
baculovirus
Autognapha
catijor-
BPviabilityfollowing
exposure
to a
tria!impregnated
onto filter paper
variety
ofphysical
andchemical
agents StrIPS.
selected
for theirpotentialusein the
eradication
of thisvirusfrom
oms hrimp
culturepondsandfacultiesLeBlanc Lu etdaL 991! screenedtissues of
sk' .pforviruses
usinga vari-
andOverstreet,
1991a,
b!.
ety of fishcelllines.ln tissueextracts
imilarly,
SimilarlMomoyama 988! from
andSano these
P. BAJliNstrks
andP. zPNrIrrgrrrei,
investigators
successfully
isolated
reported
transmission
andspread of
BMNV viawater- a newrhabdmrirus
inlarvalP.juponims on an estabbshed
borneexposure of tissues fish
to extracts ceQ line,epithelioma
cyprini EPC!.
papulosurn
containinginfectiveBMNV.These
StudieS
demOnStrated
thata COnSpeCifIC
AU of the above
P.japcmicus
larval
bioassay
syst
y system
e examples represent
could be
be used
u toassess
thepresence
of creative
approaches
that research
ers
have app
liede oto the problemof virus
 Current Shrt Die nostra Methods 217

detection
in shrimptissuesor shrimp andabnormal
culturedshrimpP. vart-
farms. While there have been soxne rurmei, P. stylimshxs,P. manodorx,
P.
gains reportedin developingshrimp chixtartsis,P. japonicus!,
Thehistological
cellculturesforisolatingshrixnpviruses changes
thatsignalthe presence
of an
Chen and Kou, 1989!, these systems infectionof the lymphoidorganare
are not ready to be usedfor the routine easyto recognizemicroscopically.Pre-
detectionof penaeidviruses. Thus, the cisexnicroscopicdescriptions
of subtle
need remains current for the use of differencesin thesestructuralchanges
shrixnp bioassaysand the other tech- are now beingdocumented,but they
niques foi' detecting shrimp viruses. may not allow diagnosis
of specific
viruses,due largely to the nonspecific
Detection is problematicalfor the reo- natureof the shrunphost'sresponsein
like viruses because these agents re- the lymphoid organto viral invasian.
quire direct visualization with a Thus, ultrastructural studies wi11 be
transmissionelectron microscope. His- necessary to distinguishwhichvirusor
topathologicalstructuralfindings, the virusesmay be present,or not present
magenta-staining
cytoplasmicinclusion in pathological lymphoidorgantissues.
bodies within hepatopancreasepithe- Culture of the penaeidrhabdovirusin
lium Lightner and Redman, 1991!at- EPC cells is an effectiveapproachfor
tributed to reo-like virus infection in detectingthis lymphoid organ agent
shrimp, are subcellularlesionsof un- Lu et al., 1991!.Furthercomplicating
known sensitivity and speci6cityfor the issue is the dearth of inforxnation
the detection of these viruses. Thus, concerningthe lymphoid viruses as
determinationthat a group of shrimp agentsof disease.
In thisvolume,de-
are free of a penaeid reo-likevirus tailed informationis presentedabout
cannotbe presentlyassuredin a tixnely severalrecentlyrecognized
lymphaid
or reliablysensitivefashionseeLotz, organvirusesof P.monodori
culturedin
this volume!. ThailandseeFlegeletal., thisvolume!.

A similarenigmaexistsfor a galaxyof The continuedfocuson developingim-

viral agentsthat haverecentlybeen proved
diagnostic
methodologies
for
identified associatedwith histopatho- penaeid
viruses
isaclear
priority
need.
logical
lesions
in thelymphoid
argan
of A reliable irxvitro ceHculture systemis
shrimpOwenset al., 1991;Lightner, atopissue,
aswellascontinued
efforts
In press;
Hegel
etal.,thisvolume!.
The ta evolvehighly sensitive
molecular
cellular changesfoci of hyperplasia methodsfor detectionof viral antigens
and hypertrophywith cytoplasmic or nucleic acid.
vacuolationand variousforms of inclu-
sion bodies!of the lymphoidorgan, Rickettsia
which aresuggestive
ofviralinfection,
have been frequentlyencountered Rickettsia-like
andchlamydia-like
agents
normal areknown from farmedshrimp in some
rock, unpubl.!in clinicaUy
218
regions Chong As a futureresearchpriority in penaeid
inAsiaandtheAmericas
and Loh, 1984;Lightneret al., 1985;
Andersonet al., 1987;Brock,19N; Krol
et aL, 1991;Lightner, In press!. Eco-
nomically significant syndromes of
pond-reared
P. rrsmdonAndersonet
aL, 1987!and pond-rearedP. tamrMmei
Lightner,In press!have beenattrib-
utedto infectionby rickettsia-hkeagents.
Thepenaeidrickettsia-like
agentshave
not been cultured iri vitro on arti6cial
media or in cell culture systems. Nor
havemolecularmethodsbeendeveloped
for the identificationof these agents in
shrimptissues.
Histopathological
meth-
ods are used to detect penaeid rick-
ettsia-likeagents,but the sensitivityof
thisprocedure is limited.to recognifion
of moderateto heavy infections for the
agentsthat form microcolonieswithin
the cytoplasmof specifictarget tissues,
or by thechamteristiccellularpathologic
response,suchas thatwhich occurs in
shrimp affectedby the Texasnecrotiz-
inghepatopancreatitis syndrome.When
the abundanceof rickettsia-likeorgan-
ismsis low, histopathologicaldetermi-
nation is uncertain, and detection by
transmission electron microscopy ex-
aminationis the current method sug-
gestedfor theseinfections Krol et al.,
1991!. Special stains for bacteria,in-
cludingtissue Gram stain or Giemsa
methods, enhancethe visibility of the
penaeid rickettsia-like agents in his-
topathologicalpreparations,and alsoin
smears from infected tissues. Giemsa-
stainedimpressionsmearsof selected
target tissues may have potential as a
clinicaltechniquefor rapid detectionof
penaeidrickettsia-likeorganisms.
Brock
diagnostics, development of immu-
nologicreagentsare neededfor rapid
detectionof early or subdinical infec-
tions by the rickettsia-likeagents of
farmedshrimp.
Bacteria
Bacteria are associated with endemic to
epidemicdiseasesof cultured shrimp
Sanoand Fukuda, 1987;Liu, 1989!.On
the basisof morphological appearance,
several forms of bacterial infection are
recognized in farmed shrimp. These
include filamentous and nonfilamen-
tous cuticle fouling Lightner et al.,
1975!; shell disease Cipriani et al.,
1980! and colonization of internal or-
gansby bacteria that range from focal
lesions to fulminating septicemia
Lightner, 1988!.In manyfarmingre-
gions, diseasesattributed to infection
by Vibrio spp. are consideredto be the
most common and significant infec-
tiousproblemsimpactingshrimpfarm-
ing Oohnson, 1978; Lightner, 1983,
l985, 1988; Takahashi et al., 1985; An-
derson et al., 1988; Baticados, 1988;
Nash, 1988, 1990; Boonyaratpalin,
1990; Lavilla-Pitogo, 1990; Lin, 1989;
Mohney et al., 1991!.
Vibriosis is reported to be caused by a
varietyof Vibriospp., but other Gram-
negativegenera have also been impli-
cated as causative agents of septic
syndromes of penaeid shrimp Light-
ner, 19N!. Other classesof biotic agents
viruses, rickettsia, fungi and/or proto-
zoa!; multiple speciesof bacteriarecov-
eredfrom moribund shrimp in a given
 Current Shri
episode;nutritionaldeficiencyi.e.,vi-
tamin C deficiency!;toxic syndromes
i.e., hemocytic enteritis!; crowding;
transfer; or handling may be concur-
rent featuresin shrimp populationsaf-
fectedby vibriosis.Thus,preciseand
timelyetiologicdiagnoses
canbeprob-
lematical asthe recoveryor demonstra-
tion of bacterial infection may not, in
somecases,provide a comprehensive tologicalpreparationsby specialstains
etiologicdiagnosis.Moreover,subdini- that demonstrate the acid-fast nature of
cal nutritional deficiency and environ-
mental conditions are suspected, but
unproven,factorsthat may precede
and lead to outbreaks of bacterial dis-
easein cultured shrimp populations.
The presenceof bacteriain diseaseepi-
sodes can be demonstrated rapidly by
microscopic inspection of wet-mounts
of whole larvae or tissue biopsy speci-
mens Lightner and Lewis, 1975;Light-
ner, 1983, 1988; Nash, 1990!. Also,
bacterial agents and host inflammation
can be visually confirmed in his-
topathologicalpreparationsBrockand
Lightner,1990!.Standardmicrobiolagi-
cal methods are applied to detect bac-
terial agentsin shrimp tissues Lightner
and Lewis, 1975;Lightner, 1983,1985,
1988; Dempsey and Kitting, 1987;
Lavilla-Pitogo, 1990!. The majority of
bacteria associated with shrimp dis-
eases are nonfastidious and are identi-
fied, following in vitro isolation an
artificial media, on the basis of their
morphology, staining characteristics
and biochemical test reactions Light-
ner, 1983,19SS;Nash, 1990!.Severalof
the pathogenic vibrios are biolumines-
cent. This aids recognition of this group
of organisms, both in culture settings
Dia nostic Methods 219
aswellasoncetheorganismisisolated
on agarmedia Pitogo,19SS;Mohney
et al., 1991!.
The filamentous bacterium Leucothrir
rrrrrcoris a fastidious organism and re-
quiresa specializedmedia for i' vitr0
culture Lightneret al., 1975!.Acid-fast
bacterial infections are identified in his-
the bacterial cell wall Lightner and
Redrnan, 1986; Krol et al., 1989!.
Antibiotic sensitivityprofilesare often
determined for bacteria isolated from
outbreaksof shrimp diseasesto help
shrimpculturistsselectthe mostappro-
priate drug for treatment. Standard
proceduressuch as the Kirby-Bauer
Method are characteristically used to
make these determinations.
The development of specific immu-
nalogicreagentsfor detectingthe com-
mon bacterial pathogens of shrimp
would aid the speedand sensitivityof
identification of these bacterial patho-
gensin casesof shrimp disease.
Fungi
Fungiareimpartantpathogens
of cul-
tured shrimp. Of primary concern are
membersof severalphycomycetefami-
lies lagenidium
sp., Sirolpidium
sp.and
Haliphthoros
sp. are encounteredmast
often! and the imperfect fungus genus,
Fusariurrr
spp., in particular, Fusariirm
solaniEgusaand Ueda, 'l972;Lightner
and Fontaine, 1973; Lightner, 1981!.
The diseaseof penaeidsassociatedwith
 attackby the phycomycete
fungi, tory thatroubnelyidenbfiesmembers
termed larvalmycosis,
isa clinical
prob- ofthegenusFasariumisrecommended.
lernlimitedtolarval
through postlarval
stages.On the otherhand,Fusarium Boththephycomycete
fungiandFusar-
diseaselsa eh@% issue
primarily
of iranspp.canbereadilyisolatedin vitro
intensively
cultured,
olderpopulationsfromchnical
material
onstandard
my-
of certainsensitive
penaeid
speciescobiologicalmedia Baticadoset al.,
i.e.,P,japoniars
andP. stylirostris!
1977;Colorni,1989!. SaboraudDex-
Egusa
andVeda,1972;Hoseet al., troseAgaror PYG peptone-yeast
ex-
1984!.
tract-glucose!
Agarsupplemented
with
2.5%NaQandpenicillin-streptomycin
E6agnceis
oflarvalmycosis
isthrougharesuitabLe
for recovery
of theagents
microscopic
demonstration
ofthevege- of larval mycosis.SaboraudDextrose
tative
hyphae,
whichareinvariablyAgaror Potato DextroseAgarsupple-
abundant
throughout
larvaethat have mented with 2.S%NaC1andpenicillin-
diedor are dyingfromthe disease. streptomycinare acceptable
mediafor
Identification
ofthefungus
togenus in vitrocultureof Fusarism
sp. from
canbe determined
directiy
in wet- shrimp tissues.
mountpreparationsof infectedlarvae
if thespecialLzed
sporangia,
whichdif- Protozoa
ferbetween
genera,
canbefound.
Protozoafrom several distinct orders
Firsariuni
solani
infection
results
inlarge, arereported
aspathogens
of penaeid
variable
sized,
irregular
shaped,
heav- shrimp.Commonlyencountered
in
ily melanized
ulcerated
to nodular
cu- shrimpfarm environmentsare the ses-
ticular
lesionsHoseet al., 1984!.
In sileprotozoans
andothersthatcolonize
susceptible
penaeid
species
andtheap- the cuticlesurfaces,
inc1uding
the
propriate
agedshrimp,
clinical
diagno- peritrichsZoothamniumsp., Epistylis
sisofFusarium
disease
canbemadeon sp.,Vortirsllasp. andIagenophrys
sp.;
the basisof thegrosslesions.
Confir- thesuctorians
Acineta
sp.andEphebta
mation through demonstrationof sp.; various
flagellates
andan apos-
branching,
nonseptate
mycelia
andthe tome ciliate Overstreet, 1973, 1990;
canoe-shapedmacroconidia,charac- Couch,1978,1983;Johnson,
1978;
teristicfor the genusFusariutn,can Lightner,1983,1985!.
often
bemade
bymicroscopic
exarr6na-
tionof wet-mountimpression
smears Othergroups includethe gregarine
oflesion
material
Lightneretai.,1979!. generaNematopsis
and Qyhalolobgs
However,speciesidentification
of the LotzandOverstreet,
1990!;the mi-
fungususuaoyrequiresin vitr0culture crosporidan
genera
Agetasoma,
Ameson
andisolationof the agent.Confirma- andPleistophora
Lightner,1988!,one
tionof theidentification
bysubmissionor morepresently
unclassified
sporo-
of a representative
isolateto a labora- zoansconsidered
to belongto the Or-
quits possiblethat the significanceof Experimental growthtrials carlevaluate
abio6cfactorsin shrimp diseasecausa- the competencyof a diet to support
tion is underestimatedBrock, 1991!. shrimpgrowthto determineif reduced
growth rateis diet-related.Thesetrials
NutritionalSyndrt:eneaQiseases are time-consumingand must be car-
ried out with proper controls, but, if
Nutritional syndromes/diseases of done correctly,will clearly identify or
farmedshrimp are pririmily problems elinunatefeedquality as the causefor
encounteredin groups of shrimp cul- the problem. Vogt et al. 985, 1986!
tured indoors, in tanks that have a advocate histological or transmission
minimum of natural productivity electron microscope evaluation of the
and/or in intensive rearing conditions hepatopancreasfor early recognition of
with a highbiomassor standingcrop dietary deficiency states in cultured
of shrimp. Deficiency diseases/syn- shrimp. However, inonitoring proto-
dromesare much lesslikely to occurin cols such as those described by Vogt
extensivelyfarmedshrimp.In part,this and co-authors are not apparently in
reflectscontributionof naturalproduc- wide use.
tivity to the diet. Hence, as with the
infectiousdiseases, the importanceof Four diseases are attributed to nutri-
dietarydeficienciescloselyparallelsin- tional deficiency or nutrient imbalance
tensification
of shrimpfarming. of growout cultured penaeid shrimp,
blackdeathor ascorbicacid deficiency,
Nutritionaldiseasesare not reported body cramp,blue syndromeand soft-
for shrimplarvae.Thislikely reflects shell syndrome.The etiologic agents
our inabilityto recognizethe early attributedto thesediseases/syndrornes
stagesof deficiencydiseasesin larval areascorbicaciddeficiency Lightneret
populations, rather than the absolute al., 1977!;
tissuecationimbalance,par-
absence of nutrientdeficiencysyn- ticularly hypokalemia low K'! with
dromes in hatchery populations. Once hypercalcemia highCa"! Lightneret
larvaelosecondition,theyrapidlyfall al., 1989!;dietarydeficiencyof carote-
preyto bacterialattack.Thus,it is pos- noid pigments such as astaxanthin
sible that some bacterial diseaseout- Menasvetaet al.,1990!and,possibly,
breaksin penaeidhatcherieshavetheir lowvitaminA Lightner, In press!;
and
genesisas a deficiencyproblem. Harddietaryimbalance of Ca'' and P, or
datatosupportthisspeculation
is pres- exposureto certain pesticides, includ-
ently lacking, however. ing Aquatin, GustathionA, Rotenone
or SaponinBaticados
et al., 1986!.
In growoutfarms,poor qualitydiet
may result in reducedgrowth and in- At present,
presumptive
diagnoses
for
creasedfeed conversionrate FCR!. the describednutritional diseasesof
Thesesymptoms arevague, andmay farmed shrimp
aremadebydemonstra-
be causedby non-nutritional factors. tion of a compatible
historyandthe
 Current Shri Dia nostic Methods

grossandmicroscopic changes favorableresponseto pretreatmentof

structural
Table9! that characterizeshrimp suf- water with EDTA, to be due to the
feringfrom prolongedexposureto diets presence
of "toxic" substances
in the
lackingin the speci6cnutrient s!. De- water.
velopment
andregularuseof analytical
protocolsfor monitoringselectednutri- SomepoisonssuchDDT and PCBsare
ents in shrimp tissues or processed accumulatedin the hepatopancreasof
feeds would be helpful, along with shrimp.Couch978! mdicatedshrimp
quality control programs. exposed
to 0.20-ppb
DDTconcentrated
the chemicalto 40 ppm. Thus, hepa-
Toxicoses topancreas
tissueanalysisfor selected
chemicalsshould provide a usefulap-
Clinical syndromesand diseasesare proachto detectionof pesticidesin
recognized in farmed shrimp to be suspected poisonings of farmed
causedby exposureto water-borneor shrimp.Couch978! reported nohis-
orally ingested toxic substances. In topathologicalchangesin shrimp
hatchery systems,suspectedtoxic syn- acutelypoisonedwith DDT,Dieldrin,
dromesare, at best, poorly understood. Mirex and PCBs.Vogt 987! reported
For example, incidence of protozoea P. histopathological
changes
in thehepa-
vemamei larvae with appendage de- topancreas
of P. monody,prior to the
formities can be reduced by treatment onset of behavioral impacts, after ex-
of culture water with EDTA Brock, posedto 1-ppmdimethoate.
Thus,his-
1991!. While it is easy to recognize, topathological
examinationhas been
microscopically, larvaewith appendage suggestedto be of someutility for
deformities, the exactcause s!for these diagnosing
pesticide
poisoning
in cul-
lesionsare not clearlyknown, but are turedshrimp.However, thesensitivity
presumed, largely on the basis of a andspecificity,
and,thus,usefulness
of
histopathologicsl
methods arelikelyto ture or pH extremes; low dissolved
belowforthedetectionofmostpesti- oxygen; exposure to an abrupt, large
cidepolsonings
in shrimp, change in sa1inityand dissolvedgas
supersaturation.
~FK4s of diseases
or syndromesof
keavrnor suspected
toxiccauseare Diagnoses
of diseases
resultingfrom
derived
bydemonstration
ofa compat-environmental
factorsareapproached
iblehistory,presence
of characteristicby interpretation of enviranmental
structuralchanges,and, for selected monitoringfindings,a compatiblehis-
agents,Mentification
by analytical tory,andspeciestructuralchanges
chemistry
methods of theinjurious found
in affected
groups
of shrimp.
agentin shrimptissues,feedsor the
Thesalient
features
forseveral physical
cultureenvtrorunent.
1be morecom-
montoxic
conditions
offarmed andchemical
shrimp disease factors
relevantto shrimp
andthecurrent
means
fortheir
recog- causation
arelistedin Table11.
nitionarelistedin Table10.
Human Factors
Environmental
Extremes
Individuals
formulate
andimplement
Physical
andchemic' in the husbandry
factors procedures
on shrimp
bethe arms.
Decisions
onstocking
density,
f
shrimp
culture
environment
can
direct
orcontributing ofdiseasesbrands
causes ortypes
offeeds,
feeding
rates,
ofcultured
shrimp.
Themore
impor-
tant
ofthese
factors
include tionandenvironmental
tempera- parameter
monitoring,
water
use,
techniques
ap-
 Current Shri Dia noetic Methods

'Table
11.Diagnostic
findings
forsome
shrimp
diseasestsyndromes
caused
byenviron-
mental factors.

plied in transfers of shrimp stocks, etc. the error can be frequent or sporadic;
are tremendously important mis- with other factorsbeing equal,this will
judgmentshavethe potentialto sigrufi- influencethe patternof occurrence
of
cantly impact shrimp health and vibriosis in hatchery tanks.
hatchery or farm productivity. Also,
workers may make errorsin carrying Human decisionaland implementation
out procedures
setup by hatchery/farm errorsand the "disease"eventsthey
management. Implementation errors lead to may be separated in time by
can directly influence diseaseoccur- daysto weeks.'Ihis titnelag compVi-
rence, and survival or growth of catesrecognitionof causeand effect
shrimp. For example,on the manage- relationshipsbetweenfailuresof hus-
ment side, the farm managerwho pur- bandryproceduresand diseaseout-
chasesa batchof processedfeedthat is breaksor otherimpactsonproduction.
months old because the cost has been Forexample,
duringnurserytransfers,
reduced,maybebuyinga lot of trouble prolongedout-of-water
exposure
of
harvested
for the shrimpponds. Importantly, it juvenileshrimpmayresult
shrimp in highpost-stocking
maybe a monthormorebefore mortality,butlow
fed the rationdisplayclinicalevidence survival of the populationmaygo un-
of de6ciency.
On the workerside,if recoyuzedforseveral
months
untilthe
fromhatch- pondis harvested.
Artemianaupliiharvested
ingtanksarenotproperly
rinsed,high
numbersof Vibriospp. can be inocu- Shrimpproduction loss dueto em-
lated into larval rearingtanks, and, ployeeandnonemployee theftisan-
within a fewdays,larvalvibriosismay other aspectfor consideration.A
occur. Dependingon circumstances, common patternof presentation
is
 Current Shri Dia restic Methods

Conclusions The detection methods for agents of

shrimp diseasesare similar to those
Diseases are a major factor impacting used in other animal husbandry sys-
the productivity of marine shrimp tems. However, molecular methods
farming in regions where semi-inten- have only recently been developedfor
sive to intensive farming practicespre- identifying biotic agents associated
vail. The causesof shrimp diseasesare with diseasesof farmed shrimp. These
numerous. Possibly, and more often techniques will likely becomeincreas-
than is realized, disease outbreaks in ingly availablein theyearstocome.The
farmed shrimp are the outcome of an lack of shrimpcell culture systemsfor
interaction of multiple etiologic factors. isolatingviruseshaslead to the appli-
In this regard, diseases of farmed cation of a range of shrimp bioassay
shrimp paralleldiseaseproblemsin in- techniquesas interim methodsfor the
tensive fish or land-based animal hus- study and/or the detectionof shrimp
bandry. viruses.Clearly, a priority research
needis the developmentof shrimpcell
The diagnosticprocesscanbe thought culture systems.
of ascomprisedof two interactivecate-
and2! priori- Abiotic determinantshavereceivedless
gories;1! agentdetection,
tization of agentsas to their relative attention than their biotic counterparts
contribution to a givencaseor outbreak in the studyof shrimpdiseases. Nev-
of disease. Obviously, if detection ertheless,there are severalsignificant
methods applied in a given case can clinical syndromesthat have been
onlyrecognize
certaintypesof deter- linked to nutritional, environmentalor
minants, the interpretation of the im- toxic causes.It is possiblethat these
portance of these agents may be represent
the"tipof theiceberg"
and
skewed, and, in some instances, mis- thesignificance
maybeunderestimated
leading. for theroleof abioticagentsassubciini-
 PenseesmctnodonFabricius, from brackishwa-
cal, contributing factorsin diseaseout- ter ponds.Aquaculture.56: 271-285,
breakson shrimp farms,'I%ereis dearly Baticados, M.C.L., R.M, Colaso and R,C.
a need to routinely inciarporatenutri- Duremdez. 1987. Histopathology of the
chronic soft-shell syndrome in the tiger
tional and environmentalparameteras- prawnPeaaeas
moriadon.
Dis. Aquat.Org,3;
sessmentwhen investigatingoutbreaks 13-28.
of shrimp diseases,particularly on in- Bell, T.A, and D,V. Lightner. 1984. IHHN
virus: Infectivityand pathogenicitystudies
tensiveshrimp farms. in Peewee styiirostrisand Periarus
iziniiamef.
Aquaculture. 38: 185-194.
Bell,T'.A.,D.V.LightnerandJ.A.Brodh.1990.
AcknovAedgments A biopsyprocedurefor the non-destructive
determination of IHHN virus infection in
Supportwasprovidedby TheActuacul- Peareusxziiinamei.J. AquaticAnimal Health.
ture Development Program, Depart- 2: 151-153.
Boonyaratpalin,S. 1990. Shrimp larval dis-
ment of Land and Natural Resources, eases.Irc M.B. New, H. de Saran and T.
Stateof Hawaii for the preparationof Singh Eds.!. Technical and Economic As-
the manuscript.Mrs. JanetYasamasuis pectsof ShrimpFarming,Prca~ings of the
Aquatech'90 Conference,Kuala Lumpur,
thankedfar assistance
in obtainingref- Malaysia,11-14June,1990.pp. 158-163.
Brock,J.A.1988.Rickettsialinfectionof penaeid
shrimp. Iic C.J. Sindermann and D.V.
Lightner Eds.!. DiseaseDiagnosis and Con-
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the penaeid shrimp. In: J. McVey Ed.!. CRC
Handbook of Mariculture. Volume 1. Crus-
taceanAquaculture,CRC Press, Inc., Boca
Raton. pp. 289-320.
Lightner, D,V. 1985.A review of the diseases
of cultured penaeidshrimps and prawns
with emphasis on recent discoveries and
developments, In: Y. Taki, J.H, Primavera
and J.A. Llobrera. Proceedingsof the First
International Conferenoe on the Culture of
PenaeidPrawns/Shrimps, Iloilo Cit, Phil-
ippines. pp. 79-103
Lightner, D.V. 1988. Diseases in cultured
penaeidshrimps and prawns. In: C. Sinder-
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Lightner, D.V. 1990.Virosessection;introduc-
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in Lightner,D.V., andC.T.Fontaine,1973.A new
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Lightner,D,V., C T. Fontaineand K, Hanks.
1975,Someformsof gill diseasein penaeid
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Lightner, D.V. and D.H Lewis. 1975. A sep-
ticemic bacterial disease syndrome of
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V Methods. 31: 189-196.
26.
M~ DV Ll.~c ~~DA Martin, .WrsA.H. MeekandP. Wiileberl.
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ar- rkius!coloration
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mineral
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Diseases
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Hosts, FishFarming
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Prawn
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Llu,C.i.1989.
Shrimp
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Ove rstreet,
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Antipodean
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treatment.
Irr:D.M.AkiyamaEd.!.Pro- agents.Int. J. Parasitol,20: 551-564.
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Management
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Publishedthe Ovenureet,
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W.E.Hawkins.
1988.
Experimentalinfecti
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1990.Parasites Perrarru
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Irr:J.C.Chavez
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 New Developmentsin PenaeidVirology:
Applicationof Biotechnology
in Researchand
DiseaseDiagnosisfor ShrimpVirusesof
Concern in the Americas

DV Ughtne' B.T.R~ LBruce, RMRe~nand J.Mari

Degertnnent
of Veterinary
S~
University
ofArizona,Tucson,
arizona
85721,U.SA

J.R. Bmarri
Dboratoire de Pathckgie Cream, lNRA-CNRS
UniM.rsitehlh~tpellierII, Scienceset T~ques du Langue'
Race E. Bataillon,34C95MontpelherG&ex 5, Rance

!CIStraCt

Twentyyearshaveelapsedsincethe 6rst shrimpvirus,Baculotxrus penaeiBP!, wasdescribed

fromGulf of Mexicopenaeids.Today,a dozenor morepenaeidvirusesarerecoguzed,andall
exceptBPhavebeen discovered" within the past12years.Many of theseviruseswerefound
becauseof their adverseeffectson culturedshrimp.Despite the considerable economic
importanceof penaeidvirusesto the world'saquacultureindustry,relativelylittle is known
aboutthesepathogens.Thereare probablya numberof geographic strainsof virusessuchas
infectious
hypodermaland hematopoietic necrosisvirus IHHNV!, hepatopancreatic parvo-like
virus HPV!, and the baculoviruses,Penaeusmonodon
baculovirus MBV!, BP, and baculoviral
midgutglandnecrosisvirus BMNV!. However,until recently,no toolswere available
with
whichto investigate
this question.

Likewise,untilrecently,thediagnostic
methodsavailable
to shrimppathologists
weretraditional
procedureswhichusedlightmicroscopy, histopathoiogy,
electronmicroscopy,
directserological
methods,enhancement, andbioassays!thathavebeenemployedin otherareasof animaland
humanpathology. Only recentlyhaveadvanced biomedicalmethodsbeenappliedto thestudy
of penaeidviruses
and to the development
of improved
diagnostic
procedures.
Monoclonal
antibodies
for IHHNV and genomicprobesfor IHHbK and BP havebeendeveloped.
These
antibodies
andgenomic
probesarethe firstof theirkind developed
asresearch
toolsand
diagnostic
reagents
in crustacean
aquaculture.

~hddressee
forcorrespondence,
 InirodUt;tkl
mmses
have
beendeterrruned,
perrrut
Atthistime,nearly viraldis- tingthetaxonomic
adozen placement
or tenta-
easesof culturedand wQdmarine tiveplacement
oftheseagentsTables
penaeid shrimpareknown.Knowl- 1 and
2! Couch,
1981,1991;Mathews,
edgeofthese andthediseases1982;
viruses Johnson andLightner,1988;
theycause rangesfrom"extremely BrockandLightner,
1990;Lightnerand
scanty"to"considerable."
Someofthe Redman, 1991,
1992;andAdams and
penaeid viruseshavebeendescribed Borutmi,
1991!.
Includingthe viruses
in
~olelyonthebasis or penaeids,
ofthediseases morethan30viral diseases
disease
syndromes
inwhichthey were arenowknownto occurin the Class
found,ontheir inhostceHs Crustacea
location Johnson,1983,1984;
and
an on their morphological
charac- Bonami
andLightner,
1991;Adams and
teristics.
Many char- Iionarru,
ofthebiochemical 1991;
Lightner
andRedma
of a fewof thepenaeid992!.
Majorgroups
ofviruses
repre
1 n,
acteristics
sented
inthecrustacea
include
theRe
 Biotech in Disease Dia nosis

oviridae, Picornaviridae,Parvoviridae, details are yet available on the nature

Togaviridae,BaculoviYidae, Paramyxo- of the "yellow-head syndrome" virus.
viridae, Rhabdoviridaeand Iridoviridae
Adams and Bonami, 1991!.Hence, as Several of the penaeid shrimp viruses
the culture of penaeid shrimp in- infectseveralhostspeciesg able3! and
creases,the discoveryof other viral havea vast geographic distribution.For
diseasesfrom these groups and possi- example,BP is present in wild and
bly others!seemsa virtual certainty. culturedpenaeidshrimpon bothcoasts
of the Americas and in Hawaii. It seems
Consistent with this prediction is the inconceivable that the same virus type
recent recognition that yet another dis- or strain could be on both sides of the
ease syndromeof cultured penaeid American continents and in a native
shrimp, "yellow-head syndrome," is wild penaeidin Hawaii.Similarcom-
causedby a virus. The name of the rnents
maybemadeaboutthehostand
disease
syndromereflects a consistently geographic
rangesof HFV andMBV in
present clinicalsign of a diseasethat the Western Pacific and Indian Ocean.
has caused serious losses of cultured Hence,it is verylikelythat eachofthe
Penaeusmonody in Thailand. Yellow-knownpenaeid virustypesis,in real-
hasbeendemonstrated ity,composed
headsyndrome of several
distinct strains
experimentally
to havea viraletiology which mayvary in their virulenceat-
in studies in which the disease was tributesand in otherways.The devel-
transmittedby injectionof healthy opmentof molecularor serological
shrimpwith 0.22-pmfiltratesof tissue methodsfor detectionand differentia-
homogenatespreparedfrom affected tionof geographical
strainsof viruses
shrimp T. Flegel, AquacultureRe- like the BP group will advance the
searchCentre, Songkhla,Thailand, understandingand managementcapa-
pers.comm.,June1992!.However,
no bilitiesfor thesepathogens.
++
i ~
very
serious
disease
due
tovirus
reported
inone
ormore
lifestages
ofspecies.
+ + Sgnlarant
+~
diSeaae
SOmetimeS
Inreciions
due
tOviruS
byvirus
repOrted
known
inOne
Or
mOre
tooccur
lifeStageS
inwith
OfSpecieS.
species,
butwith
Insignificant
effects.
4>assification
~l esperlrnentai
infections
according
achieved,
toHolthuis
but
fl980!
Insignificant
effects.

Thecurrent
diagnostic
methods
forthe
penaeid
viraldiseases
haverecentlyviruSeS,
>sdiffiCult,
microscopy
These
atbeSt.
are
~»ng ~gh
diagnosed
with
been
reviewed
Lightner
andRedman,
1992!,
andthese
methods
havegener-certaintyonly with transxnission
aHy
been
dependent
upon: and electron
1!gross microscopy TH4i!.
Directhis-
dinical
signs,
and2!light topathology
microscopic ofDavidson'sAFA-pre-
demonstration
ofunique served
cytopathol- tissues
hasbeenthemethodof
ogyasdemonstrated micros- choice
bydirect foracute
andmostsubacute
copy
oftissue
impression orby forms
smears, ofIBIDdisease
Bell
andLight-
histopathology.
Diagnosis
ofinfectionner,
1988;
Lightner
andRedman,1992!.
caused most However,
bya fewoftheviruses, fordetection
of asyrnpto-
notably andthereo-hkemabc
thetogavirus IF6iNV
infections,
enhancement
andbioassay
techniques
weredevel-
 Biotechnol
oped Lightner et al., 1983b; Brocket
al., 1983;Bell and Lightner, 1984,1987;
Lightner and Redman,1992!.
The purpose of the enhancementtech-
nique is to increase the prevalence
and/or severity of infection within a
captive population to increase the
chancesof a positivediagnosis
in popu-
lations from which direct samples
might otherwiseprovidea negative di-
agnosis.In the applicationof the en-
hancement technique for IHHI'A', a
population of postlarval shrimp P.
stylirostris or P. vannamei!are reared
under relatively crowded and stressful
conditions to about PL60. Samples for
histopathology are taken at intervals
throughout the 30- to 60-day enhance-
ment period, and if IHIP/ is present,
its prevalenceand/or severity of infec-
tion will be increased to diagnosable
levels.Enhancementhas, at best, only
limited usefulness as a diagnostic
method for demonstration of IHI~P
infection in subadult or adult P. styli-
rostrisor P. vannameithat are asympto-
matic carriers of the virus.
Infection by IFB~K in asymptomatic
carrierswas made possibleinitially by
use of a bioassay-based diagnostic
method Lightner et al., 1983b,1987;
Lightner and Redman, 1992!.In the
bioassayprocedure, potential carriers
of IHHIM are bioassayedwith sensi-
tive "indicator" shrimp, which aretypi-
cally0.05-to 2-g juvenileP. stylirostris.
The "indicator"shrimparemost effec-
tivelyexposedto the "test"shrimp in
a bioassay for IHHNV by feedingarebeingdeveloped
or gnatho- shrimpdiseases
minced,pooledcarcasses
in Oisease Dia nosis
thoracies
of the "test"shrimpto the
"mdicator"
shrimpovera 14-day
pe-
riod.If IHI~P is presentin the"test"
shr'mp,theindicator
shrimptypicaUy
dispiayreadilydiagnosableII.694 dis-
easein histopathological
samplestaken
on or afterday 14of a typical28-day
bioassay.
The presentdiagnosticproceduresfor
the penaeidviral diseasesare largely
dependentupon history,clinicalsigns,
direct light microscopyof tissueor fecal
squashes,or on histopathology Table
4!. Electron microscopyis important in
some diagnostic applications. Tech-
niques like enhancement and bioas-
says,when coupled to histopathology,
add to the sensitivity of these proce-
dures. The practicalapplicationof these
proceduresis sometimesdifficult; how-
ever, and their sensitivity is often very
limited. Examinationof relatively small
samplesis one important limitation; the
length of time and the amount of spe-
cialized equipment required to carry
out routine histopathology and/or elec-
tron microscopyare other factorslimit-
ing their practical usefulness.Also, the
cost of histopathology and electronmi-
croscopy,and of maintainingisolation
labs for enhancementand bioassayadd
to the list of reasonswhy better, more
rapid, moresensitivediagnosticproce-
dures are needed.
Diagnostic
proceduresusingtissuecul-
ture, serologicmethods,and DNA
probes,whichhavebecome stateofthe
art in human and veterinary medicine,
for somepenaeid
Table
4!.Thispaper
is
7abte
4 DtagrtOStlc
metb0dg
forthepftnaatd
viral
disftases:
thetradition
t me0048
fANIE
ods

'Deflnlt4mssnd key nAnmces for «achvirus:

no known or publlshoi appHcattonof technique.
applkationof technktueknown or published.
++ ~ appQcatlon of technktueconskleredby authorsof presentpaper to provide reasonablediagnosac
sensitivity.
+ t + technktue rovklesbest avaatblediagnosUcsensitivity.
C co available
kitsunderconsideration
by a manufacturer
of diagnostickits.
UghtnerandRedman,
1992;Ughtner,unpuMshed.
Owens et al1991
Motnoyama,
1983,1988,1%9a,b, c, d; Momoyama
andSano,1988,1989
1991;Ughtner and Redman,1992;Johnson,1990;Overstreetet al., 1988
Adamsandsonaml,1991;TsingandRonamt,1987;Andersonet al., 1987;Hashet al., 1988;Krol et al., 1990
sAdams
andRonaml,
1991
sonamlet al., in presa
Note;
8F~ brightiieldexamination
of issueimpression
smears
andwetmounts,
LM lightmicroscopy,
RM electronmkToacopy,RUSA- enayme-linked
lmmunosorbent
assay,
Fhbs- polyclonal
antibodies,
hMe - monoclonal antibodies.

a summary of recent efforts in our acterization studies. Infection in this

laboratoryto apply biotechnologicalbatchof broodstockwasconfirmedby
techniquesusing gene probesand direct histological examination of
monocional antibodies
to developnew Davidson'spreservedspecimens and
diagnosticmethodsand researchtools by bioassaytestsusingO.> to O.l-gP.
for penaeidshrimpviruses. stylimsfrisas the indicator for IM.BA'
Lightner et al., 1983b,19m'; Bonarni et
Characterizationof IHHNV al., 1990!.Usingproceduresoutlinedin
Bonamietal. 990!, IHHNV waspuri-
A singlebatchof adult P. vffrinlerfei,
the fiedfromhomogenates
of thegnathot-
progeny of cultured broodstock, was horacies of the IHHNV-infected P.
used as the source of If9~l for char- vstiifmfifa
in a SeriesOf StepSin WhiCh
 Biotech
differentialcentrifugationof homoge-
nized tissuewas followed by sucrose
and CsCI densitygradientcentrifuga-
tion. IHHNV full virions! formed a
discreteband at a densityof 1.405g/ml
in the final CsC1gradient. The virus
was pelletedand utilized for sub-
sequentstudies in which we deter-
mined the virion size, the polypeptide
compositionof its capsid, and its nu-
cleicacid type.
IHHNV was found to be most like
members of the Parvoviridae in its char-
acteristics.E9~F is a nonenveloped
icosahedral particie averaging22 run in
diameter Rg. 1!; havinga meanbuoy-
ant density of 1.405gtml in CsCl den-
sity gradientcentrifugation;havinga
linear, single-stranded DNA ssDNA!,
genomeapproximately4.1 kbp in size,
with the majority of its ssDNA strands
beingminus -! strandsthat are incor-
porated into the capsids separately
from the complementary plus +!
strands;and havingfour polypeptides,
VP1 to VP4, with molecularweights of
74, 47, 39 and 37.5 K-daltons Fig. 6!,
respectively, making up its capsld
Bonami et al., 1990; Mari et al., In
prep. [aj!.
Infectivitystudies, using semipurified
fromsucrose
gradients!and purified
from CsCI gradients! II~K injected
into juvenile P. stylimstris, confirmed
that the 22-nm particles isolated from
infectedshrimp are the causeof IHHN
disease. Further confirmation that
IHI~ is caused by the 22-run virus
particlewas obtainedby immunizing
micewith highly purified preparations
in Oisease Dia nosis
from CsC1gradients!of IHHIA', and,
subsequently,
testingtheresultantanti-
IHHNV serum with tissues from
known IHHNV-infected and known
uninfected
P. stytimstris.Thehyperim-
munizedserafromthesemiceprovided
a dear distinction between IHI~F-in-
fectedand uninfectedshrimp in fluo-
rescent antibody FAB! tests. This
serologictest, the infectivity trials run
with purified virus, and subsequent
tests with a gene probe for IHE~Q
DNA discussed later in this review!
confirmed that the virus isolated and
characterized was indeed IHIP.
These findings contradict Lightner's
earlier opinionon the taxonomicplace-
ment of IEGER JV Lightner, 1988!,and
findings reported by Lu et al. 989! and
Loh et al. 990!, in which the authors
reported that IHHl'A' was a picor-
navirus or even a rhabdovirus Lu et
al., 1991!. Inspection of the data pre-
sented in the papersby Lu et al. 989!
and Loh et al. 990! revealedthat the
virus-like particles they isolated were
12 nm in diameter not 19 nm as the
authors reported!, are morphologically
identical to penaeid hemocyanin van
Bruggen,1986!,and that the chemical
characteristics reported for the pre-
sumedvirus particles Lu et al., 1989!
are those expectedfor hemocyanin
Bonami et al., 1990!. These same
authors claimed Loh et al., 1990! to
have cultured IHHNV in a fish cell line.
However,subsequentstudiesshowed
that the virus grown in the EPCfish
cellswas a rhabdovirus Lu et al., '1991!.
Likewise,infectivity studiesrun with
both the 12-nmparticle Lu et aL, 1989;
240 Li htner et al.

