PCR + - Central Institute of Brackishwater Aquaculture

,I
Expert Consultation on Rapid Diagnosis of
Shrimp Viral Diseases
12" - 14" June 2002, Chennai
Workshop Programhe and Powerpoint Presentations

Expert Consultation on Rapid D i e hf ~
Shrimp Viral Diseases
12* - 14* June 2002, Chennai
urn ICAR
Programme
12"' June 2002
1 OOOh Inauguration ceremony
1 lOOh Tea break
1130h Session 1 Indian scenario and perspectives on I'CR and shrimp
health management
Session Chair.$: Dr T.C S~intingo and F'rof: Tint Flegel
Brief introductory remarks on workshop organization. object~ves and outputs
I)r Michael Phillips, NACA
Application of PCR technique in the detection of shrimp pathogenic viruses ir
lndia - Ittyn Karunnsagar and Indrani Kurunmagar, College o f Fisheries,
Mangulore
. Shrimp health management in lndia with special reference to viral diseases an
PCR - K.K. Vjuyan and T.C Santiagu, CIIjA, Clzennrri
Role and activities of MPEDA in the Rapid Diagnosis of shrimp d~seases
through PCR - B. Vishnu Bhrrt, MPEDA
Discussion session
1300h
1400h
Lunch break
Session I (continued)
Issues and implications of PCR results to management of WSSV Lessons
from DFID white spot syndrome epidemiology project C. I'. Mohnn, College
of Fisheries, Mangalore

Epidemiological study of viral diseases of shrimps from culture
ponds of North Coastal Andhra Pradesh Application of
histological and molecular diagnostic techniques -R Madhavi, Andhra
Unhversity. Visakhnpainam
Private sector comments and presentations - D.Ramraj, Padmanabha Labs.
M..Surlarshan Swanry, Andhro Pradesh Shrimp Hatcheries Association, Ms
Subhnshni, Bangalore tienn'
Discussion session
1530h
Tea break
1600h Session 2 Regional experiences
Sess~ori C'hnrrs: Prof R Madhatpi and Dr Richard Hodgson
The development of PCR and other diagnostic tests for control of
viral diseases of shrimp in Thailand - Tim Flegel, Mahiilol University
Practical application of PCR testing in Thailand and the Impact
on shrimp disease management on farms - Roonsirn~ Withycltunnnrnkul,
Mnhirlol University
Discussion sesslon
1700h
Close for day
13"' June 2002
1900h
Session 2 (continued)
PCR test formats and the future ofPCR technology for shrimp v~ral
detection -Richnr~l Hodgson, CSIRO, Australia
The appl~catton ofPCR for control of shrimp viral disease -
strategies for rtsk management - Peter Walker, CSIRO, Awralin
- Nattonal and rey~onal harmonisation of WSSV PCR testlns protocols
- Peter Walker, CSIRO, Australia
Recent NACA experiences, including lessons on shrtmp d~sease dtagnost~cs
from the MPEDA-NACA technical assistance -Micltrrcl Phillips, Vishnu
Hhat and Arun Parliyar
Discusston sesston
1030h
Tea break

1100h Session 2 (continued)
Application of PCR diagnosis in WSSV - K. K Rnjendrnn, CIFE. Munthni
Use of dot blot tests for white spot diagnosis - K.M. Shnnhnr, College of
Fisheries, Mangnlore
Some emerging shrimp viruses - Peter Walher, CSIRO. Austrnlin
1230h Session 3
Working groups
.Ycssion Chnirs: Peter Walker nnd Indrani Knrunacagnr
Introduction to working group sessions -Michnel Phillips
. Working group 1. Practical application of rapid diagnostics (lihrrry)
Working group 2 PCR and rapid diagnostic technologies (confererrce roonr)
o Working group 3. Researchable issues (G'BII lahorntory - roont 206)
(see separate document on working groups)
1300h
Lunch break
Working groups continued
1700h
1930h
Close for day
Dinner at Vedika Hall, Savera Hotel
14"' June 2002
0900h Session 3 Presentation of working group findings and
recommendat~ons by Workrng Ciroup Charrs
1030h
l lO0h
Tea break
Summary of workshop recommendat~ons followed by clos~ng
ceremony