Figure 1. Transmission electron micrograph of purified IHHNV prepared from Penaeus vannamei by
density gradient uitracentrifugation. 2% PTA staining. Bar = 20 nm.
Figure 2. Immunoblot of purified IHHNV reacted with monoclonai antibodies, Spots 1-6 are IgM class
monocional antibodies; spot 7is a mouse MAb made to a nonviral antigen; and spot 8is a pool of MAbs
1-6. All single and pooled MAbs, except the MAb in spot 7, show a strong reaction with IHHNV.
Figure 3. IVestern blot of puNied IHHNV reacted with L-5 MAb. Lane 1 contains molecular' weight
markers placed with transfer for reference!; lanes 2 and 3 contain IHHNV polypeptides V1-VP4
arrowheads! that are demonstrated by their reaction to the L-5 MAb.
Figure 4. Lane 1:A silver-stained gel of IHHNV pofypeptides. IHHNV has four polypeptides, VP1, VP2,
VP3 and VP4 arrowheads!. Lane 2 contains molecular weight markers.
 Biotechno in Disease Oia enosis 241

a
b
C

Figure5. IHHIV
V DIVAlanes2 and3! compared to molecularweightmarkerslane1!.Benda located
at about6.5-7kbp!corresponds
to partiallyassociated+ and- ssDNA;bandb estimatedat 4 kbp!is
dsDNA;and diffusebandc locatedat 2.6 - 1.5kbp!is ssDIVA.Ethidiumbrorniabstain.5 arm/!
incorporated in the gei.
Figure6. IHHNVDIVAfollowingtreatmentwithRNaseA lane4!, MungBeannucleaselane3!, heat
treatment minat 95 C andchilledquick/yin anice bath;lane2!, andseparationby agarosegel
electrophoresis.Bandsvisfb/einlanes 1and 5 aremolecularweightmarkers,lane 2is ssDNA,lane3
is dsDIVA,
andlane3 showsnodegradatfon
by RIVase.
Ethidiumbromidestain.5 g'mi!inaxporated
in the gel.
Figure 7. Dot blot hybridizationwith the DIG-labeledBS4.5probe of Log di/utionsof IHHNV,ihfected,
uninfected
homogenized
shrim tissues,Labeledrowsare:+C= positivecontrol;-C= negative
control;
7A,BA,3Aand92-7= +forIHHNV;and29-1 bothrows!is a LogdilutionofpurifiedIHHNV preparation
from 10' to 10 . Thefirstpositionin 7Aand 3A containno samp/e.
Figure8. Hybridizationof theDIG-labeled
probeBS4.5afterSoutherntransferfromtheagarasegelto
IHHNVDNAbands left two lanes;arrowindicatesbandc of ssDNA! and withrestrictiondigestsof
itself 8931! and withothersmallerIHHNVDNAinserts BA401,Ba402,DR22,andBG45!.
 Loh et al., 1990!and with the rhab- mediumdesignedto promoteactivated
doviruswerereportedas "inconclu- B-lymphocyte growth Kowalskiet al.,
sive"in the challenged juvenileP. 1990!. Spleen cells were fused with
styfieettfa.
Hence,thisseriesof contra- SP2/0myeloma cellsafterdays0, 1, 5,
dictoqr
reIireteby Lu et al. 989and and8 in culture.Culturescontaining
1991!andLohet al. 990!Indicatethat grcnving
colonieswereassayedfor the
tNsgroup
lsnotworldng
withIHHIA/. presence
of virus-specific
antibodyby
indirect enzyme-linkedirnmunosor-
Mono:lonalAntibodiesto bentassayELISA!.
lHHNV
LISA test~
ToInitiate
production
ofmurine
mono- Pl,teswere
i~b tedoverMght
at4 C
clonal
anHbxBes MAbs!
toIHH'&, with crudesupernatantfluids from
highly
purified
virus
was
prepared
ac- ~JV-infectedoruninfected shrimp.
cording
topreviously
described
meth-
Afterwashing
withphosphate-bu8ered
odsBonami
etal.,1990!.
SpecScaily,
saline
withTween-20 PBS-Tween!,
wells
IHIP%waspurSed frominfected
P. wereblockedwith PBS-Tweencontain-
sfiJI/rostr/s
collected
froma commercial
shrimpfarminHawaii.Crude ing10%nonfatdrymilk.Supernatant
shrimpfluid
homolenate
was ciarSedbya series
of addedfrom hybridornacultureswas
low<peedcentrlfugations
andthe
virus tothewellsfor1h at37 C.Goat
waspelieted g.Thevirus anti-mouse
ot145,000 IgG,IgM,lightchainanti-
suspension
was withactivatedbody
treated conjugated
tohorseradish
peroxi-
charcoal,
filtered bed, dase,
ona Celite-23S andthesubstrate-color
reagent
extracted andthevirus ABTS
withfreon, ,2-azino-bis--ethyl
benzthia-
again
pelleted g,Thevirus zoline-6-sulforuc
at14S,000 acid!!
wereusedto
suspension
wasfurther ona15 develop
purified the
reactions.
Theoptical
den-
- 40%
sucrose
gradient bya 25sities
followed were
read
at405nmina BT2000
- 45%
CsCI
gradient. corre-Microkinetics
Fractions plate
reader
Fisher!.
Hy-
sponding
toa density
of1.405g/ml bridomas foundbyELISA todisplay
contained
Intact
virionsasconfirmed highspecificity
and good intensity
in
bytransmission
electronmicrosc
croscopy
theirreaction to IHHNV-infected
TXM!.
BALB/c8yJ
strainmice The shriinp
tissuesand a veryfaint
orno
jackson
Laboratory,
Inc.,
Bar Harbor, reaction
touninfected
shrimp tissues!
Maine!wereiminunizedintraperi-were subclonedthreeormore time
toneaQy
withpurSed IHHIW erst y limitingdilution
toobtain mono-
s

injection
inFreund's
CompleteAdju-clonal cultures.
Theclonesfrom these
vant!.
Three
days
afteraanalintrave-y 'domas provided six"working"
nousimmunization,
themouse with clones.The sixMAbs selectedwere
thehighest
titer
waseuthanized
andits subsequently characterizedandall
spleen
removed.
Spleen cells
werewere found tobeofthemu-kappa
maintained
inculture
ina conditioned lgM!
isotype Table
5!Poulos etal.,
In
prep ! ~
 Bioiechno in Disease Dia

Table 5. Characterization of

Optical densityat 405 nm.

Mhb obtainedfrom hybridomatissueculturesupernatantfiuid.
s Case
itig7-l02;
hotnogenate
ofIHHNV-infected
p.styh'rosttis,
con9rmed
byhistology;
collected
inf987
and
stored at -70'C.
s Case
883-2448;
hornogenate
ofuninfected
P,styffrostris,
confirmed
byhistology;
collected
in1983
andstored
at -70'C.

To further characterize these MAbs, werefound to reactstronglywith puri-

they were assayed with purified
IHHNV in immunoblots and in West-
ernblotassays.
Irnmunoblotswerepro-
ducedby dotting purified IHHNV onto
nitrocellulose membranes. Western
blots were prepared by electro-transfer
of SDS-PAGE gel bands onto nitrocel-
lulose membranes. The membranes
were baked at 37'C for 2 4 h, blocked
for 1 h with PBS-Tweencontaining10%
nonfat dry milk, and baked againfor 30
min. The membranes were incubated
with hybridorna supernatant fluid,
which was semipurified and concen-
trated by ultrafiltration for 2 h at 37'C.
After washing in PBS-Tween,the rnern-
branes were incubatedwith goat anti-
IgG, IgM, light chain antibody
conjugatedto horseradishperoxidase
HRP! for 1 h at 37'C. After washing in
PBS-Tween,the reaction was amplified
using the BLAST HRP Blotting Am-
plificationSystemfrom NEN-DuPont
Corp, Colordevelopment was achieved
using 4 CN/HN2 -chloro-1-naphthol
/hydrogenperoxide!.'Ihe six IgM MAb
fied IHI~F in immunoblots and in
Westernblots Pouloset al., In prep.!
Figs. 2 and 3!.
The six MAbs were used successfully
in indirect ELISA to distinguish be-
tween BIHNV-infected and uninfected
shrimp of known diseasestatus in ar-
chivedsamplessamplesthat hadbeen
stored at -70'C for morethanoneyear!
Table 6!. However, with fresh bssue
samplesof known IHI&1V status from
history, histopathologp or bioassay!,
more variable results were obtainei8. It
wasdeterminedthat freshclinicalspeci-
mensof uninfected shrimp may elicit a
nonspecificresponseof variablein-
tensityin the indirect EL1SA.This
finding indicates that there are sub-
stances in fresh, crude, shrimp ex-
tracts that bind with murine antibodies
non-specifically,
but that arenot found
in archivedsamplesafterlong-termfro-
zen storage Pouloset al., In prep.!.
 Tabie
6,Cotnpatfson
ofotinkal
resuits
using
dNerent
diagnostic
tnethods
forthedetection

' EUSA
~es;assay
DNAwas
run
using
MAbto
lHHNV;
hybridization
was Histology
performed
with was
IF694V read
clonefor
presence
8Q3t. ofCowdry
A inclusion
CAI!
Mari
et.
aL
Partially Of
the
purified 5%iNVSenOrne
vtrions
from and
the
uae
infected ofprObeS
shrimp. indiagnOSis.
ManuSCript
inpreparatiOn
[a].
+ ~ negative
re@tivein~ ofposittve colorimetric
colorimetricreaction.
reactions.
1he
number
of+ indicates
's positive
reaction
intensity-
NT not tested.
Nh ~ not apphcable.

Preliminary
studies
intothenatureof ducea positiveEUSA reactionwhen
thesubstance infresh
s! shrimptissues assayedagainstuninfected
shrimpho-
thatbinds munne antibodies
suggestmogenates usinga secondary
antibody
thatthesubstance may
s!belectins, thatdetects
all classes
ofmouseimmu-
which areanunportant
component of noglobulin,
but notwhen usinga sec-
the "immune"or defense
mechanism ondary antibody specific
onlyfor the
ofcrustacea
Olafsen,
1986;
1988!.Lect- gamma chain
of mouseIgG;thisactiv-
insarelarge,giycoprotein
molecules itymay beduetothebinding
ofshrimp
thatbindtospecific
polysaccharides
on lectinsonthecarbohydrate
side-chains
othergbjmproteins.
NormalandIHI~- of theIgMmoleculePouloset al.,In
4nmune
mouse
sera
werefound
topro- prep.! P'able7!.
 ner ef al.

SmaI siteof the pUC 18vector.Trans- DNA inserts selected as potential

forrnation
wasdoneaccording to stand- probesfor IHHIM detectionwere la-
ard methods Seidxnan,1989! using beledusingthe nonradioactive
GeniusI
competent
E.coli-DH5
cellsMarietal., IGt Boehringer
Mannheim, Inc.!, which
In prep.[a]!. contamsdigoxygenin-11-dUTP as the
DNA label and uses an ELISA-based
About5,000transformed F. coh white systemfor final detection. To verifythat
colonies;unalteredcoloniesareblue in selected inserts were derived from
thiscioning system!colonieswere iso" IHEBWFDNA, Southern transfer Ma-
lated,and, of these,500colonieswere niatiset al., 1982!and dot-blottechniques
randomly selected forfurtheranalysis. wereperformedon nitrocellulose mem-
Alkalinelysisminipreps Birnboim, branes BA85 Schleicher k Schuell,
1983!wereusedfor screening trans- lnc.!. A Southern blot of the IHIP/
formedplasmidsisalatedfrom the DNA showing the three different
white E. ooticolonies.After digestion bands A, B and C! was probed with
with different restrNionenzymes,the DIG-11-dUTP-labeledBS4.5. Figure 8
size of the II~if dsDNA inserts was shows dearly the hybridizationof the
found in a largeproportionof the trans- probe with the most visible band C,
formedcolonies to be lessthan 1 kbp; ssDNA! of the three bands. This con-
only5%of themexhibiteda sizegreater firrns that the clonedDNA corresponds
than1 kbp;and onlyone done, BQ31, to at least a part of the viral genomeof
hadan insert of 4.5 - 4.7 kbp. Because 59ihPF Mari et al., In prep. [a]!.
the insertsize of the clone BQ31 corre-
sponded apparently to a full-size The specificity of the probe BS4.5was
IHHl'R genome,it was chosenfor in- further investigated by reacting the
itial investigation.
Other cioneswith probe with a variety of insect and
inserts2 kbp ar largerwere alsose- shrimp parvoviruses.BS4.5 was re-
lected for further studies. These ciones actedwith dot-blots prepared from ex-
hadinsertsof 2.3, 2.0, 3.2 and2.2 kbp, tracted insect parvovirus DNA; with
andweredesignated asBG45,BA401, purified IHHNV DNA; purified
BA402,andDR22,respectively. After IHIP/ virions; homogenized tissues
digestionwith different restriction en- fram known MHIM-infected shrimp;
zymes, the size of the insert of BQ31 healthy shrimp; and purified HPV =
wasestimatedtobe4.5 -4.7 kbp,which hepatopancreatic parvo-like virus
larger than the TKM-estimatedsize [Lightner and Redman, 1985a]!; and
of theIHHI'4Vgenome, 4.1kbp.This tissues hepatapancreas!of HPV-in-
was confirmed by agarosegel electro- fectedshrimp. The results Fig. 7 and
phoresis: theB bandof the viral dsDNA Table8! indicate dearly that this probe
migratesa little farther than doesthe has a high degree of specificity for
BamHI/Sacl-digested insertof BQ31 IHI-INV no cross hybridization with
w"ichis coded asBS4.5!. the shrimp DNA from healthy animals
or with HPV!. The detection limit in
 Biotech
Table8, Resultsof assaysvriththe DNAprobeBS4.5for IHHNVagainsta varietyof insect
thesedot-blotassayswith Logdilutions
of IHHNV DNA was estimated to be
about0.1 pg of MEilM DNA.
The BS4.5probewas testedin sita on
paraf6nsections af IEB~Y-infected and
healthyshrimp.Infectedand uninfected
P. stytirtostns
and P. vunttameiwere fixed
in Davidson's fixative, embedded, and
sectioned at 5 pm using standard histo-
logicalproceduresBeH and Lightner,
1988!.Somesectionswere stainedwith
HkE for use as a histologicalreference
Fig, 9!. Otherswere used for in sita
hybridization usingDIG-11dUTP-labeled
probes.No reactionwas found in un-
infectedtissues.Conversely,a strong
positivereactionwas obtained in in-
fectedshrimp sectionsFigs. 10-12!.
Known targettissuesfor IHI-INV were
positiveat the ceHularlevel, and exhib-
ited labeledvirus-containing
areasin
both the cytoplasmand nucleus of
manycell types Figs. 10-12!.
In Disease Dts 247
EspeciaHy
significant
in the in situ hy-
bridization tests is the intense reaction
of the Cowdry type A inclusionbodies,
which are the principaldiagnostichis-
tologicalcharacteristic
of II&iN disease
Figs.%12!. Histopathologyof parallel
HRE-stained sections from the same
infectedand uninfectedtissuesFig. 9!
was in agreementwith the previously
publisheddataon the diseaseLightner
et al., 1983a;Bonamiand Lightner,
1991; Lightner and Redrnan, 1991,
1992!.Perhapsmoreimportantwasthe
recognition by the probeof IIBINV-in-
fected tissues that were not detected as
readily in routine H6iE-stained sec-
tions. The fact that the IHI-iNV probes
tested reactonly with IHHIA'-infected
tissues, and not with insect parvovirus
DNA, and, moreover, not with HPV-
infected tissues, underlinestheir high
specificity to IHHI'A'. 'Bus definitively
resolves the controversy of the real
causative agent of IHI~ disease,in
whichanotherresearch
grouphascon-
tended that IHI~K might be a picor-
 11

y~% W. M' ~ i!
thatlsdiagnae5c
forIHHNV
infection.
Section
is ofthegwsofa Juvenile
PenaeUs
stylirostris.
H8F stain.
Bsr~ tope
! ii i .Ai
stained
darkpvrp/e
bythegenorrsc
pebeBS4.5
toIHHNV.
Nostain.
Bar= 10pm,
F'gnat11and12.Hetolot
ice!secticvwsof
giNs
andantennal
gland
from
a P.stylirMris
witheveryheavy
IHHNV
infection.
Manyfocal
casions
aredsvnonetrated
bythe8$4.5
probe
thatconsists
ofCAlsanode!,
cykphsmic
masses
ofIHHNV,
andvirus-centalnfng
celidebns
fromnecrotic
cellsarrowheads!.
Nostain.
BsIs= 20@m.
 Biotechnol
navirus or a rhabdovirus Lu et al.,
1989;Loh et al., 199G;Lu et al., 1991!.
Baculoviros BP and MBV! Gene
Probes
BP is an important virus of the Arneri-
canpenaeidsP. zennamei
and P. styli-
rostris,while MBV is its counterpart in
Asian,African and Australianpenaeid
shrimp Tables1-3!.Thediagnosticpro-
cedures for detection of acute or sub-
acuteBPandMBVinfectionsaresimple
and straightforward Table 4!. How-
ever,the current diagnostic procedures
for BPand MBV do not provide diag-
nosis of latent carriers of these viruses,
nor do they provide the needed re-
searchtools to determine how many
virus types makeup each of the BP and
MBV groups.Furthermore,important
research and disease management
questions i.e., latency, transovarial
versus horizontal transmission, etc.!
cannot be answered with the available
methods for detecting these viruses.
Hence, the development of specific
DNA probesfor these viruses has been
initiatedby our laboratory.BecauseBP
is of more immediate interest to the
shrimpcultureindustry of the Ameri-
cas, most of our work has been concen-
trated it, and the results of that work
will be reported here. The status of our
workwith MBVwill be reportedelse-
whereMariet al., In prep.[bj!.
IF Virus
BP DNA was obtained from virions
purifiedfrom BP-infected
juvenileP.
vannameiobtamedfrom pond-reared
stock in Hawaii. Methods used for BP
in Oiseaae Dia nosis
purificationand DNA extractionhave
beendescribedpreviously Bruce etal.,
1991;Mariet al.,In prep.[b]!.Confir-
mationof the purity of purifiedBP
preparations
was determined by UV
spectrophotometerand TEM ex'uiuna-
tion of 2%FI'A-stained
samples
of BP
fromCsC1
fractions
afterdensity
gradi-
ent centrifugation.
Following
extraction
of DNAfrompu-
rified BP virions, the dsDNA was di-
gestedwith the restrictionenzyme
BamHI,and the resultantfragments
were separatedusing agaroseelectro-
phoresis.Bandsof theBamHI-digested
DNA wereextracted fromthe geland
ligatedinto the Sari6il-digested
and
dephosphorylated vectorpUC18.The
recombinant plasmids were then used
to transform E. coli DH5 cells. Trans-
formed cells were frozen back to create
a library of BP DNA, and initial screen-
ing of the recombinantplasmidsin the
transformedcellswasperformedto be-
gin restrictionenzymemappingof the
BP DNA inserts. The larger BP DNA
insertswereselected
aspotentialgene
probes, labeled with the Amersharn
nonradioactive enhanced chemilumines-
cencekit, and tested againstknown
BP-infected
shrimpsainplesand known
negativecontrols. One 3.%kbpinsert of
BP DNA HQ-15! was selected as a
promising BPgeneprobe.HQ-15reacts
well with the BP positive controls
tested,and it doesnot displaya reac-
tion against negative control or unin-
fectedshrimptissues.Thesefindings
were verified using the samepieceof
DNA asa probebut labelingit with the
BoehringerMaru&eimGeniusI system,
Ta5e
9,SiNlmary
Ofvaults
oitrain whiCh
theBPprobe
HQ-15
waS
teSted
withva6ous
~pica,

Sphd&df m&6mph moor

IN'~ pooled
lAerSro,
mhofehrimp
HPhomogeoetee
ofwSd
p.oeoMmi,
p,stylirortrr'r
andP.califorrricnsis
from
MbaOar~ SP
errrehedrel
Oerhreioo
bodice,
HF~ hepetapanCreee.

Samples
of shrimpwith knownBP no.91-7in Table9!. In the ciir6cal tests,
infections
andwith unknownBPstatus theHQ-15probedisplayed
a positive
fromdifferent
geographical
regions reactionagainstEcuadorian-derived,BP-
were testedin cRniciiitrials with the infected,larval and juvenile P. ven-
probeHQ-15to determinethe useful- rienei, and with feces collected from
nessof theprobeasa diagnostic
re- infectedP. emrlmei from Ecuador. The
agentandto investigate
thepossible probeexhibitedno reactionagainst
useoftheprobe
todetect
potentiallypolyhedrapurified from Bp-infected
differentgeographicalstrainsof BP wildP.aiztecus
fromtheGulfof Mexico;
Table9!. In preliminary trials,the withpresumedBP-infected
P. vennamei
probedisplayed a positivereaction fromBrazil;
or with a pooledmix of
againstDNAextracted fromail BP
components i.e.,purifiedBPDNA, shrimp hepatopancreas
horriogenates
purified
nucleocapsids,
purified ofwildP.variriamei,
poly- P. P.sfylirostris
and
hedra,andsupernatant
froma ho- calijbrriierisis
from Mexico. As ex-
mogenizedpreparation!of the pected,
HQ-15did nothybridizewith
BP-infected
P.tgiinena Hawaii,IHMA'. However,
from HQ-15did react
from
which
theprobe
wasmade
case witha sample
of P.shjlirvstris
froma
population
that hadbeenrearedin
 251

Guam and which had no prior history
of BP. Additionally, the probe exhibited
no reaction against known, unin-
fected, homogenized hepatopancreas Bell, T.A. and D.V. Lightner.1988,A Hand-
bookof NormalShrimp
preparations, which served as negative
controls gable 9!.
The initial results with the probeHQ-15
suggest that there may be differences
in Atlantic and Pacific strains of BP,
which are distinguished by this particu-
lar probe.Results also suggest that the
probe may detect latent BP infections
in populations like the Guam-rearedP.
stylirostris population, which had no
prior history of patent BP infections.
Acknowledgments
Grant support for the shrimp disease
studies reported was from the U.S.
Department of Agriculture's Gulf Coast
ResearchLaboratory Manne Shrimp
Farming Consortium; Sea Grant, U.S.
Department of Commerce; and Grou-
pement de Cooperation Scientifiquesur
les BasesBiologiques de 1'Acluaculture.
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Crustacea,Vol. 6. AcademicPress,N.V. andR.M.Redman,1987,Glyceroltolerance
Johnson,P.T. 1984. Viral diseases of marine of IHHN virus of penaeidshrimp, J. World
invertebrates.HeigoI6nderMeeresunters. Aquacult. Soc. 18: 196-197.
37: 65-98. Lightner, D.V. and R.M. Redman.1991.Hosts,
Johnson,P.T, and D.V. Lightner.1988.The ographicrange and diagnosticprocedures
rod-shaped nuclearviruses
of crustaceans: or the penaeid virus diseasesof concern to
t-infecting
species.Dis.Aquat.Org.4: shrimp culturists in the Americas. Irc P.
141. DeLoach, W.J.Dougherty,andM.A. David-
Johnson,
S.K.,1.990.
Handbook
of ShrimpDis- son Eds.! Frontiersof Shrimp Research.
eases. Sea Grant Publ. No. TAMU-SG-90- Elsevier,Amsterdam.pp. 173-196.
601,TexasAkM Univ.25p. tner, D.V. and R.M. Redman. 1992. Geo-
Kowaiski,
M., K. Hannig,G. Klock,P. Gess- graphicdistribution,hosts, and diagnostic
nsr, U. 7immermann, G.A. Neg and D.W proceduresfor the penaeidvirus diseasesof
Sammons. 1990. Eiectrofused manunaiian concern to shrimp culturists in the Ameri-
cells
analyzed
byfice-flow
electrophoresis. cas. Irc A,W. Fast and L.J. Lester Eds.!
BioTechniques.9: 332-341. Cultureof MarineShrimp:Pnnclpiesand
Krol, R,M., W. Hawkins and R.M. Over- Practices.
Elsevier,pp. 573-592.
sttert.1990.
Reo-Iike
virusinwhiteshrimp Loh, P.C.,Y. Lu and J.A. Brock 1990.Gmwth of
Perseus sasneneiCrustacea:Decapoda!: thepenaeid shrimpvirusinfectious hypoder-
co-occurrence
with Ba.uiooiruspnaai in malandhematopaietic
nezm~is
virusin a 6sh
experimental
infections.
Dis.Aquat.Org. cell line. J. V ' Methods.28: 273-280.
8: 45-49.
Lu,YP,C. Loh an J.A.Brock.1989.Isolation,
Laster,R.J,G.,A. Doubrovsky,
J.L.Paynter, purification and characterization of infec-
S.K,Sambhi andJ.G.Atherton.1987,
Light tious hypodermaland hematopoieticne-
andelectron
microscope
evidence
of bacu- crosisvirus IHHNV!frompenaeidshrimp,
Iovirusinfection
in theprawnPesaeas
plde- J. VirologicalMethods.26:339-344,
jas. Dls.Aquat.Org.3: 217-219. Lu,Y., E.C.B.Nadala,J.A.Brockand P.C.Loh.
Lightner, D.V. 1988. Diseases of Penaeid 1991. A new virus isolate from infectious
Shrimp. In: C.J. Sindermannand D.V, hypodermal
andhematopoieticnecrosis
vi-
LightnerEds.!,Disease
Diagnosis
andCon- rus IHHNV!-infected
penaeidshrimps.J.
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Molecular
cioning.
A laboratory
man-
Lightner,D.V,, R.M. Re@man and T.A. Bell. ual,C.S.H.,545p.
1983a.Infectioushypodermaland he- Mari,J.,J.R.Bonami
andD.Lightner.
In prepa-
matopoietic
necrosisIHHN!,a newlyrec- ration a!. Structure
andcloningof the
ognized
virusdiseaseof penaeid
shrimp.J. genome ofIHHNV,anunusualparvovirus
Invertebr. Pathol. 42: 62-X.
Lightner,D,VR,M. Redman,T.A. Bell and
pathogenic
forpenaeid
shrimp.Diagnosis
of
J,A. Brock, 1983b.Detectionof IHHN virus
thedisease
usinga highly
specific
probe.
Mari,J., J.R.Bonarni,B.T.Poulosand D.V.
in Perseus
styIinufrisand P, nasmunei
im- Lightner.In preparationb!. Partialcharac-
 Biotech
terlzationand cionlngof the genomeof the
PmSNPV MBV ~ monodon baculovirus!
from Penaeus monakm.
Mathews, R.E.F, 1982. Classification and nomen-
clahue of viruses.In~ogy. 17: 1-199.
Momoyama, K. 1983. Studies on baculoviral
mid-gutglandnecrosis
of Kurumashrimp
Peimus jsponicus!-III,Presumptive diagnos-
tic techniques.Fish Pathol. 17: 263-268.
Momoyama, K. 'l9N. Infection source of bacu-
Ioviral mid-gut gland necrosis BMN! in
massproductionof Kurumashrimp larvae,
Penaeus
jaiponicus,Fish Pathol, 23; 105-110.
Mornoyama,K. 1989a.Virucidaleffectof some
disinfectants on baculoviral mid-gut gland
necnaslsvirus BMN!. Fish Pathol, 24; 4749,
Momoyama, K. 1989b. Tolerance of baculoviral
mid-gut gland necrosisvirus BMNV! to
ether, NaCI concentration and pH. Fish
PathoL 24: 175-177.
Momoyama, K. 1989c. Survival of baculoviral
mid-gutglandnecrosis
virus BMNV!in
infected tissues and sea water. Fish Pathol.
24. 179-181.
Momoyama, K. 1989d. Inactivation of bacu-
loviral mid-gut gland necrosis BMN! virus
by ultraviolet irradiation, sunlight exp'
sure, heating and drying, Fish Pathol. 24:
115-11S.
Momoyama, K, and T. Sana. 1988.A method
of experimentalinfectionof Kurumashrimp
larvae,Penaeus
japorucus Bate,with baculovi-
ral midgut gland necrosis BMN! virus. J
Fish Dis. 11: M5-111,
Momoyama,K. and T. Sano.1989.Develop- Tsing, A, and J.R. Bonami. 1987.A new viral
mental stagesof Kuruma shrimp, Penaeus
jsponicusBate, susceptibleto baculoviral
mid-gut gland necrosis BMN! virus. J, Fish van Bruggen,E.F.J.1986.Electronmicrciscopy
Dis. 12: 585-589.
Nash, M., G. Nash, I.G. Anderson and M,
Shariff. 1988, A reo-like virus observed in
iri Disease Dia nosis
thetigerprawn,Penueus inotmkmFabricius,
from Malaysia.J. FishDis. 11:531435.
Olafsen, J,A. 1986. Invertebrate lectins: bio-
chemicalheterogeneity
asa possible
keyto
theirbiological
function.ChapterS.In: M.
Brehelin Ed.!. Immunity in Invertebrates.
Cells, Molecules,and Defense Reactions,
Springer-Verlag,
Berlin.pp. 94-111.
Olafsen,J.A. 1988.Roleof lectinsin invertebrate
humoraldefense.In: W.S.Rsher Ed.!.Dis-
easeprocessesin marine Bivalve Molluscs.
Am. FisheriesSoc.Sp,Publ. 18;189-205.
Overstreet, R.M., K.C. Stuck, R.A, Krol and
W,E. Hawkins. 1988.Experimentalinfec-
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penaeiin the white
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iMnnamei
as a bioassay,
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WorldAquacult.Soc.'I9; 175-187.
Owens, L., S. De Beer and J. Smith. 1991.
Lymphoidalparvo-likevirus in Australian
prawns.Dis. Aquat.Org. 11:129-134.
Poulos,B.T.,B.Trumper,J,R.BonamiandD.V.
Lightner,In preparation.Monodonalanti-
bodiesto IHHNV of penaeidshrimp.
Sano,T., T. Nishirnura,K. Oguma,K. Mo-
moyama and N. Takeno. 1981.Baculovirus
infection of cultured Kuruma shrimp
Penaeus jsponicusin Japan.FishPathol.15:
185-191.
Seidman,C.E. 1989,Introductionof plasmid
DNA into cells. In: F.M. Ausubel, R. Brent,
R.E. Kingston, D.D. Moore, J,G. Seidman,
J.A, Smith, and K. Struhl Eds.!. Current
Protocolsin MolecularBiology.Vol. 1. John
Wileyk Sons, N.Y. pp. 1.8.1-1.8.8.
diseaseof theshrimp,Penaeusjaponicus
Bate.
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ContributedPapers

SPF Stocks
 Selective
Breeding
ofSpecific
Pathogen-Free
SPF!Shrimp
forHighHealth
and IncreasedGrowth

Jams A. @+an
The Oceanic Institute
Ma4apuuPoint
Waimanalo,Hawai 96795,U.SA

World
shrimp
farming
depends
onseed
thatiseither
gathered
from
thewildorproduced
in
hatcheries
bywildwaught
spawners.
This
approachisinherently
unreliable.
Incontrast,
great
success
hasbeen
achieved
through
selective
breeding
inmeat
production
technologies
such
as
poultry,
cattle
andswine.
Itislikely
thatshrimp
farming's
future
willinciu
deselective
breeding
anddomestication.
Shrimp diseaseshavehada devastating
effect
onshrimpfarming
inboth
theUnited
Statesandaround theworld.Inother
meatproduction
industries,
modern
growers
relyoncertified
virus-free
stock toavoidvirus-caused
diseases.
Continued
expansion
ofthe
global
shrimpindustry
willrequirereliable
supplies
ofvirus-free
shrimp.
Basedonthese
assumptions,
theU,S.Marine
Shrimp
Farming
Program
initiated
a project
in
1989
todevelop
reliable
supplies
ofspeafic
pathogen-free
SPF!
P.arnnmnei
fortheU.S.
industry.
Theproject
includes
a selective
breeding
program
toimprove
thequality
oftheSPF
stocks,
This
paper
describes
ourapproach
andinitial
results
indeveloping
a selective
breeding
program
for
improving
theSPFstockproduction
performance.

Introduction mendous success

ofbreedingprograms
in othermeatproductiontechnologies,
Worldshrimpfarmersproducedover shrimpfarming'sfuturewill likelyin-
633,000h4Tof penaeidshrimpin 1990 dudeselectivebreeding
anddomestica-
Rosenberry,1991!, Worth more than tion.Shrimpfarmers
will useimproved
$2.5billion,farmedshrimpsupplied stocks,bredforoptimalperformance
in
over 25% of the world's demand. culture.
Nearlyall of this productionwas de-
rivedfrom seedeithergatheredfrom ln the last severalyears,shrimpdis-
thewildorproduced
fromwild-caught easeshavehada devastating
effecton
spawners.Becauseof the inherent in- U.S. and worldshrimpfarming.Dis-
stabilityof this approachand the tre- easesincreaserisk, deterringinvest-
mentandcommseialdevelopment. In In 1990,a coQaboration
with Dr. James
addition,exoticdiseases
carriedby non- Lannan,OregonStateUniversity,was
nativeshrimp asenviron- undertakento developa management
areperceived
mentalrisksthat, in the U.S., could planfor the SPFstock Lannanand
precludetheuseof ~native shrimp Wyban,1990!.Recogni'kgthe genetic
withthebestcommercial such implicationsof breeding the small
potential
as Pemus oenenciand P. monody!. closedpopulationof SPFshrimp,the
Manyoftheseproblems arecaused by planwasdesignedto avoidinbreeding
viruses, for which there are no thera- and conservethe geneticintegrity of
peuticcures Kalagayanet al., 1991; the SPF stock. As the success of the SPF
Lightner,In press!.In othermeatpro- programemerged,opportunitiesto ex-
dvcthnindustriessuchaspoultry,cat- pandthe broodstockmanagementpro-
tle andswine,moderngrowersrelyon gram into a breeding program for
certified virus-free stocks to avoid vi- domestication
and improvementof the
ruacaused disease.Similarly,sustain- SPF stock of P. aarrnumei increased.
abledevelopment of thegloM shrimp
industrywN requirereliablesupplies A breedingadvisorygroupwas con-
ofvirus-free
shrimp. venedat The OceanicInstitute Ol! in
July1991.Thegroupincludedbreeding
Building
onthese assumptions,theU.S. experts from the poultry, swine and
Marine Shrimp FarmingProgram salmon industries whowerebrought to
USh4SFP!initiated
a projectin 1989to Hawaiito helpplanthebreedingpro-
develop
specificpathogen-free SPF!P. gram for SPF shrimp. The establish-
tennnmsi.
Thegoalof the USMSFPis to ment of foundingstocks,quarantine
stimulateexpansion of theshrimp andscreeningprotocols,nuclearherd
farming
industry in theUnitedStates. management,facility requirements,
Therefore,
thepurpose
ofitsSPFpro- breeding
strategies
andcommercial
op-
gram4'toestablish
reliable
supplies
of portunities
for the SPFbreedingpro-
SPFP.omnensi
fortheU.S.industry. gramwerediscussed.
The breeding
Following
establishment
ofSPF
shrimp advisory
groupincluded:
Drs. James
stocks,
theUSMSFP planned
toinitiate Lannan,OregonStateUniversity;Txy-
a selective
breeding
program to im- gveGjedrem, Institute
of Aquaculture
provetheproduction
quality
oftheSPF Research, Norway; Fred Shultz, Ani-
shrimp.
mal BreedingConsultant,
California;
andMr.Andrew
Coates,
PigImprove-
Theestablishment
andperformance
of mentCompany,
Kentucky.
theSPF
stock
hasbeen
described
by
Wyban
et al. 992!.Thispaper
de- Following
several
days
offormalpres-
scribes
ourapproach
andinitialresults entations
andlively discussions,
the
indeveloping
a selective
breeding
pro-
gramforimproving
theSPF pro- breeding
stock experts
provided
ommendations
detailed
ondeveloping
rec-
a breed-
duction
performance.
ingprogram
for SPFshrimp.This
 af SPF$

manuscript is a synthesis of the recom- Tabis

1.A worldng
listofexcfudabie
paiho
mendationsprovided by the ad~iexy
group. It alsodescribesprogressto date
toward establishing a selective breed-
ing program for SPFP. vannenei.

Goals of the SPF Breeding Program

The goal of the breedingprogram is to

develop a faster-growing cultured
shrimp P. canna'! through selective
brewing.