Introductory remarks
Network of Aquaculture Centres in Asia-Pacif
aquatic animal health programme
Support to diagnostic capacity build~ng is an important
part of the programme
m PCR has been promoted extensively to detect shrimp
viruses, in India and elsewhere, but problems noted
AClAR supported project "Diagnostic tests and
epidemiological probes for prawn viruses In Thailand

Cooperation with NACA
Financial support provided by AClAR
Cooperation with MPEDA, India and CSIRO, Australra
Working group to assist in technical aspects of
programme development
ClBA organisatlon committee
Thanks to all concerned!
procedures In lndla
To ~ntroduce recent regional development In PCR and rapld
diagnostic techniques and their application In shrimp health
management elsewhere In Asla
To develop practrcal recommendations for effective use of PCR
and rapid diagnostic techniques in shrimp health management
procedures with~n lnd~a
To initiate a process of identifying re
disease diagnosis and shrimp health

information on current PCR method
techniques) in use in shrimp culture in lnd~
diagnostic) techniques as part of shrimp health management
procedures in India.
m Participants exposed to recent regional development in PCR
and rapid diagnostic techniques and their application in shrimp
health management elsewhere in Asia.
Consensus on a set of recommendations for effective use of
PCR and rapid diagnostic techniques in shrimp health
management procedures within India.
Preliminary consensus on research II,,~:, ;UI h3mrmat
disease diagnosis and shrimp health management
Plenary sessions
Indian experiences from Univer
8 Regional experiences from Thailand and Australia
Three working group sessions to discuss specific
issues and develop recommendations
=Plenary session to present and finalise
recommendations

APPLICATION OF PCR FOR DETECTION OF SHRIMP VIRAL
DISEASES-INDIAN EXPERIENCE
Indrani Kamnasagar and Iddya Karunasagar
UNESCO Center for Marine Biotechnology and Department of Fishery Microbiology
University of Agricultural Sciences. College of Fisheries, Mangalore-575002
Tel/Fax: (0824) 246384, email: mircen@sancharnet.in
Shrimp viral diseases are a major problem for shrimp aquaculture in India and other
parts of Asia. Since there is no effective treatment for viral diseases, avoidance is the
major strategy in health management in aquaculture The major routes of entry ofthe
viral pathogens into the aquaculture systems are through infected broodstock and
through infected larvae. For detection of pathogens, we need a sensitive method that can
detect infection, before it can lead to any signs of disease Polymerase chain reaction
(PCR) is a highly sensitive technique that is based on amplification of the nucleic acid In
our laboratory, we have been working on PCR protocols for detection of shrimp vlrsses
such as whitespot syndrome virus WSSV) and monodon baculovirus (MBV)
Before performing PCR it is important to know what samples are to be used Appropriate
tissue should be taken (eg hepatopancreas for MDV and gills, stomach, cuticle, pleopod
for WSSV) This requires a knowledge of the target tissue for the virus The samples
should be preferably live animals or fixed tissue In the case of larvae, whole larvae may
be preserved in ethanol In the case of adults, the target organs/ tissues may be dissected
out and placed in ethanol Ethanol preserved samples are suitable, but care should be
taken to see that the preservative penetrates the target tissue before there IS degradation oi
nucleic acid due to tissue enzymes.
In animal tissue the viral pathogen is present in cells Before performing PCR. ~t is
important to process the tissue and extract DNA There are different methods for
extraction ofDNA and these methods vary in their efficiency Therefore resuits of PCR
may vary due to different extraction methods used In some shr~mp tissue, there may be
inhibitors of PCR These need to be removed by appropriate purification steps