The speMc objectivesof the SPFbreed-

ing program are:
andphysically
excluded
froma facility
~ To maintainthe SPFstatusof stocks; are considered.Diagnosable
microbes
thatcauseeconomically
siyd6cantdis-
~ To avoid inbreeding; and ease in P. vmmamei and that can be
excluded
froma facility<yeM patho-
~ To improve shrimp growth and gens!are listed in TaMe1.
survival to marketsize 0 g!.
Development of a historical record
Establishing the Founder SPF Stcek through ongoing screeningis neces-
sary to insure SPFstatus. Thmughout
Building on the SPFconceptdeveloped this manuscript,broedstock
shrimpthat
forlivestockindustries,theSPFshrimp havepassedthroughthis rigorousproc-
program was initiated in 1989 Wyban essare referred to as "SPF brexhtcek."
et al., 1992!. Since P. 6ennameiis the When SPF bnxxhtock are transferred
principalshrimpspeciesculturedin the to a commercial facility, they and the
United States and the rest of the West- nauplii and postlarvae derived from
em Hemisphere Rosenberry,1991!,it those broodstock are referred to as
waschosenasthe targetspeciesfor the "high health" shrimp, becausetheir
progfarxL SPF status is no longer certain, and a
new historical record for that facility
The de6nition of what constitutes an must be established.
SPF shrimp population follows the
guidelines developedby the Interna- In June 1989,postlarvalP. amenti
tionalCouncilfor the Explorationof thewereimportedfroma hatchery in Mex-
Seafor working with exoticspecies ico.FollowingpreliminarySPFdiagno-
ICES,1988!.Only diseas~using mi- sis by histology, these shrimp were
crc4es that can be reliably diagnosed shippedto Hawaiiandmaintained in
 that A genetic
~srtine Iioa~y confirmed diversity
analysis
comparing
these
shrimp
were~94flee andre- P. emursmei
within its natural range to
p
KonaPopulation
1 wasrecorrunended
dicatad theywerealsofreeaftheother Larrnan,
1980!and hasbeeninitiated.
excludable pathogens.
Theywerethen Researchers
from Tufts University and
~hipped toOI'sSPFshrimp quarantine WorchesterPolytechnicalInstitute are
facilityOl-Keahuolu!on theislandof usingmolecular
techniquesboth nu-
Hawaii. Postlarvaeproducedfrom clearand mitochondrialDNA! for this
these -tentative" SPF broodstock were purpose,
Similartechniques
have been
diagnosed asSPF,thuscon6rrning the used in other marine invertebrates
stock'sfull SPFstatus.Thisfounding Brownand Paynter,1991!.In addition
groupof SPFP. rarrrrrsrrlci
is referredto to providing measures of diversity
~ sKonaPopulathn1. within the SPF population s!, the mo-
leculartechniques used for diversity
AcMitlon of New Genetic Material analysismayalsoyield"markergenes"
that can be usedin the brmding pro-
Theoriginal
population
ofSPFshrimp gram, if they can be correlated with
probablyrepresentsa narrowgenetic growth,
samplhg af thespecies.
To avoiddet-
rirnental
founder effects,
thebreeding A comparisonof genebcvariationin a
programwas advisedto startwith the farmedpopulation
of P. rammrmei
with
widestpossible
samplingafthespeciesthree naturalpopulationsacrossthe
Shultz,19B6;Gall,1990!.However, speciesrange using allozyme tech-
rigorous
screeningof newstockaddi- niquesfoundvery low levels of vari-
tions
mustbeemployed
toprotect
the ationand heterozygosity
in all four
valuable
SPFstatus
ofthefirstpopula-stocks,andverylow levelsof differen-
tion.
tiation
between
thewild populations
Sunden
andDavis,1991!.Theauthors
Numerous attempts
toacquire
addi- concluded that allelefrequencies
tional
samplesofSPFpostiarvae
from amongpopulations
thewildtoexpand
thegene
poolofthe speciesrange have throughout the
little variation.
SPFstockhavebeenunsuccessful.
IHHNvirus
isnow inwild However,
widespread traits
ofeconomic breedin
un portancemay be under different
!
Se-
l ective
pressures
d across
therange, an
range
Lightner
etal.,1990;
Pantoja-differentiation
betweenpopulations
or
Moraies
andLightner,
1991;
Lotz
etaL, locales
couldbesignificant.
1991!.
A new
approach
todeveloping
SPFpopulations
involving
nondestruc-
tiveindividual
broodstock Breeding
screening Program
Design
using
gene
probe
diagnostic
proce-In rnainta' '
ures
dures iscurrently
is beingtested
Light- maintaining
captive
broodstocks,
ner,tNsvolume!. there
aretwogoals
that
impose
conflkt-
ingrequirements
formanagement.
The
 of SPF

fxrstgoalrequiresproceduresthat avoid toimprove

shrimp
perfc~~ byse
detrimental inbreeding of the captive lective
~~ng willbeverified
bysys
broodstock.The secondgoalrequires tematic,
scientific
xnethods
tooptimize
geneticmanipulation
to improvepro- theeffectiveness
ofthebreeding
pro
ductionperformance.
To precludecon- gramto ixnprove
economically
impor
flict, the SPFbreeding program consists tanttraitsfor shrimpculture.
af two levels: multiple discretepopula-
tions and multiple maternal lineage The objectives
of thesefoundation
ex-
familieswithin eachpopulation Fig. 1!. perimentsare;
A similar two-levelbroodstockmanage-
ment program is used in Norway to ~ To determine male and female
breed Atlantic Salmon Refstie,1990!. contributions
to the quantitative
traits, growthand survival;
Previous selectivebreeding efforts for
reproductivequality indicatedthat P. ~ Toestixnate
+fiancecomponents
xenmrxtrei
respondsto selection,and re- additiveandnonadditive
genetic
productivequality canbe improvedby variation, and nongeneticvari-
selectivebreeding Wyban et al., sub- ation! and phenotypic,genetic
mitted!.Realizedheritability estimated and environmental correlations.
for several quantitative variables in-
cluding naupliitspawn,spawmng fre- If thereis additivegeneticvariance
for
quency and hatchrate were compared Ipmvthrate, individualselection
will be
in two selectedfamilies againstnonse- applied.Responses
from eachgenera-
lected controls. Two different full-sib tion will add to previousgains as
famiTiesfrom the two outstanding
re- steps in a stairway.If there is heterosis
productive females with the highest for growth,it will beutiIizedto addto
hatch rates, matingtspawningfre- the effects of individual selection.
quency,and lifetimenaupliiproduc- Whilecrossbreeding
mustbe repeated
tion were reared to adult size. Two eachgeneration,it has the advantage
independent experiments were con- that one'scompetitorscannotduplKate
ductedcomparing
the selected
families the specificcrosses.
against nonselectedcontrols.Selected
femalesfar out-producedthe nonse- A "sub-line"
systemwiDbeusedin the
lected controlsin both experiments, breedingprogram.A populationre-
indicatingthatP. mnmmxti will respond sultingfrom one ixnportation!
will be
to selection
for a quantitative
character. subdividedinto multiple, genetically
isolated,
maternallineagefamilies.
The
Because much of the fundamental systemis basedon the theorythat
knowledge about inheritance of eco- becauseof inbreeding,geneticvariance
nomictraitsin shrimpis unknown,a V! within a family will go to zeroas
number of foundationexperiments theinbreeding coefficientF! goesto 1.
were recommended.Bestopportunities As the variance between families in-
262 Wn

SPF Shrimp BreedingScheme

1990 1991 1992 1993 1994

Featly
i
~J -
S~~-- ----e-
Featly
9
Fi~at~C
t Fcatty4 I -e
Population [~FIII
Fe~at9 I e-
L Featly
9 ~ - -- -e-
F eat~1D
[ Featlyl l
l~e
Generation
PO Generetton
Pl Generation
PX Generation
F3 Generation
P4

Popula

Generation
PO Generetton
Pi Generation
P3

Generation
PO Generation
E'1 Generation
P2

Figuret. SPFshrinpbreedingscheme.Genetically
discrete
populatensaresubdivided
andmaintained
in 12 individual families.
 weeks.Figure3 illustrates
growthof
three familiesspawnedon the same
day sameage!andgrownunderiden-
tical conditions. The difference in
gnmrthamongthe three familiesindi-
catesthereis signi6cant
diversity
for thesefamiliessuggeststheremay be
growth among thefamiTies
in Popula- sufficientgeneticvariationto improve
tion 1.
As of April 1992,sevenfamiliesthat
reached selectionsize 0 weeks in
growout! have been harvested. 'Iheir
relative
production
performance
isplot-
ted in Figure4. Familieswere ranked
by meansize at 20 weeksand coeffi-
cientofvariationCV!, foodconversion
ratioFCR!,
andsurvival
werealsoplot-
tedusingthe samefamilyranking. beginning.Basedon theseprelimi-
Thesesevenfamilieswerenot aUreared
F/gun<
4. Growthpefamanceat sevenSPF
famNes
tanked
bysizeat20 weeks
«iymmet.
Ua'ttgthesamerante'ng,
coeds/snt
ot win'aton
CVJ in indiMdo4size, feed canwrsian ratio
FCR!and svnmelare atsoksted,
simultaneouslyas in Fig. 3!, so envi-
ronmentaldifferencestemperature!
could have contributed some of the
variationin growth. Nonetheless,the
substantial
variabilityin growthaxnong
P. mmnamei growthrate by selective
breeding.In addition,the signi6cant
negativecorrelationsof CV p .Ol!
andFCR p <.05! with growthsuggest
thatthe bestfamilyin termsof growth
is alsothe best in other production
criteria.
The breedingof SPFshrimpfor high
health and improved growth is only
nary results and knowledgeof the suc-
cess enjoyed by breeders of other
meat animals, significantopportuni-
tiesto improveshrimp performance
and advancethe industry are wait-
ing.
Conclusions
~ A singlepopulation
of SPFP. rsiti-
enneihasbeen establishedin Hawaii
andis calledKonaPopulationl.
~ A breedingprogramdesignedto
protecttheir SPFstatus,avoidun-
necessaryinbreeding,
andimprove
growthand survivalthroughse-
lectivebreedinghas been estab-
lished.
~ Kona Population1 has been sub-
dividedinto 12 full-sib families.
 Selective Breedi
~ Significant variation in growth
among families was observed.
a A strong correlation between
growth to market size and size
uruformity and feedconversionef-
ficiency was observed.
Acknowledgments
The advice of the breeding advisory
group is sincerely appreciated. It in-
cludes Drs. James Lannan, Fred Shultz
and Trygve Gjedrem and Mr. Andrew
Coates. Mrs. Betty Sonoda provided
office support. Funding for this project
from CSRSof the U.S. Department of
Agriculture to the U.S. Marine Shrimp
Farming Program Grant No. 91-%838-
585l.
Literature Cited
Brown, B.L. and K.T. Paynter. 1991.Mitochon-
drialDNAanalysis
of nativeandselectively
inbred ChesapeakeBay oysters, Crassostms
virginica. Mar. Biol. 110: 343 '52.
Gall,C.A.E.1990.
Basisforevaluating
breeding
plans.Aquaculture.85: 125-142.
Gjedmn,T. 1985.Improvementof productivity
through breeding schemes.GeoJournaL
10!: 233-241.
ICES.1988.Codeof practiceto reducetherisks
of adverseeffectsarisingfrom introduction
of non-indigenousmarinespecies. Proceed-
ingsof the 1979StatutoryMeetingof the
InternationalCouncilfor the Explorationof
the Sea ICES!. In: C.J. Sindermann and
D.V. Lightner, Eds.!. DiseaseDiagnosis
and Control in Marine Aquaculture in the
Americas. Elsevier, New York, N.Y.
Kalagayan, H., D. Godin, R. Karma, G.
Hagino,J. Sweeney,J. WybanandJ. Brock.
1991.IHHN virus as an etiologicalfactorof
runt deformity syndrome RDS! in juve-
of SPF Shri 267
nile Gems amneeci culhmdin Hawaii.J,
WorldAquacultureSoc.22!: 235-243.
Lannan, J. E. 1980.Broodstockmanagement of
Crussssfnl galasI. ~ and environmental
variationin survivalin the Larval rearing
system.Aquaculture. 21:323-336.
Lannan,J. E. andJ. Wyban.1990,Broodstock
management programfor SPFshrimp,Un-
publishedmanuscript.The OceanicInsti-
tute. 24p.
Lightner,D.V. In press.Diseases of cultured
penaeidshrimp.In: J.P.McVey Ed.!. CRC
Handbook of Mariculture: Crustacean
Aquaculture.CRCPress,BocaRaton, Fia.
Lightner, D.V., R.R. Williams, T.A. Bell, R.M.
Redman and L.A, Perez. 1990. A collection
of casehistoriesdocumentingthe introduc-
tion and spreadof the virus disease
IHHN
in penseid shrimp culture facilitiesin North-
western Mexico.In: C.J. Sindermann Ed.!.
Proceedingsof the InternationalSympo-
siurn on the Effects of Introductions and
Transfersof AquaticSpadeson Resources
and Ecosystems.Special Publicationof the
WorLdAquacultureSociety.pp. xx-xx.
Lotz,J.M.,RM. Overate<,D.V. Lightnerand
R.M. Re&n Ln. 1991,Occunenm af IHHN virus
in pe~id shrimpfrom wild ulationsof
the eastern Paci& Ocean. Annual Meet-
ing of WorldAquaculture
SocietyAbstract!.
Pantoja-Morales,
C. and D.V. Lightner.1991,
Statusof the presenceof IHHN virus in wild
penaeidshrimp from the coastof Somera
Mexico.Societyfor InvertebratePathology,
Abstract!
Refstie, T. 1990. Application of breeding
schemes.Aquaculture. 85: 163-169.
Rosenberry,R. 1991.World ShrimpFarming,
1990.AquacultureDigest,SanDiego,CA,
55 p.
Shul', F.T. 1986.Developinga commenial
Iweedingprogram.Aquaculture.57:65-76.
Sunden, S,L.F. and S.K. Davis. 1991. Evalu-
ation of genetic variation in a domestic
populationof PenaeususnnmndBoone!:a
comparisonwith threenaturalpopulations,
Aquaculture. 97: 131-142.
Wyban, J.A., J.N. Sweeneyand R.A. Karma.
1988,Shrimpyieldsandeconomic potential
of intensiveround pond system.J. World
AquacultureSoc.19!: 210-217.
Wyban,J.A., J. Swingle,J.N, Sweeneyand
G.D. Pruder. 1992.Developmentand com-
mercialperformance
of high healthshrimp
from SPF broodstock Penane oanennei. In:
J, Wyban Ed.!. Proceedings
of the Special
Sessionon Shrimp Farming,World Wyban,J.A.,R. OyamaandR. Deupree. Sub-
Aquaculture
Society,
Baton
Rouge,
Lh.pp. mitted.Response toselection
forreproduc-
254-260.
tivequalityin thePacificwhitesheep,P.
usnnsma.Aquaculture.
 Developing
SpecificPathogen-Free
SPF!
Animal Populationsfor Aquaculture:A Case
Studyfor IHHNVirusof Penaeid
Shrimp
JefheyM. Latz
Gulf~ Reaeart& Laba3tory
P.O.Bax 7000,703 E. BeachHvd.
Ocean Springs,1%s.issippi,
U.SA

To securespecificpathogen-freeSPF!broodstockfor integrationinto nuclearbreeding

operations,the utmost caremust be usedto detectpaihogensand to preventthe introduction
of psthogens. Confidencein the SPFstatusof a founderpopulation increaseswith the
development of a lengthyhistoryof negativediagnostic
resultsandincreases
with thevariety
snd sensitivityof the diagnosticmethodsemployed.Therelativelysinallnumberof mumats
neededandthe finite time framemakesfounderpopulationacquisition easierthansecuring
SPFseed.Wildstockscanbesources of SPFanimals;however,aquaculture
activities
havespread
pathogenstowild stocks,diminishingthesupplyof wild SPFanimals.
Theprocedures employed
to secureSPF populations from wild stocks can, in principle, be extendedto extractSPF
individuals from contaminatedpopulations.

Introduction BIF&JV is a parvovirus Bonamiet al.,

Since the discovery and subsequent
descriptionof infectious hypodermal
and hematopoietic necrosis virus
IHHNV! Lightner et al., 1983a!, the
virus has been a major force in deter-
mining the activities and directions of
the U.S. shrimp farming industry. In
fact, the widespread use of Penaeus
vannamei
ratherthan the fastergrowing
P. stytirostrisis largely becauseP. mn-
namei is more resistant to IHRN-in-
ducedmortality.
1990! and its course of infection in P.
stylirostris has been weil documented
Bell and Lightner, 1984;1987!.Juve-
niles with acute IHI-IN disease exhibit
reduced feeding, mottling of the cuti-
cle, unusual swimming behavior, and,
ultimately, 80 - 95%mortality. The in-
fection in P. vannamei, on the other
hand, induces no clearsignsof disease
and no dramatic increasein mortality.
Hence, P, vennaeei was considered an
asymptomaticcarrierof the virus Ught-
ner et al., 1983b!,
 Lotz

However,
recently
therehasbeenevi- release
of the exoticvirus into U.S.
dencethatIHKA' infections
cancause coastalwatersgrew.
disease
«yndromes in P.mnemreiun-
dergipicalaquacultureconditions.
In Thesetwofactorsresulted
in aneffort
1989,
anextensiveepizootic
of56ihPl' within theUnitedStatesto controlthe
infectionin P. rianrtamei
occurred spreadof IFB~l. TheGulf CoastRe-
throughout
theU.S.shrimp
farmingsearch
Laboratory
Consortium
GCRLC!
industryaswellaselsewherein the isresponsible
forthiseffort.TheGCRLC
WesternHemisphere.
Atthesametime, was formedin 1984to accelerate the
therewasanincrease in reportsof developmentofmarine shrimp farming
nmting,deformities
anddemised pro- in theUnitedStates through research
ductiononfarms.In 1991,
Kalagayan andtechnologytransfer.It iscomprised
etaLlinked99PWtorunt-deformity ofsixinstitutions
working cooperatively
syndrome RDS!
bydemonstrabng the Fig.1! andis fundedby theUnited
presence
af RDSin IHHlA'-infected
animals
but not in IHHl'Ã-freeani- States
Departmentof Agriculture.
mals.
Although theprecise
relationship
betweenIHHl'&andRDS hasyettobe In
the
1989, thesixinstitutions
GCRLC undertook
comprising
to disinfect
detailed,
it is dearthatIHFiMFcan contaminatedfacilities at member in-
have
a severe negative
impactoncul- stitutions four of the six had been
turedP. mnnsssei.
Concemitant
with
thatIHI-INVcan contaminated
thedemonstration with IHI-BA'!, and re-
cause
serious m aquaculturedstockthemwith IHIP' -free P. risrt-
diseases
penaeids, overthepossiblenamei.
concern A source of IHHNV-free P.
vannmtrei
hadbeenlocatedin Mexico
by

THE ocKHvc
 Deve n SPFSh 27$

researchersat the University of Ari- occidcntalis

from the coast of Ecuador.
zona. The strategy was to relocate Finally,Lotz et al. 991! alsofound
IHIP/-free postlarvaeto an isolation 99~1 in wild P. vatitiltiei and P.
facility, grow them to broodstock,and stylimsttispostiarvae
in theGulf of Cali-
producepostlarvae.The IHHNV-free fornia and throughoutthe Pacific
postlarvaecould then be provided to coastal
waters
ofCentral
America
Figs.
member institutions. In the summer of 2 and 3!.
1989, the sources of IKKV-free ani-
mals in Mexico were infected with No aniinals whose status relative to
IFBihW, Lightner et al. In press!have If9~F is unknown are allowed into
documented the introduction and clean
facilities.
Hence,
a domestic
sup-
spread of IHHNV throughout the ply of certified IHI~F-free animals
aquacultureindustry in the northern wasneeded.The shrimp wouldneed
half of Mexico. In addition, Pantoja- to besubjected.
to acutequalitycontrol
Moralesand Lightner 991! have con- measures,anda lengthy historyof ag-
firmed the introduction of IEIIiNV into gressive
and repeateddiagnostic test-
the wBd stocks of P. vmvurmei,P. styli- ing for IHHNV would need to be
rostrisandP. calijornietisis
of the Gulf of developed.Eventually,a planwas de-
California. Lightner pers. comm.! has velopedfor obtaining
andmaintaining
also determined the existence of a centralsupplyof96&Pif-free animals
IHHNV in wild P. eetmamei and P. that would be protectedfrom IHI~P

Rgute2. A49pOfO'Ndco,CensedAlnmhs, and Figure3, The distdrutionof IHHNV and 8P ii

northern
SouthAmeemshowing
thedistnbution penaeidsINNP. The viruseshave co-extenafve
ofPenaeuavannarnei
andPenaeusstyliroatris, disttkefiam ShadedeIIss taipreavgtheoaois-
Thetwo speaeshave co-extensivedistribusans rtsnoeof IHHNV snd 8P. Unahadedat@asnpny-
ii the ales shown. sentregionswhkh havenotbeensurveyed.
 Lalz

~ sweUasotherpathogens
andfromthe biology
oftheshrimp,andtheexperi-
dangersofgenetic
inbreeding.
Theplan encesof animal breeding prograinsin
hasbeeninitiatedby the GCRLC,and poultry,salmon,and swinecausedus
the resultingprogrammay be viewed to seekapproximately12 separaternale-
astheinitialphasein thedomestication female crossesfrom each of three to ten
of P. ismnumi. sites. Therefore, the total number of
animalsneeded if collectedfrom ten
FounderPopulations sites! is 240 20 males and 120 fe-
males!.Oncethese animals arein place,
The nudeusof the protected
stocksis no moreimportationsare necessary.
keptin strictisolation
and mattuiged
under~ protoccll
to malnt84lsuRdent The small number of animals to be
geneticdiversityandallowfor the ju- secured and the finite number of foun-
didousselection of desirableproduc- der populationsnecessaryto accom-
tiontraits,Theoperation
of thenudear phshthegoal setsthis typeof activity
breeling
fadhtyis detailed
in Nyban apartfrom the more extensiveandcon-
thisvolume!andwN notbedealtwith tinuousimport of animalsfor general
furtherhere.Instead,I will focusonthe seedacquisition
orproduction.
Supply-
effortsof the GCRLC to secureSPF inganimals
tofoundanuclearbreeding
populations for the startor enhance- facilityentailslowvolume,but necessi-
mentofstocks
inthenuclear
breeding tateshigh confidence
in assuring
that
facUity.
Populations
destined
tobein- pathogensof interestare absentfrom
mq',orated
intothenudear
breeding
fa- all 240 animals,
cility are referred to as founder
populations.
FounderPopulation
Thegoalof anyfounder
population Acquisition
acquisition
istoprovide
spedfic
patho- Theinitialstepin securing
founder
gen-free
shrimpthatcanincreasethe populationsis to determinewhich
genetic
diversity
ofthestock
heldatthe pathogens
andpotential
pathogens
nuclear
breeding
facility.Therefore,
it should
beexduded.
Thenext
step
isto
wouldbe desirable
to secureseveral locatepotentialsourcesof founder
founderpopulations ofshrimp from populations,
andthethirdstepiscol-
distinct
geographic locationsthrough-iectmg
thepotential
founderpopula-
outthe naturalrangeof P. ttsnnamei.
tionsThefourth stepistoensurethe
Themoreanimals
thatareobtained
frommoresites,thegreater the in- ofndidate
all
founder
pathogens
populations
that are
tobe
arefree
excluded.
crease
in geneticdiversity
atthenuclear
breeding
facility.However,onemust Pathogens to be Exctoded
also
considerwhat canactually
beac-
quired
andsubsequently maintained.It is essential
to ite~
Attention
totheresourcesavailable,
the th pa ogensto be excludedfrom the
 Oev SPF Shrt P latkes

founder populations. Goals such as thanthesame agent in a farmpond

"disease-free"are too general to be Theinvestmentinthenuclearbreeding
useful. No animal populationscan be stock
is toogreattorisknotexcluding
maintainedfree of disease;the term poorlyunderstood
parasites.
"disease" includes maladies of unknown
andunforeseen
causes.It is not possi- It is important
thatallpathogens
onthe
ble to exdudeagentsthat areunknown. exdusionlist canbe exdudedfromthe
Because new agentsarebeingdescribed populations.
Attemptingto exclude
or-
from penaeidshrimpregularly, it is ganisrns
suchasV briosp., facultative
probably
unavoidable
thata newagent parasites
of shrimp and hutrutns
that
will eventuallybe detectedin estab- are ubiquitous
in shrimpgrowout
lishednuclearbreedingstocks. ponds and tanks, would be futile.
However,it might be possible
to ex-
Further, no animal populationcan be dudea serotype ofa particular
species
maintainedfree of a pathogenthat is ofVibrio,e.g.,oneof thehumanpatho-
undetectable
or ef'fectively
undetectable. genicVibriochalet biovars.
For example,the reo-likevirusespres-
entlyareonlydetectablewith the aid of On the otherhand,somepathogens
a transmission
electronmicroscope.
To canbeeasilyexcluded
froma popula-
undertakethe developmentof reo-free tionwhile in quarantine.Forexample,
shrimpstocks wouldbepremature.Nev- tetraphyHdean cestodelarvaearecom-
ertheless,
it isof paramountimportance mon in wild penaeids.The definitive
that all pathogensin the stockbe known, hosts for these animals are elasmo-
including those that may not actually branchfish;skates,
for exatnple.Since
be on the list of pathogensto be ex- shrimp ponds do not have skatesin
cluded.As the nudear breedingstocks them, the life cycleis not completed
becomemorevaluableto an industry, and the parasite is eliminated.
Grega-
expensive measures maybe justified to rines, which often use bivalves asinter-
rid thesestocksof a newagent. mediatehosts,area potentiallyserious
problem. While some intermediate
Known obligateparasitesof unknown hostsmay be found in shrimpponds,
effectshould be excludedif possible. our experiencewith the gregarine¹
Pn thispaper,"parasite"is definedas matopsispenaI from penaeidshrimp
anyorganism thatusesanotherorgan- in the Gulf of Mexicosuggeststhatthe
ism as its habitat. This includes bacteria shrimp lose their infections several
and viruses!.In general,it is not good days after relocation to a quarantine
practiceto leave a parasite off the ex- tank.
clusionlist simply becauseits effectis
unknown;suchan agentmaybecome The actuallist of pathogens
to be ex-
a problem
in thefuture.A parasite
that cludedalsodepends
uponthe species
turns out to be a problem in a nuclear of focusand the geographic
areaunder
breeding stock is much more serious consideration for stock acquisition.
 With the extensive movement of he~ca or parvo-like viruses in P.
shrimp species around the world for mepramei.The reo-like viruses Krol et
aquaculture purposes,the geographic al., 1990!werenot placedon the exclu-
rangeof a parhzdarpathogen maybe sionlistbecause noreasonablediagnos-
leseiinportant
thanthespecies
ofshrimp tic methods exist.
under consideration.
Bacteria.Most shrimpbacterialprob-
Pathogens Ot Penaeus vannamei lemsarecaused by secondaryinvasion
by free-livingbacteria.Targetingthese
ln thissection,
I willoutlinethepatho- for exclusion would be fruitless. Fur-
gensconsidered
fortheexclusion
listby thermore,it is extremelydifficult to
the GCRLC.More detailed accountsof distinguish between bacterialinfections
thepathogens
maybefoundin Light- acquired in quarantine from those ac-
ner983! orSindermann andLightner quired in nature. Were bacteria to
988!. Althoughviral pathogensare becomea problem,it is likely that we
our primary concern, we considered could have eliminatedthem during
manyotherdisease
agents.
Despiteour quarantine by the judicious use of
attention to numerousagents, how- antibiotics.
ever,prospectivefounder populations
wereInvariablydestroyedasa resultof The only bacterialagentswe consid-
contammationwith one of two viriuies, eredas potentialprimaryparasitesof
IHK'& or Bacalovims
penaeiBP!. shrimp are the rickettsia-likeorgan-
isms. We therefore screened animals
Viruses.Themaingroupof organisms for hepatopancreatic
granulomasthat
targetedfor exclusionfrom the founder might have indicated an infection with
populationsare the viruses. Virusesare rickettsia-likeorganisms Krol et al.,
the most widespreadof the serious 1991!.
Founder
populations
containing
pathogensof P. eermamri,and there are rickettsia-likeorganismswould have
no proven therapeuticsto eliminatevi- beendestroyed.
rusesfrom infectedanimals. The initial
focus was on IHHICT and 8P because Fungi.Althoughfungi e.g., Fusarium
theseareseriouspathogens
andthey solano!
areofconcernin shrimpculture,
were hkely to be encountered fre- theyarenotconsidered
primuy patho-
quently. They were, in fact, the inost gensof shrimp Lightner,1988!andwere,
common pathogens encountered. We therefore,
not to be excluded.However,
alsoconsideredthe other known bacu- thepresence
ofa fungus
in a sample
may
lovinLses
occluded
andnonocciuded! haveprecludedits incorporationinto the
and the parvo-like~~s to be unac-
nudgerbreedingstockas it may have
ceptable.
However,with the possible portended
otherstress-related
problems.
exceptionof HPV hepatopancreatic
parvo-likevirus!, it was unlikelythat Protozoa.The fouling protozoancili-
we would find other known bacu- ates peritrichs such as ZoofhamniMm
 Deve n spF sh 275

sp., suctorianssuch as Aci~ sp., and nematodes!

neededtobe excluded.The
apostoxnessuchasHyalophysasp.! were exclusion of intermediate hosts from
not listed for exclusion. However, a the quarantineand nuclearbreeding
heavyinfestation could indicatestress facilitieswould prevent transxxussion
in the quarantine facility and might and eliminate most helxninths.
haveprecludedthe inclusion of a sam-
ple into the nuclear stocksfor general Crustaceans. Crustaceans were not
health considerations. placedon the list. Crustaceans
suchas
bopyridisopodscouldhavebeenelimi-
Microsporanse.g., Amesotxsp., Agma- natedby removalof infectedindividu-
mna sp., Pleistaphomsp. and Thelohania als. These parasites also utilize an
sp.! were placed on the exclusionlist, intermediatehost e.g., copepods!,
However,they occurat low prevalence making transmission more difficult in
in wild populations,and infectedindi- quarantine.
viduals are easily detected and re-
moved from a population during Locating PotentialSitesfor
quarantine.In addition, there is some
evidencethata piscineprimer hostmay Acqoisition
be necessaryto allow transmission Oncea listof pathogensto be excluded
from one shrimp host to another was developed,the processof screen-
iversen and Kelley, 1976!. Microspo- ing possiblesourcesof founderpopu-
rans, therefore, could have been elimi- lationsbegan.The two optionswere
natedfroxn a contaxxunatedpopulation culturefadlities andwild populations.
during the quarantine period because Culture facilities are often contaminated
transxnission would have been pre- with IHI~P aswell asBPthroughout
vented by the absenceof fish. The the range of culture of P. mtnamei.
removal of affected individuals would Therefore,we surveyedwild popula-
have preventedpathogensfrom leav- tionsfor the presenceof the two agents
ing the quarantinefacility. of primaryinterest.

Gregarineprotozoansmay be of con- Detection of Viruses

cemin soxneaquaculture settings.How-
ever, they are apparently elixninated
spontaneously from shrimpduring the
quarantinephase.The exclusionof in-
termediatehostsfrom quarantinefacili-
ties prevents transmission. Therefore,
gregarineswere not placedon the ex-
clusion list.
Helxninths. We did not consider that
helminths cestodes,trematodes,and
All anixxxals were screened for viruses
by meansof standard histological ex-
amination BeU and Lightner, 19%!.
IHHNV was detectedby the presence
of Cowdry Type A intranuciearinclu-
sions in several tissues of ectoderinal or
me.+dermalorigin Lightneret aL,1983a!.
BP was deb~ by the preside of in-
tranuclear polyhedralocclusion
bodiesin
ceUsof the hepatopancreasCouch,1974!.
 276

'Ihe sampling
protocol
premed that infected P. stytitostnswill show xnortal-
initially
a sitebescreened
bycollecting ity andtheCowdryTypeA intranuclear
a smailgrabsaxnple
of arum;xls
froxnthe inclusions characteristic of IHIiNV infec-
area,fbdng
theanimals
forhistological tion.
examination
while stillin the field,and
subsequently
examining
theinfor the Assumingaxuinalswere negativefor
preset efIHHIM andBP.If a sample IHHIM aftera "stylirostris-bioassay,"
was not positiveafter the examination the nextstepwasto shipthe remaining
oftento20animals,
thena largebatch animaLsto a quarantinefacility in Ha-
of live animalswas to be obtainedand waii for further examination and clear-
placedinto quarantine.
In practice, ing. From there, introductioninto the
havrever,
thegrabsamples
andthelive nuclearbreedingfacility wasto begin.
samples
weretakensimultaneously
wherever
possible.
Therefore,
samples Directintegration
of the founderpopu-
for examinationwere a combinationof lationintothe nuclearbreedingfacility
directsamples
ofwild postlarvae
and is anoption.However,in general,it is
adults, and a large numberof wild moreprudentto growthe new popu-
pox4arvae held in quarantine
for 30 to lation into broodstock and introduce
60 days. tested offspring as families into the
nuclearbreedingfacility,
Neusedpastlarvaeinstead
ofjuveniles
oradultsforseveral
xeasons.
Rrst,post- Theabsence
of a pathogen
froxna po-
larvaeareeasiertohandleandxnaintain tentialfounderpopulationcanonly be
in quarantine,
allowingus to screena assured "to-the-best-of-our-abili-
largernumberof animals.Second,be- ties." Thus, for certification,it is nec-
cause animals xnust be sacrificed for
essary to specify the methods of
emmination,weneeded a large
enough detectionused,the numberof times the
sampleto insure that we would have diagnosis was applied,the numberof
animalsleft over.Third,postlarvae
or axumalsto which the diagnosiswas
youngjuvenilesoftenshowdiagnostic applied,and the length of quarantine.
signsof the two ~LLses better than Confidencein the absenceof a patho-
olderjuveniles
oradultsBellandLight- genincreases with increased
sensitivity
ner,1987;
LeBlanc andOverstreet,
1990!. of the diagnostictechniques, greater
numberof testsperforxned,greater
If IHHIM was not detected after 60 number of animals checked, and
daysin quarantine,a bioassay
diagno- longerperiodsof quarantine.
sis for IHHIM was performed.An
IHI~P bioassay diagnosis consists
of QUarantine Facilities and
feedinga sampleof P. exxxnunxsi
thatis
suspectedof carryingIHI~P to the Procedures
moresusceptible
P. sglimstns Lightner Theprimary
meansof assuring
that a
et al,, 1983b!.After nineto 30days,any pathogenis not presentin a founder
 Dev in SPF Shri P htions

population is to develop a lengthy his- ties are preventing the contamination

tory of negative diagnostictest results. of samples with pathogens from sur-
Quarantine is the crucial step in devel- rounding areas and anim &, and pre-
oping an appropriate history. venting the contamination of
surrounding grounds and waters with
Quarantine serves three functions. An exotic speciesand their pathogens.
individual may havebeeninfectedwith
the pathogen of interest only recently, The quarantine facilities at GCRL con-
and, therefore, may not have devel- sist of a large greenhouse 5 m x 9 m!
oped the signsof infection. In this case, sited on a concrete slab with an 8- cm
the quarantine period should provide high lip completely surrounding the
the time necessaryfor the shrimp to slab Fig. 4!. The greenhousecontains
develop signs of infection. Second, 15 2,000-L circular tanks. Each 2,000-L
some infectious agents such as BP are tank has an in-tank biofilter and can be
more likely to be found if an infected drained individually into effluent PVC
host is stressed Couch, 1974!. Quaran- drain pipes. The effluent pipes empty
tine is usually stressfuland can provide into septic tanks equipped with chlori-
such a stimulus. Third, the quarantine nators. From the chlorinating septic
procedurecan amplify a diseaseagent tank, the effluent canbe pumped to the
within the quarantined sample. Infec- municipal sewer line. Contamination
tious agents present in a small number between tanks is prevented by opera-
of arumals wiH eventually be transmit- tional procedures and the quality of the
ted to uninfected individuals. The para- maintenance crew. The crew is weH
site becomesmore prevalentand canbe trained in isolation, sanitation, and dis-
detectedby examining fewer individu- infection procedures.
als.
The other facility used for quarantineis
The danger in quarantine is, of course, a3 mx 5m isolation room that can
that the quarantined populations are accommodate four 650-L circular tanks.
open to exposureand subsequentinfec- Eachtank is separatedfrom the others
tion with important pathogens.The use by shower curtains and each tank has
of quarantine requires extremely tight its own in-tank biofilter. The tanks are
quality control and precautions to pre- not drained during the holding period
vent contamination of samples. and the water is chlorinated at the end
of the holding period. Eachtank has its
Quarantine Facilities own maintenance equipment: ther-
mometer, nets, beakers, etc. The room
The quarantine facility and procedures has a center floor drain that empties
used at the Gulf Coast Research Labo- into an outside septic tank equipped
ratory GCRL! in Ocean Springs, Mis- with a chlorinator. From the chlorinat-
sissippi, U.S.A., will be examined. The ing septic tank, the effluent can be
two main goalsof the quarantinefacili- pumped to the municipal sewer line.
278 Lotz

Cross contamination is prevented by ppm chlorine for 1 - 24 h prior to

operationalprocedures, dischargeinto the municipal sewer
lines that terminate at a landfill. All
We employ sentinel tanks containing dead animals, molts, feces, etc. are
uninfected animalswhenever possible. disinfected with chlorine- or iodine-
For example,we presentlyusesentinel containingdisinfectants
ar by autociav-
tanks and aquaria containing IEGiNV- ing. Surfacesarecleanedwith chlorine-
free P. stylirostris as a check against or iodine-containing disinfectants.
contamination. The sentinel tanks are
treated as the other quarantine tanks To ensure that infectious diseases are
and are subjectedto the routine proce- not introduced into the quarantine fa-
dures employed for the other quar~- cilities or transferred between tanks,
tine tanks. we established routine sanitary work
practices.
Theseincluderestrictedac-
Quarantine Procedures cess, the use of foot baths at the en-
trances to all doors, regular cleaning
To minimize the risk of releasing exotic and disinfecting of equipment and
organismsduring quarantine activities, rooms, disinfection of shrimp waste
all effluent water is disinfected. Disin- and debris, and dean food preparation
fectionis accomplishedwith 100-to 200- areas.

Figure4. Hoorplanof thegreenhouse

quarantine
facilityat theGulfCoastResearch
Laboratory.
 Deve in SPF Shrim P Uiations 279

Most introductions and transfers of stylimstris anywhere throughout its

pathogensoccur asa resultof the trans-
port of shrimp or shrimp parts. There-
fore, specialattention is paid to prevent
the transfer of tank contents from one
to another; similarly, equipment is seg-
regatedby tank with no overlap in use.
Dry feedis keptawayfrom shrimpand
shrimp debris. Each tank has its own
nets, and nets are disinfected after each
use and allowed to dry completely be-
tween daily deanings. Surgical gloves
are used during routine tank cleaning,
and gloves are disinfected and dis-
carded after each tank is cleaned. Natu-
ral water is used in the facility; it is
routinely settled, filtered and disin-
fected with chlorine.
The SPF Status of Wild
Penaeus vannamei
The geographic range of P. vunnamei
extends from the northern Gulf of Cali-
fornia to the northern portion of Peru
Fig. 2!. Through the efforts of the
GCRLC, the status of wild P. aNtnumei
hasbeen documentedalong most of its
range Lotz et al., 1990;Lightner et al.,
In press!.The mostwidespreadof the
agentsof interest is, unfortunately, the
virus of most crucial interest: E6&W.
Figure 3 shows the distribution of
IHIP/ in wild penaeids throughout
the Pacific Coast of the Americas. The
highest density of II-IHN virus is in the
Gulf of California, where all of the
specimensin a sampleof wild, adult P,
stYlirostriswere found to be IHI~K-
positive. It is significant that this area
has one of the highest densities of P.
range. The range of P. stylirostris is
coextensive with that of P. vunnarnei.
The Gulf of Panama also appears to
have high levels of IIiHNV, but lower
than the Gulf of California. This area
yielded fixed-in-the-field samplesof P.
vannamei postlarvaewith IHHNV infec-
tions, The lowest density of IHHIAT
appearedto be in the northern portion
of Central America. No positive fixed-
in-the-field samples were found from
Guatemala, Nicaragua, or northern
Costa Rica; however, virus was de-
tectedin samplesfrom Nicaraguaand
El Salvadorafter 30 to 60 days of quar-
antine. Southern Mexico has not been
surveyed; its status is unknown, The
waters surrounding Ecuador have
yielded evidence that IHHNV is pre-
sent in wild populations, but surveying
has not been as extensive as that in
Central America and Northern Mexico.
The survey of shrimp of the Pacific
Oceanrevealedthe presenceof numer-
ous parasitesin addition to the viruses.
Peritrich and apostome ciliates were
common on the gills. Gregarines were
seen in the intestines. Finally, nema-
tode, cestode, and trematode larvae
were observed in the hepatopan-
creases, musdes and nerve cords of
infected shrimp.
Acquiring SPF Stock From
Contaminated Sources
Acquiring SPF animals from geo-
graphic areasfree of the pathogens of
interest is simply a matter of collecting
 Lotz
samplesand subjectingthem to the
processof certification.However,if no
pathogen-freesites are found, acquir-
ing SPFanimals is more complicated.
The data acquired from the survey
throughout P, vannamei'sgeographic
distribution revealed that no area was
unaffectedby IHHNV. Sincethe wide-
spreadoutbreak of IHHNV in 1989,no
IHHI'PV-freepotential founder popula-
tions have been found, despite exten-
sive efforts and the quarantining of
numerous samplesof wild postlarvae.
Therefore, our original objective of lo-
cating BVPFl-free geographic sites
had to be reformulated. Instead of seek-
ing IHHNV-free populations of
shrimp, we now wish to securea cer-
tain number of IHHhAl-free individuals
from populations of shrimp which are
known to carry the virus.
When we conducted our survey using
quarantine, it was desirable to collect
as large a sample of shrimp as possi-
ble in order to be certain that the
region was free of the virus. Our
target for sampling postlarvae under
the initial survey objective was to
quarantine 10,000to 20,000animals
for eachsample. If one of those 10,000
to 20,000 animals was infected, then,
in time, a large proportion would
become infected and the virus would
be easily detected after 60 days of
quarantine.Presently,thereis no way
to select individual postlarvae from a
contaminated sample. Consequently,
if one animal in a sample is positive
for the virus, the whole sample is
destroyed.
Our inability to detectvirus from some
areas prior to 60 days in quarantine
suggeststhat only a few animalswere
carryingthe virus. If only a fewanimals
from a given wild population carry the
virus, a small sample containing 1,000
to 5,000shrimp may be virus-free. If the
prevalenceof an agent in the wild is
known, securinganimals free of the
agentis a statisticalsamplingproblem.
What is the optimum samplesize to be
certain no infected animals are present
in a given samplers
The above problem is related to the
problemfish inspectorsfacedetermin-
ing how many fish to examine from a
particularbatch to have a certaindegree
of confidence that they will find the
pathogen. The American FisheriesSo-
ciety "blue book" provides a table that
recommendshow many fish should be
examined Amos, 1985!. For example,
if a parasite is present in 10% of fish
and the lot being evaluated contains
4,000 Gsh, 27 should be examined in
order to be at least 95% confident that
the pathogenwill be detected,if pre-
sent. The answerto the fish inspectors'
problem is determined from the hyper-
geometric statistical distribution and
appliesto samplingsmalltargetpopu-
lations.
When the target population is very
large e.g., a wild population of
shrimp! then the related binomial dis-
tribution applies.Table1 providesthe
maximum sample sizes that ensure at
least a 50% chance that no animals in
the sample are infectedwith the patho-
gen of interest. Once the maximum
 Deve l SPF Shrt P Uhtions 281

Table 1. Sample size that ensures a Mio chance of a parasite

acceptablesample size is determined, of the time an areais sampled, one of

how many separately packaged sam- the five samples will be free of infected
ples should be obtained to be 95% individuals. The likelihood of finding
certain at least one sample is free of uncontaminated packagesincreasesas
infection? more packagesare collected.

If there is a 50%chance probability = Once the size of the sample and the
0.5! that an infected animal is present number of samples to be collected is
in a sample, the probability that it is determined, then the collection of ani-
present in both of two such samplesis mals and the lengthy procedure of
quarantine and subsequent develop-
0.5 x 0.5 = 0.25 5%! ment of the crifical SPFhistory begins.
Becauseshrimp wiD be selectedfrom
and, therefore, contaminated areas, the development
of SPF histories is critical.
100% - 25% 75%.
The ability to select animals from the
Hence, there is a 75% chance that the wild rests on the assumption that not
pathogen of interest is absent from ail animals from a contaminated wild
either one or both samples. If three population carry the pathogen.In prin-
such samplesare collected,the chances cipal, obtaining SPF individuals from
are that 87.5% of the time one of the contaminated culture facilities is the
three samples will have no infected same as obtaining them from wild
individuals. Through this process, it is sources. However, the likelihood that
clearthat if a parasiteinfects50%of the there are specific pathogen-free indi-
packagedsamples, one should coQect viduals in a facility is reduced because
five separately packaged samples to the animals in culture are at higher
ensurethat at least95% actually 96.8%! densities than in nature. Hence, the
 Deve In
Bell, T.A. and D.V. Lightner. 1988.A Hand-
book of Normal PenaeidShrimp Histology.
SpecialPublication No. 1. World Aquacul-
ture Society, Baton Rouge,Louisiana, U.S.A.
Bell, T.A., D.V. Lightner and J.A. Brock.1990.
A biopsyprocedurefor the non-destructive
determination of IHHN virus infection
Penaeus
vannarnei.
J. Aquatic Animal Health.
2: 151-153.
Bonami, J. R., B. Trumper, J. Mari, M. Brehelin
and D.V. Lightner.1990.Purificationand
characterization
of the InfectiousHypoder-
mal and HaematopoieticNecrosisVirus of
penaeid shrimps. J. General Virology. 71:
2657-2664,
Couch,J.A. 1974.An EnzooticNudear Polyhe-
drosis Virus of Pink Shrimp; Ultrastructure,
Prevalence, and Enhancement. J. Invertebr.
Pathol. 24: 311-331.
Iversen, E.S. and J.F. Kelly. 1976.Microspori-
dosis successfullytransmitted experimen-
tally in pink shrimp. J. Invertebr. Pathol.27:
407-408
Kalagayan,H., D. Godin, R. Karma,G. Hayno,
J. Sweeneyand J.Wyban. 1991.IHHN Virus
as an etiological factor in Runt-Deformity
Syndrome RDS! of juvenile Penaeusvan-
namei cultured in Hawaii. J. World
Aquacult. Soc.22: 235-243.
Krol, R.M., W.D. Hawkins and R. M. Over-
street.1990.Reo-likevirus in white shrimp
PenaeusvannanreiCrustacea: Decapoda!; co-
occurrencewith Baculoviruspenaeiin experi-
mental infections.Dis. Aquat.Org. 8: 4549,
Krol, R,M., W.D. Hawkins and R,M. Over-
street. 1991. Rickettsial and mollicute infec-
tions in hepatopancreaticcellsof cultured
Pacific white shrimp Penaeus vunnanrei!.J.
Invertebr. Pathol. 57: 362-370.
LeBlanc, B.D. and R.M. Overstreet. 1990. Preva-
lenceof Baculoviruspenaeiin experimentally
infected white shrimp Penaeusvannarne'j
relative to age. Aquaculture. 87: 237-242.
Lightner, D.V. 1983. Diseases of cultured
penaeid shrimp. In. J.P. McVey Ed.! CRC
Handbook of Mariculture, Volume I, Crus-
SPFShrlm P Uhtioris
tacean Aquaculture. CRC Press, Inc. Boca
Raton, Florida, U.S.A.
Lightner, D.V. 1988.Fungus Fusariurn!disease
of juvenileand adult penaeidshrimp.pp.
64-69.In: C.J.SindermannandD.V. Light-
ner Eds.!. DiseaseDiagnosis and Control in
in North AmericanMarine Aquaculturend
edition!. Elsevier, Amsterdam, Nether-
lands.
Lightner, D.V., R.M. Redmanand T.A. Bell.
1983a.Infectious hypodermaland hemato-
poieticnecrosis IHHN!, a newlyrecognized
virus diseaseof penaeidshrimp, J. Inver-
tebr, Pathol. 42: 62-70.
Lightner, D.V., R.M. Redman, T.A, Bell and
J.A. Brock. 1983b. Detection of IHHN virus
in Penaeusstylirostris and Penaeusvannarnei
imported into Hawaii, J, World Marie. Soc.
14: 212-225.
Lightner, D.V., R.M. Redman, T.A. Bell and
L.A. Perez. In press. A collection of case
histories documenting the introduction
and spread of the virus IHHN in penaeid
shrimp culture facilities in northwestern
Mexico, In: C.J. Sindermann Ed.!. Pro-
ceedings of the International Symposium
on the Effects of Introductions and Trans-
fers of Aquatic Specieson Resourcesand
Ecosystems. Special Publication of the
World Aquaculture Society, Baton Rouge,
Louisiana, U.S.A.
Lotz, J.M., R.M. Overstreet,D.V. Lightner and
R.M. Redrnan. 1991, Occurrence of IHHN
virus in penaeidshrimp fromwild popula-
tions of the easternPacificOcean,J. World
Aquacult, Soc. 22: 37A.
Pantoja-Morales, C. and D.V. Lightner. 1991.
Status of IHHN virus in wild penaeid
shrimp from the coast of Sonora, Mexico.
Program and Abstracts of the XXIV An-
nual Meeting of the Society for Inverte-
brate Pathology.
Sindermann, C.J. and D.V. Lightner Eds.!
Disease Diagnosis and Control in North
AmericanMarineAquaculturend edition!.
Elsevier, Amsterdam, Netherlands.
Latz
 Growth and Survival of Virus-infected and SPF
Fbnaeus vannameion a Shrimp Farm in Hawaii

NickCarpanter
Amxierk Aquahrm, Inc.,P.O.Box131
KaM'u, Hawaii 96731, U.SA,

James A. Brock
&qartmertt of lard ard Natural Resources
Aquaculture De4opment Program
335 MevcharcStreet, Rm. 348
Honolulu, Harm 96813, U.SA

In late 1982,Amorient Aquafarminitiatedwork with &naeusvannamei at their maturationand

hatcherysite locatedin Kahukuon the islandof Oahu, Hawaii. From 1983to 1989,laboratory
testsdetectedno known virus diseasesor other obligatepathogensin shrimpculturedon the
Amorientfarm. In early 1989,however,infectioushypodermaland hematopoieticnecrosisvirus
IHHNV! was discoveredin stocksof P. vannamei at the farm. The effectof IHHNV infection
on shrimpproductionwas dramaticand was expressedas a markeddecreasein growththat is
characteristicof runt-deformitysyndrome RDS! of P, vannamei,In the IHHNV-infected RDS
groups,the coefficientof variationin size CV! increasedfrom 10 - 20%to 40%, and pond yields
decreasedaccordingly. In mid-1990, Bacubmruspenaei BP! infectionswere also found at low
prevalence
andseverity
levelsin samples
of shrimpexamined
fromAmorientponds;h~r,
no negativeimpacton productioncouldbe attributedto the BPinfection.In January1992,the
farm was stockedwith the progenyof SPF P. vannaniei
broodstock.This report considersthe
resultingdisappearanceof RDS and the productionand yield improvementsobtainedwhen
IHHNV-free shrimpwere againculturedon the Amorientfarm.

Introduction the culture of the freshwaterprawn,

Macrobrachiumm ~bergii. On the main
Amorient Aquafarm, Inc. is a 175-acre farm site, there are 142 1-acre .46-ha!
9.6-ha! shranp and prawn farm lo- earthen ponds and one 0.5-acre.23-
cated on the North Shore of Oahu, ha! concrete-sidedround pond. There
Hawaii. The farm was constructed in are also 10 0.25-acre .11-ha! brood-
1977 and was originallydesignedfor stock ponds at an adjacent area 0.5
 Ca
miles from the main farm site.
maturation/hatchery facility is located
at a third site dose to the broodstock
pond area.
In late 1982, 78 adult, wild-caught
Penaetfsvannameiwere shipped from
Ecuador and introduced in quarantine
at the Amorient Aquafarm matura-
tion/hatchery site in Kahuku, Oahu,
Hawaii. The intent of the introduction
was for the Amorient staff in Hawaii to
develop appropriate technology and
gain experiencein shrimp maturation
and larval rearing, and to eventually
transfer this knowledge to the com-
pany's 1,000-acre 55-ha! commercial
shrimp farm in Ecuador.
In mid-1986,a subpopulation of adult
P. trumodon
obtained from the Aquacul-
ture Development Program, State of
Hawaii, were stocked into the Amori-
ent Aquafarm maturation facility. This
group of shrimp originated asoffspring
from a wild-caught spawner collected
in waters off Sabah, Malaysia. After
introduction to Hawaii and repetitive
direct histopathology and shrimp bio-
assayevaluation during the 18 months
of quarantine following introduction, a
small group of adult shrimp was trans-
ferred to the Amorient Company.
On the basisof initial successin repro-
duction,spawning,productionof post-
larvaeandfavorable
resultsin growout
with P. vunnamei, the decision was
made to engage 80% of the Kahuku
farmin shrimpculture.Over the next
The term "high health" animalsis preferredby Wyban this volume! when referring to animals that have
beenremovedfrom an SPFquarantinefacility.
iNer arid Brock
The several years, shrimp production was
rather consistent, ranging between
2,400and 3,000lbs/acre/yr,400- 3,000
kg/ha/yr!. Starting in late 1986,P. mono-
donwere also stockedfor growout, but
by early 1988culture of this specieswas
limited due to poor pond performance
relative to P. vannamei under the envi-
roninental conditions and husbandry
practices in use at that time oo. the
Amorient farm.
In mid-1987, an outbreak of infectious
hypodermal and hematopoietic ne-
crosis virus II~V! disease occurred
on a shrimp farm nearAmorient Aqua-
farm. The origin of the I'D JV in this
outbreak is not determined. However,
by late 1988,IHHNV was detected in
samples of F6 generation shrimp col-
lected from the Amorient matura-
tion/hatchery area, and by mid-1990,
the virus was widespread in growout
ponds on the farm. Also, in August
1990, Bacutoviruspenaei BP! infection
was found in shrimp sampledfrom the
Amorient farm.
In December 1990, The Oceanic Insti-
tute provided Pl generation, Mexican-
derived, specificpathogen-free SPF!P.
vunnamei broodstock to Amorient
Aquafarm from which SPFpostlarvae
were produced and stocked into
growout ponds.
MgteI jgls apd Meth !
Comparisonbetween non-SPF-derived
and SPFshrimp pond productionand
 Performance
Table 1. A summaryof the tHHNVand BP histopathologyresultsfor Penaeusvannamei
growth performancewas done by de-
termination of the total weight of
shrimp harvested from ponds, percent
survival, weekly growth rate, feed con-
version ratio, and size distribution
mean, standard deviation and coeffi-
cient of variation! for shrimp weight
from random samples minimum N =
100!of different populations.
From 1983 onwards, diagnostic exami-
nation for the detection of
penaeid viruses and other obligate
pathogenswere periodically conducted
on the offspring of P. vannameiand P,
rnonodon cultured at the Amorient
and on stocksproduced from the Am-
orient maturation/hatcheryfacility that
were distributed to other locations
Hawaii.
For diseasemonitoring, shrimp were
sampled at various sizes/ages,indud-
ing postlarvae PL6-12!, 0.5- to 1.5-g
nursed juveniles, 4- to 1Dg subadults
from growout ponds, and 5-g
broodstock.Specimenswere either fro-
zen for the P. sglirostris bioassay test
Lightner et al., 1985!or killed by injec-
tion and immersion in Davidson's
tive Huma son, 1979!
histopathology evaluation. Histological
of SPF P. vannamei in Hawaii 287
processing and slide preparation fol-
lowed standardprocedures.Tissuesec-
tions were stained with hematoxylin
and eosin Luna, 1968!.
Prior to restocking the ponds in 1991
with the progeny of SPF broodstock,
the ponds were dried for two weeks,
and 800 lbs of agricultural limestone
was spreadover the pond bottoms. The
bottom gravel of the round pond was
known dried and then partially fiUed to cover
the substrate, which was treated with
10 mg/L of chlorine overnight before
the pond was refilled.
site,
In the growout trials, a 25% protein,
locally produced pellet was fed to
in shrimp stocked into earthen ponds,
and a 45% protein, imported pelleted
ration was provided to the shrimp in
the round pond.
Results and Discussion
The histopathologyexaminationresults
for P. vannameisampled from the Am-
orient farm and maturation/hatchery
areasfor the period from 1987through
fixa- 1991 are listed in Table 1. In addition,
for P. stylirostris bioassay trials were con-
ducted on shrimp that originated from

the Amorient populationup to mid-
1987. For example, between January
and May 1987,indicatorshrimpbioas-
say tests were carried out on tissue
samplesfrom threegroups N = 50! of
Amorient subadult to adult P. emnamei.
Prior to 1989,infections by either BP or
IHI~ viruses were not detected
bioassaysor by direct histopathology
evaluation of shrimp from the Amori-
ent farm. Direct histopathologytests
conductedon juvenile through adult N
= 30! P. mottokm sampled from several
pondsin August 1987were alsonegative
for known obligate shrimp pathogens.
However, IHHN virus infection was
detected histologicaily in samples of
postlarvaecollectedfrom the Amorient
hatchery in early 1989 Fig. 1!. As the
year progressed, the frequency of
IHHN virus-positive groupsincreased,
Figure t. Peroentageof IHHNV-positiveindiMduais
er and Brock
and by May of that year, IHHI'W was
detected in 100% of the postlarval
groupssampled.Within IHHNV-posi-
tive groups, the averagenumber of
individualswith histologicaliy
diagnos-
able IHHNV infection increased
through1989and 1990.Averagepreva-
in lence of infection in early 1990 was
26.2%,but duringthe summerof 1990,
prevalenceof infectionincreasedto an
average of 43.1%. Coincidentally,
broodstockwere replacedeveryfourto
six months,and the increasedpreva-
lence of infection may have reflected
higher levelsof IHI~V infection in the
older broodstock.
In late 1990,The OceanicInstitutepro-
vided severalgroups of SPF nauplii
Mexicostock!to AmorientAquafarm.
The postlarvaeproducedwith these
nauplii tested negative by histological
withingmqpsof postlamaetestedfor IHHNV.
 Performance of SPF P. Osn/Mmei in Hawaii

Non-SPF Qrowoor
criteria for IHHNV infection, whereas 1/2S/IS
postlarvaeproduced in sister tanks us-
ing nauplii from IHHNV-infected
broodstock Amorient's Ecuador stock!
continued to test positive for IHHNV
Fig. 1!. At no point during this period
did any of the postlarval populations
N = 71! harvested from the hatchery
test positive for BP.

Unfortunately, histogram assessments ~S S~ IS ~I ~~ IS II IS e

were not carried out for shrimp cul- Iioivl0LNL
woioiITQI
tured on the Amorient farm before
IHHNV was detected, from 1982 Moo-SPF QrowoIIr

through 1988.RDSwas not apparentin 10/6/QO

early 1989;this is demonstratedby ran-

dom histograms of populations from
1-acre earthen ponds in which the size 1S
a
coefficient of variation CV! was only 11
1794 Fig. 2a!. However, a nursery har- S Ia
vest at that time contained an unusu-
ally high number of "small" juveniles 5~
a large percentageweighed 0,3 g or
less Fig 3a!. As time passed,on aver-
age, the CV slowly increased,peaking ~I ~~ I IS iS I~ i ~ SS e SS SS SS
at 46% in late 1990 Fig. 2b!. The in- NolvolML waoHTto|
creasingCV was also apparent in suc-
cessive crops harvested from the SPF Orowor/r

intensive, 0.5-acreround pond Figs.

4a, b!. As a result of RDS, the average
harvest size decreasedfrom 11.9 g to
8.5 g in the 2 growout trials conducted ss

during this period. ss

In late December1990,Amorient Aqua-

farm received SPF P. mmnamei brood-
stock. These shrimp were founder-
generation stock collected by the U.S.
Shrimp Consortium as postlarvae in ~1 I~ ~ 92~ I~ Is Is I I % & ss IS
Mexico and grown to broodstock at the
quarantine facility of The Oceanic Insti- Rgure 2. Size I/aria@on in Panaeua vanrIamei
tute in Hawaii.
dUIKg gRwHDlk
290 Ca enter and Brock

NOG-SPFNursery ROUND POND, Hoo-SPF

! 000 7/27/00

24
2 44 28
K
Cl O 18
O It 2
2
IV I
II 8

08 2 4 8~ 181214'I ~ 18 28 2224
.'I 4 J J I4 44 4 l I,l IJ lJ
MEAN
WEIGHT
!8!
SIZEIS!

ROUND POHD, No!I-SPF

Non-SPF Nursery 11/! 0/00

1090
28
24

24
0 14
2 X 44
Cl
I 12
8

I MEAN
WEIGHT
8!
~~ .I I IJ I 44 4
SlzEIe!

ROUHO POND, SPF

SPF Nursery 3/15/0 1
!aa! 28

4
IL. 2
a 2
18
JI O
O III 12,
I
2

I I I 14 IJ
slzE !8! VEANWEIGHT
8!

Figure8, Size !/Esnhtion

in PenaeuevannarT!eiin Fig!/red. Size variat!onof Penaeusvannerneiin
the nursery phase. round pond gro!/!/outtrials.
 Performance of SPF P, vannamei in Hawaii 291

Improved production was noted imme- harvests. In the non-SPF harvest on

diately with the progeny of the SPF Nov. 16, 1990, 8% of the shrimp were
broodstock.Note, for example,the size below marketable size and 53'%%d
were
distributions from two consecutive under 8.5 g; theseanimalscommanded
nursery harvestsdepictedin Figures3b a low price. In comparison, 100% of the
and 3c. Both ponds were stocked at a SPF crop was sellable, and only 3%
similar density and reared for the same weighed less than 9.5 g. Similar results
number of days. Figure 3b represents were obtained for the earthen ponds.
non-SPF postlarvae; many of the har-
vested shrimp were 0.25 g or less. IHHN virus was detectedhistologically
Figure 3c gives the data for the SPF in samples collected from ponds on the
animals; their average size at harvest Amorient farm in 1989 Table 1!. In
was approximately 1 g, 1989, the average prevalence of
IHHNV-infected P. vannamei was 61'%%d,
We also observed a dramatic reduction but increased to 89'%%d
in 1990. Since SPF
of RDS in the earthen semi-intensive shrimp havebeen stockedat Amorient,
ponds and in the intensive round IHHN virus infection has not been de-
pond, Figure 2c is a size histogram for tected his tologically in P. vannameisam-
an earthen pond stocked with SPF P. pled N = 270! from ponds on the site.
vannamei.The CV for this pond was However, more study is required be-
19'/o.Figure 4c contains similar data for fore the status of IHHN virus in the
a population harvested from the round earthen ponds of the Amorient farm
pond the CV was 9%. will be understood.

The production data for the growout Furthermore, the prevalence of BP in-
ponds discussed above are in Tables 2 fection declined from 20'%%d
to 4'%%d
in
and 3. For the IHHNV-infected shrimp, pond-rearedshrimp between 1990and
there was a generaldecreasein growth 1991. In laboratory experiments, Le-
rate, mean harvest size and Blanc and Overstreet 991! demon-
lbs/acre/crop.However, once the SPF strated that BP is inactivated by
shrimp were stocked, there was less desiccation.Perhapsthe two-week dry-
size variability and production im- ing period between crops partially in-
proved. activated infectious BP in the pond
sediments, Further study is required to
As indicated by the data, one benefit of clarify this issue.
the SPF broodstock was a reduction in
the level of RDS in growout and nurs- In summary, stocking the progeny of
ery. This was extremelyimportant from SPF broodstock on an IHHNV-contami-
a marketing point of view. Using three nated farm where RDS was a serious
successive round pond harvests as an problem resulted in the virtual elimina-
example Table 3!, the SPF crop yielded tiori of RDS and improved production
a 62.5'%%d
higher return than the non-SPF and profitability.
292 Ca er and Brock

Table 3. Marketi im act of usi non-SPF versus SPF shrlm

Count Size g! 4 Shrimp Percent Pounds Price Value !

IHMEP1-positive
roundpondcrop July 27,1990
UnseUable 4 62 75 0.00 0

71-110 4-6 23 6 227 3,75 851

51-70
46-50
6.5 - 8.5
9.0 - 9.5
47
20
13
5 496
189
4.00
4.25
1,984
803

41-45 10.0 - 11.0 45 12 458 4.50 2,061

3640 11.5 - 12.5 63 17 649 4.75 3,3
31M 13.0 - 14.5 83 22 841 5.25 4,415
2~
21-25
15.0 - 17.5
18.0 - 21.5+
55
32
15
9 561
342 6.25
3,226
2 137

Total 374
IHHN-positive round pond crop Nov 16, 1990
UnseUable 4 32 8 248 O.N 0
71-1'10 4-6 67 17 531 3.75 1,991
51-70 6.5 - 8.5 1N 28 876 4.00 3,504
4680 9,0- 9.5 33 8 248 4.25 1,054
41M 10.0 - 11.0 69 18 562 4.50 2,529
3640 11.5 - 12,5 48 12 374 4.75 1,776
31M 13.0 - 14.5 23 6 184 5.25 966

2&30 15.0 - 17.5 11 3 90 5.75 517

21-25 18.0 - 21,5+ 1 1 27 6.25 169

Total 392
SPFround pond crop Mar. 15, 1991
51-70
46-50
6.5 - 8.5
9.0 - 9.5
3
1
21 77
35
4.00
4.25
308
149

4145 10.0 - 11.0 38 27 1,145 4.50 5,152

36-40 11.5 - 12.5 74 52 2,212 4.75 10,507
31-35 13.0-14.5 27 19 802 5.25 4 210

Total 4 271
 Performance of SPF P. vannamei in Hawaii 293

References Lightner, D.V., R.M. Redman,R.R, Williams,

L.L. Mohney, J.P.M. Gerx, T,A. Bell and
Brock,J.A., Unpublished. J.A. Brock. 1985. Recent advances in
Humason, G.L. 1979. Animal Tissue Tech- penaeid virus disease investigations. J,
niques. 4th ed., W.H. Freemanand Com- World Marie, Soc. 16: 267-274.
pany, San Francisco, Luna, L.G. Ed.!, 1968.Manual of Histologic
LeBIanc, B,D. and R.M, Overstreet, 1991, Effi- StainingMethodsof the Armed ForcesIn-
cacy of calcium hyperchlorite as a disinfec- stitute of Pathology. 3rd ed., McGraw-Hill,
tant againstthe shrimp virus Baculovirus New York, N.Y.
penaei.J, Aquatic Animal Health. 3: 141-145,
294 Ca nter and Brock
296 Jaenike, Gre
more than 4.5 MTlhalcrop. These in-
consistent yields have resulted from
low survival and growth rates.The low
growth rates have been impacted by
runt-deformity syndrome RDS!,
which has been causally linked with
IHHNV Kalagayanet al., 1991!,but the
low survival ratesremain unexplained.
In an attempt to achieve more consis-
tent yields, a cooperative research
agreementto utilize high-health stocks
of shrimp was entered into with the
Gulf Coast ResearchLaboratory Con-
sortium GCRLC! in September1990.
The GCRLC supplied Harlingen
Shrimp Farms with enough specific
pathogen-free SPF! broodstock to pro-
duce postlarvae for commercial-scale
comparisonswith selectedfarm stocks
" Texasbroodstock source," or 'VBS"!,
that were IHHNV positive. SPF brood-
stock, by definition, are free of IHHNV,
hepatopancreatic parvo-like virus,
Baculovirus
penaei,microsporidians,gre-
garines, nematodes and cestodes.
Ponds stocked with postlarvae pro-
duced from the SPF broodstock here-
after referred to as "high-health"
animals! outperformed the offspring of
the TBS in terms of survival, overall
yield and decreasedsize variation.
Methods and Materials
The hatchery at Harlingen Shrimp
Farm is housed within a 3,600-m con-
crete building and includes a water
treatment facility, two maturation
rooms, broodstock holding and accli-
anation areas and over 500 tons of larval
rearing capacity. The maturation area
and Ham er
consistsof two light- and temperature-
controlled rooms, each containing 12
8-ton circular fiberglass tanks. The
tanks are plumbed in groups of four;
each group has a biofilter. In prepara-
tion for receiving SPF broodstock, one
of these four tank systemswas physi-
cally isolatedfrom the rest of the tanks
with a plastic curtain. A new biofilter
was installed, and the whole area was
carefully sterilized, A protocol for re-
ceiving and isolating the SPF brood-
stockand their progeny was developed
in conjunction.with Dr. Paul Frelierand
the Gulf Coast Research Laboratory
Consortium Frelier, pers. comm!, The
protocol was strictly followed, Stand-
ard maturation methodologies utilizing
unilateral eyestalk ablation were used
with both groups of broodstock. Nau-
plii produced from the SPFbroodstock
were isolated from the TBS nauplii, and
initially were reared in an isolated part
of the larval rearing area.
The larval rearing area consisted of
rows of fiberglasstanks that drain into
trenches for harvesting. One of these
rows of tanks was physically isolated
from the others with a plastic curtain.
Identical, standard hatchery methods
were used for both groups, exceptthat
the high-health postlarvae were in-
itially stocked at lower densities be-
cause fewer broodstock were sourced
for nauplii. The high-health nauplii
were initially isolated from the TBS
nauplii. After several million postlarvae
were produced in isolation, the larval
rearing area was no longer physically
segregated. The nauplii produced koan
the two broodstockgroups were segre-
 SPF Stocks
Table 1. Comparisonof the averagegrowth rate,feed conversionratio FCR!, survivaland
'Data not available.
gated into separate tanks whenever
possible;however, a number of mixed
tanks were stocked to maximize tank
space and optimize production. After
six weeks of completely isolated pro-
duction, the two broodstock groups
were mixed in massspawning tanks for
severaldays.
Eleven ponds of varying sizes,totaling
50 ha, were stocked with either high-
health postlarvae or TBS postlarvae
Table 1!. Although the hauling tank
and transfer baskets were sanitized
with chlorine prior to stocking with
high-health postlarvae,no attempt was
made to use segregatedequipment or
supplies in the management of the
growout ponds. Management strate-
gieswere applied accordingto stocking
density. For example, aeration rates
ranged from no aerationat the stocking
density of 18 postlarvae/m to nearly 15
hp/ha for the smaller ponds stocked at
75 postlarvae/m. The average daily
in Texas 297
water exchangerates ranged from ten
to 45% per pond, depen.ding on the
biomass estimate. All ponds were fed
two to three times per day using 45'%%d
protein crumbles until shrimp reached
1 g; thereafter,30%protein prawn pel-
lets were used. Feedingrateswere cal-
culated using a standard feed curve
based on percent biomass, and were
adjusted according to observed con-
sumption rates monitored with feed
trays. Ponds were evaluated on a
weekly basis. Each week, 100 to 200
shrimp from each pond were sampled
using cast nets. The average weight
was determined, and shrimp were in-
spectedfor state of health and vigor,
signs of stress, feeding activity, de-
formities, size variation and shell le-
sions, Samplesof stocked postlarvae,
30 day-old juveniles and adults nearing
harvest were collected and fixed in
Davidson's solution for diseasetesting.
All samples were examined at Texas
A&M University, where diagnosesfor
298 Jaenike, Gre
Table2. Average
number
of naupliiproduced
persourced
IHIP and other disease agents were
madeby directhistology.The sizedis-
tribution in eachpond was determined
by individually weighing samplesof
shrimp and by examiningfinal process-
ing packout reports.
Results
Over 85million naupliiwereproduced
by 140 female, SPFbroodstockfrom
March through June, 1991. The per-
centage
of femalesmatedper day and
the average number of nauplii per
spawnfor both broodstockgroupsare
listed in Table 2. The averagenumber
of nauplii per spawn takesinto account
all femalesthat were mated and placed
into spawning tanks sourced!,
The SPF and TBS broodstock per-
formed similarly in terms of percent
females mated per day; however, the
SPFbroodstockaveragedmorenauplii
per spawn.
Not all of the nauplii producedfrom the
SPF femaleswere segregatedin larval
rearing. Overall, 36 million pastlarvae
were produced from segregatedSPF
nauplii. Survivalfrom nauplii to post-
larvae was better in the high-health
postlarvaeTable3!. The survivalrates
and Mam er
fernaleandpercentage
oftotal
listed in Table 2 represent only the
results from 9,000-L tanks. The size,
vigor and appearanceof the high-
health and TBSpostlarvaewere similar.
Approximately11.5million high-health
postlarvaeweresegregated in growaut
ponds.The resultsfrom elevenponds
stackedwith either high-health or TBS
pastlarvaewill be discussed
here Table
1!. Growth rates did not differ greatly
between the high-health and TBS
ponds. Average time from PL5 to 1 g
averageweightwas32d forhigh-health
animals and 38 d in the TBS ponds.
Furthermore,growth from 1 g to har-
vest weight averaged.87 g/wk and .84
g/wk in high-healthand TBS ponds,
respectively Table 1!.
A dramatic difference, however, was
observedin the degreeof sizevariation
observedin the high-healthand TBS
groups.A typicalTBSpondpopulation
averaging 1 g in size contained some
shrimp that were less than 0.1 g and
others that weighed more than 3 g, A
typicalhigh-healthpopulation,by con-
trast, had a size distribution ranging
onlyfrom 0.5 g to 1.5 g, This difference
became mare pronaunced as the
growout period progressed the size
distribution in TBS ponds increased
 SPF Stocks
~TBS or mixed.
weekly, whereasthe high-health popu-
lations maintained a tight size distribu-
tion throughout the culture period. As
a result, a much more uniform product
was harvested from the high-health
ponds Fig, 1a, b!.
The level of rostral or tail deformities
detected during weekly samples and
at harvest typically ranged from 15 to
25% in TBS ponds, but was less than
3% in the high-health animals Table
1!.
Survival rates were higher in high-
health ponds as compared to the TBS
ponds. Average survival from PLS to
harvest for high-health animals was
51%; the best pond had 72% survival at
harvest. Average survival for the TBS
animals was only 40%; the best pond
had a 46% survival rate. Feed conver-
sion rates FCRs! were also much better
in SPF ponds Table 1!.
Discussion
The SPF broodstock and their high-
health offspring outperformed the TBS
broodstock and their progeny in the
hatchery; however, the differences be-
in Texas 299
tween the two groups were even more
pronounced during growout.
The SPF broodstock produced more
nauplii per spawning female than the
TBS broodstock; however, the SPF
broodstock were much larger, and
there is a positive correlation between
broodstock size and spawn size. The
percentage of females mating and
spawning per day was good for both
groups and did not appearto be differ-
ent. The sourcing pressure on the SPF
broodstockwas slightly more intensive
due to fewer femalesproducing.
Overall survival of the high-health nau-
plii was higher than that of the TBS
nauplii, The greatestdifference was in
April, when survival of the high-health
and TBS nauplii was 72% and 59%,
respectively Table 3!. At that time,
however, the high-health tanks were
stocked at lower densities, and in our
hatchery, lower densitiesin larval rear-
ing often translate into increasedsur-
vival. In May, the two groups were
similar in terms of survival from nau-
plius to postlarva, but in June and July,
when densities were similar, the high-
health tanks yielded better survivals
 Jaenike, Gre ard Ham er

than TBStanks.Finally, the appearance ences between broodstock groups and

of postlarvaewas similar between the between families within broodstock
two groups. groups, entailed feeding BP-laden
Artemiato mysis larvae and monitoring
Additional comparisons between the mortality. No differences in resistance
high-healthand TBSnauplii and post- to BP were detected Lester, pers.
larvae were conducted at two universi- comm.!.
ties. Individual families of nauplii were
sentto the University of Houston Clear- Four groups of postlarvae from each
lake for comparison of resistance to broodstock source were sent to Texas
Baculovirus
penaeiBP!. The experiment, A&M University for a replicatedexperi-
which was designedto comparediffer- ment conducted in a controlled envi-

Figure 1a. Comparisonof size distributionat harvestfor fourponds,

 SPF Stocks in Texas

ronment tank system CastiHeet al., growout. Initial sampling revealed a

1992!.This comparisonevaluatedpost-
larval performance for 30 and 60 days,
beginning with PL6. Survivals were
similar; however, high-health postlar-
vae posted significantly better growth
rates and also had a significantly
smaller size variation than
postlarvae,
The differences between high-health
and TBS animals were more evident
uniform size distribution in high-
health ponds and a wide size variation
in the TBS ponds. These differences
increased as growout progressed Fig.
1a, b!. The uniform size distribution of
the high-health animals allowed for
the TBS more accurate average weight and to-
tal biomass estimates and resulted in
more efficient feed management, as
revealed by the FCRs Table 1!. Feed is
in the largest operational cost in produc-

Figure tb. Corrparison of size dl'stributionat harvest for four ponds.

302 Jaenike, Gr
ing shrimp the lower the FCR,the
better the profit margin.
The more uniform size distribution was
also advantageousat harvest.Shrimp
buyersprefera uniformproduct;runts
areregardedasliabiTities,The uniform
distribution of the high-health animals
benefited fresh or heads-on marketing
where grading is more labor intensive.
Batches that could be sold either as
wholeshrimpat pondside,or as"bulk-
ungraded" at market,had lower han-
dling and processingcosts,
Theaveragesurvivalrateof high-health
animals was better than that of the TBS
shrimp Table1!. Historically,our farm
site has experienced wide ranges in
pond survival rates, which are still
largely unexplained.The high-health
shrimp ponds also exhibited a wide
range of survival rates 5% to 72340!.
There is increasingevidencethat the
low survival rates at our farm are due
to unknown factors related to the
growout ponds and are not the direct
resultof postlarvalquality.The higher
averagesurvivals of the high-health
animalssimplyindicatesthat that they
havea greatercapacityto toleratethese
unknown factors. In addition, the
growth rates of high-health shrimp
were slightly higher than the TBSani-
mals.
The average growth rate in the SPF
ponds, .87 g/wk after 1 g, was lower
than anticipated.This may havebeen
ard Ham er
due to underfeeding resulting from
higher-than-expected
survivals.
In summary,with better survival, more
uniform size, easier management re-
sulting in lower FCRs and more mar-
ketingoptions,thehigh-healthanimals
performedbetter than the TBS prog-
eny.
Acknowledgments
HarlingenShrimp Farmsis gratefulto
The Oceanic Institute and the Gulf
CoastResearchLaboratoryConsortium
GCRLC!for providing the SPFshrimp
stocks. It is important for research
groupssuchasthe GCRLCto be at the
forefront of production-oriented pro-
jects. Dr. Paul Frelierand his staff at
Texas A&M University spent hours
analyzing the samples and helping to
develop the protocol for maintaining
the SPF stock as disease-free. Finally,
the entire staff at Harlingen Shrimp
Farms was instrumental in producing
the shrimp and being conscientious
about the SPF project,
Literature Cited
Castille, F.L., T.M. Samocha, A.L. Lawrence,
H. He, P. Frelier and F. Jaenike. 1992.
Variabilityin growth and survivalof early
postlarvalPenaeas vannamei Boone,1931.
Aquaculture.In Press.
Kalagayan,H., D, Godin, R. Karma, G.
Hagino,J. Sweeny,J. Wybanand J. Brock.
1991.IHHN Virus as an etiologicalfactor in
runt-deformitysyndromeRDS!of juvenile
Penaeus vannamei cultured in Hawaii. J.
World Aquacult. Soc.22: 235-243.
 ContributedPapers

Research,Regulations,and Health
Management
 Shrimp CultureTechnologiesInc.: Researchto
ImproveShrimp Geneticsand Health

Rolhnd Lalamole
ShrimpCultureTechnologiesInc.
Harbor Branch Oceanographic Institution
5600Old DixieHwy.,R. Pierce,Rorida34964,U.SA.

Abstract

This paper presents the results of three pastlarvaevaccinestudies and a broodstockvaccination

study. Vaccineswere administered to postlarvae and evaluatedin nursery and growout ponds.
Survival in the nurseries and direct-stockedgrowout ponds improved by an averageof 19% and
33%, respectively.Vaccinatedanimals that were harvestedfrom the nursery ponds and placed
in growout did not have a higher survival rate than the control group; however, survival was
more than 80% in both the vaccinatedpond and in the nonvaccinatedcontrol pond. The results
of the vaccination of broodstock were disappointing with respectto survival; however,improved
production in the first two months suggeststhat researchshould be continued. The results of
a survey for infectious hypodermal and hematopoietic necrosis virus IHHNV! on six shrimp
farms in Honduras are also presented.

Introduction ~ Stability testing of a concentrated,

live, packaged algae for use in
ShrimpCultureTechnologies
Inc, SCTI! hatcheries;
is a company newly formed by Shrimp
Culture Inc. to improve the geneticsof ~ The use of probiotics to control
cultured shrimp through selective bacterial infections in larval cul-
breeding. Selection will be imposed ture tanks; and
upon a pool of shrimp that has been
certified as specific pathogen-free SPF!. ~ Experiments toward the develop-
ment of polyploid shrimp.

Other areas of research are:

i Efficacy testing and licensing of a
water-administered Vibrio
for larvae, and the testing of an
injectablevaccinefor broodstock;
Broodstock
A selection program, under the direc-
vaccine
tion of Dr. JamesLester, University of
Houston, Clear Lake, Texas, was insti-
tuted at Agromarina de Panama in
306 Laramore

Table 1, Penaeus vannamei vaccination studies.

tAverageof four replications.

Averageof two replications,
Difference
tn survivalis not significantlydifferent,Increase
in yielddueto larger
size shrimp at harvest.
Studiesconductedin the dry seasonunder adversegrowing conditions.
Conductedin the Ministry of Agricultural Developmentof Panama'sresearchponds.