Different groups have been using different primers for detection of WSSV. In our
Laboratory, we have compared efficiency of different primers In our experience, samples
showing clinical signs are positive with primers yielding laiger amplicons (more than 1
kb), while samples without any sign of disease are positive with primers amplifying
smaller fragments. Further, a number of carrier animals were positive only in nested
PCR. From our experience, we would recommend one step PCR with primers amplifying
Fragments of about 500 bp and nested PCR with primers internal to the product of first
step PCR This nested PCR is also useful for screening apparently healthy larvae for the
presence of WSSV.
There are different aspects affecting the results of PCR besides primers. Sometimes,
excess host DNA might inhibit PCR. Sometimes PCR may be negative in animals
showing clinical signs. This may be due to inhibition of PCR by excess template DNA
and by excess host DNA Such problems may be overcome by dilution of DNA extract
Since PCR is a highly sophisticated technique, it is important that the test be performed in
properly setup laboratories by well trained personnel who are aware of the intricacies of
the technique. There could be false positives due to contamination of reagents and
pipettes There could be false negatives due to inhibition ofPCR either due to improper
extraction of DNA or due to excess template or host DNA
Our Laboratory has been offering training in setting up and operation of PCR
laboratories These are intensive one week courses with maximum of five to six trainees
All trainees are given hands-on experience with PCR with simulation of trouble shooting
so that they can setup and operate PCR laboratories

Monodon Baculovirus
Monodon baculovirus (MBV) is the first virus
reported from lndian shrimp farms
The virus, widely distributed in the culture
populations are well-tolerated by the shrimps,
as long as rearing conditions are optimal.
Presently MBV is enzootic in lndian
hatcheries and farms as in other south east
Asian countries

Hepatopancreas parvo virus
(HPV)
Infections resembling Yellow
head virus (YHV)
-recorded from Indian farms using
histopathology
-Need confirmation using PCR
diagnosis
WSSV-PCR diagnosis
. PCR was developed for the WSSV
isolates from
. Taiwan (Lo ef a/. 1996)mested
. Thailand (Nunan and Lightner,
1996), nested
. Japan (Takahashi, 1996), single
step
. China (Kimura, 1996), nested

WSSV-PCR, India
Mangalore fisheries college,
Mangalore, India-nested PCR
(Karunasagar n karunasagar)
w Central Institute of Brackishwater
aquaculture, (CIBA) Chennai, Indianested
PCR and nested PCR kit
(Vijayan n santiago)
CMFRI, CIFT, CIFE, TNAU, AP GOVT,
CFDDM-Cochin University, Anna
University
MPEDA -PCR labs
Rs 5001- ($10) nested PCR per sample
Private Laboratories (imported PCR Kits)
Rs 12001- ($25) nested PCR per sample
CIBA's PCR kit marketed by GENE1
Rs 2001- ($5) nested PCR per sample
PCR labs in Shrimp hatcheries

Practical issues with PCR-detection
No common standards in the PCR diagnosis in the
country, however ell the labs follow standardised
(published) PCR protocols, or PCR kits developed in
India, Taiwan or Thailand.
. Lack of standardized methodologies can produce
inconsistent results which can hamper the reliability
and comparability of the DNA-diagnostics, affecting the
decision making process in health management.
The use of PCR technique for health management
necessitates a higher level of validity because of the
therapeutic and management decisions rests on the
outcome of the test
Areas that require
standardization are:
Sample collection
live
frozen
fixatives
70% to 95% Ethanol
SED buffer
Long term storage:
Frozen sampes at -70%

The proper selection of DNA sources
(selection of tissue)
WSSV infects tissues of ectodermal and
mesodermal origin
Tissue has to be selected on the basis of
successful PCR amplification and
consisitency
8 At our laboratory, DNA template from the
tissues, gill, epidermis, stomach wall, eyestalk
without compound eye and pleopod gave
consistent PCR amplification
At our lab, gill or pleopod is preferred
PCR inhibiting substances- Compound eye
DNA template
H The amount of template DNA required for
a successful PCR amplification has to
be standardized with respect to the size
and tissue of the shrimp
H An excess quantity of DNA template can
inhibit the PCR reaction, causing false
negative results.
Pooled samples