1988. Progeny from the selected ani-
mals produced an average increase in
weight of 7.4% at harvest, when com-
paredto theprogenyof the nonselected
maturation animals. In addition, the
uniformity of size was enhanced.Un-
fortunately, poachersdrained and har-
vested the pond containing the second
generationanimalsreservedfor brood-
stock. The infectious hypodermal and
hematopoieticnecrosisvirus IHIP/
status of the animals in this selection
program is unknown.
The Oceanic Institute, located in Hawaii,
reported improved growth rates and
uniformity of size in selectedSPFani-
mals see Wyban, this volume!. Also,
postlarvaepurchasedfrom LagunaMa-
dre's hatchery in Texasand shipped to
Honduras outproducednonselectedani-
mals during the dry seasonof 1990 Un-
published report, Dr. Bill MacGrath,
Shrimp Culture Inc.!.
There is little doubt in my mind that
within the next decadethe larger, more
progressiveshrimp farms will be stock-
ing ponds with postlarvae produced
from selected SPF broodstock.
Vaccine Postlarvae
Efficacy testing of a killed Vibrio bac-
terin, grown on agar plates, was con-
ducted in Panama in 1989/90 Table 1!.
Survival increased in nursery pond
evaluations, conducted over a 36-day
period, from 64.8% to 77.4% average
of four replications!. Yields increased
from 567 lbs/acre to 699 lbs/acre. Stock-
ing density was 600,000/acre.
 Shrirn
Growout evaluations were conducted
on four 10-acreponds two vaccinated
and two controls!, stocked with juve-
niles from the nursery trial, Survival
was excellent in both cases; 83% for the
vaccinatedponds, 85%for the controls.
However, the yields from the vacci-
nated ponds were higher than the con-
trol yields; 1021lbs/acrevs. 869lbs/acre.
The higher yields were due to a 17%
increase in the size of the
shrimp.
The reason for the size difference
readily apparent. Some have suggested
the possibility of less stress from micro-
bial assault or, perhaps, nonspecific
protection against one or more viruses
neither group was tested!. While the
difference was significant, there were
only two replications.
A third study was conductedin ponds
at the experimentalstation of the Min-
istry for Agricultural Development
MIDA! in Aquadulce, Panama. The
ponds were direct stocked at
Culture Techno ies inc.
35,000/acre. Survival increased from
50.8% to 67.6%, and yields increased
from 372 lbslacre to 519 lbs/acre aver-
age of four replications!, Harvest was
after 114 days.
Tests are currently underway in Ecua-
dor and Honduras evaluatinga vaccine
produced from a broth culture using
three strains of Vibrio. Antigens pro-
harvested duced in broth culture are less expen-
sive than those produced on solid
media.
is not
Vaccine Maturation
Two prototypes of an injectable Vibrio
vaccineemulsified in an oil-basedadju-
vant were tested against unvaccinated
controls, Six tanks were tested for each
treatment in this three-month study.
Evaluation was for survival and nau-
pliar production. Only females were
vaccinated see Table 2!. Survivals for
the "RED" vaccine, "BLUE" vaccine,
and "YAH'E" control groups were 68%,
72% and 84%, respectively Fig. 1!.
 l.ara more
Naupliar production per treatment
group was 27.2 million, 29.8 million
and 27.8 million, respectively. These
differencesare not significant.
It was interesting to note that both
vaccines outproduced the nonvacci-
nated control the first month: Red, 7.1
mullion; Blue, 8.9 million; and the Con-
trol, 4.8 million. Production was essen-
tially the samethe secondmonth; 15.1
million, 14.8 million, and 16.2 million,
respectively, but dropped slightly be-
low the control the third month: 5.1
million, 6.1 million, and 6,8 million,
respectively; although, the difference,
again,was not significant Fig. 2!.
If naupliar production is correctedfor
survival, that is, number of females
remaining in the tanks after removing
mortalities, the vaccinated groups out-
produced the nonvaccinatedgroup
slightly Fig. 3!. The averageproduc-
tion of nauplii per female over the
three-month period was: Red, 679,000;
Blue, 725,000; and Control, 556,000.
Figure f. Number of vaccinatedfemale brood-
stock survivingaNerone, @Noend three months
in production,
Higher mortalityin the treatedgroups
mayhaveresultedfrom the traumaof
vaccination no sham injection was
madeon the controls!. It is alsopossible
that the greaternumber of femaleshan-
dled during the first month could have
increasedmortality in the more produc-
tive tanks. In any case, the early in-
creases in production dictate that
additional research be conducted.
IHHNV Survey
At the request of the US/AID shrimp
program in Honduras, six Honduran
shrimp farms were surveyed for IHHN
virus. All farms tested positive for
IHHNV. The rate of infection ranged
from 20% to near 100% Fig. 4!. The
shrimp selected for the survey were
juveniles weighing approximately 3 g.
A severity index scores from one to
four based on the prevalenceof Cow-
dry A inclusion bodiesfound in suscep-
tible tissue! was positively correlated
with the percentage of animals in-
fected.
Figure2, Numberof naupliiproducedper treat-
ment at the end of the first, second, and third
months. "Slue" and "Red" indicate two different
vaccine formulations.
 Shrim Culture Technolo ies IrL.

This survey was conducted in the dry infection, to survival. Pond 19 was free
season April 92!. Pond harvest data of detectable IHHNV but had a lower
was not available at the time this report survival rate 9%! than pond 21 8%!.
was prepared. During the dry season, The latter had the highest severity score
growth rates are normally depressed, of the six ponds studied, but was also
declining rapidly after about 7 or S g, among the highest in survival Fig. 5!.
and virtually stopping at 12 - 13 g. No records were available on runt-de-
Deformities were less than 5% in all the formity syndrome RDS!.
ponds sampled. Attempts will be made
to obtain data on stunting and deform- In practice, I have been unable to cor-
ities following harvest. relate RDS with E~l on extensive
and semi-intensive farms in Latin
It is interesting to note that wild-caught America. I also believe there is evidence
postlarvae were stocked into virgin that RDS is not the result of a single
ponds ponds not previously stocked! agent or stress, at least in Latin Ameri-
on farm No. 12S. Yet, this farm had an can ponds. This is not to say that we
88% incidence rate and a severity score should not strive to produce SPF
of one, indicating that IHHNV came in broodstock. Common sense dictates
with the wild postlarvae. that virus-free animals are desirable.
However, very little is understood
An additional, more extensive survey, about the mode of infection and sus-
was made on farm No. 132, testing ceptibility of P. varrnamei to IHI-IN'If.
animals closer to harvest. A compari- One farmer asked, "If SPF postlarvae
son was made between survival at har- were available, and I went to the ex-
vest, severity of infection, and the pense of stocking them, what would
percentage of wild postlarvae in the prevent wild stock from entering my
population. There was no correlation
between the percentage of wild ani-
mals in the population or severity of 100
90

70
60
50
40
30

Figure 3. Average number of nauplii produced Figure 4. The percentage of IHHNV-positive ju-
per female after adjusting for survi val. venile shrimp and the r elative seventy ofinfection
based on the number of inclusions observed for
six farms i n Honduras,
 Drugsand Chemotherapeutants
for Shrimp
Diseases: Their Present Status in the
United States, with an Overview of
Researchand ApprovalProcesses

m~sA.~
Unifier.ityof Arizona
DepartnmCof VeterinaryScient
Tucson,Arizona85721,U.SA

Diseases
areplayingan increasinglyimportantrolein thecultureof shrimp.Controllingdiseases
has been, and wiH continue to be, essentialto the expansion of the industry. At present, only
onecompoundhasbeenapprovedby eitherthe EnvironmentalProtectionAgency KPA!or the
FoodandDrug Administration FDA! for usein shrimp culturewithin the United States.This
paper summarizes the presentstatusof drug researchand the basicrequirements for drug
approvalin the United States.

Introduction substantially. Although data is lacking

As most researchers in the field
shrimp pathology are quite aware, in-
ternational shrimp culture is in a strong
growth phase.The 1991world produc-
tion of shrimpwas approximately 2.4
million MT, with 28% coming from
aquaculture. By comparison, the total
world production in 1983was approxi-
mately 1.8 miHionMI, but only 7%was
contributed by aquaculture Rosen-
berry, 1991,1992;Food and Agriculture
Organization, 1989!. Kith increased
production, the prevalence of infec-
tious and noninfectious diseases in cul-
tured shrimp has also increased
to quantifythis increase, a rise in the
of volume of shrimp disease-related litera-
ture, along with more conferences,
workshops and higher diagnostician
caseloads are all indicators of the in-
creasedimportanceof shrimp diseases.
Many shrimp diseasesfall into the po-
tentially treatable category, primarily
those with bacterial, protozoan and
fungal etiologies. Further discussion in
this paper will focus on treatable dis-
eases.The actual mechanics of drug
therapy will not be discussed here;
readers should refer to Bell 992! for
that information,
312 Bell

Drugs Approved in the may be left in the water during treat-

ment and will coincidentally be cleared
United States of any att~ed algae or filamentous
bacteria. There is at least one generic
In the United States, the process for
copy of Aquatrine4; however, its use in
approving food animal drugs and
shrimp ponds, tanks or raceways
chemotherapeutants is extremely
within the United States would be con-
complicated, time-consuming and ex-
sidered illegal,
pensive. Approval processdetails will
be discussed later; the process is so
In May 1991, public notification was
complex that only five drugs/chemo-
officially made, via the FederalRegister,
therapeutants are approved for use in
that the data package submitted for the
aquaculture in general, only one of
use of Formalin on shrimp had been
which can be used in shrimp cul-
acceptedby the U.S. Food and Drug
ture.
Administration FDA!. Such notifica-
tion permits any commercialcompany
The one chemotherapeutant for
to submit a New Animal Drug Applica-
which the approval process has been
tion NADA! for approved use of For-
completed for shrimp use in the
malin in shrimp culture. It is our
United States is Aquatrine4 more
understanding that Argent Chemical
commonly referred to as Cutrine4-
Laboratories Inc. Redman, Washing-
Plus, Applied Biochemists Inc., Mil-
ton! submitted a NADA in January
waukee, Wisconsin!. Aquatrine4, a
1992; an FDA decision is expected
chelated copper sulfate compound, is
within six months from the submission
registered and approved by the U.S.
date. At least one other company may
Environmental Protection Agency
be preparing a Formalin NADA pack-
EPA! for usein fish and shrimp ponds,
age for the FDA.
tanks and raceways.It is registeredfor
use as an algaecide against common
BesidesAquatrine4 and Formalin, no
algal species such as Chara, Spirogyra,
other compounds have formal FDA or
Ctadophora, Microcystis, Spirulina, En-
EPA approval for use on cultured
teromorpha, Oscittatoriaand other algae.
shrimp within the United States.There
Its label also permits use against the
are, however, severalcompoundspres-
filamentous bacteria Leiicothrix. Fish
ently consideredby FDA as "low regu-
or shrimp in water treated with Aqua-
latory priority chemicals." This means
trine4 can be harvested immediately
that FDA has reviewed all relevant lit-
after treatment. Depending upon the
erature and data on the use of these
SpeCifiCCirCumStanCeS,Aquatrine4
compounds and has decided that their
can be applied at levels up to 1.0 ppm
use posesminimal risk if the following
for up to approximately 24 h. Aqua-
conditions are met Guest, 1992!:
trine4 is nOntOxiC at the reCOm-
mended dose rates; therefore, shrimp
 roval of Shri
~ The compoundsareusedonly for
indicated conditions;
~ The compounds are used only at
prescribed rates;
~ The compounds are used accord-
ing to good managementpradices;
~ The compound is of appropriate
grade; and
~ Thereis not likely to be an adverse
effect on the environment,
Unfortunately, these compounds are
seldom effective in combating shrimp
diseases.This list indudes Geyer,1992!:
~ Sodium sulfite improves hatcha-
bility of fish eggs not used with
shrimp!;
~ Sodium chloride osmoregulatory
aide and parasiticide not
with shrimp!;
~ Sodium bicarbonate anesthetic in
fish not used with shrimp!;
~ Acetic add parasiticide rarely
used in shrimp culture!; and
~ Carbon dioxide gas fiish anes-
thetic rarely used with shrimp!.
The FDA is reviewing a long list of
compounds that may eventually be
placedin this category.They have also
reserved the right to remove com-
pounds from this list as new informa-
Chemalhe eutants in the U.S. 313
tion becomesavailable indicating the
compound's lack of safety and effec-
tiveness Geyer, 1992!,
Groups Involved In
Research and Approval
Process
The entire drug approval processhas
traditionally been monetarily sup-
ported by the pharmaceutical firm
manufacturingthe drug for which ap-
proval is being sought. Unfortunately,
even an investment in drug research to
assist a large industry relative to
shrimp! like farm-raised catfish seldom
holds the potential for required profit
marglrls.
Therein lies our dilemma; an industry
in a significant growth phase and with
the genuine need for chemotherapeu-
tants, but still too small to provide
used sufficient incentive for pharmaceutical
firms to investin aquaculturedrug re-
search.Fortunately, a few drug compa-
nies are either investing in aquaculture
drug researchbased on the perceived
potential of aquaculture in the United
States,or they are investing in drug
research in the United States with a
worldwide market in mind.
The majority of existing shrimp drug
workisbeingsupportedby publicfunds.
In particular, support is being provided
by the U.S. Department of Agriculture
USDA! through two separate agen-
cies. The Regional Aquaculture Cen-
ters, and more specifically, the Center
314
for Tropicaland SubtropicalAquacul-
ture CTSA!, has provided funding for
the past five years. CTSA normally
funds projectswithin Hawaii and the
Pacific Basin protectorates.
The USDA also supports minor animal
drug work including aquaculture!
throughthe InterregionalProjectNo. 4
IR-4! program.The IR4, through its
four regional offices and via USDA
funds, helps groupsother than phar-
maceutical firms obtain minor use ap-
provalsfor new animal drugs. h6nor
use is defined as that intended for use
on minor animal species anything
other than the major species: cattle,
swine, chickens,turkeys, horses, dogs
and cats! or that intended for use on
minor diseases of major species.
There are several groups involved in
drug researchfor aquaculture species.
Only threegroupsareinvolvedin for-
mal shrimpdrugresearchin theUnited
States: the Gulf Coast Research Labo-
ratory GCRL! in Mississippi, Texas
A8zM University's Collegeof Veteri-
nary Medicine TAlvfU!,and the Uni-
versity of Arizona's Departmentof
Veterinary Science UA!.
The GCRLgroup has concentratedon
researchnot necessarilyintended to
supportapplicationsfor drugapproval,
such as chronic toxicity studies and
preliminarydrug screening.Theyhave
also been involved in the initial screen-
ing of previouslyuntestedcompounds.
Much of their work is most applicable
as supplemental data to NADAs.
Belt
TheUA group,on the otherhand, has
concentrated its efforts on research
aimed specificallyat fulfilling NADA
requirements.FDA-requiredwork con-
ductedby the UA group indudes: in
vita efficacytrials minimum inhibitory
concentration, or MIC testing!, in vivo
safetytrials, in vinoefficacytrials, effi-
cacyfield trials, medicatedfeedmanu-
facturing stability trials, and human
safetytrials shrimp tissueresiduetri-
als!. In addition, the UA group has
conducted studies that were prereq-
uisite to the FDA-required studies or
that would be consideredsupplemental
studies. The prerequisite and supple-
mental studies include: palatability,
pharmacokinetic and feedmanufactur-
ing studies.The UA group played a
majorrole in the datapackagegenera-
tion for both Aquatrine+ and Formalin.
TheUA groupinvolvedin drug studies
comprisestwo aquaculturepathobiolo-
gists,one aquatictoxicologist,one mi-
crobiologistandseveraltechniciansand
graduate students.
The TAMU group has only recently
become involved in formal drug testing
studies. They have been assisting
and/or coordinatingthe UA efficacy
field trials for the past two years and
will be doing the samefor at leastthe
next two years. They have also been
involved in protocol development for
field trials. The T/dufU group comprises
primarilya certifiedveterinarypatholo-
gist specializing in aquaculture spe-
cies!, several advanced degree
veterinarians and technicians.
 roval of Shrl
Present Drug Researchto
Support UltimateApproval
Severalshrimp-usecompoundsare at
variousstagesof investigation.The ma-
jority of the researchis aimed specifi-
cally at FDA or EPA approval for use in
shrimp culture. A few of the com-
pounds listed have been dropped from
further consideration.The list of prom-
ising compounds includes:
~ Oxytetracyciine;
~ Romet-30
~ Sarafine sarafloxacin!;
~ BaytriP enrofloxadn!;
~ Florfenicol;
~ Unnamed fluoroquinolones from
Parke-Davis; and
~ Treflan. trifluralin!.
Oxytetracycline approval studies have
been underway for approximately 15
years. The original funding was pro-
vided the National Marine
Service Corliss et al., 1977; Corliss,
1979!and later by Pfizer the manufac-
turer! and Marine Culture Enterprises
a business partnership between the
Coca-Cola Co. and the F.H.
Co.!. The initial funding and work con-
ducted by the UA ceasedin approxi-
mately 1986. Work began again in
approximately 1988,with funding from
the CTSA; this work continues Bell et
Chemothe ulants in the U.S, 315
al., submitted; Mohney et al., In press;
Williams et al., In press!.This latter
periodof work hasfotmsedon efficacy
field trialsand hasbeenconductedby
TAMU and UA. PFizerofficiallyspon-
sored the studies, but provided only
minimal support, Pfizer's support has
been primarily in the areas of medi-
catedfeed assays,protocolreviewand
joint meetingswith FDA. Futurework
on efficacyfield trialswill alsobe par-
tiallyfundedby anotherUSDA-funded
agency, the U.S. Marine Shrimp Farm-
ing Programoperatingunderthe Gulf
CoastResearch
Laboratory
Consortium!.
The potentiated sulfonamide Romet-
30 has been the subject of required
FDA studies since approximately 1988.
CTSA began funding preliminary MIC
and larval toxicity studies in 1988
Mohney et al., In press;Williams et al.,
In press! and continues to fund other
Romet-30' studies. Since approxi-
mately 1990, Hoffmann-LaRoche has
alsoprovided financial support, specifi-
cally in the areas of larval toxicity,
juvenilepharmacokinetics,field bioavail-
ability and field feed manufacturing
studies. Hoffmann-LaRoche has indi-
cated an interest in continuing penaeid
Fisheries
shrimp studies in the areas of juve-
nile/adult palatability, toxicity and resi-
due, The work on Romet-30 has almost
exclusively been conducted by the UA,
A portion of upcoming work, clinical
Prince
efficacytrials, may be supported by the
Marine Shrimp Farming Program,
which will provide funds to TAMU.
316
Sarafin4, generically referred to as
sarafloxacin,is a fluoroquinolone com-
pound. CTSA began funding prelimi-
nary MIC and larval toxicity studies in
1988 Mohney et al., In press;Williams
etal., In press!andcontinuesto finance
other Sarafin4 studies. Since approxi-
mately 1990,Abbott Labs has also pro-
vided financial support, specifically in
the areas of larval and juvenile toxicity
trials, juvenile/adult palatability and ex-
ploratory residue studies. Abbott initi-
ated FDA Investigational New Animal
Drug Application INADA! submission
for Sarafin4 and has agreed to fund
continuing penaeid shrimp studies in
the areas of infectivity model develop-
ment and testing. The work on Sarafin4
has been conducted solely by the UA.
Abbott Labs has expressedinterest in
having the UA test a similar fluoroqui-
nolone in a like fashion.
Another fluoroquinolone compound,
Baytril4, generically referred to as en-
rofloxacin, was investigated preliminar-
ily by the UA with funding by CTSA
that was received in 1989-90. The work
completedto dateincludedpreliminary
MIC trials and in viv0 larval toxicity
trials Mohney et al., In press;Williams
et al., In press!. The manufacturer of
the compound, Mobay ShawneeMis-
sion, Kansas!,was approachedfor spon-
sorship and possiblemonetarysupport
of future work; they declinedany major
participation, but agreedto provide the
UA with research quantities of the
drug. The UA has approachedthe IRX
WesternRegion! for possiblesponsor-
ship and funding, and verbal approval
Bell
has been granted. The IRA Eastern
Region! is presently sponsoringand
funding Baytril4 studies on trout.
Florfenicol a cMoramphenicol analog
manufactured by Schering-Plough,
Kenilworth, New Jersey! was tested
preliminarily. All work on the com-
pound was conductedin 1988-89by the
UA and funded by CTSA. Preliminary
tests induded in vitro MIC studies and
iri vivo larval toxicity studies. The re-
sults of thesestudieswere presentedto
Mobayin a request
for sponsorshipand
potential funding of future studies.
Schering-Plough declined to provide
sponsorship or funding for future
work. Further florfenicol work was
dropped from the list of CTSA-funded
work; however, IR-4 may be ap-
proached for financial support and
sponsorship. It is our understanding
that Schering-Ploughhas been concen-
trating their efforts on acquiring Japa-
neseapprovalfor use of florfenicolin
yellowtail Seriolaqumquemdiafa!
culture.
Severalfluoroquinolones produced by
Parke-Davis Ann Arbor, Michigan! were
tested by the UA. These compounds
are not commerciaHyavailable for any
animal use and, thus, were only iden-
tified by a codenumber. Both MIC and
larval toxicity studies were conducted
Mohney et al., In press;Williams et al.,
In press! and two of the compounds
provided the highest estimated Mar-
gins of Safety of any drugs tested to
date. This work was funded by the
CTSA, but ceased early in 1991. Parke-
Davis has been approached for spon-
sorship and funding, but has declined
 roval of Shri
in both regards.One of the compounds
has sincebeen licensedto Upjohn, Inc.
Kalamazoo, Michigan!; a similar re-
quest for sponsorship and assistance
has been presented to Upjohn and a
response is pending.
Treflan+ is a commercially available
product manufacturedby Elanco Indi-
anapolis,Indiana!. It is Elanco'sformu-
lation of trifluralin that is traditionally
used in agriculture as a pre-emergent
herbicide. Over the years, it has been
found by the aquacultureindustry to be
an effectiveand safefungistatic /fungi-
cidal compound used against Ugeni-
dium caltinectesand Simlpidium sp. Both
of these organisms cause the ubiqui-
tous disease known as larval mycosis.
Elancohasbeenapproachedto sponsor
and fund research to verify empirical
findings, but has declined the offer.
CTSA has begun 992! to fund UA
research in this area and the IRA West-
ern Region! has agreed to sponsor and
provide additional funds for the re-
search.A formal argumentfor the clas-
sification of trifluralin as a pesticide
and, thus, under the jurisdiction of
EPA! as opposed to a drug and, thus,
under the jurisdiction of FDA! hasbeen
submitted to FDA. FDA has verbally
informed the UA and IR-4 that the use
of trifluralin in shrimp culture is, in
their opinion, consistent with that of a
pesticide and not a drug. Preliminary
work is now underway to seek EPA
approval for Treflan~ use in shrimp
culture.
Chemothe eutanfs in the U.S. 317
As was noted earlier, total drug devel-
opment in the United States is ex-
tremely complex and expensive. The
costs can be substantiaIly reduced if
application processesand requisite test-
ing can be abbreviated. One of the
easiest means to achieve this is to seek
a "label extension" for a product al-
ready approved for other species, in
particular, other aquatic species.This
procedure aHowsfor the referencing, in
the application for shrimp use, of infor-
mationgeneratedfor the originalspecies.
Such data would most often indude en-
vironmentaland animal safety data. Be-
causenearly all, if not all, new animal
drug development costs are either
dearly outside the limits of public fund-
ing and/or are unattractive to the phar-
maceutical firms, the UA group has
decided to pursue compounds for
which only a label extension is re-
quired. Unfortunately, the drugs in this
category may not be the safest and/or
most efficacious drugs available. How-
ever, severalavailableapproved drugs
of moderateefficacy/safetyare of more
use to a farmer than no drugs, even if
they have excellentefficacy/safety.
Need For Approved
Therapeutants
The need for approved chemothera-
peutants in shrimp culture seemsobvi-
ous. The prevalence of diseasesand
concomitant mortalities appears to be
increasing exponentially relative to the
growth of the industry.
318
The use of drugs and chemotherapeu-
tants within shrimp culture falls under
the jurisdiction of the local, state, pro-
vincial and/or federal government in
which it is being conducted. Unfortu-
nately,thereis no uniform setof inter-
national codes governing the use of
such compounds. Some governments
cannot control their use, and this is
where shrimp culture has flourished.
In countries like the United States,
where shrimp culture is inherently
marginal due to basicenvironmental
and climatic constraints, strict proce-
dures for the approval of drugs has
hindered the growth of the shrimp
culture industry.
Within the United States,shrimp pro-
duced with only FDA-approved drugs
and chemicals are perceived by the
consumer to be of higher quality. Pro-
viding shrimp culturists with a useful
set of tools with which to combat dis-
easeswill discouragethe illegaluseof
chemotherapeutants.
Approval Requirements
There are two requirementsfor FDA or
EPA approval for the use of drugs or
chemotherapeutants in aquaculture,sci-
enti6c research and administrative tasks.
Research Studies
The studies required by the FDA are
outlined below. These basic studies are
dictatedby commonsense the use
of chemotherapeutants
in any situation,
eeI
be it in aquacultureor with humims,
can be allowed only when there is
requisiteinformationin four basicar-
eas.
~ Efficacy. Does the compound
achieve the desired results?
~ AnhmnalSafety. Can the results be
attained without further jeopard-
izing the health of the patient,
whether it be a shrimp, a cow or
a human?
~ Human Safety. Does its use pose
potential dangers to humans
other than the patient in the case
of human medicine In the treat-
ment of food animals, will there be
unsafe drug residuesin the edible
portionof the animalwhen a per-
son consumesthe product?In any
situation, does the use of the com-
pound posea dangerto the person
administering it to the patient?
~ Environmental Safety. Does the
therapeutant harm the environ-
ment? In the caseof aquaculture,
when a drugis administeredto the
cultured species, does it eventu-
ally makeits way into the environ-
ment at harmful levels?
For terrestrial animals, there are rela-
tively well-defined protocols for gener-
ating informationin eachof the four
areasnoted above. Extrapolation from
terrestrial animals to shrimp has not
beensimple, however. The following is
a brief summary review of such proce-
dures as they apply to shrimp.
 roval af Shri Ghemothe Utants in the U.S. 359

Efficacy or Effectiveness latter procedureshave yet to be devel-

One of the first stepsin establishingthe oped for shrimp, primarily due to the
effectiveness of a prospective com- lock of truly obligate bsiWrial patho-
pound is to test it against potential gens.
pathogensto demonstratethat they are
sensitive to the drug. This is accom- Safety When Used on Test Animal
plished by determining, via in vitro The lowest dose toxic to the test animal
testing, the Minimum Inhibitory Con- must be established.Toxicity is not just
centrations, or MICs, for the the lowest level causing mortality;
drug/pathogen combinations. rather, toxicity is the level that causes
death and any other deleteriouseffect
To accomplish this, a "standardized e.g., lethargy, poor growth, aesthetic
test battery" of shrimp disease-associ- considerations, etc.!. Mesc values are
ated isolates was assembled Mohney normally establishedby means of one
et al., 1992!. The test battery comprises or more of the following stand-
18 gram-negativeisolatesrepresentative ardizedprocedures:Lethal Concentra-
of the numerous geographic regions, tion LC!, Lethal Dose LV!, Effective
shrimp genera,bacterialgeneraand an- Concentration EC! or Effective Dose
tibiotic profiles. It also includes two ED!.
reference bacteria, Escherichia coti and
Pseudomonasaeuroginosa, from the As one might expect, the toxicity test-
American Type-Culture Collection. In ing proceduresestablishedfor cattleare
our studies, we use the standardized not directly applicable to shrimp.
test battery to determine an MIC profile Therefore, a standardized protocol for
for each prospective compound as the the irutial testing of any prospective
first screening test. Acceptable MICs drug against shrimp has been devel-
are normally less than 2.0 ppm. oped Wilhams et al., 1992!.The proce-
dure calls for an initial 48-h EC trial to
Assuming the drug is determinedto be be run on protozoea III - mysis I Z3/Mz!
safe see Animal Safety Section below!, larvae. This trial is actually begun 24 h
the same drug must be established as before the protozoea stage metamor-
being effective at approximately the phosesinto the mysis stageand contin-
same dose levels in in vino trials. These ues for 24 h into the mysis stage.
latter trials entail the use of dose-titra- Assuming these EC values are accept-
tion studies where the disease is inten- able a Therapeutic Index greater than
tionally induced with the pathogen!, 4.0!, additional larval toxicity studies
followed by administration of the drug are conducted. These include a 24-h EC
at varying levels.If the drug is effective, trial during the first 24h of the nauplius
there should be a positive relationship stage, followed by a 48-h KC trial for
betweenthe level of drug administered the first 48 h of each of the remaining
and the level of disease reduction. These larval stages protozoea, mysisand post-
320
larvae!. All larval toxicity trials are con-
ducted at a commercial shrimp hatch-
ery or a commercial-sized research
hatchery. A complete set of stand-
ardized test containers and supportive
equipment and supplies are brought to
the hatchery; the host hatchery need
only provide larvae, algae, electricity
and a work area of approximately 2 - 3
m.
A combination of the toxic level and the
efficacious level yields the Margin of
Safety differencebetween values! or
Therapeutic Index ratio between val-
ues!. This index is the relationship be-
tween the highestlevel of drug required
to inhibit the growth of the pathogen
and the lowest level toxic to the shrimp.
A five-fold Margin of Safety is generally
required before our group investigates
a prospectivecompound further.
Hulrian Safety
If it is assumed,from the previous tests,
that the drug is nontoxic to shrimp at
levels that are effective againstthe tar-
get pathogens, it must then be further
assumed that effective levels will be
achieved in the shrimp muscle. Of
greatestconcernto the consumeris the
question, "How long are theseeffective
levels retained within the muscle of the
shrimp?" It is expected that no com-
pound considered for use in shrimp
culture would be retained within the
shrimp tissues indefinitely. The drug
shouldbe degraded and excretedvia
the shrimp's metabolism and the com-
pound's inherent half-life. Typically, a
culturist must halt treatments of a given
Bell
drug for a spedfied period af time
before marketing the product for hu-
man consumption. That period of time,
known as the "withdrawal period," is
the amount of time a given drug per-
sists in the edible flesh of treated
shrimp at detectablelevels.
The studies used to establish the with-
drawal period are referred to asresidue
or depletion studies. They are time-
consuming and expensive,requiring a
significant amount of detailed labora-
tory analyses.An extremelyrigid set of
analytical guidelines, referred to as
Good Laboratory Practices or GLP!
must be followed for the analyticaldata
to be acceptable.A laboratory must be
certified by the FDA as being able to
conduct GLP; hence,there arevery few
GLP labs in the United States. Typi-
cally, the majority of these data are
generated by the pharmaceutical com-
pany making the compound.
In the past, our activities in this area
included feeding medicatedfeeds up-
take! and holding shrimp after medica-
tion depletion!. Samples of shrimp
collected during uptake and depletion
were typically frozen and shipped on dry
ice to the drug manufacturer for analy-
ses of drug levels in the edible portion.
The most important aspectof any prod-
uct is its inherent safetyto the end user
or consumer. In response to the U,S,
consumer, the FDA appears to have
become more sensitive to the issues of
contamination in food products.The
recent formation of the FDA's Office of
Seafood, which is responsible for en-
 val of Shri
suring the final purity of domesticand
imported products, is indicative of this
concern for consumer safety. Hence,
this aspectof the drug approvalprocess
may be most important, and surely is
the most expensive step.
Environmental Safety
The FDA is primarily concernedwith
reviewing information to support the
premisethat the prospectivedrug does
not harm the environment. A data
packagefor an aquatic animal should
include information that demonstrates
rapid degradation within the cultured
animal, short half-life within the cul-
ture system, a low effluent volume, a
highly diluted effluent, a further dilu-
tion of the effluent once it enters natu-
ral water systems,etc.
Although, to our knowledge, the FDA
is only concernedwith the prospective
drug harming the environment as a
direct toxicant, there are other factors
that should be of equal concern to
shrimp culturists. Probably highest on
this list is the direct or indirect effects
on the microbial flora inside and out-
side of the shrimp facility. The use of
antimicrobials,especiallyat suboptimal
levels, can unnaturaGyshift the bacte-
rial composition toward a resistantspe-
cies. Theoretically,each successiveuse
of the compound could increase the
proportion of drug-resistant microbes.
Such changes would be considered a
direct aquaculture-specificimpact.
Chemothe eutante in the U.S,
The environment can also be indirectly
affected, As the number of drug-resis-
tant microbes increases, the chances for
this characteristic to be transferred to
previously drug-sensitive bacteria re-
lated or unrelated to the original! in-
creases. Drug resistance may be
transferred via genome or extraMro-
mosomal plasmid exchange. The effec-
tivenessof that particular drug can thus
be further reduced.
Administrative Procedures
Unfortunately, the scientific studies in-
volved in the new animal drug clear-
ance are not the only requirements.
Administrative tasks can be more diffi-
cult than the science.The following is
a brief summary of various types of
FDA applications and procedures that
need to be followed to acquire drug
approval in the United States.
Protocol Review
Prior to undertaking any experiments
required for ultimate drug approval,
FDA strongly recommends but does
not require! that you submit protocols
for their review. Their rationale is that
it will be much easierfor you to change
a protocol on paper than to repeat an
experiment becauseof inadequate or
inappropriate procedures. It is often
prudent for investigatorsto follow pro-
tocol review submissions with a visit to
FDA to discuss the protocols.
By law, the FDA must respond to
nearly every type of submissionwithin
a set period of time; unfortunately,

from the investigators point of view,
these time frames are quite liberal up
to 180days!and the responsedoesnot
have to be definitive. For example,180
days after you have submitted an
INADA, FDA may respond by telling
you that a particularrequired.item was
not included with your application.
Again,frequentcontactswith FDA fol-
lowing a submission, either by phone
or in person, may hasten the process.
InvestigationalNew Animal Drug
Application9NADA!
An approvedINADA allowsfor theuse
of an unapproved drug; however,
INADAs are intended to be used for a
specificpurposeand with severalre-
strictions:
~ Meaningful Data. Use of the pro-
spectivedrug under the authority
of an INADA will only be permit-
ted when such use will generate
data applicableto the submission
of a New Animal Drug Applica-
tion NADA!. Therefore,the drug
can only be used under conditions
specifiedin the INADA protocols.
For the INADA to be approved,
theseprotocolsmust be reviewed
either prior to INADA submission
or during the INADA review,
~ Human Safety. Use of the com-
poundshouldpresentvirtually no
hazard to consumers. Reasonable
information must be available
indicatethat rapid depletion of the
drug does or will occur in treated
animals.
Bell
~ EnvironmentalSafety.The useof
the compoundshouldcausemini-
rnumimpacton the environment,
Information must be presented
that supportsthe rapid naturalor
induced artificial! degradation,
innocuous nature and/or minimal
effluent of the prospective drug.
~ Investigators.
INADAsmaybesub-
mittedby any quaiifiedresearcher
or entity; however, FDA is now
encouraging producer groups,
state agencies, universities, re-
gional aquaculture centers and
othersuchorganizations to submit
the INADA insteadof everyindi-
vidual desiringto use the drug.
Such entities can supervise the use
of the drug and aGowonly those
individuals agreeingto follow pro-
tocol to use the compound, thus
mmimizing the chancesof gener-
ating meaningful data.
New Animal Drug Application
NADA!
NADAsprovidefor the submission
of
requireddatain supportof a requestto
gainthe approvalof a newdrugfor use
with animals. As noted before, the
submission,excludingthe actualcost
of scientificinvestigations,can be ex-
tremely high and time-consuming.
Typically, NADAs are submittedby
the pharmaceuticalfirm manufactur-
ing the drug; often a specializeddivi-
to sion within the firm handles this task.
As notedpreviously,the NADA must
contain supportivedata in four spe-
cific areas:
 Bell

Coriiss,J.P.,D.V. LightnerandZ.P. Zein-Eldin. Mohney, L.L., T.A. Bell and D.V. Lightner.
1977.Someeffectsof oraldosesof oxytetracy- Submitted.Shrimpantimicrobial testing.I.
cline on growth, survivaland diseasein Irt vitrosusceptibility
of thirteenGram-nega-
Penseus
aztecus.
Aquaculttue.11: 355 '362. tive bacteria to twelve antimicrobials. J.
FoodandAgricultureOrganization.1989.Fish- AquaticAnimalHealth.
ery Statistics;
CatchesandLanding,Vol. 68. Roedel,P.M. 1973.Shrimp'73 - A billiondollar
Food and Apiculture Organizationof the business.Mar. Fish. Rev. 35 M!: 1-6.
United Nations. Rosenberry,B. 1991.World ShrimpFarming
Geyer,R.E.1992.FDARegulation
ofDrugsfor 1990.AquacultureDigest,San Diego,Cali-
Use in Aquaculture; Law and Policy.Pre- fornia.
sentedat theNationalAquaculture
Extension Rosenberry,B. 1992.World ShrimpFarming
Workshop, Ferndale,
Akansas.3 ~ 1992, 1991.AquacultureDigest,SanDiego,Cali-
Guest, G.B. 1992. RegulatingDrug Use in fornia.
Aquaculture.
Presented
attheCatfish
Fartn- Williams,R.R,, T.A. Bell andD,V, Ligbtner.Submit-
ers of America National Convention, Or- ted.Shrimpantimambialtesting.II. Toxicitytesting
angeBeach,Alabama.28 February1992. and safety dettmnination for twelve antimicrobials
withpenaeidshrimplarvae.J.Aquatic~ Health.
326
cultured and wild stocks due to acci-
dental introduction of nonindigenous
pathogens.
Precautions During the Initial
Phases of an Introduction
Precautions for introducing non-native
species shouldbegin long before any
actual movement of animals, The spe-
ciesproposedfor introductionshould
be subjected
to a detailedriskassess-
ment based on these areas of concern:
~ A review of geneticinformation
about the species,especiallyfrom
viewpoints of selectiveforcesin
the new environment and poten-
tial hybridization with native
stocks if escapes occur.
~ A review of ecologicalcharac-
teristicsof the speciesespecially
from viewpoints of competi-
tion/predationor colonizationpo-
tential in recipient waters if
escapes occur.
~ An examination made of disease
problems
in naturalpopulations
of
the speciesproposedfor importa-
tion, and the history of disease
outbreaksor unexplainedmortali-
ties encounteredduringany pre-
vious cultureattemptsshouldbe
reviewed.
~ Examinationsfor the presenceand
effects of pathogens of shrimp,
which concentrates on viruses-
but not to the exdusion of other
pathogengroups, If viral patho-
Sindermann
gensarefoundin naturalpopula-
tions,pathogen-free
stockswill be
soughtfor introduction.Addition-
aHy, the potentialinfectivityof
each pathogento other shrimp
species should be determined
dearly, as should carrierstatere-
lationships
and possibleviral syn-
ergisms. The search for viral
pathogens should indude carrier
state bioassays and stress en-
hancement tests Lightner et al.,
1985!.
If a decisionbasedon adequatescien-
tific information is made to introduce a
non-native speciesof shrimp, then an
adequate facjTiitymust exist in the re-
cipientcountrya fail-safequarantine
systemimmuneto accidents
thatcould
lead to escapes,into which the im-
portedanimals can be receivedand
maintained throughout one entire life
cyde and preferablylonger.'IMs, ide-
ally, should be a facility createdex-
presslyfor the purposeof quarantine,
and not merelya convertedcommercial
hatchery, or even worse! a collection
of assorted tanks in the basement or on
the grounds of a university marine
laboratory.
PrecautionsDuring Culture
of Non-native Shrimp
Once the quarantineperiod has been
completedand closecontinuousexami-
nation of at least one entire life cyde
has not disclosed the presence of
pathogens,the F> generationof the
introduced speciescan becomea possi-
ble base for aquaculture production in
 Precautions
the recipient country. Sevenadditional
precautions apply to shrimp culture
generally,but to the introduced species
in particular:
~ If feasible, the facility rearing the
introduced species should be spa-
tially isolated from facilities rear-
ing other species, preferably for
several generations of the
stocked species, and any un-
usual mortalities should be ex-
amined carefully.
~ If other speciesarebeing produced
in the samefacility, they should be
kept completelyisolatedfrom the
introduced species,which may be
susceptible to pathogens carried
by but not lethal to the other spe-
cies. This isolation means
exchangeof species,staff, equip-
ment or water.
Figure 1. Somegrossindicationsof stressin shrimp.
for Non-native ies
~ A routine disease monitoring pro-
gram should be developed,based
principally on gross observations,
histopathology, stress enhance-
ment tests and carrier bioassays.
This activity should be augmented
by a stress monitoring program
based on the kinds of gross signs
shown in Figure 1. A competent
virologist should be available on
retainer, to confront perceived
emergencies,
~ Introducing a non-native species
must be considered as a single
circumscribed event, designed to
create a broodstock population.
Successfulcompletion of the pro-
posed steps does not imply that
subsequentimportationsof that
no speciescanbe madewithout con-
trolsor restrictions,
particularlyin-
sofar as disease risks are
concerned.
 Sindermann

~ Batches of shrimp of unknown A Proposed Code of

origin or uncertain diseasehistory
should never, at any life stage,be
brought into the culture facility.
~ Detailed records shouM be main-
tained on the source,performance
and dispositionof everybatch of
shrimpthat entersor ishatchedin
the culture facility.
~ Every effort should be made to
achieve and maintain specific
pathogen-free status for the cul-
ture operation. A classification
system for shrimp hatcheries
should be developed that is com-
parableto the one usedto classify
and certify salmonid hatcheries in
the United States.
Practicefor introdUcing
Non-rlativeShrimp
How does all this come together' It
does so in a code of uniform practices
for introducedspedes, modified and
expandedfor shrimp from the ICES
InternationalCouncilfor the Explora-
tion of the Sea! Code of Practice
Regarding Introductionsof Non-in-
digenousMarine OrganismsI'ig. 2!. I
offerthis adaptationfor your examina-
tion and comments. I know that some
will say as others have already said!
that the code is too extreme or too
idealistic that producers can't live with
it. Sucha codemay meanthe difference
betweenviability and failure of shrimp
culture enterprises and, if ignored, it

PROPOSED STEPS TO REDUCE DANGERS OF DISEASE

IN THE INTRODUCTION OF NON-INDIGENOUS SPECIES

INTRO
LOTS
WATE
DI SEA
CLOSED
IN RECIPIENT

INTAIN AND
UDY CLOSED
STEM POPULATION

OD STOCK
YSTEM

Figure 2. An iIIustration of some major features of the ICES Code of Practice Concerning Introductions
of Non-indigerous Species from Sindennann, 1986!.
 Precautions for Non-native Ies

may also result in actions that can affect potential competition with
productivity of native wild stocks. species in the recipient envi-
ronment.
Several clarifications need to be stated
early: b! Appropriate governmental
authorities, state and federal
~ The proposed code applies to intro- including fishery management
ductions planned for either inten- agencies!,should examine the
sive, semi-intensive, or extensive information on the "candidate
culture,sinceexperiencehasshown for admission" to assess: the
that escapesfrom any coastalcul- rationale and justification for
ture facility are inevitable, the introduction, its relation-
ship with other membersof the
~ Some of the guidelines may seem ecosystem,and the role played
overly restrictive, but introducing by parasites and diseases.In-
a newspeciesis a pre-emptiveand adequateinformation should be
often irreversible ecological step; grounds for rejection of the ap-
therefore, the decision-making plication.
process governing introductions is
critical. Some details of the pro- c! The probable effects genetic,
posed code follow. diseaseand ecological! of the
introduced species in the new
area should be assessed care-
Recommended procedure for all spe- fully, including examination of
cies of shrimp before reaching a deci- the effects of any previous in-
sion regarding new introductions. troductions of this or similar
species in other areas.
a! Individuals, companies,govern-
mental entities and research d! The appropriate governmental
groups contemplatingany new entities should consider all data
introduction should assemble and the possibleoutcomeof the
and presentto appropriatestate introduction, and reach a deci-
and federalagencies,at an early sion to approve or disapprove
planning stage,information on the proposed introduction.
the species, stage in the life
cycle to be introduced, area of If the introduction is approved, the
origin, proposedplaceof intro- following actions should be taken:
duction, and objectivesof the
introduction, with detailed in- a! A broodstock population
formation on its environmental should be establishedin an ap-
requirements, epifauna, dis- proved fail-safe quarantinefa-
eases,associatedorganisms and cility. The progeny of the
 Sjndermann

introduced species may be period,the introducedanimals

transplanted
to the naturalen- and their offspringshouldbe
vironment if no diseases or immediately destroyed,
thefa-
parasites
becomeevidentduring cilitysterilized,
andtheapproval
the quarantineperiod,but not for introduction withdrawn.
the original import. '&e quar-
antineperiodwiH provideop- f! Everyeffortshouldbe made,in
portunityfor observation
for the United States and elsewhere,
diseasesand parasites.Duration to developand use certified
of the quarantineperiodshould specificpathogen-freebrood-
be at leastonecompletelifecyde, stocks and certified hatcheries,
regardless
of the stageat which modeled on the highly success-
the shrimpwere introduced. ful Conwy Wales!programfor
molluscan shellfish.
Any unusualmortalitiesat any
life cyclestagein nativestocks
of After an introduction has been effected,
the countryof origin or in quar- a continuingstudyshouldbe madeof
antine,or low averagehatchery the introducedspeciesin its new envi-
survival rates - 10%!, should ronment,and progressreportsshould
be consideredas possiblyin- be submittedto the authorizing govern-
ducedby hithertounidentified mentalagenciesannually.
viral infections, and moribund
specimens shouldbe examined Discussion and Conclusions
closelyfor occlusionbodiesor
ultramicroscopic viral arrays. A codeof uniformpracticessuchasthis
oneis a necessary
firststep,but it must
c! Diagnosticproceduresfor sus- be followedquicklyby protocoldevel-
pectedviral diseases shouldbe opment by detailedproceduralin-
extended to include carrier strudions.We need detailedprotocols
statebioassays with susceptible for everythingrelatedto new introduc-
species and stressenhancement tions inspection, certificationand
overcrowding!tests Lightner quarantine. Additionally,we needto
et al., 1985!. develop protocolsfor repeatedintro-
ductionsof speciesthat have become
d! All effluentsfrom quarantine part of established commercialpractice
facilities must be sterilized in an a reducedprogramof continuous
approvedmanner,whichmeara disease monitoringand inspection.We
killing all living organismsin alsoneedto developprotocolsfor spe-
the effluents. cies that have been introducedpre-
viously and then reintroducedafter
If evidence of disease is ob- passingthroughculture facilitiesin a
tained during the quarantine third country where new pathogens
 Precautions
may have been acquired. These and
other protocolsgive the necessaryform
and substance to any proposed pro-
gram to reduce risks from introduced
diseases.
A code of uniform practices and the
development of detailed protocols are
not enough, however. We need a regu-
latory framework to ensurecompliance
with the codes and protocols~nd
is not easy,sincethe last thing that any
aquaculturistwants is moreregulations.
All of this activity takes place on a
national level, but to be reaDyeffective,
such a code must achieve international
acceptance,sinceshrimp culture is truly
global, with a pathogen transfer net-
work that ahnost defiesdescription al-
though Lightner has madean excellent
attempt in his recent [1990]paper!. The
best vehicle to faciTitate communication
is not dear either, although it could be
proposed as an early initiative of the
newly formed North Pacific Marine Sci-
enceOrganization a Pacificcounterpart
of the Atlantic-oriented ICES! Ste-
wart, 1991!, Alternatively, FAO,
through its various regional fisheries
commissions, could form a nucleus for
the network that would be requiredas
could the OIE International Office of
Epizootics! through its four regional
groups.
This final step of international accep-
tance is a difficult one. In the North
Atlantic, where the celebrated ICES
Code has existed for almost 20 years,
some movement toward acceptance of
the Code has been seen in many mem-
for Non-native ice
ber countries. I know of no comparable
activity in the Pacific,exceptfor a work-
shop on exotic aquatic organismsheld
in Australia in 1988.
There is some reason for optimism,
however. Even in the absence of a
strong legal structure to control intro-
ductions, the risks from exotic patho-
gens can still be reduced significantly
this by the ready availability and utilization
of specific pathogen-free SPF! stocks.
The desirability and utility of SPF seed
sources seem dear, and we have sev-
eral examples salmonid culture in the
United States and bivalve mollusc cul-
ture in Britain! of the effectiveness of
the approach. Every effort should be
made to develop and use SPFtechnol-
ogy, induding provision of certified
broodstockas well as postlarvae.Pana-
ceas are rare, however, and SPF stocks
are not always as advertised. Probably
the best recent example of this melan-
choly conclusion is the spread of cray-
fish plague in Britain since 1981,
principally becauseof shipments of an
infectedintroducedcrayfishspeciesfrom
a supposedly "disease-free" hatchery
Thompson,1990!.The shipmentswere
carrying the plague fungus, Aphunomy-
ces astaci.
In addition to possible deficiencies in
SPF technology, other disease prob-
lems may develop from unknown or
unrecognizedpathogensin the imported
populations pathogens that have not
been described in the technical litera-
ture, but that may have the potential for
outbreaks in stressed populations or in
susceptible native species of the recipi-

ent country. An excellentrecent exam-
ple is the introduction and dissemina-
tion of the protozoan pathogen,
Perkirisus karlssoni, together with its
host, the bay scallop,Argopecten
irradi-
rirts, in waters off eastern Canada
after three generations in quarantine,
during which time the pathogenwas not
recognized, except as an "idiopathic
granuloma" McGladdery et al., 1991!.
So the risks from unknown pathogens
are never zero and this may be espe-
cially true for shrimp, in which new
disease agents are being discovered
with distressingfrequency.
In conclusion, it is obvious from the
present status and stature of disease
problemsin shrimp culture that precau-
tions must be takento reducethe spread
of pathogens.Regulatorymeasurescon-
stitute one approachto risk reduction,
but development and use of SPF stocks
offer a complementary approach. In my
opinion, to make a significant impact
on disease risks from introductions
shrimp, we needa combinationof read-
ily availableSPFstocksand acceptance
of national and international uniform
codesof practicewhen we move those
stocks, or any non-native stocks.
SirKIermann
The forces encouraging the chaos of
indiscriminate introductions can be over-
come, and a rational conservativesys-
tem can be assembled to ensure that
shrimp introductions will be made ac-
cording to agreed-upon uniform proto-
cols, preferably from certified SPF
sources.With such a system in place,
risks from introduced pathogens can
eventually be reduced to manageable
levels.
Literature Cited
Lightner, D.V. 1990. Viruses section: intro-
ductory remarks. Irr: Perkins, F.O. and
T.C. Cheng Eds.!. Pathology in Marine
Science.AcademicPress,New York. pp.
3-6.
Lightner,D,V., R.M, Redman,R.R. Williams,
L.L. Mohney, J.P. M. Clerx, T.A. Bell and
J,A. Brock, 1985, Recent advances in
enaeid virus diseaseinvestigations, J.
orld Marie. Soc. 16: 267-274.
McGladdery,S,E., R,J. Cawthorn and B.C.
Bradford, 1991. Perkinsuskarlssoriin. sp,
Apicompiexa! in bay scallops, Argopecten
irradiarrs.Dis. Aquat. Org. 127-137.
Sindermann,C.J.1986.Strategiesfor reducing
of risks from introductions of aquatic organ-
isrns:a marineperspective.Fisheries.11!:
10-15.
Stewart, J.E. 1991. A brief review of the Inter-
national Councilfor the Explorationof the
Sea ICES! on the occasion of the formation
of the North Pacific Marine Science Organi-
zation. Can.J. Fish. Aquat. Sci. 48: 2543-
2550.
Thompson, A,G, 1990, The danger of exotic
species.World Aquaculture.21!: 25-32.
 Issues Relatedto Regulationof PenaeidShrimp
Diseases in Texas, U.S.A.

Sterling K. Johnsy
Extension Fish Dime Specialist
DepartmiW of WiklNe and Fisheriesfences
TexasA 8 M Urger.ity,CollegeStation,Texas77843,U.S.A.

Regulation of exotic shellfish has been stated in Texasregulatory code for almost two decades.
Requirements and implementation of the regulation was rather orthodox, Those involved in
shrimp aquaculture and other interests, however,had to addressperceptions and issues arising
from the influence of a number of factors.An abbreviatedanalysisof influences and impacts is
offered, aswell as suggestionsfor thosewho must addressthe generalissueof diseaseregulation
for penaeid shrimps.

Introduction of government. Regulation, therefore,

Regulation by government has become
more prevalent in the world and espe-
cially in the United States. Texas has
had a part in this and to some degree
provides an example of how people
have approached regulations related to
penaeid shrimp diseases,
Becausethis is a discussion of regula-
tion, it will be helpful to briefly explain
the form of government that is present
in the United States. The United States
is a federal union composed of na-
tional, state,and local governments.Its
constitution specifiesthe authoritative
power that each segment of govern-
ment possesses. The people control
that authority through their elected
representatives in the different places
comes from either national, state or
local government,
Protection for the common good has
always beena function of government.
The continual problems are: who de-
fines and determines the common
good? How will protection be
achieved? As governments enlarge and
societies become more complex,
authority to formulate and enforce
regulation is normally delegated to
more remote places in bureaucracy. As
regulators approach a "setting in
stone" of regulation, they often find
that the political processis not inclusive
of enough suitable viewpoints. They
must make extra effort to obtain peo-
ple's input in order to avoid unbal-
anced results.
 Johnson
Issuesare what people believeto be
importantat a particular
time.Theim-
portance
of a particularissuevariesas
time takesits courseand accordingto
a varietyof influences.
Shrimpdisease
regulationcouldbe describedas a sin-
gleissuefromtheperspectiveofvalid- In 1991, Texanscultured shrimp inten-
I havechosen sivelyin morethan 350ha of ponds
ity or currentresponse.
insteadto acceptthe issue'sexistence
as fact and consider the issues that
relate to its presentstate of develop-
ment in the state of Texas. The view-
pointis that of an extension
specialist
whose role is education and service in
theareaof aquaticanimalhealth.That
role includesno exerciseof regulatory
authority.
Thispaperwill givea briefbackground
on importantaspects
of development
and statusof shrimpdiseaseregulation
in Texas,speakto the key influences
and impacts, and mention some gen-
eral and particularhelps. In regardto
theparticularhelps,theyareaddressed
primarilytotheprincipal
component of
this conference'sparticipantsthe
research scientist.
Developmentand Status
ShrimpAquaculturein Texas
Interest in marine aquaculture in-
creasedin Texasin the early1970s.By
the 1980s,severalmarine facilitieswere
established.
The emphasis
on shrimp
aquaculture
in Texaswaspartlydueto
theexistingand well-developedshrimp
fishery, with its establishedprocessing
and marketingchannels.During the
developmental
phase,a numberof na-
tive and exotic nonindigenous!species
werecultured.Of the species
used,one
emergedas the commercial
species
of
choice: Penaeus vannamei.
distributedamonglessthan ten farms,
These were clustered mid-way up the
coast near Palacios, Texas and in the
southern-most
countyof the statenear
BrownsvNe, There were two small in-
land efforts. Two hatcheries existed,
with a productioncapacityof 60 to 80
millionpostlarvaeper month.The esti-
mate for importedseedlast year was
40% of total stock. One or two more
hatcheriesare plannedfor 1992-93.
RegulatoryAuthority
Forregulations
to exist,theremust be
someseatof authority.Regulationsthat
relate directly to shrimp diseasein
Texas are found within the statutes of
the naturalresourceagency,the Texas
Parks and Wildlife Department
TPWD!.Theyrelateto theuseof non-
indigenous
speciesnowcalledharmful
or potentially harmful species!.The
TexasDepartmentof Agricultureand a
commission on animal health do not
presently
regulateshrimpdisease.
The
former does issue a fish farming li-
cense,which is necessaryfor general
operation.
On a federalor nationallevel, regula-
tion is still rather indirect. The U.S. Fish
and Wildlife Service USFWS!. a re-
sourceagencyof the Departmentof the
 Shri Disease
Interior could, in certain instances, as-
sure compliance to state regulations
based on a legislated act Lacey Act!.
That agency also requires an import
license for wildlife, including shrimp
introduction. A simplehealth certificate
or statementof health for port-of-entry
check is a requirement of the U.S.
Department of Agriculture.
Environmental Concerns
Diseaseis only one of severalenviron-
mental concerns.This is important for
diseasespecialiststo recognizebecause
regulation of disease,especiallyif intro-
ductions are at issue, is often inter-
woven with regulation of other
concerns.The developmentand status
of other concerns influence the exist-
ence of diseaseregulation. Regulation
in Texas reflects more the concern for
the natural environment than for
aquaculture.
Regulation Relating to the Control
of Shrimp Disease
Regulation of exotic shellfish imports
was added to the Texas Regulatory
Code in 1973. The licensed producer
was to fulfiG certain requirements in
accordance with the permit issued.
Later, when it was recognizedthat ex-
amination of nauplii for viruseswas not
practical, new rules were adopted.
The crawfish industry convinced the
resource agency to exdude crawfish
from this regulation. They did this by
showing the need to import animals on
a regularbasisfrom neighboring states
Re ulation in Texas
and noting that the movementswould
occur within the natural range of the
species.Subsequently,introductions
and distributions of Australian crawfish
without disease inspections have been
a common occurrence.
Shrimp farmers took the opportunity
early in the development of regulation
to influence how regulationswere to be
implemented. The decision for imple-
mentation was to examine subsamples
af each batch of imported stock for
diseases. The examination was to fol-
low a setof generalguidelines and the
inspectorwould be someoneapproved
by the agency.
Current rules indude the stipulations
that imported nauplii must be quaran-
tined and examined monthly as they
reach postlarval sizes, and that hatch-
eries from which the nauplii come must
be certified as free of disease. Regular
shipments of postlarvaeand lar'gerani-
mals are inspected on a shipment-by-
shipment basisand held in quarantine
until they are declared dean by the
regulatory agency.
The focus of the regulation relating to
introduction of exoticshrimps and their
diseasesis the protection of the natural
resource,in particular, the shrimp fish-
ery, which is valued at $500 million.
The regulations emphasizeprevention
of escapeof exotic speciesand inspec-
tion of exotics for disease.
Key items of regulation intended to
control escapes are: an acceptable de-
sign capable of stopping effluent, a
 Johnson
screen barrier between stocks and dis-
charge capable of containing any life
stage,dikes around farms within a 100
year Hoodplain, and adequatesecurity
to prevent removal by people.
Impacts
What are some important influences to
the development of shrimp disease
regulation in Texas?I will discussfirst
the nature of the influence and then its
impact.
Societal, Agricultural and Fisheries
Development
Will development of shrimp culture
regulations proceed too fast? Will un-
necessary regulations emerge under
pressure from strange places, as regu-
lators say, "better safe than sorry." Or,
because of stalemates, will there be too
little too late? The general state and
pace of development of our society,
agriculture, and fisheries, impacts
everything, induding regulation,
Impact: It is difficult to obtain a dear
reading on regulatory expectations
when change is rapid and structures
are slow to develop, Regulatory con-
cepts and stipulations may be in writ-
ing but implementation is vague.
Early diseaseregulations for exoticsin
Texaswere drafted primarily with the
intent to control oyster diseasesand to
have a mechanism i.e., permit! to keep
track of where animals were coming
and going Terry Leary, GCSMC, per-
sonal communication!. Shrimp were
induded, but expectationswere stated
on the permit and on a case-by-case
basis. Expectedbeneficiariesand haz-
ards were not dearly defined,
Governmental Mandates
The basic mission of an agency is re-
flected in the regulation it directs. For
example, agriculture agenciestend to
support regulatory disease programs
that would enhance or safeguard the
production and marketing of an agri-
cultural commodity. Natural resource
agenciesfocus more on protection or
conservation of natural systems.
Most governmentagenciesthat control
aquaculturewere not instituted to serve
aquaculture.Yet, government agencies
will, under pressurefrom without and
within, tend toward an expansion of
their roles to include things such as
aquaculture if financial resources are
available and when efforts can be justi-
fied. When conflicts arise, however,
they generaHyretreat to the mandates
that originally established their role and
the will of the clientele they were origi-
nally intended to serve.This is no small
problem for aquaculture.
A call for action often follows a per-
ceived need for regulation or a concern
about how to implement existing regu-
lation. But an agency's effort toward
aquaculture including regulatory!
often suffersin terms of its priority and
the conviction and darity of delivery.
This is becausesocietal leadership relies
on agencies or agency groups whose
fundamental mandates make them
 Shrim Disease
more or less inappropriate for particu-
lar regulatory applications.
This is one of the reasons that private
production interests and their repre-
sentativesdo not give hearty support
to government initiatives concerning
regulation, even when they are told
that they will be beneficiaries. Private
production interests tend to favor a
suppression of regulation by govern-
ment. In the United States, there are
fewer regulations in states with large
aquacultureindustries. Consumer and
environmental groups, in contrast,
tend to favor adoption of regulation.
Impact:There is essentiallyno regula-
tion of diseasein Texasby the national
government. This is not to imply that
there needs to be. National government
does little to reinforce state regulatory
laws concerning aquatic animal dis-
ease. The U.S. Fish and Wildlife Serv-
ice is active in control of movements of
endangered species, but shrimps are
not considered endangered. National
guidelines and policies are undevel-
oped in regard to aquatic animal intro-
ductions. Only recently has legislative
mandatedevelopedto deal with harm-
ful nonindigenous species. The legisla-
tion is so targeted toward a single
speciesthat its regulatory mandate may
be too weak to form the basis for devel-
opment of meaningful regulation from
the national level,
The state government of Texas has fo-
cused on regulation of aquatic animal
disease through its natural resource
agency TPWD!. Due to an emerging
R Ulation in Texas
environmental awareness, an environ-
mentally consciousTexanperceivesthe
introduction by release of nonindi-
genousanimalsor their diseasesasvery
detrimental. Concern is also expressed
by thosewho are dependenton exploi-
tation of natural shrimp stocks. The
agency'smandateis to protectthe natu-
ral resource, so the focus is on protec-
tion against organisms perceived as
biological pollutants.
Research
The desire for reliable information in an
expanding industry is great. Knowl-
edge of disease is popular. Mystique is
often king in shrimp culture where
biologists vie for attention and disease
is a subject by which sweeping state-
mentshave the ability to gain attention.
Disease also is often an easy answer for
failure and failure is common in the
rush to adapt new and unproven tech-
nologies.
Researchbrings us information based
on acceptedscientific methods. Accu-
rately documented and presented, it
will hopefully pass the scrutiny of
peers. Scientific information is sup-
posedto eclipseexpert opinion as "the
best information available" and be a
notch above "rumor has it."
Impact: Researchhas had a consider-
able impact on the regulatory level of
decision making in Texas and other
places.During the past few years, gov-
ernment agency personnel wanted to
know whether or not the time was right
to take prudent action by regulatory
 Johnson
control. They also wanted to know
which endpoints to establish for dis-
eases considered to have significant
harmful effect. For certain viral diseases
of shrimp, research had stated that
danger existed and caution was
needed. Warnings had been made by
respected scientists, at least from the
theoretical and predictive point of
view. Texas regulation of shrimp dis-
ease, at last reading, does show an
indusion of what research has said on
the matter. It has an implementation
design that is workable for aquacultur-
ists even though the regulations are
basedprimarily on protection of natural
resources.
Perception
Perception = how things are seen or
understood; reality = how they really
are. Science as a method helps us to
gaina bettergraspof reality.And peo-
ple who do science have their own
perception.And, obviously,much of
what I am sayingis my own perception.
Regulationis basedon the gifts good
and bad! of research, but only to a
degree.Environmental problemsin our
societygain responsemore on the basis
of public perception than scientific re-
ality. In the areaof shrimp disease,the
perceptionof the public,managers
and
regulators of what scientific data says
may be somewhat different from what
it said. Yet it is that perceptionthat has
had more forcein bringing issuesto the
regulatory table. Oncethere, defenders
of reality usually have a chanceto fend
for themselves.
Impact: I haveno opinion polls that can
document the history of the influence
of perception on Texas regulation. I
have by experience,however, seen its
importance time and again and have
spent a considerable amount of time
relating research results to decision-
makersto help them do their best.
Political Process
People and groups have particular
views concerningwhat is important for
the common good. If they are active
and yet open to compromise, their ef-
forts will servethe progressof all.
Impact: Several groups have engaged
in the political processof formulating
Texasdiseaseregulations and the way
they are implemented. The Texas
Aquaculture Association has been the
primary advocate group for aquacul-
ture. The Texas Shrimp Association
shrimp fishermen, or "shrimpers"!
has beenthe primary group advocating
protection of the fishery resource.En-
vironmental and conservation groups
have had some influence, usually in
support of the shrimpers' positions.
Those opposed to the use of nonindi-
genousspecieshave not failed to wave
the banner of virus danger before a
public already sensitized by media to
the perception of harmful effects of
human viruses.
The shrimpers have had an important
influence on bringing regulation to the
tablebecausethey representthe largest
fishery, with a value many times that
of shrimp aquaculture. Authority has
 Shri Disease
been rather considerate to aquaculture
in Texasin this regard. It has respected
and involved the aquacultureposition
even when aquaculture was compara-
tively weak in force and organization.
Several state agenciesmade efforts to
be responsive even when those agen-
cies had limited staffing and resources
devoted to aquaculture. TPhi%3facili-
tated common forums for shrimpers
and aquaculturists and met with
aquaculturistson multiple occasionsfor
input.
Producer Actions
In respectto regulation, shrimp grow-
ers are expectedto comply and make
attempts to provide a good example.
They are not expected to engage in
illegal actions or to otherwise greatly
offend the perception of what the pub-
lic considersacceptable.If they do what
is noncompliant or offensive, it can
result from malice, accident, ignorance
or somewhere in between.
Impact: On the whole, Texasaquacul-
turists have presenteda goodexample.
Most of what is good and workable in
our regulations are a result of sensible
and accurate inputs from this group,
whose membershipis highly regarded
by public officials. Mistakes, however,
have fueled the need for tighter con-
trols, especially in regards to escape-
ment. Escapes of small proportion are
anticipatedin time, but toward the end
of the 1991 growing season, several
hundred pounds of juvenile shrimp
were released into natural waters in
South Texas. Public awareness was
R Uiation in Texas
roused throughout the state and new
and more restrictive regulations were
printed, as adopted in the TexasRegis-
ter in March 1992. Environmental activ-
ists havetargetedthe responsiblefarms
and are searchingfor someway to find
fault in comphanceso that someonecan
be brought to justice. CameronCounty
has filed chargesagainst a number of
people, partnerships and corporations
with production units at the release
site.
Helps
Regulation is praiseworthy in many
respects,but it is greatly weakened if
from the view of the complier or others
it is vague, politically motivated, inef-
fective, or unpredictable. Time, effort
and expenseof producers and others
are often wasted at adversarial forums
where the focus is merely a defenseof
self interest. It would be much better
for all if the mind-set and environment
were conducive to conflict resolution..
Much anxiety could be avoided on the
subject of regulation if the need for
good endpoints is seen and efforts
structured so that endpoints could be
properly established.
Endpoints
Endpoints are the descriptors used to
determine the fulfillment of objectives.
They are expressedby measuresin the
form of criteria fixed standards! or
guidelines broad performance stand-
ards!, Guidelines are more appropriate
measures for shrimp disease regulation
becausethey better reflect the reality of
340 Johnson

industry, the dynamics of biology and and money! consideringwell-founded

the use of professionaljudgmen.t.They but predictive approaches.The consid-
have a flexibility that is concurrentwith eration of the distinctive realities of the
changing but definable circumstances, diverse problems are ignored in most
There has been a tendency in shrimp cases. It is worthwhile again to note
diseaseregulatoryeffortsto focuson that both scientific and unscientific fac-
the criterionof presenceor absenceof tors influence the selection of end-
a virusagent.This is becauseavoidance points and objectives.
is a favored managementstrategy for
aquatic animal viruses, and because Common Sense
unique biologicalcharacteristicsfavor a
technological approach based on host In a technological society, the use of
specificity. common senseis often bypassed.Dis-
ease regulation should recognize the
Regulation is shaky and unclear to a often vast differences in species biol-
great extentbecauseof the lack of ac- ogy, regional settings, and types of
ceptable endpoints. Good and better culture systems. It should recognize the
endpoints areneededif shrimp disease true complexity of problems associated
is to be regulated. Also needed is agree- with diseaseand that natural systems
ment on the commonly derived objec- have, at any point in time, a unique
tives that state what people want. They dynamism.
form the basis for endpoint selection.
Common objectives are reached by
communication, political process,con- A recognition of actions of scaleis also
sideration of valid information, scien- helpful when considerationsare given
tific input, and other means.Time and to protecting systems of nature or
effort must be devoted to derivation of aquaculture.Can, for example,a small
common objectives so that good end- producerrespondto requirements that
points can be selected. present unbearable economic hard-
ship?
Regulatoryendpoints should be effects-
based because if there is no effect, there Level of Implementation of
is no problem. In practice,however,we Regulations
often have regulations before we have
data that determines harmful effects. Certainly the global nature of shrimp
Predictionis very important to the proc- disease introduction will necessitate in-
ess of endpoint selection,but predic- ternational cooperation. From where
tion is dangerous if acceptedwholly will implementation of regulations be
and without question. This generally directed once there is basic agreement
happenswhen sciencebecomesregula- oncommon.
objectivesand discernment
tion. Supporters of shrimp aquacul- of recognizable differences from the
ture, in.my view, have spentmuch time global to local level? I think, for effec-
 Shri Disease
tiveness sake, it should be directed
from the lowest level possible.
Helps From Scientistsin
Reaching Regulatory
Endpoints
Wise Use of Limited Resources for
Research
There are few people researching
shrimp disease. More people, along
with more support, of course, are
needed. As a specialistresponsiblefor
transmitting research-based knowl-
edge, my opinion is that we need
"more of this and more of that" instead
of "a little of this and a little of that."
Someresearchneedsto be repeated;in
some cases, several times.
It is fortunate that many researchers
have a strong degreeof solidarity with
what would be called aquaculture re-
search. Those that say they are in
aquaculture because of the research
money usually do not have enough
acquaintancewith aquacultureto make
wise choices on research direction. This
is not to say that there is less need for
viewpoints and active involvements
from as broad a baseas possible.
Balancing Reductionist and Holistic
Approaches
Reductionist or "bottom-up" ap-
proaches try to rely on the simplest
R Ulation in Texas
laboratory data. They are helpful in
prediction and diagnosis but give a
view that is remote from real world
situations. Holistic or "top-down" ap-
proachesgive a doserview of reality by
direct measurementsof problem im-
pacts i.e., field data basis!. They are
not as predictive or diagnostic. Both
approaches,however, are needed for
proper determination of endpoints
used in regulatory controls.
Honesty in Recommendation of
Technology-based Standards
As specialists, we have a basic respon-
sibility to explain to decision-makers
the array of'possiblestrategiesavailable
to reach solutions on regulatory issues
of shrimp disease.This includesthe use
of proper endpoints. We must, to the
best of our ability, communicate the
actual strengths and weaknesses of
each, and not be afraid to take stands
against those that are ill-founded or
unscientific. And if the field of choices
is bleak and there are areas with little
or no answers, then we must say so.
This is one of the best things that we
can do. To say that more researchis
required may not be appreciated,but it
may be just the kind of help that is
needed.
Appreciation of Impact of Perception
It has beensurprising to me to find that
researchers have been just about as
clumsy in respecting the value of per-
ceptions as anyone else. Perception,
however, does steer the ship. It fuels
the development of issues. Poor per-
ception does not do much for direction
or choice of action, Who will speakfor
scienceand what wiH they says Why
do somerespectedaquaculturespokes-
personsgivecredenceto what couldbe
nothing more than rumor in conversa-
tions with managers, regulators and
the public7
Careful attention by specialists to por-
tray repeatedly and with conviction
what is accurate will do much to aid the
process toward good regulation. One
only has to look to recent strugglesin
quelling divergent and confused per-
ceptionsof the U.S. public about infec-
tious viruses HIV! to see the need for
accurate information.
I have seen perception along with
"facts" aired in a vehement and adver-
sarialmannerin regulatory hearingson
shrimp introductions in attempts to
sway opinion. Socialsciencegiveslittle
help in those sessions.This sayssome-
thing for the need of more socialscien-
tists to be involved in aquaculture,
especiallyin social settings beset with
factions.
Appreciation of Various Roles
We are often asked to make contribu-
tions from a scientificperspective.Most
of us welcome the chance to help in
new and different ways. The call for
contribution often comes from unfamil-
iar people beyond our ranks as disease
specialists.It comes becausewe were
perceived to have certain competence
an attribute which we may confirm
by self-perception. Someoneelse with
similar competence may have a role
that better 6ts the need. If this is the
case, and we are aware of it, then we
as professionalsshould redirect the re-
quest of our inquirer. Also, we usually
have a perspective on problems that
allows us to identify others in auxiliary
roleswho could be very supportive. We
should identify them to our inquirer
and get them involved, Appreciation of
rolescanhelp to reachappropriateend-
pointsfor regulation.
Resource Person to Coalitions
We must not be afraid to engage as
resourcepersonsin coalitions, I'actions
are present and a few resourcepeople.
Coalitions are formed in order to re-
solve conflicts, often in time of crises
and often because of the need for, or
the reaction to, regulation. If we are
involved, we can do much good, and
personalrisk is minimized if we stay on
track in our role. It is important to
remember to stay within one's role in
conflict resolution. Our position is
often flattering, and momentary aban-
donment of role can mean that credibil-
ity will be lost in an instant.
Summary
Shrimp aquacultureand knowledge of
shrimp diseasehave developed during
the past two decades.It is difficult for
government to provide proper regula-
tory responsefor this new and expand-
ing development because of the
necessityto cope with a variety of im-
pacts. These include societaldevelop-
ment, insufficient government
Pantoja-Morales,
C. andD.V. Lightner.1991.
Statusof the presence
of IHHN in wild
penaeidshrimpfromthe coastof Sonora,
Mexico. abstract!. Society for Invertebrate
Pathology,
XIV AnnualMeeting.Flagstaff,
Arizona,August4-9, 1991.
Producer Actions
Dailey,R. 1991.Tougherregulations
sought
after release of exotic shrimp. Valley
Morning Star, Harlingen, Texas, Decem-
ber 20.
Garza-Trejo,H.F. 1991.Alien shrimpthreaten
LagunaMadame.
The Brownsvilie
Herald.
Brownsville, Texas, November 15.
Waits, M. 1990. Shrimpersfear farms' exotic
species
will taintbay.ValeyMorningStar.
HarlingenTexas,November27.
Waite, M. 1991. Experts, shrimpersdebate
quarantine
system.Valky MorningStar.
Harlingen,Texas,August29.
Waite, M. 1992. Chargesfiled againstshrimp
farms, Valley MorningStar. Harlingen,
Texas,May 8.
General Helps
Chapman,P.M.Environmental
qualitycriteria:
What type should we be developing'PEnvi-
ron. Sci. Technol. 25 8!:1353.
Elston. R. 1981. Discussion of shellfish certifi-
cation issuesaimed at formulating sensible
solutions.AquacultureMagazine.May-June
Issue. pp. 28-33.
Glaze, N.H. 'l991. Good scient and the scien-
tist. Environ. Sci. Technol. 25 8!:1341.
Plumb,J. 1982.Influenceof regulations
on the
spread
offishdisease,
In:Aquaculture:
Pub-
lic Health, Regulatoryand Management
Aspects,SeaGrantPubl.No. TAMU-SG42-
119.TexasARM Univ. pp. 87-97.
 ShrimpHealthManagementProcedures

Brad R.LeaMaster
The Gxt'vie Institvte
N'Ialapuu Poirk
Waimanah, Hawaii, 96795, U.SA

Increasing
demandformarineshrimpwillpressure shrimpproducers
to intensifytheirculture
techniques
to increase
production.
Intensive
shrimpproduction
systems
will invariably
encounter
healthanddisease
obstacles.
Thispaperdescribes
generalpreventive
healthrecommendations
and
approaches that can be usedto developa preventivehealthprogramfor amrineshrimp.
Specifically,
the textdemonstrates
how preventive
healthmeasures canbe appliedto specific
shrimp diseases.