DNA extraction procedure
Time consuming DNA extraction procedure cannot
be suggested in rapid diagnostics
I Need simple and time saving extraction
procedure to produce quality DNA template
The extraction procedure should minimize the use
of PCR inhibitory substances viz. phenol, SDS, etc
I A simple, rapid and cost effective DNA extraction
procedure developed at CIBA using alkaline lysis
coupled with boiling gave good quality DNA for
PCR, in just 15 minutes, PCR using this DNA
template was comparable to the PCR, where DNA
extracted using standard methods (Maniatis, 1989),
were used
PCR process and risk of contamination
m
Risk of contamination
PCR facility should include separate sample preparation
room and amplification room
PCR facility must be clean, and strict discipline should
be followed in handling using dedicated micropipettes
and disposable tips
To prevent cross contamination between samples,
disposable tissue homogenizers, centrifuge tubes, and
pipette tips have to be used for DNA-template
preparation
Positive and negatiie controls should be run along
each reaction to evaluate any false positive and false
positive reaction.

I PCR screening and the hatchery factor
perators adopts the truthful
creening of spawners on the basis
f PCR, the testing of seed from the

Threat of horizontal transmission
of virus
I
creeks and estuaries
potential agents in horizontal
transmission of WSSV: dogs, foxes,
crows..
Human factor

ROLE AND ACTIVITIES OF MPEDA IN THE
RAPID DIAGNOSIS OF
SHRIMP VIRAL DISEASES THROUGH PCR
B.Vishnu Bhat
The Marine Products Export Development Authority
(Ministry of Commerce & Industry, Govt. of India)
Regional Centre
32-2-10, Prrjasakthi Nagar
Vijayawrdr - 520 010
E-Mail: viwviwmaeda@sanchrrnet.in
- Aquaculture is one of the developmental activity in Asia to provide
food security to the growing population.
- Shrimp aquaculture has become an important economic activity in
maritime states ofIndia, particularly in the state of Andhra Pradesh.
- Shrimp farming has shown a rapid stride during early 1990's with
high capital input from public and private sector Estimated potential
area identified for brackishwater aquaculture in lndia - 1.2 million
- Area under shrimp farming in lndia - 1.562 lakh hectare
- About 85,000 hectare under shrimp farming in Andhra Pradesh alone

- Prior to 1980's shrimp aquaculture has been practiced in certain
coastal states of India in traditional way
- Scientific farming has been developed with series of demonstrations
through various promoting agencies like MPEDA. CIBA. Dept, of
- With the establishment of commercial hatcheries, feed mills and other
allied activities the technology has been standardized Govt of India
identified shrimp aquaculture as an activity of 'extreme focus'
Cont..
Short production cycle of 4 to 4 % months, high rate of return
Investment s~gnificantly contributed massive and rap~d growth
shrimp aquaculture sector
- Aquaculture brought out vast changes in rural economy due to
Utilization of large expans of waste and unutilized lands
Generat~on of more elnploymetit and Income
Uplifiment of rural poor
Food security to the nation
lmprovernerit of quality of life in rural Iblk and
Valuable foreign exchange

~p~
'
Though several remedies have been advocated for control of
bacterial, protozoan, fungal diseases etc Viral diseases were
not adequately controlled
Since 1994 - 95, the crop loss due to disease was estimated as
99.753 M T in terms of quantity and Rs 27,300 million in terms
of value In Andhra Pradesh alone. During the 2001 - 02, a crop
loss of 20,133 M T valu~ny Rs 503 50 crores was assessed due
to disease in Andhra Pradesh
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Major shrimp diseases identified:
- For every type of diseases an array of pathogens in tht
environment are found in aquaculture operation
Type of diseases:
- Bacterial
Viral
- Parasitic
- Fungal and nutritional diseases
1 Viral diseases:
Viral diseases have been most devastating in shrimp aquaculture
- White Spot Syndrome Virus (WSSV) affects all age groups In
shrimps
- WSSV affects shrimp through vertical or horizontal transm~ssion
- Disease outbreak due to WSSV causes heavy economic loss to the
farming sector
- Monodon Baculo Virus (MBV) affects mostly in brood stock and
larvae in hatcheries
- MBV affects during grow out culture and stunts the growth without
much mortality