lntloductiori of shrimp farming. Sano and Fukuda

The modern era of shrimp culture be-
gan in the late 1970sand early 1980s.
Technologiesto enhance productivity
combined with the lucrative Japanese
and North American marketsprovided
the necessaryeconomic returns to es-
tablish a viable industry. Today, the
world shrimp industry produces 28%
of the shrimp placedon world markets.
In 1991, the world's shrimp farmers
produced an estimated 690,100MT of
shrimp from approximatelyone million
ha of ponds Rosenberry,1991!.
The profile of shrimp farming is shift-
ing from extensiveculturesystems0
- 500 kg/ha/yr! to semi-intensive00-
5,000 kg/ha/yr! and intensive ,000-
10,000kg/ha/yr! farxning Brock, 1991a!,
Unfortunately, diseaseshave accompa-
nied the expansionand intensification
987! reported that the annual losses
related to disease in 1984 totaled 70.2
MT, 3.4%of the entire production from
kuruma shrimp hatcheriesand farms in
Japan.Lightner ln press!reported that
in most of the major shrimp farming
regionsof theworld, hatcheryandfarm
production losses due to diseasesare
increasing.In Taiwan, diseasescontrib-
uted to the near collapseof the industry
Lin, 1989!.Indeed, a discerrungshrimp
farmer must be knowledgeable in the
area of diseasesand preventive medi-
dne, This paper summarizes preventive
health measures and demonstrates how
they can be applied on shrimp farms.
EtiologicAgents
A variety of biotic and abiotic agents
causeshrimp diseases Lightner, 1983,
1988;Brock, 1991b!.The important viral
 LeaMaster
diseasesof penaeidshrimp are listed in
Table 1, and the principal bacterial,
fungal and parasitic diseasesof shrimp
in Table2. An important consideration
is the dynamic interplay between hus-
bandrypractices,stress,and the causal
agentsin many diseaseoutbreaks.These
interactionsincreasethe difficulty of an
accuratediagnosisand proper control.
Infectiousagents,environmentalfactors
soil and water, chemicals, biotoxins
andpesticides!,
hostcharacteristics,
and
husbandrypracticesall contributeto the
causation~mplex of shrimp diseases.
PreventiveHealth Principles
The production of healthy animals is
the goal of any commercialenterprise.
The goal of a preventive health pro-
gram is to minimize loss due to clinical
and subdinical disease and to maximize
reproduction and quality of shrimp.
Providing conditions to insure the good
health of cultured shrimp indudes the
period from hatchingto marketing. Ad-
ditionally, as production methods are
intensified, and the efficiency of con-
version of feed to meat product is in-
 Shri Health Mana
creased, stresses are introduced that
increasethe potential for disease.
Preventing diseaseis much more eco-
nomical than providing expensive
treatments following a disease out-
break.Thereis not a single,ideal,uni-
versal preventive program that can be
applied by every producer. Specific
considerations must be taken into ac-
countfor individualenterprises;how-
ever,therearesomegeneralpreventive
recommendations that can be made in-
cluding after Fraser, 1986!:
1. Provideadequateand dean water.
2. Provideadequatespace.
3. Provide adequateand properly bal-
anced feed.
temper
turchang 15.Isolate
ordestroy
diseased
5. Alwaysuse sanitaryprocedures.
6. Removefecesas often as practica-
ble, removedeadfish, prevent the
accumulationof otherorganicmat-
ter such as uneaten feed and the
accumulationof a biofouling com-
munity;i.e., algaeand slime.
7. Followan "all-in, all-out" concept
when feasible.
8. Thoroughly clean and disinfectbe-
tween crops.
9. Separateage size! groups as much
as is practical.Avoid mixingspe-
emant
cies. Polyculturepracticesare an
exception.
10. Avoid unnecessary
handling.
11. Immunizeagainstdiseases
common
to the geographicarea if feasible.
12. Control internal and external para-
sites.
13. Providediligentsurveillance
to rec-
ognizeearlysignsof disease.
14. All new incoming shrimp should
be quarantined&omresidentstock,
Movementof shrimpshouldbe re-
strictedfrom a suspectedor un-
known disease status area.
Detention time should be at least as
longasthe incubationperiodof the
suspecteddisease.
shrimp.
16. Provideadequatenursingfor dis-
eased shrimp.
17.Begintreatmentof diseasedanimals
as soon as possibleafter diseaseis
diagnosed.
Developinga preventive health pro-
gram entails assessingthe current
needsand objectivesof the producer,
the current management and hus-
bandry practices, the current health
and diseaseproblems,educationof the
producerif appropriate,and the future
anticipatedneeds.The basicprinciples
used to protect the producer, as well as
customers,
are 1! beginby establishing
healthy, disease-freeanimals; 2! estab-
lish barriers to prevent disease from
entering the farm; 3! surveillanceand
monitoringfor the presenceof disease;
4! have disease-fighting programs in
place and initiate prompt action when
disease does occur; and 5! continuous
effort to upgrade the overall health
status of the program.
Application of Preventive
Health Principlesto Shrimp
Diseases
Options for prevention and control of
shrimpdiseases
haveevolvedfrom tra-
ditional veterinary and animal hus-
bandry methodology. For diseasesof
biotic origin, quararttineand restriction
of movement, disinfection and sterili-
zation, enhanced species resistance,
disruption of the parasite/pathogen For P. stylirostris and P. agneamei,
life cycle,chemoprophylaxisand che- IHI~I/' is extremely threatening. In
motherapy, enhancementof host's juvenile through adult P. shjlirostris,
defenses, and stock management prac-
tices have been applied on a commer-
cial ot experimentalbasis.For diseases
of abiotic origin, removal of the oppor-
tunity for exposure,feed supplementa-
tion, altering or improving stock
managementpractices,and utilization
of resistant species have been used
Brock, 1991a!.Table3 summarizesthese
various methods.
Biotic Agents
BarrierRearingand Restricting
Movement
An example of preventing diseaseby
utiljzing the principles of restricting
movement quarantine! and barrier
rearing would apply for the infectious
hypodermal and hematopoieticnecrosis
virus IHHMif!. IHIP/ is a parvo-like
virus that is highly contagious to all
species
of penaeidshrimptestedBonami
et al., l990; Lightner, 198S!.IHE~F is
widely distributedin cultured penaeids,
but its range in wild shrimp has not
been fully determined Brock, 1991a!.
IHIP/ occurs as a rapidly disseminat-
ing diseasecharacterizedby high mor-
bidity and mortality Bell and Lightner,
1987a; Brock, 1991a!. In P. oannamei,
infection by IHHNV during early devel-
 Shn Health
opmental stages results in a disease
characterized by poor growth, various
cuticle and appendagedeformities,and
reduced survival during later stagesof
culture Kalagayan et al., 1991!. This
phenomenon has been termed "runt-
deformity syndrome." Larval stagesof
both P. stylirostrisand P. mnnameiare
commonly infected; however, B6&W
is not associated with clinical disease in
these younger life stages.IFB~P in-
fections are persistent in shrimp, so
once a group of shrimp becomes in-
fected, the virus remains in the popu-
lation and is transmitted between
generations Brock, 1991a!,Severalre-
ports have indicated that IHFINV has
probably been widely disseminatedas
a result of the movement of live shrimp
associated with aquaculture practices
Lightner, 1983;Brock et a11983; Col-
orni et al., 1987!.
Improved diagnosticshave contributed
to the preventionand controlof II-IHNV.
Specific pathogen-free SPF! stock are
showing promiseas a meansto prevent
IHHNV. The Gulf Coast Research
Laboratory Marine Shrimp Consortium
at The Oceanic Institute has SPF P.
vannameiderived from a founder popu-
lation of postlarvaefrom a commercial
hatcheryin Northern Mexico and in-
troduced into Hawaii in 1989 Brock,
1991a!. Strict enforcement of rigorous
quarantine protocols and routine sur-
veillance viz., barriers and restriction
of movement! have kept these shrimp
free of IHF-PW to date.
Mana ement
Disinfection
Bacterial infections result in severe eco-
nomic losses in shrimp hatcheries
Nash, 1988; Brock, 1991a!. Bacterial
disease in hatcheries can be associated
with sudden, high mortality and almost
complete loss of production. The types
of bacteria associated with these infec-
tions are facultative pathogensthat res-
ervoir in the water, larval feeds or the
hatchery environment Lightner, 1983,
1985; Brock, 1991a!. Disease occurs
when bacterial populations increase,
and high densitiesof potentially patho-
genic speciesdominate the hatchery
tank microflora. Unfortunately, antibi-
otics have been indiscriminately used
in shrimp hatcheriesas a quick control
measure instead of changing poor hus-
bandry practices.Besidesbeing costly,
the routine use of antibiotics has led to
the emergence of antibiotic-resistant
bacteria as well as posing health risks
to hatchery workers Brown, 1989!.
Successfulprevention of bacterial dis-
easescan be achieved by a properly
balancedculture medium coupled with
an "all-in, all-out" practice that allows
for routine disinfection and sanitation
of equipment, pipes, and tanks be-
tween uses Brock, 1991a!.
Disruptionof Pathogen/Parasite
Life
Cyde
The Microsporidaareprotozoathat have
been associated with the disease "cotton
shrimp." Microsporida are common
parasitesof wild shrimp Lightner, 1988!
and have been identified from penaeid
shrimp in the Eastern and %western
hemispheres Anderson et al., 1989;
Lighter, 1988!. Wild-caught adults
stocked into maturation systems can be
infected. Moderate to heavy Microspor-
ida infections are diagnosed by the
gross appearanceof involved organs
which appear fuzzy or "cotton-like."
Even though Microsporida infection of
cultured shrimp is not common, infec-
tions of growout shrimp populations
result in reduced survival and eco-
nomic loss related to rejection of the
product at the packing plant.
The life cycle of these protozoa is com-
plex; shrimp serve as an intermediate
host and a predatory fish is the final
host. Vertical or horizontal transmis-
sion does not occur Lightner, 1988!.
Prevention of microsporidosis involves
disruption of the parasite's life cycle.
This can be achieved by screening to
exdude infected shrimp from the farm,
or by removing the predatory fish that
serves as the final host from ponds
Brock, 1991a!.Shrimp with microspori-
dosis should not be transported; infected
shrimp should be destroyed.
Chemoprophylaxis
The strategic and responsible use of
chemical products can be important in
a preventivehealth program, In shrimp
hatcheries, larval mycosis is a ubiqui-
tous problem that can lead to devastat-
ing losses if not properly controlled.
Motile zoospores are responsible for
disseminating larval mycosis infec-
tions. These fungi are saprophytes and
are thought to reservoir in the hatchery
water system and possibly in the brood-
stock shrimp. Lagenidiumsp. is nor-
mally associatedwith diseaseoutbreaks
in the nauplii and protozoea stages,
while Simlpidiani sp, infection is com-
mon in the late protozoea to mysis
stages.Older shrimp are rarely infected
because their thicker cuticle inhibits
penetration of the zoosporegerm-tube
Brock, 1991a!.
The use of trifluralin Treflan ! is very
efficaciousin the prevention of larval
mycosis. Trifluralin is a herbicide that
effectively destroys the motility of the
fungal zoospores and prevents wide-
spread infection Bell and Lightner,
1987b; Brock, 1991a, 1991b!. In addi-
tion, proper disinfection of hatchery
equipment helps reduce the presence
of the fungal reservoir in the environ-
ment Baticados, 1988!.
Chemotherapy
Chemotherapy has been used to suc-
cessfullycontrol rickettsial infections in
shrimp. A pleomorphic, intracellular
infectious agent has been associated
with a serious disease syndrome of
pond-cultured P. vannarneiin localized
geographicareasof Texas.This disease
has been termed "Texas necrotizing
hepatopancreatitis
syndrome" TNHPS,
formerly known as "Texas pond mor-
tality syndrome"! Bell and Frelier,
1991!. The presence of the infecting
organism can be verified by histologic
demonstration of the characteristic mi-
crocolonies in the hepatopancreas of P.
vunnameiusing special stains or electron
microscopy. The use of oxytetracycline-
medicatedfeed has been reported to be
 Shn Health
effective in controlling ThlHPS Bell,
1991; Bell and Frelier, 1991!.
ResistantSpecies/Strain
Fusarium disease is a fungal disease
that can be a serious affliction of subadult
to adult cultured penaeid shrimp. The
causativeagentsare Fusariumsoli and
possiblyother Fusarium
spp. F. solartiis
a ubiquitous organism with a world-
wide distribution. It is a saprophytic
fungus and can be abundant in culture
systemswhere excessdecayingorganic
matter is presentor where culture prac-
tices are conducive to cuticular wound-
ing. F. solaniinvades dead or damaged
tissue such as cuticular wounds. Any
site is susceptible, but gals, append-
ages and uropads are most often in-
volved Brock, 1991b!. There is a strong
correlation between species and age
and susceptibility to infection Light-
ner, 1988!,Among Asian penaeids,P.
japonicusis highly susceptible Light-
ner, 1988; Brock, 1991b!. Practical pre-
vention measures recommended in
culturesystemswhereFusariumdisease
is a problem include using a resistant
speciesof shrimp suchas P. monodon, It is generally understoodthat consci-
reducing organic matter, and reducing
wound-creating husbandry practices
Brock, 1991a!.
Enhanced Host's Defenses
Increased understanding of the im-
mune responseand mechanismsof re-
sistance to disease by marine shrimp
has led to the developmentof immuno-
prophylaxisregimensfor certainbacte-
rial diseases. In juvenile and adult
shrimp, the bacteria that invade shrimp
Mana ement
tissues are predominately gram-nega-
tive, oxidase-positiverods belonging to
the genus Vibrio Brock, 1991a!. Sep-
ticemic vibriosis and locaBzed internal
Vibriosp. infections are associatedwith
different dinical signs. Gross signs in-
dude opaque abdominal musculature,
anorexia, and darker pigmentation. In
larval and early postlarval shrimp,
signs of vibriosis indude melanization
and necrosisof appendagetips Light-
ner, 1988!.
Studies have shown that immunization
protected shrimp from experimental
challengeto bacterial pathogens itami
et al., 1989!. Further, Lewis and
Lawrence 985! reported that vaccinat-
ing penaeids with a killed Vibrio sp.
bacterin resulted in improved protec-
tion againstvibriosis in pond-raised
shrimp. Vaccinatingto enhancethe
shrimp's defensesagainstbacterialdis-
easesmay become a practical preven-
tive health technique for shrimp
farmers in the future.
Stock Management Practices
entious and responsible husbandry
practices are essential for successful
production. Proper stock management
practiceshavea majorimpacton low-
ering stressorpressure.Severaldis-
eases associated with biotic agents can
be prevented by adhering to sound
stockmanagementpractices.For exam-
ple, bacterialfouling is a frequentprob-
lem in penaeidhatcheries.It is a disease
condition that results from the heavy
colonization of cuticular surfaces by
noninvasivebacteria.All age classesof
shrimpcanbe affected,but the disease
is commonlyassociated with larvaland
postlarvalmortality.At low densities,
bacterialcolonizationcausesno appar-
ent harm to the shrimp. When nutri-
ents are plentiful in the culture water,
multiphcation of bacteria is stimulated
and the microbial numbers increase to
levelswhere physiological functionis
impaired. When fouling organisms
heavilycolonizethe gill lamellae,respi-
ration is compromised Lightner, 1988;
Brock, 1991a!.
Therapeuticantibioticor chemicaltreat-
mentmay be indicatedin certaincases
of bacterialfouling to lower bacterial
numbers; however, successfulpreven-
tion is closely related to the proper
managementof the predisposing con-
ditions.Theseincludeimprovedwater
quality,increasedwaterexchange,lower Feed Supplementation
stockingdensity,and dosesurveillance Supplementingfeed for potentialdefi-
to miniixuzeoverfeeding Brock,1991a!. cienciesis a commontechnique.Vita-
AbioticAgents
Remove From Exposure
Formulatedfeedssubjectedto storage
under humid tropical conditionsare
commonlyinfested with the fungus
Aspergillus
jhvus. This mold species
producesseveralclassesof aflatoxins.
Of these, AF Bi is one of the most
potent naturally occurringhepatocar-
cinogens. Aflatoxicosisis not consid-
ered a significantdiseaseof cultured
penaeids.However, mortalitieswere
inducedin a populationof juvenileP.
vannameiafter feeding diets containing
50- to 300-ppmaflatoxinB for 28 days
Wisemanet al., 1982!.The pointis that
the mechanism for aflatoxicosis to be-
comean importantdiseasein shrimpis
in place, becausepenaeidsreared in
intensiveand semi-intensivesystems
are fed diets that are typicallyformu-
lated with ingredientsthat occasionally
contain aflatoxins. Aflatoxin could also
be producedin feedsimproperlystoi'ed
under the warm and humid conditions
typicalof the regionswhere penaeids
are cultured Lightner, 1988!.
Practical preventive measures would
includeremovingthe opportunityfor
exposureby properhandlingand stor-
age of feeds,and the raw ingredients.
Feeds and feed ingredientsthat appear
moldy should be avoided.
min C deficiency, or "black death
disease"can affect all penaeid species
and is known to affect juvenile to
subadultshrimpculturedintensivelyin
tanks. Black death disease results from
a low level of vitamin C in the diet
Lightner, 1988!. Shrimp with clinical
vitaminC deficiencymay experiencea
1 - 5% daily mortality.More typically,
exposureto otherstressorstriggersmas-
sivelossesLightner,1988;Brock,1991b!.
The extent of the disease in cultured
crustacean populations is unknown.
Therefore, subclinicalvitamin C defi-
ciencymay be quitecommonin inten-
sive culture establishments,
 Shri
Control and prevention of black death
diseaseis achievedby proper diet for-
mulation to supplement sufficient vita-
min C required for metabolic needs.
Shrimp tissuecontent of 0.03mg ascor-
bic acid/g is recommended Lightner,
1988; Brock, 1991b!.
Stock Management Practices
As previously mentioned under the
biotic agentsof disease,stock manage-
ment strategiesthat promote an overall
decrease in culture-related stressors
will greatly increase production effi-
ciency. Again, some of these strategies
include adequate and unpolluted water
supply, proper stocking densities,ade-
quate and a properly formulated feed,
undue temperature changes,and dili-
gent sanitation at all times.
Resistant Species/Strains
In temperate regions, seasonallycold
temperaturesareoften a limiting abiotic
factorfor farming shrimp. The commer-
cial aquaculture community has ex-
pressed an interest in identifying a
shrimp that could be cultured in the
United States during periods when P.
vannameiculture is temperaturelimited.
Data suggestthat P. chinensisis a cold-
resistant species,Besidesits good growth
at low temperatures,P. chinensisis easy
to culture, has broad salinity tolerance
and could be readily marketed in the
United States,Europe and Japan Main
and Fulks, 1990!. Future research in the
area of penaeid genetics may yield va-
rieties of shrimp that are resistant to a
broaderrangeof disc~musing agents.
Health Mana ement
Summary
Preventive health is not just the routine
application of diseaseprevention pro-
cedures; it encompassesa way of think-
ing, philosophy, and goals.Preventive
health programs must be customized
for each farm. Successfulpreventive
health systems require input from
many areas,induding nutrition, envi-
ronment, health and physiology, dis-
ease microbiology and pathology!,
epidemiology, genetics, management,
and economics.
Meticulous planning and constant sur-
veillance are required to decreasethe
danger of disease.The routine day-to-
day data that is generated by system
monitoring should be stored in an or-
derly fashion to allow for easy and
frequent analysis.Such analysisof data
can provide advancedwarning of dis-
easeproblems in many instances.The
application of computer software pro-
grams to aid in preventive health is in
its infancy. Without question,computer
programswill becomeincreasinglyuseful
to help monitor and prevent disease,
thereby increasing the efficiency and
production of shrimp farms in the fu-
ture.
The aquaticenvironment, aswell asthe
physiology of marine shrimp, offer dis-
tinctive challengesfor the producer and
health professional. However, these
challenges can be met with innovative
application of traditional veterinary
preventive health principles.
 LeaMaster
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Anderson, I.G., M, Shariff, M. Nash and G.
Nash. 1989. A hepatopancreatic mi-
crosporidianin pond-rearedtiger shrimp,
Penaeusmonodon,from Malaysia.J. Inver-
tebr. Pathol, 53: 278-280.
Baticados,M.C,L, 1988.Dise~s of prawns in
the Philippines. SEAFDECAsian Aquacul-
ture. 10: 1-8.
Bell, T.A. and D.V. Lightner. 1987a,IHHN
disease of Penaeusstylirostris: effects of
shrimp sizeon diseaseexpression.J. Fish
Dis. 10: 165-170.
Bell, T.A. and D.V. Lightner. 1987b.An outline
of penaeidshrimpculturemethodsindud-
ing infectiousdiseaseproblemsandpriority
drugtreatments. Vet.Hum.Toxicol.Suppl.
1!. 29: 3743.
Bell, T.A. and P.F. Frelier. 1991.The treatment
of TexasPond Mortality Syndrome TPMS!
with oxytetracyclinemedicatedfeeds. 1990
field trial results, World Aquaculture '91.
San Juan, Puerto Rico Abstract!. p. 18.
Bell, T.A, 1991.Overview of diseasesand drug
needsfor major aquaculturespecies:shrimp.
Vet. Hum. Toxicol, Suppl. 1!. 33: 19-23,
Bonami,J.R.,T. Bronwen,J. Mari, M, Brehelin
and D.V. Lightner. 1990.Purification and
characterizationof the infectious hypoder-
mal and hematopoietic necrosis virus of
penaeidshrimps.J. Gen. Virol. 71:2657-2664.
Brock, J.A., D.V. Lightner and T.A. Bell. 1983.
A review of four virus BP, MBV, BMN and
IHHNV! diseasesof penaeid shrimp with
particular
referenceto clinicalsignificance,
diagnosis,andcontrolin shrimp aquaculture.
Int. Council for the Explorationof the Sea,
C.M. 1983/Gen;10/Mini-Symposium.
Brock, J,A, 1991a.An overview of infectious
diseasesof cultured penaeidshrimp with an
emphasis
onthosecausedbyobligatepatho-
gens.U.S. Marine ShrimpFarmingPrograin
Shrimp Breeding Workshop. July 16-18,
1992.The Oceanic Institute, Waimanalo, HI.
Brock, J.A. 1991b.An overview of diseasesof
cultured crustaceans in the Asia Pacific re-
gion.Reporton a Regional
StudyandWork-
shop on Fish Diseaseand Fish Health
Management, Asian DevelopmentBankAg-
riculture DepartmentReport SeriesNo. 1.
June1991,pp. 347-395,
Brown, J.H. 1989. Antibiotics: Their use and
abusein aquacultuie. World Aquaculture.
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Colorni, A,, T. Samocha and B. Colorni. 1987.
Pathogenic viruses introduced into Israeli
mariculture systems by imported penaeid
shrimp. Bamidgeh.39: 21-228.
Fraser, C.M. 1986. Management,husbandry,
nutrition, introduction. In: C.M. Fraser Ed.!.
The MerckVeterinaryManual. Merckk Co.,
lnc., Rahway,N.J., U.S.A. pp. 1022-1025.
Itami, TJ. Takahashiand Y, Nakamura. 1989.
Efficacy of vaccinationagainst vibriosis in
cultured kuruma praw ns, Penaeus
j aponims.
J. AquaticAnimalHealth.1: 238-242.
Kalagayan, H., D. G odin, R. Karma, G,
Hagino, J, Sweeney,J. Wyban and J. Brock,
1991.IHHN virus as an etiologicalfactor in
runtAeformitysyndromeRDS!of juvenile
Penaeus uannamei cultured in Hawaii. J.
World Aquacult. Soc. 22: 235-243.
Lewis, D.H. and A.L. Lawrence. 1985. Immu-
noprophylaxis to Vibriosp. in pond reared
shrimp. In: G.L. Rogers,R. Dayand A. Lim,
Eds.!. Proceedingsof the First International
Conferenceon Warm Water Aquaculture-
Crustacea.BrighamYoung University, Laic,
HI. pp. 304-307.
Lightner,D,V. 1983.Diseasesof culturedpenaeid
shrimp. In: J.P,McVey Ed.!. CRCHandbook
of Mariculture.Vol. I, Crustacean Aquacul-
ture. CRCPress,BocaRaton,FL. pp. 289-320.
Lightner, D,V, 1985.A review of the diseases
of cultured penaeid shrimps and prawns
with emphasison recentdiscoveriesand
developments.In: Y. Taki, J.H. Primavera
and J.A. Uobrera Eds.!. Ptceeedingsof the .
First International Conference on the Cultuie
of Penaeid Prawns/Shrimps. Aquaculture
Dept., SEAFDEC,Iloilo, Philippines. pp.
79-103.
Lightner,D.V.1988.Diseases
of cultutedpenaeid
shrimpsandprawns.In: C.J.Sindermann and
D.V. Lightner Eds.!. DiseaseDiagnosisand
Control in North AmericanMarine Aquacul-
tuie, 2nd ed. Elsevier,New York. pp. 8-127.
Lightner,D.V. In Press.Diseases
of cultured
penaeidshrimp.In: J.P.McVey Ed.!, CRC
Handbook of Mariculture. Crustacean
Aquaculture.
CRCPress,BocaRaton,Florida.
Lin, C.K. 1989.Prawn culture in Taiwan. What
went wrongl World Aquaculture. 20: 19.
Main, K.L. and W. Fulks. 1990. The Culture of
Cold-tolerant Shrimp: Proceedingsof an
Asia-U.S. Workshop on Shrimp Culture.
The Oceanic Institute, Waimanalo, Hawaii,
U.S.A. 215p,
Nash,G. 1988.Diseasesof shrimp and prawns.
Fish Farming International. 15:30-31.
Rosenberry,B. 1991.Five billion shrimp cock-
tails to go! World Shrimp Farming. Novem-
ber 1, 1991.pp, 1-19.
 Shri Health Mana ement

Sano,T. and H. Fukuda.1987.Principalmi- Wiseman,M.O., R.L. Price,D.V. Lightnerand

crobial diseasesof mariculturein Japan. R.R. Williams.1982.Toxicityof aflatoxinB
Aquaculture.67: 59. to penaeidshrimp.Appl. Environ.Micro-
biol. 44: 1479-1481.
DiscussionGroup
Summaries
~ I I Il II r l I
 Discussion Grou Summaries

possible quarantining of transported Other key avenues of research men-

stocks and the certification of stocks tioned were developing bacterial vac-
and hatcheries as SPF, cines for nauplii, postlarvae and
broodstock, and "probiotics", benefi-
In Discussion Group F, participants cial bacteria that displace or offset
prioritized both the overall concerns pathogens.
and the research concerns that were
raised during DiscussionGroup A. The Japan
need for SPF stocks and more diagnos-
tic techniques were identified as two of In Japan, P, japonicusis the dominant
the most important issues for re- species of cultured shrimp. Kazuo Mo-
searchers and farmers alike. moyama noted that the most economi-
cally important diseasesof P. japonicas
in Japanare causedby Vibrio spp. and
Discussion Group A: Fusariamsp. Vibrio diseaseshave be-
Country-by-Country come increasingly important in recent
Concerns years; they are estimated to cause 20%
mortality during growout. In particu-
To begin the discussion group sessions, lar, there is a new, highly pathogenic
participants were asked, simply, what Vibrio strain called "V>briosp. PJ" that
their primary concernswere with re- is the subjectof much concernin Japan.
gard to diseasesof cultured shrimp. As While there are two antibiotics sold in
one might imagine, responses were Japan to combat Vibrio diseases,their
varied, reflecting each individual's beneficial effectsare only temporary.
home country and experience, in addi-
tion to their role as scientists, farmers Among penaeids,P.j aponicus
is known
or extension agents. to be particularly susceptible to Fusar-
ium disease, especially under intensive
Central and South America culture conditions. Reducing shrimp
densitiesis the only effective meansof
Becausethe majority of his experience controlling this fungus. Baculoviral
has been with the shrimp culture in- midgut gland necrosis virus BMNV!
dustry in Central and South America, used to be a major problem, but, as a
Rolland Laramore focused on the situ- result of effectivepreventive measures,
ation there. Topping his list was the it is now relatively rare.
problem of poor growth during dry
seasons. Noting that several factors In more general terms, Tokuo Sano
probably contribute to the phenome- discussed the need for a dean culture
non, Dr. Laramore was optimistic that environment to discourage facultative
genetic selection of robust and disease- pathogens and the problems associated
resistant strains of Penueus uannamei with developing bacterial vaccines,
could alleviate some of the problems, while Kazuo Momoyama aired con-

cerns about the international transport
of shrimp stocks and feeds.
Malaysia
Speaking on behalf of both Malaysia
and Asia in general, Mohd. Shariff
called for more ecological studies of
shrimp culture situations studies
that would enable researchers
tor changes in potential pathogens. He
further noted that chemotherapeutants
are not necessarily the best means of
combatingshrimp diseasesnot only
can they harm the environment, but
they are not always effective. There-
fore, immunological studies should re-
ceive a high priority. Finally, Dr. Shariff
called for the standardization of shrimp
research methodologies. Perhaps a
manual similar to the American
ies Society"Blue Book" could be issued
for shrimp disease workers, Such a
project would benefit from cooperation
between the Asian Fisheries Society
and the American FisheriesSociety.
People's Republic of China
In what was to become
theme,Dou Chenjoined Mohd. Shariff
in cautioning against the use of
chemotherapeutantsin shrimp culture.
Stating that the most serious diseases
of cultured shrimp in China are those
causedby Vibriospp., Prof. Chen listed
severaldisadvantagesto using antibiot-
ics, including the possibility of promot-
ing fungal diseases and fostering
resistant strains of bacteria.
ple, as a result of overuse/abuse,
oxytetracycline is now completely use-
Discussion Grou Summaries
less. Since not all bacteria are harmful,
Prof. Chen believes that the best way
to control the potentially pathogenic
species is by ecological or biological
control.
Although there is concern about vi-
ruses in China, viral diseases are not as
economically significant as those
to moni- causedby bacteria. Anxiety is increas-
ing, however, over a number of dis-
eases of unknown etiology, induding
black-white spot disease see D. Chen,
this volume!. Finally, epicommensal
diseases also significantly impact
shrimp culture in China.
Philippines
Fisher- JosdNatividad was another who spoke
out against the indiscriminate use of
antibiotics in shrimp culture. He also
shared his concerns about the current
widespread use of probiotics and other
drugs in the Philippines. Becausethe
impact of probiotics is largely un-
known, and because great quantities of
probiotics, as well as other drugs, are
a common present on the farms, Dr. Natividad
called for some sort of government
control or clearanceprocessfor probi-
otics and other compounds that are
now unregulated. Secondly, Dr. Na-
tividad said that he needed field diag-
nostic techniques to help him quickly
assessproduction problems. Finally,
some diseases that affect the market-
ability of shrimp, most notably "black
For exam- meat disease" and "tail rot disease",
have resulted in rejection of shipments,
alarming shrimp farmers.
362 Oiscussion
South Korea
Most shrimp culture in South Korea is
extensive; as a result, relatively few
disease problems have been encoun-
tered. Myoung Ae Park did, however,
list some disease agents that had been
encountered in farms and hatcheries
Korea: BMNV in larval P. japonicas!,
hepatopancreatic parvo-like virus
HPV; in P. chmensis!and Vibrio spp.
Ms, Park pointed out that better water
quality managementcan aHeviatesome
disease-related
problems.
Taiwan
When it comesto shrimp diseases,Tai-
wanese farmers and researchers have,
unfortunately, gained a great deal of
knowledge through experience.Ac-
cordingto S.N. Chen, the most impor-
tant problemis environmentalimpact.
How does shrimp culture impact the
surrou.nding environment, and, in
turn, how do environmental changes
affectcultured shrimp?Vibriosisis also
a key concern, and while progress is
being made in the area of vaccines,
deliverytechnologyhasbeenproblem-
atic. Shrimp virusesalso need to re-
ceive more attention, as does the Recognizingthat many diseasescan be
problemof drugresidues in harvested avoided by improved husbandry tech-
shrimp in some Asian countries. niques,a numberof the expertsfrom
As noted by Cheng-Fang Chang, inten-
sification is responsiblefor many of the
problems encountered on shrimp
farms. In intensive systems, epicom-
mensal diseases are important; water
quality managementis key to control-
ling fouling organisms. Interestingly,
Grou Summaries
Mr, Chang also listed gregarines as a
major concern. While their affect on
shrimp performanceis unknown, a re-
cent survey in Taiwan found that 80%
of cultured P. monodon were infected
with theseintestinal parasites seeLiao
et al., this volume!. Finally, Mr. Chang
in reminded the group that the relation-
ship between the culture environment
and diseasesshould not be ignored.
Thailand
TimothyFlegelcalledforrapiddiagnos-
tic techniquesthat can be usedin the
field. Noting that suchtechniquesare
needed for abiotic as well as biotic
diseases,Dr. Flegelrecountedsomeof
his experiences with pesticides that
were toxic to shrimp at levels in the
range of pg/L. A second concernis the
need for better preventives, including
vaccines, probiotics and management
techniques.Finally, Dr. Flegel advo-
cated using specific pathogen-free
SPF! broodstock as a way to simulta-
neouslyimprove the diseasesituation
and breed better strains of shrimp.
United States
the United States called for studies to
address issues such as sustainability,
the ramifications of intensification, site
selection/environmental planning,
feeds, preventive health maintenance,
and the impact of certain culture prac-
tices on the environment. Carl Sinder-
mann challenged reseaichers to help
 Discussion
farmers "manage around pathogens,"
wMe James Wyban said the shrimp
culture industry had a great deal to
learn from other meat production in-
dustries such as the cattle, poultry and
swine industries.
The two commercial representatives,
Nick Carpenterand FritzJaenike,were
amongseveralparticipantswho shared
concernsabout the spread of viral dis-
eases. The movement of stocks threat-
ens efforts to estabhsh disease-free
facilities and may also impact popula-
tions of wild shrimp. In a related topic,
SPFtechnologywas mentionedby sev-
eral membersof the U.S. contingent as
a means of improving the shrimp in-
dustry's image, increasingproduction,
and beginning the processof domesti-
cation.
Bacterial diseases, however, were also
listed as concernsby three U.S. partici-
pants, For example, Donald Lightner
pointed out that farmers need to have
effective, government-approved
chemotherapeutants, and Nick Car-
penter wondered whether vaccines
could be a realisticsolution to the prob-
lem of bacterial diseases,Finally, Carl
Sindermann strongly encouraged re-
searchersstudying shrimp diseasesto
quantify the economic impact of dis-
easeusing standardized measures, This
is the only way researchersare going to
receive needed support, said Dr. Sin-
dermann; scientists need to convince
the shrimp industry, governmentsand
funding agencies that their work is
economically significant. He encour-
agedconsultation with economistsand
Gro Summaries
expertsin population managementand
epidemiology,
and the uniformstatisti-
cal treatment of data.
DiscussionGroup B:
Prevention and Treatment of
Diseases in Growout
Although the varied means by which
viral, bacterial and protozoan diseases
are preventedand treatedwas the topic
of this session, most of the discussion
centeredaround two issues:preventing
viral diseases,and poor performanceof
animals in growout in many areas, pos-
sibly as a result of 10 - 15 years of
culture activities.
Virus Prevention
As evidenced in Discussion Group A,
viruses are not the only important
pathogensof penaeidshrimp. The rela-
tive importance of viruses to cultured
shrimp depends on a number of fac-
tors, including the type of shrimp cul-
tured, the type of viral and nonviral
pathogens present, the stressors pre-
sent in the culture environment, and
how heavily infected a given shrimp
population is. There are environmental
and regulatory issuesas well. If a cer-
tain viral pathogen is absent from a
given area, it is important to exclude
that virus from nearbyculture facilities.
In many cases,the best way to prevent
viral diseases is to use certified SPF
broodstock. An SPF program for
Penaeusvannameihas already been im-
plemented in the United States see
 Discussion
Wyban, this volume!. Maintaining SPF
P, monodon in Southeast Asia may
prove much more difficult, however.
Steve Psinakis, a shrimp farmer from
the Philippines that participatedin the
workshop, asked about the possibility
of breeding disease-resistantstrains,
thereby eliminating the need to keep
animals isolated from some pathogens,
What about the problem of introducing
the progenyof SPFbroodstockhereaf-
ter referred to as high-health animals!
into ponds that once held virus-in-
fected shrimp? How do these animals
perform?Do they becomeinfectedwith
viruses during the growout cycle?Pre-
liminary results from several shrimp
farms in the United States indicate that,
in these situations, the incidence of
viral disease is greatly reduced, signifi-
cantly improving production. Factorsto
consider are the treatment of the pond
bottom prior to stocking, and the na-
ture of the virus es! of interest. For
example, it is likely that occluded vi-
ruses such as MBV will be more difficult
to eliminate from ponds than nonoc-
cluded viruses.
In Hawaii, growout ponds were dried
for 10 to 14 days and treated with 800
lbs CaCO3/acreprior to being stocked
with high-health postlarval P. van-
namei.Bacutovirus penaeiBP! and infec-
tious hypodermal and hematopoietic
necrosis virus IHHb&! were the vi-
ruses of concern. Shrimp yields were
Grou Summaries
very good, and the incidence of runt-
deformity syndrome was greatly re-
duced. Some of the shrimp, however,
did test positive for BP see Carpenter
and Brock, this volume!.
In Taiwan, growout ponds are rou-
tinely driedbetweenharvestsand then
treated with chlorine 0 - 20 ppm for
48 h!. Theselevelsof chlorine are effec-
tive against some viruses and other
pathogens; however, they probably
also harm the natural environment.
The results of a number of studies were
discussed with regard to eliminating
pathogenic viruses, For example, a
treatment of 0,1 ppm iodine for 6 - 7 s
eliminated 99.9% of the trout virus,
IHN, from water Batts et al., 1991!.
Studies on BP were performed at the
Gulf Coast Research Laboratory Le
Blanc and Overstreet, 1991a,b!. BP-in-
fected hepatopancreases were sub-
jected to a variety of treatments:
desiccation, calcium hypochlorite,
heating, pH extremes,etc. Though the
results are not directly transferableto
treating pond bottoms, the researchers
found that BP could be inactivated
rather easily using several methods.
Finally, it was mentioned that the mi-
crobial activity in the pond bottom
could be a significant factor in destroy-
ing infective viruses in the sediment,
In a related question, participants dis-
cussed the best means to test sediments
for the presence of viruses. Bioassay
studies are presently being used in
Mississippi for BP. Gene probe and
 Discussion
PCR polymerasechain reaction! tech-
nology may also be applied to this
problem in the future.
The impact of Shrimp Farming on
Postlarval Quality
During the course of the discussion,
Steve Psinakis, a shrimp farmer from
the Philippines, submitted the follow-
ing hypothesis for discussion: "In
many placesaround the world over the
past 10 - 15 years, shrimp farming
activities have done something to re-
duce the quality of both wild-caught
and hatchery-reared postlarvae, Farms
are experiencing higher feed conver-
sion ratios, lower growth rates and
lower survivals, and the problem is
getting worse. The animalsseemmuch
more sensitive to perturbations than
they used to be." As evidence to sup-
port his hypothesis,Mr. Psinakisnoted.
that despite improved culture tech-
niquesand better trained staff, produc-
tion declines year by year.
Furthermore, the phenomenon has
beenobservedin ponds of varying ages
using both wild seed and hatchery-
raised seed from wild-caught spawn-
ers. Other participants related
experiencesthat seemedto support the
above hypothesis. For example, P, van-
namei was once considered asympto-
matic for the IHI~ virus; now it is
dearly not asymptomatic. Is this a re-
sult of deterioratingstocks?Also, while
postlarval quality problems have not
been observed in Panama over the past
15 years, farms in Guayaquil, Ecuador,
Grou SunNna ries
have experiencedlower production in
recent years, and increased incidence
of disease. Problems are worse in areas
with a high density of farms.
What specific activities might be re-
sponsible for harming wild stocks of
penaeids?Two possibilities were dis-
cussed,the useof chemotherapeutants,
and the releaseof hatchery-rearedpost-
larvae into the natural environment.
Chemotherapy in the hatchery allows
animals that are naturaHy weak and
susceptible to disease to survive,
thereby selecting for shrimp that are
lessfit. Theseanimalstypically perform
poorly in growout. Most hatcheryman-
agers,moreover, have no incentive to
produce animaIs that will perform well
in growout, they profit by producing
large numbers of healthy-appearing
postlarvae. This makes it difficult to
obtain animalsthat will remain healthy
during growout,
In a related problem, it is a common
perception in. many parts of Asia that
countless numbers of diseased, hatch-
ery-reared postlarvae are regularly re-
leased into coastal areas. If some of
these shrimp survive and reproduce,
these artificially selectedanimals may
alter the composition of the gene pool
of the wild population.Secondly, be-
cause of the widespread practice of
transporting stocks without regard to
their disease status, viruses have been
introduced to previously"clean" cul-
ture facilities. Furthermore, we are
learning that a number of wild shrimp
populations now carry pathogenic vi-
ruses see Lotz, this volume!, possibly
366 Discussion Grou
as a consequence of nearby shrimp
culture activities. The impact of these
viruses on natural stocks is unknown.
The above hypothesis has not been
proven it has not even been tested.
Becausethere are so many interrelated
factors,finding answerswill be diffi-
cult. Clearly, though, more emphasis
should be placed on determining the
capacityof a given culture areato sup-
port shrimp. Also, precautionsshould
be taken to avoid new viral introduc-
hons, and chemotherapeutantsshould
be used more judiciously, Finally, the
practice of releasing cultured animals
into the natural environment should
involve consideration of animal health
and genetic diversity.
DiscussionGroup C:
Prevention and Treatment of
Diseases in Hatcheries
A key conclusion from Discussion
GroupB wasthat the performance
of
shrimp in growout dependsgreatly on
the treatment the animals receive in the
hatchery.Therearea numberof differ-
ent "hatchery philosophies" whereby
the water and the animalsare managed
to encouragegrowth and prevent dis-
ease.In general, hatchery diseasesare
prevented and treated by
~ Managing the culture water;
~ Adding chemicals to the culture
water or, more rarely, to the
feed!;
Summaries
~ Monitoring the culture environ-
ment; or
~ Managing the animals.
Prevention
Viruses. No one in the group reported
usingspecificwatermanagement
tech-
niques to prevent viral diseases.Many
culturists, however, pretreat culture
water, either with chemicals such as
chlorine or iodine, or by other means
such as ozonation or ultraviolet radia-
tion Table 1!. Often culture water is
filtered beforehand to increase the ef-
fectiveness of these other sterilization
methods.
In some places, cultured shrimp are
routinelymonitoredfor the presenceof
viruses. Such measures,depending on
the sensitivity of the assayused, could
help prevent the spreadof a virus from
infected tanks to uninfected tanks. Ani-
mal management,however, is the best
way to preventviral diseases.Simply
put, stocking with high-health postlar-
vae is the most effectivemeansof keep-
ing viruses out of the hatchery.
Secondarily,eggsand/or nauplii can be
rinsed or chemically treated to remove
external virus partides. This has effec-
tively prevented BMN outbreaksin Ja-
pan, and is also used to lower the
incidence of MBV in P. monodon hatch-
eries and BP outbreaks in commercial
shrimphatcheriesin SouthandCentral
America. Finally, many hatcheries
these days are using batch methods;
drying out their system in between
cycles.While this is quite effective for
 Discussion Grou Summaries

Table $. Preventingdiseasesin hatcheriesin the Americasand Asia .

Potential Water Chemicals Monitoring Animal Other
pathogens management management

Viruses None. Pretreatment of Periodic Begin with Use batch

water with diagnostic hishhealth techniques
ozone~ screening, arumals, rinse with dry-outs
chlorine, broodstock or treat shrimp in between.
iodine, UV history eggsat3d/
or
filtration will nauplii
enhance
effectiveness of
above!.
Bacteria increase Disinfection Daily Rinse shrimp Rinse Artemis
exchangerate, with chemicals monitoring naupliiand nauplii, use
use microalgae listedaboye, with agar eggs,optimize batch
and other antibiotics, lates, e.g., stocking techniques
means to malachite CBS counts density. with dry-outs
condition green, with water and in between,
water, optimize vaccines, larval optimize
temperature. EDTA ppm homogenates. nutrition, add
in Taiwan, 10 microalgae that
R minthe have inhibitory
ifippines!. effects.
Protozoa fncrease Formalin, Observe water Rinseshrimp Rinse Artemia
exchangerate, malachite with naupiii and nauplii,
optimize green, copper microscope. eggs- optimize
temperature, sulfate, EVI'A. nutrition.
Fungi Treflan, Observation Optimize
malachite with nutrition.
n EDTA microsco e.
~Preventive
methodsappearingin this tableareusedeithersinglyor in combination
in hatcheries
producing
P. vannamei,P. mosokm,P. chinessisor P.japonicus.Some may be effectiveonly for speci6cagentsor shrimp
speCie, and somemay not be effectiveat all.
Sano and Momoyama, this volume; Liao et al., this volume.
The prophylacticuse of antibioticswas strongly discouragedby most of the workshop participants.

preventing bacterial problems, it may The chemical pretreatments used to

also destroy viruses,
Bacteria.Increasing the rate of water
exchangecan prevent the build-up of
high levels of bacteria in a hatchery
system. It is also important to maintain
optimum temperatures Table 1!, Fur-
thermore, many believe that healthy
microalgae blooms can inhibit the
growthof potentiallypathogenicbacte-
ria.
prevent bacterial problems include
those listed above for preventing vi-
ruses. Additionally, a number of differ-
ent chemicals may be added to the
culture water to inhibit bacterial
growth, including malachite green,
EDTA and antibiotics.
Most of the participants were strongly
againstthe prophylacticuse of antibiot-
ics because of the dangers of selecting
 Discussion
for and disseminating antibiotic-resis-
tant strains of bacteria, and because it
may be detrimental to the shrimp in the
long run. However, antibioticsareused
regularly in P. monodonhatcheries in
the Philippines, Taiwan and elsewhere
to prevent bacterial diseases. S.N.
Chen noted that much effort is being
expendedin Taiwanlooking for alter-
nativesto antibiotics. In general,hatch-
eries that do not use antibiotics produce
animals that perform very well during
growout, but production is reduced in
thesehatcheries.Timothy Flegeladded
that oxytetracycline OTC! is used suc-
cessfully to improve production in P.
rnonodon hatcheries in Thailand, even
though most of the bacterial strains
isolated from diseased shrimp are resis-
tant to OTC. He also noted that the
addition of very small amounts of OTC
improves the hatching rate of P. mono-
don eggs. He hypothesized that OTC
was having some other effect; one that
was unrelated to its antibiotic activity.
Penaeusvannarnei, in contrast to P. mono-
don, appearsto be much more tolerant
of high levels of bacteria.Whereasan-
tibiotics are sometimes used prophylac-
tically in Central and South America,
their use is not ubiquitous. Antibiotics
cannotlegally be used either to prevent
or to treat bacterial diseases in the
United States,and the commercialrep-
resentativespresentindicated that, un-
der most circumstances, they would
not consider using antibiotics prophy-
lactically even if such use were legal.
In the People's Republic of China,
where the use of antibiotics is not regu-
Gro Summaries
lated, P. chinensis hatcheries do not
routinely add antibiotics to the culture
water. Finally, in Japan, the use of
antibiotics is strictly regulated. Prefec-
tural extension agentsinstruct farmers
on the proper usageof antibiotics only
a few of which have government ap-
proval! and antibiotics are not used
prophylactically.
Many hatchery managershave pinned
their hopes on vaccinesto prevent bac-
terial diseases, While bacterial vaccines
for penaeidshrimp are not now widely
available, several researchers at the
workshop had developed and tested
vaccinesthat could eventually be useful
in hatcheries e.g,, see Laramore, this
volume!.
There are a numberof ways to monitor
hatcheriesfor bacteria,including doing
total plate counts or TCBS counts of
water and/or larval homogenatesTable
1!. This is usually only practiced rou-
tinely in areas where there is a high
density of farms and/or the water avail-
ableis not of optimal quality. The wide-
spread practice of rinsing or treating
shrimp eggs andlor nauplii to remove
bacteriabefore stocking falls under the
category "animal management". Simi-
larly, Arternianauplii, a common feed
in shrimp hatcheries, are routinely
rinsed before use to minimize the
spread of bacteria.
Employingbatch techniquescan also
help minimize bacterial problems Ta-
ble 1!. And, sincebacteriaare ordinarily
considered facultative pathogens of
shrimp, another good way to prevent
 Discussion
infection is to minimize stress. This can
be accomplished, in part, by providing
adequate quantities of high-quality feed
and by optimizing stocking density.
Finally, there is some evidence seeD.
Chen, this volume! that certain species
of microalgaeinhibit the growth of Vi-
brio spp. and other potential pathogens
adding these microalgae to the cul-
ture water should, therefore, lower the
density of thesebacterialspeciesin the
tanks.
Protozoa.Many of the methods used to
prevent bacterialdiseasesalso apply to
diseases caused by protozoa Table 1!.
In addition, chemicals such as Formalin
and copper sulfate are also used in
hatcheries,and it is important to moni-
tor the culture water regularly with the
aid of a microscope to monitor proto-
zoan populations.
Fungi. Larval mycosis is the most well-
known fungal diseaseafflicting larval
penaeids. This disease is usually pre-
vented by adding Treflan+ to the rear-
ing water, although chemicals such as
EDTA and malachite green have also
been used Table 1!.
Treatment
Viruses.Oncea shrimp population has
been infected with a virus, there is no
way to get rid of the virus. There are,
however, techniques that can maximize
the performance of virus-infected ani-
mals in culture. The most commonly
cited method is simply to minimize the
stress placed on the animals. For exam-
ple, in the past, Nick Carpenter has
Grou Summaries
effectively "managed around" BP at his
P. vannamei hatchery by lowering
shrimp densities Table 2!. In addition,
monitoring the level of infection can
help in making managementdecisions.
Bacteria. Increasing water exchange
and optimizing temperature are water
management techniques that can be
used to treat diseases of bacterial etiol-
ogy Table 2!. Antibiotics, of course,
may also be effective, but, as Tokuo
Sano noted, treatment should be pre-
ceded by sensitivity analyses and deter-
mination of minimum inhibitory
concentrations. In Ecuador and else-
where, hatchery managersare adding
1 - 10 ppm sucrose to culture tanks to
encouragethe growth of bacteria that
might outcompete potentially patho-
genic species and strains. Although the
technique is still being refined, shifts in
the types of bacteriarevealedin TCBS
counts have been documented. Sucrose
is also being used in growout, but its
effect in ponds is more difficult to docu-
ment. Similarly, some culturists in Asia
and in the Western Hemisphere are
adding so-called "beneficial" bacteria,
or "probiotics" to hatchery tanks, and
even to growout ponds, in the hope
that they will prosper and outcompete
Vibrio spp. and other potential patho-
gens.
Protozoa. Flushing to lower the densi-
ties of microbes on which epicommen-
sal protozoa feed is one management
method for protozoan fouling disease
Table2!. Alternatively, chemicalssuch
as copper sulfate, malachite green and
EDTA can be added to tanks. In fact,
 Discussion Gro Summaries 371

Table 3. Recornrnendations.
1. Developpriority pathogens
list containingpathogens
that areeconomically
or ecologically
significant, that can be diagnosedwith existing technology.
- Should we app!y certification?Where?
What will be t'he criteria for sensitivity what level is acceptableor should it be zero
tolerance!?
2, Selectand organizecommitteescomprisedfrom membersof the following: the World
AquacultureSociety,the AsianFisheriesSociety,theOfficeof Internationale
Epizootiesand
theEuropean
Association
of FishPathologists.
Committees
will reviewandstandardize
diagnosticprocedures.
Committee must be representative,
Take advantage
of computernetworkingto alleviateneedfor manyformalmeetings.
Handbooks need to be developedby committeesand so do SPFprocedures.
3. Reference labs. S cimen exchan will be ve hei ful in the dia nosis of certain diseases.

ing one or more representative commit- be impracticalto excludethat pathogen

tees of experts to: from that region.

~ Develop or endorse handbooks Research and Development on

that contain detailed diagnostic
procedures; and
~ Develop or endorse guidelines for
certifying diagnosticians, shrimp
stocks and culture facilities Table
3!.
Perhaps committees could be formed
within each of the following organiza-
tions: the World Aquaculture Society,
the Office Internationale des Epi-
zoohes, the European Association of
Fish Pathologistsand the Asian Fisher-
ies Society.
Regional committees, it was decided,
would probably be needed to develop
lists of pathogensto be excluded &om
stocks. Different species and various
regions are expectedto have different
lists. For example,if a certainpathogen
is endemic to an area, that is, present
in the wild shrimp population, it would
New Diagnostic Techniques
Tissue Culture. There was general
agreementthat much more needsto be
done in the area of shrimp tissue cul-
ture. The lack of progress in this area
has hindered the development of new
diagnostictechniques.
Idiopathic Syndromes. Some partici-
pants believed that the phrases "idi-
opathic lesions" and "idiopathic
syndromes" are overusedin the scien-
tific literature. Furthermore, diagnosti-
cians would benefit from a standard set
of steps to be used to investigate idi-
opathic observations in cultured
shrimp.
Multiple Pathogens. Similarly, diag-
nosticiansoften encounter shrimp that
contain multiple pathogens. In this
case,it is very difficult to determine if
one pathogen is more important than
 Discussice
establishing reliable domestic supplies
of SPF stocks prior to the adoption of
regulations. Another issue raised was
the need for international cooperation
in establishing workable, reasonable
quarantine procedures.
Furthermore, aswas pointed out in the
previous discussion, before one can
begin to certify stocks and hatcheries
on a large scale, standard diagnostic
procedures must be in place, and there
must alsobe qualified diagnosticiansto
use those procedures.
In a related problem, how should we
develop priority pathogen lists? This
issue was touched on in the previous
discussion. Some researchers won-
deredwhether enough is known about
the geographicrangesof pathogensto
develop pathogen lists. Furthermore,
any pathogens on an exclusion list
must be able to be diagnosed with
certainty. Other participantswere wor-
ried that priority pathogen lists would
be misused by governments in some
countries and becomeregulatory lists.
The SPF Issue
Motivations. Were are a number
reasons countries or groups might want
to develop SPF shrimp stocks. One is
the shortage of broodstock in some
areas. Domesticated stocks are needed
becausewild sources of high-quality
broodstock are becoming scarce.Logi-
cally, if one is going to begin a domestic
stock program, he or she should begin
Gro Summaries 373
with SPF animals. One could also argue
that domesticated stocks are needed so
that genetically "superior" strains of
animals can be developed. Alterna-
tively, some companies simply want to
have a reliable supply of high-health
animals for growout. Finally, other
companies may be motivated by the
economic incentive of selling high-
health seed to other farms.
Approaches to Certification. Finally,
when it is time to certify animals and
hatcherieswith regard to their patho-
gen status, what approach should be
taken? Some fish hatcheries are classi-
fied based on the number of pathogens
present in their stocks.For example,a
ClassA hatchery may contain SPFani-
mals, whereas animals in a Class 8
hatcherymight carry one known patho-
gen, and so on.
It may also be desirable to categorize
the pathogensthemselves.For a given
area, diseaseagents might be divided
into groups basedon 1! the presenceor
absence of the agent in the natural
environment, and 2! the threat posed
by the agent in question. Finding an-
swers to the above mentioned ques-
of tions for all of the known shrimp
pathogens affecting all the various spe-
cies and culture regions will certainly
require a great deal of study. The Fish
Disease Commission of the Office Inter-
national des Epizootieshas alreadybe-
gun to developa list of excludablefish,
shrimp and mollusc pathogens see
Sano and Momoyama, this volume!.
374 Discussion
Table 4. Overall disease-related concerns,
in order of decreasing importance,
DiscussionGroup F: Open
Session and Wrap-Up
This sessionwas devoted to prioritizing
the concerns raised in Discussion
Group A. Two summary lists were
made that reflected 1! the participants'
overall disease-related concerns, and 2!
their priorities for future disease re-
search. There was some overlap; for
example, the need for SPFstockswas
both a researchpriority and an overall
concern. Everyone was asked to indi-
vidually prioritize the issueson the two
lists. The results are in Tables 4 and 5.
The need for SPF stocks was ranked
highest among the overall shrimp dis-
ease-related concerns and was also sec-
ond on the researchpriority list. This
reflects, in large part, the perceived
threat of viral diseasesto the global
shrimp culture industry, and the need
for a reliablesupply of seed The use or
abuse of chemotherapeutants was the
Gnu Summaries
TableS, Researchpriorities, in order of
decreasing importance.
secondmost important overallconcern.
In particular, the prophylactic use of
antibiotics and the presence of drug
residues in harvested shrimp were at
issue. Third on the list was the need for
rapid diagnostictechniques.Thosepre-
sent emphasizedthe need for two dif-
ferent types of techniques, those that
could be used on farms and others that
could be applied at "minimal clinical
labs."
The need for rapid diagnostic tech-
niques was also the highest research
priority Table5!. SPFstockswere the
secondpriority, and the need for more
studieson probioticswas third. Finally,
the commercial representativesstated
that they wanted more researchefforts
to be directed toward developing more
and better chemical preventives, in-
cluding vaccines number eight and
four on the overaH concerns and re-
searchpriority lists, respectively!. Bet-
ter disinfection methods are needed, in
addition to antibioticsthat are designed
for aquaculture, and immunostimu-
lants.
 Discussion Grou Summaries
Literature Cited
Batts, W.N., M.L. Landolt and J.R. Winton.
1991.Inactivation of infectioushematopoie-
tic necrosis virus by low levels of iodine.
Appl.Environ.Microbiol.57!: 1379-1385.
LeBlanc, B. and R. Overstreet. 1991a. Effect of
desiccation,
pH, heatandultravioletirradia-
375
tion on viability of Bacrrlooirrrs
penaei,J.
Invertebr, Pathol, 57: 277-2S6.
LeBlanc,B. and R. Overstreet.1991b.Efficacy
of calciumhypochloriteas a disinfectant
againstthe shrimpvirus Bacuhpirus penaei.
J. Aquatic Animal Health. 3: 141-'l45.
Sindermann,C. 1990. PrincipalDiseasesof
Marine Fish and Shellfish. VoL 2. 2nd edi-
tion. AcademicPress,SanDiego.516p.
376 Oiscussion Gro Summaries
Appendices
 Appendix I:WorkshopParticipants

Japan Sovth Korea

Dr. Kaxuo Momoyama Ms. Myoung Ae Park

YamaguchiPref. NaikaiFisheries PathologyDivision
Exp. Station National Fisheries Research k
Yamaguchi754 DevelopmentAgency
JAPAN Shirang-ri,Kijan-up,Yangsan-gun
KyongsangnamMo626-900
REPUBLIC OF KOREA
Dr. Tokyo Sano
Laboratoryof AquaticPathology
Dept, of AquaticBiosciences Taiwan
TokyoUniversityof Fisheries
4-5-7 Konan, Minato-ku
Mr. Cheng-FangChang
Tokyo108,JAPAN TungkangMarineLaboratory
Taiwan Fisheries Research Institute

hfalaysia Tungkang,Pingtung
TAIWAN 92804

Dr. Mohammed Shariff

Facultyof Fisheriesand MarineSdence Dr. S.N. Chen
UniversitiPertanianMalaysia Departmentof Zoology
43400Serdang,Selangor,MALAYSIA NationalUniversityof Taiwan
Taipei, TAIWAN

People'e RepuNic of China

Thailand
Professor Dou Chen
Instituteof Oceanology,
AcademiaSimca Dr. Timothy Hegel
7 Nan-HaiRoad,Qingdao BPNutritionAquacultureResearch
Centre
PEOPLE'S REPUBLIC OF CHINA 577ThanadeeComplex,
Koryor-Songkhla
Road,
AmphurMuangSongkhla9000
Phliippines THAILAND

Dr. JoshNatividad
BFAR-IDRCFishHealthProject
Bureauof Fisheries4r AquaticResources
4th FfloorEstuarBuilding,
880 Quezon Avenue
QuezonCity, Metro Manila3008
PHILIPPINES
 ndix I

United States Dr. Brad R. LeaMaster

The Oceanic Institute
MakapuuPoint
Mr. Thomas Sell
Waimanalo, Hawaii 96795
Divisionof TherapeuticDrugs for Food Ani-
U.S.A.
mals
New AnimalDrug Evaluation
Foodand Drug Administration Dr. Donald V. Lightner
Centerfor VeterinaryMedicine Departmentof VeterinaryScience
7500 Standish Place Universityof Arizona
Rockville,Maryland20855 Tucson, Arizona 85721
U,S.A. U.S.A.

Dr. JamesSrock Dr. Jeffrey Lotz

Departmentof Land and NaturalResources Gulf CoastResearch
Laboratory
AquacultureDevelopmentProgram P.O. Box 7000
335 Merchant Street, Rm. 348 703 E, Beach Boulevard
Honolulu, Hawaii 96813 Ocean Springs, Mississippi
U.S.A. U.S.A.

Mr. Nick Carpenter Dr. Carl J. Sindenr4tnn

AmorientAquafarm,Inc National Marine Fisheries Service, NOAA
P.O. Box 131 Oxford,Maryland21654
Kahuku, Hawaii U.S.A.
U.S.A.

Dr. JamesA. Wyban

Mr. Fritz Jaenike The Oceanic Institute
HarlingenShrimpFarms,Ltd. Makapuu Point
Rt. 3, Box 300K Centerline Road Waimanalo, Hawaii 96795
Los Fresnos, Texas 78566 U.S.A.
U.S.A.

Dr. SterlingK. Johnson

ExtensionFishDiseaseSpecialist
Departmentof Wildlifeand FisheriesSciences
Texas AkM Univerisity
CollegeStation,Texas77843
U.S.A.

Dr. Rolland Laramore

ShrimpCultureTechnologies
Inc.
HarborBranchOceanographicInstitution
5600Old DixieHighway
Fort Pierce, Florida 34964
U.S.A.
 Appendix II: Agenda

8:30 am Dr. Paul Bienfang Introduction and 1Velcorne

The Oceanic Institute

8:45 am Dr. Mohammed ShariR' An &.emu' of theShnmpDiseaseSituation

Universiti Pertanian,Malaysia in Asia

9:15 am Dr. Donald Lightner NeutDevelopments

in Penaeid Virology:
University of Arizona Applicationof BiotechnologyinResearch
and
Arizona, USA DiseaseDiagnosisfor Shnmp Virusesof
Concern in the Americas

10:00 am Dr. JeffreyLotz Deuelopi

ng Specific-Pathogen-Free
SPF!
Gulf Coast Research Lab Animal Populations
for Vsein Aquaculture:
A
Mississippi, USA CaseStudyfor IHHN Virusof PenaeidShrimp
10:45 am Dr. James Brock Ciurent DiagnosticProceduresfor Diseases
Anuenue Fisheries Research Center of MarineShnmp
Hawaii, USA
11:30 am Dr. Josh Natividad Preucdence
and Geographical
Distributionof
Bureau of Fisheries and Aquatic Res. Penaeus monodon baculovirus PfBV! and
Philippines OtherDiseases
in Hatchery-Reared
and Pond-
Cultured Giant TigerPrate Penaeus
monodon Fahricius!in the Philippines

12:30 pm LUNCHGarden level,JeffersonHall

2:00 pm DiscussionGroupA SarimanokRoom Third floor!

CountrybyCountryConcerns
3:30 pm DiscussionGroup B Sarimanok Room
Prettenti
on and Titeacment
of Diseases
i n Grrnaout

5:00 pm RETURNTO HOTEL

6:30 pm DINNER The Oceanarium 2490 Kalakaua Ave.

Pacific Beach Hotel
8:30 am Dr. S.N. Chen Studieson the Epuootiology and Pathogenicity
NationalUniversityof Taiwan of BacterialInfectionin the CultruedGiant
Republicof China 'IlgerPraion,Penaeusmonodon,in Taiwan
9:15 am Dr. JainesWyban Selected
Breedingof SPFShrimpfor High
The Oceanic Institute Health and Increased Grinoth
Hawaii, USh
10:00 am Dr. KazuoMomoyama ViralDiseases
of Gdtttn< PenaeidShrimp
NaikaiFisheriesExp Station in Japan
Japan
10:45 am Mr. Nick Carpenter A Companson
of Vuus-Diseased
es.SPF
h morient hquafarm Penaeus vannamei on a Commemurl
Hawaii, USh Scale in Hataaii

11:30 am Ms. Myoung he Park An Overviere

of theShnmpDisease
Situation
Natl. Fisheries Res.BrDevel. hgency andDiagnosticTecluuques
andMethodsof
Republicof Korea Treatment
UsedonShrimpFarmsin Korea

12:30pm LUNCHGarden level,JeffersonHall

1:30pm GROUPPHOTOGaxden behindJefferson

Hall

2:00 pm Discussion
GroupC Sarimanok
Room
P~enti on and 1batmentof Diseases
in Hatcheries
3:30 pm Discussion
GroupD Sarimanok
Room
DiseaseDiagnosis

5:00 pm RETURN70 HOTEL

6:30 pm DINNER Camellia Restaurant 2460 Koa hve.

Waikiki Resort Hotel
8:30 am Dr. Tokuo Sano Bandoviral,Infectionsof CulturedShnmp
TokyoUniversityof Fisheries in Japan
Japan
9:15 am Mr. Fritx Jaenike ShrimpPrwhctionin TexasUsingSPFStocks
HarhngenShrimpFarms,Ltd.
Texas,USA
10 00 am Dr. Brad MaMaster
The Oceanic Institute
Hawaii, USA
10:45 am Professor Dou Chen An Ovemietoof the ShrimpDiseaseSituation,
Academia Sinica Inst. of Oceanology Diagnostic2tchniquesand Methodsof
People'sRepublicof China 7batmentUsedon ShrimpFarmsin China
11:30 am Dr. S.Ken Johnson A Residue
of theRg~uory IssuesRdatedto
TexasA & M University 7katmentof PenaeidShrimpDiseases
in 7bns
Texas,USA

12:30 pm LUNCH Gardenlevel, Jefferson Hall

1:30 pm RETURN 19 Hol'EL Free time

8;30 am Dr. Rolland Laramore ShrimpCuiture2tchnoLogies
Inc.:
Stuimp Culture Technologiesinc. ImpLementing
Resealehto ImpneeShrimp
Florida, USA Genetics and HeaLth

9:15 am Dr. Timothy Flegel Ocnrnence,

Diagnosisand Vheatment
of
BP Nutrition Aquacult Res. Center ShrimpDiseases
in Thaiiand
Thailand

10:00 am Dr. Cad Sindermann Precautionsto be Takenin Importing and

National Marine Fisheries Service Cuitunng Non-NatieeShnmp
Maryland, USA
10:45 am Mr. Thomas Bell Drugsand ChemotherrJpeutants
for Shrimp
University of Arizona Diseases: Their Present Status in the USA,
Arizona, USA 0'ith an Areruiewof &search.and ApprovaL
Processes

11:30 am Mr. Cheng-Feng

Chang Diseases
of GrassPrawn Penaeusmonodon!
Tungkang Marine Laboratory in Taiwan:A Renewfrom 1977 to 199I
Republic of China

12:30 pm LUNCH Garden level, JeffersonHall

2:00 pm DiscussionGroupE SarimanokRoom

QuarantinelSPF
3:30 pm DiscussionGroupF SarimanokRoom
OpenSessionand I'rap-up

5:00 pm RETURN TO Hol'EL

6;30 pm D1NNER Cannon Club Diamond Head Road

 endix ll

7:45 am DEPART HOTEL

8:30 am The Oceanic Institute

11:30 am MaricultuzeResearchand TrainingCenter

I:00 pm LUNCH Pat's at Punaluu

2:30 pm Amorient Aquafarm

4:00 pm RETURN TO HOTEL
ndix II
 A 153,]55, 169,182,189,216,235,249,
296
Abbreviated New Animal Drug Baculovi rus penaei
Application See BP
See ANADA Baytril, 315 - 316
Acetic acid, 313 Benthiocarb, 86, 93
Acinetasp., 5, 10,43, 51,79, 115- 116, Benzalkonium chloride,
220, 275 See BKC
Aeromonassp,, 75, 77, 127, 132, Bipenicillin, 8 -9
143, 196- ]97 BKC, 80, 118,125,]27- 128,130,132,
Aflatoxicosis, 25, 224 171, 190
Aflatoxin, 143, 352 Blackgill disease, 5 -6, 12,17,20,42,48,
Agmasomapenaei, 43, 80 52, 114, 122 - 123
Agmasoma
sp., 220,275,346 Black gill syndrome, 224
Aldrin, 86 Black spermatophore disease, 6
Alexandrium sp,, 94 Black spotdisease, 21, 23
Alexandrium tamarense, 224 Blue disease, 11 - 12, 44, 133,222
Altermonas sp., 79 BMN,
Amesonsp,, 43, 220,275,346 See BMNV
Ampicillin, 75 BMNV, 15,18- 19,38, 151,155,163,169
ANADA, 323 - 171,173,185 191,214,216,233- 235,
Aphanomyces
astaci, 331 238, 346, 360, 362, 366
Aquatin, 222 BMPC, 88
Aquatrine,312, 314 BP, 24,26, 189,212- 215,233- 235,238,
Arfemiasp., 43,49,216,225,300,367- 249 - 251, 271, 274 - 277, 282, 285 - 287,
368 289, 300, 346, 364, 366
ASCC AsianShrimpCultureCouncil!, Bubble disease, 17
61 Buthylphenyl
N-methyl-carbamate
Astaxanthin, 11, 44, 222 See BMPC

Bacillus sphaericus, 154 Cadmium, 122, 143

Bacteriainducedhepatopancreatitis,
8, 19, Calciumhypochlorite, 10,49, 54,364
23 Cancermagr'ster,86, 89
Bacterialgill disease,19 - 20 Carbamate, 88
Baculoviralmidgut gland necrosis Carbaryl, 88
vr fus Carbon dioxide, 313
See BMNV Carbophenothion,86
Baculovirus, 6, 38 39, 61, 96, 139, 146, Carchesium sp., 16 17, 43
388 Index

Carcinussp., 90 See EPA

Cephalolobussp., 51, 117, 121 - 122,220 Enzyme-linkedimmunosorbentassay
Chemotherapy, 8, 20, 28, 42, 80, 135, 348, See ELISA
350, 365 EPA, 311, 315, 318
Chitinolytic bacteria, 5 Ephelota gemmipara, 10
Chlorarnine T, 80 Ephelota sp., 220
Chloramphenicol,5, 8 - 9, 15,28, 40, 48- Epicommensal ciliates, 17, 19 - 20, 51, 114
49, 75, 77, 132 - 133, 140, 316 - 115

Chlorine, 171, 190 - 191, 277 - 279, 287, Epicommensalspecies, 24

297, 364, 367 Epidemiology, 363
Chloropyrifos, 86, 93 Epistylis sp., 5, 10, 16 - 17, 22, 43, 51, 79,
Chloroquinediphosphate, 10 - 11 115, 117, 164, 220, 346
Chronic soft-shell syndrome, 11 - 12, 43, Erythromycin, 5, 75, 140
133 Erythromycin phosphate, 8 - 9
Chrysanthemum, 91 Escherichia coli, 319
Cladocera, 85, 87 - 88, 91 Ethyl-parathion, 87
Copper, 122 Euphelota sp., 43
Crampedtails, 11 - 12, 44
Crangon septemspinosa, 86 88
Cutrine-Plus, 8 - 9, 28, 41, 312, 370
Cypermethrin, 57, 81 - 84, 88, 91 - 93 FDA, 312 - 313, 316 - 318, 320 - 322
Federal Regulations Code of!, 25
Fenthion, 87
Fentitrothion, 87
Daphnia magna, 85, 87 - 88 Fenvalerate, 91 - 93
DDT, 85 - 86,.92, 223 Filamentous bacteria, 24 - 25, 29, 40 - 41,
Decapoda, 87 - 88 50, 77, 79, 123 124, 128, 139, 146, 150,
Diaoxathan, 86 153, 155, 218, 346
Diazinon, 87 Flavobacterium sp., 127, 132, 143, 196,
Dichlorvos, 86, 89 204

Dieldrin, 86, 223 Floating head syndrome, 17, 52

DNA probe, 61, 171,213, 215, 237, 245, Florfenicot, 315 - 316
249 Flucythrinate, 87, 93
Fluoroquinolone, 316
Food and Drug Administration
See FDA
Ectocornmensal cili ates, 23 Formalin, 9 11, 17, 19, 23 - 25, 28, 41, 49
EDTA, 5, 25, 132, 223, 367, 369 - 370 - 51, l17 - 118, 124, 127 - 128, 164, 171,
ELISA, 27, 214, 238, 242 246 182 - 183, 190, 312, 314, 367, 369
Endrin, 86 Furanace, 5, 8 - 10, 28, 127, 132 - 133
Fnteromorphasp., 51, 120 Furazolidone, 5, 19 - 20, 125, 128, 132
Environmental Protection Agency Fusarium disease, 5, 20 - 21, 42, 50, 212,
 Index 389

220
Fusarium moni ltforme, 20
Fusarium solani, 5, 20, 42, 50, 212, 219- IHHN

220, 274, 346, 351 See IHHNV

I usarium sp., 6, 10, 42, 49 - 50, 52, 79, IEBVJV, 7, 24, 27 - 28, 39, 73, 141, 157,
213, 219, 351 186, 212 - 216, 226, 233 - 248, 250, 259,
269 - 271, 274 - 276, 278 - 280, 282, 285-
289, 291, 295 - 296, 298, 305 306, 308-
310, 346, 348 - 349, 364 - 365
GAB A, 92 Immunostimul a.nts, 76, 107
Gama amino butyric acid Infectioushypodermal/hematopoetic
ne-
See GABA cfosIS vjrus
Gas bubble disease, 6, 12, 20, 25, 29, 44, See IHHNV
133, 135, 143 Isopods, 133, 221
Gill rot disease, 19 21
GNS, 19, 21, 212
Gregarines, 6, 10 - 11, 121 - 122, 220, 259,
296, 346 1 agenidiumeallineetes, 5, 9 10, 42, 317
Gustathion A, 222 l.agenidium sp., 16, 42, 50, 79, 143, 219,
Gut-and-nerve syndrome 346, 350
See GNS l.agenophryssp., 5, 52, 220
Larval black spot syndrome, 13, 96
Larval rnycosis, 5, 9 - 10, 25, 42, 50, 139,
146, 150, 153, 155, 212, 220, 346, 370
Haliphthoros philippinensis, 42, 143, 155 Laspevresia pomoneDa, 154
Haliphthoros sp., 5, 9 10, 42, 219 leptolegnia marina, 10
Heavy metal poisoning, 12 leucolhrixmucor, 5, 41, 50, 143, 151,
Hemocytic enteritis, 12, 212 153, 155, ]64, 219
Hepatopancreatic parvo-li ke vi rus leucothrir sp., 9, 16, 19 - 20, 312, 346
See HPV Lindane, 86
Heptachlor, 86 LOP V, 28
HP Luminous bacterial disease, 8 - 9
See HPV Lymphoidalparvo-likevirus
HPV, 7, 15 - 16, 21 - 25, 48, 52, 73 - 74, See LOPV
141 - 142, 157, 163, 234 - 235, 238, 246
247, 259, 274, 346, 362
Hyalophysa sp., 275
Hypertrophy, 66 - 67, 70 - 72, 98, 108, Malathion, 87
142, 144, 187 188, 217 MB

Hyphomyces sp., 10 See MBV

MBV, 3, 7, 9, 14, 21 - 23, 38- 39, 41, 57,
60 64, 106, 128 131, 139 141, 144
390 Index

151, 153 - 154, 156 - 157, 169, 178, 180 See OTC
181, ] 83, 191, 212, 214, Ozone, 367
233 235, 238, 249, 346, 364, 366
Metapenaeusensis, 3, 169, 171, 185- 186
Metazoa, 21], 221, 259
Methoxychlor, 85 - 86 Paguruslongicarpus, 86 87
Methyl-parathion,81 82, 84 - 85, 87, 89- Palaemon macrodactylus, 79
91, 93, 100 Palaemonetesvulgari s, 86 - 87
Mevinphos, 87 Paranophrys carcini, 16 - 17, 51
Microsporida, 5, 10, 17,43, 50, 80, 259, Paranophrys sp., 221
296, 346, 350 Parasitic ciliate disease, 17, 51
Microsporidosis, 5 Paratya compressa
improvisa, 85, 87
Mirex, 215, 223
Parvoviridae, 39, 61, 65, 234 - 235
Moina macrocopa, 85 Penaeus a:tectrs, 88, 196, 236, 250
Muscle necrosis, 12, 17, 20, 52, 133, 200 Penaeus brasiliensis, 236
Mysidacea, 87 - 88 Penaeuscaliforni ensis, 213, 226, 236, 250,
271
Mysidopsis bahia, 86 88
Penaewschinensis, 3, 15, 17 - 18, 22, 39,
47 - 48, 50, 142, 161 - 163, 169, 171, 185
186, 212, 217, 236, 247, 353, 362, 367-
NADA, 312, 314, 316, 322 368

Nalidixic acid, 75 See also P. orientalis

Nematodes, 117 119, 259, 296 Penaeus dworarum, 87 88, 182, 215, 236
¹matopsis sp., 51, 212, 220, 273,346 Penaeus escwlentus, 3, 28, 38 39, 236
New Animal Drug Application Penaeusindictr», 3, 9, 39, 236
See NADA Penaeusjaponicus, 3, 5 - 6, 15, 18,20-
Nocti luca scinti lans, 94 21, 23, 38 42, 155 - 156, ]61 - 164, 166,
Nocti luca sp,, 94 ]69, 182 - 183, 185 - 189, 191, 214, 217,
Nosema sp., 16 - 17, 50 220, 226, 236, 351, 360, 362, 367
Nucleocapsid, 62 Penuews kerrtthwrus, 3, 38, 236
Penaeus latisulcatus, 185
0 Penaewsmarginatws, 4, 236
Penaeusme@erie!tsis,3 4, 9, 21 22, 28,
Onemonthmortality syndrome, 13,81, 97, 38 40, 42 - 43, 47, 80, 142, 151, 236
100, 106 Penaeus monodon, 3 - 4, 6 9, 11 - 15, 22,
Organochlorides,86, 89 28-29,38-44, 57-58,60-61,
Organophosphorous,86, 89 91, 93, 100 66, 68, 70, 73 - 76, 79 - 84, 91 - 92,
Oscillatoria sp., 94, 123 124, 312 94, 97, 100 - 101, 113 117, 119, 121
OTC, 74 76, 106, 127, 130, 132- 133, l24, 129 ]31, 134, 139, 141, 143 - 151,
140, 164, 315, 350, 361, 368 153 - 155, 169, 171, 178, 180, 183, 185,
Oxytetracycline 191, 195 197, 199, 201 - 204, 212, 2]7-
218, 223, 233, 235 - 236, 247, 258, 286-
 Index 391

288, 346, 351, 359, 362, 364, 366 - 368 151, 204
Penaeusmonodon-typebaculovirus Pyrethroid, 57, 85, 87, 91 - 92, 94
See MBV
Penaeusorientalis, 3
Penaeuspaulensis, 236
Penaeuspentcillatus,3, 13, 18, 22 23, 38 Quarantine, 264, 273, 275 - 278, 280, 286,
39, 47, 236 326, 329 - 330, 335, 348, 372
Penaeusplebjeus, 3, 151, 236
Penaeusplebjeus baculovirus PBV!38
Penaeus schmitit', 236
Red discoloration, 13, 114, 125 - 126, 132
Penaeus semisulcatus, 3, 13, 38 - 39, 142,
Red disease, 11 - 12, 41, 44, 146, 153, 156
169, 171, 185 186, 236
Red leg disease, 18, 22 23, 49
Penaeussetiferu.v, 6, 143, 196, 236
REO, 7, 19, 24, 40, 142, 212, 217, 234-
Penaeusstylirostris, 4, 6, 142, 213, 215-
235, 238
217, 220, 226, 236 - 237, 239, 242, 244
Reo-like virus
245, 247 - 251, 269, 271, 276, 278, 287,
3"ee REO
348 - 349
Resiguard, 10
Penaeus subtilis, 236
Rhizosolenia, 94
Penaeus vannamei, 3 4, 6, 24 - 25, 39,
Rickettsia,4, 6, 22, 41 42, 60, 211 - 212,
142, 189, 212 218, 223, 226, 236 238,
217 218, 274, 346
240, 244, 247, 249 250, 257 - 261, 266,
Romet-30, 315
269 - 272, 274 276, 279 280, 285 291,
Runt-deformity syndrome, 24, 270, 285,
295, 306, 309 - 310, 334, 348 - 350, 352-
291, 296, 309 - 310
353, 359 - 360, 363 - 365, 367 - 369
Penicillin, WO, 220
Perkinsus karlssoni, 332
Permethrin, 91, 93
Saponin, 10, 19, 23, 117, 120, 133, 222
Phloxine, 27
Saprolegmi
a parasiti ca, 10
Phycomycetefungi, 212
Sarafin, 315 - 316
Pleistophora sp., 16 17, 50, 212, 220,
Seriala quinqueradiata, 316
275, 346
5irolptdi um sp, 5, 9 10, 16, 24 - 25, 42,
Polymycin, 75
50, 143, 219, 317, 346, 350
Potassiumpermanganate,10, 51, 122
Sodiumbicarbonate, 313
Povidone iodine, 80
Sodium chloride, 313
Probiotics, 107, 360 362, 369, 374
Sodiumsulfite, 313
Protogonyau axsp., 94
Soft shell syndrome, 18, 53, 222 - 223
Protozoa, 4 5, 16, 19, 22 24, 37, 42 43,
Specificpathogenfree
211, 220, 259, 274, 346, 349, 367, 369-
See SPF
370
SPF, 26 - 27, 257 - 263, 265 - 266, 269,
Pseudomonasaeurogi nosa, 3 19
272, 279 - 281, 286 288, 291 292, 295,
Pseudomonas sp., 8, 15 16, 41, 132, 143.
297 - 299, 302, 305 306, 309 - 310, 325,
392 !ndex

328, 330 - 332, 349, 360, 362 - 364, 370, Vibrio campbellii, 49
373 - 374 Vibrio cholera, 273
Spirillium sp., 127, 196 Vibrio damsela, 125, 195 - 198, 204
Spirocamallanuspereirai, 118 Vibrio harveyi, 40, 76, 125, 140, 195 204
Spongymusclesyndrome, 13,96 Vibrio nereis, 195, 197 - 198, 204
Streptomycin, 8 9, 75, 220 Vibrio parahaemolyticus, 41, 48 49, 74,
Sulfamethazin, 8 9 125, 132, 143, 197 198, 204
Sulfamethosazole,
75 Vibrio sp., 4, 8, 15 - 16, 19 - 20, 26, 40-
41, 47, 49 - 50, 52, 54, 74 - 78, 127, 132,
143, 156, 163 164, 166, 196 - 197, 204,
2]8, 225, 273, 305, 307, 346, 351, 360-
Tail rot, 12, l26- 128, 132 362, 369
Tail rot disease, 361 Vibrio splendidus, 40, 76, 142, 198
TCBS, 369 Vibrio lubiashii, 196
Teaseedcake, 51, 54, 117, 120, 127,133 Vibrio vllniPcus, 74, 76, 195, 198
Terramycin, 5, 48 - 49, 132 Vibriosis, 5, 8, 15, 19 - 21, 40, 47 - 48, 53,
Tetracyclinechlorohydrate, 4, 8 - 9 74, 76, 163 - 164, 212, 218 - 219, 225,
Thelohaniasp,, 16 - 17, 22, 43, 50, 275 346, 351, 362
Thiothrix sp., 16, 50 Viral occlusion
Thymascarsp., 117 118 See VO
Thymascarissp., 118- 119 Vir brio harveyi, 142
Treflan, 5, 9 10, 24 25, 28, 42, 79, 315, Virions, 62 - 65, 69, 73, 96, 103 - 105,
317, 350, 367, 369-370 182, 239, 246 - 247, 249
Trichodesium sp., 94 VO, 61
Tri chodesmium erythraeum, 94 Vorticella sp., 5, 10, 16 - 17, 43, 51, 79,
Tri methopri m, 75 139, 146, l50, 153, 155, 220

U.S. Departmentof Agriculture White spot disease, 18, 53

See USDA White-black spot disease, 18, 52
USDA, 270, 282, 313, 315

Zoothamnium sp., 5, 10, 16 - 17, 22, 43,

Vibrio alginolyticus, 41, 48 - 49, 143,204 51 - 52, 79, 115 - 117, 139, 146, 150 - 151,
Vibrio anguillarum, 49, 125, 132,195-196, 153, 155, 164, 220, 274, 346
198, 204