- Lack of understanding of intricate balance between host,
pathogen and environment
- Intensive aquaculture practices results increased stress on
ce of culture technology lead to an increased production
ase outbreak and economic loss
' y of poor water quality contributed the deterioration
cological understanding and over exploitation of
goods and services beyond the carrying capacity
of farms at a particular water source causes disease
gh water due to lack of cooperation among farmer
Role of virus in disease outbreak:
- Virus is a part of eco-system
- Virus uses water as media for transmission of disease in
aquaculture eco-system
- Water is commonly used for aquaculture and other
activities
- Viral disease is not treatable and no ways tu cure.
- Like other vertebrates control of viral disease by means
of vaccine 1s not possible as shrimp does not have any
specific immune system
- WSSV affects shrimp through vert~cal 1 hor~zontal
transmiss~on

Vertical transm~ss~on
- Transmission of disease from mother (brood
stock) to offspring (seed)
Horizontal transmission
- Transmission of disease through carriers
- Carriers such as crabs, shrimps. and other
crustaceans transmits disease through virus.
- Non crustaceans such as fishes, which are
predatory to the shrimp act as carriers and
transmits the disease.
Methods adopted to contain disease :
- I'revention and control of disease is most important aspect in
aquaculture development
- Sustainability of aquaculture largely depends on implementation of
effective health management programmes

Avoidance of pathogen In environment:
Detection of pathogen is more imponant aspect in shrimp culture
- Avoidance of pathogen in brood stock in hatcheries
- Reliability and sensitivity in detection of pathogen (Virus) is more
important in prevention of disease
- PCR is found to be one of the diagnostic tool for detection of virus
in shrimp aquaculture
- Highly sensitive one and based on molecular biological techniques
- Screening of brood stock. larval and post larval by Polymerase
Chain Reaction (PCR) at hatchery level
- Detection of virus through PCR is found to be more reliable and
highly sensitive PCR test produces high yield of specific DNA
Application of PCR in shrimp health
management:
Avoidance of pathogen
Screclung of brood slock
- Scrccn~ng lanrae and post lame in Iulcl~c~y
- Screening post lmae before stocklog
- Screening and ~dentify~ng carners
Cllccklng 11,aIer and scdullcnt quallly prcscncc of vlmrcs
- Mo~utonng health of shn~np In prow out ponds
- Cl~cck~r~g lccd qu;dlly

Reduce probability of stocking WSSV positive post larvae
- Reduces the risk of crop failures due to white spot disease and
outbreak of disease epidemics. It is observed that many lease
holders (farmers) will drain the pond hurriedly and go for next crop
immediately. This is to save time so that they can do maximum
crops within the lease period
Reduces the use of hazardous chemicals and antibiot~cs for shrimp
disease treatment. White spot virus is a major disease in shrimp
farming and farmers test to try any chemical, which promises cure.
when a disease symptom or disease outbreak occurs in the
Role of MPEDA
- To make shrimp aquaculture sustainable and to contain
disease outbreak MPEDA has taken up various measures by
introducing diagnostic tool facilities for screening of shrimp brooders
and PLs and also adoption of various management measures
- Technical personnel in the field of shrimp aquaculture were
trained on screening of virus through 'Dot Blot kit' - an insitu
hybridization assay method during the end of 1996.
- Diagnostic laboratory facilities were set up at Vijayawada.
Nellore and Kakinada to detect the presence of virus in shrimp tissue
samples through Dot Blot Kit imported from "Mis Diaxotics" USA
based company during early 1997

- Awareness on importance of screening of seed through Dot Blot
Kit has been brought
- Screening of seeds before stocking has given encouraging results
- Some of the private agencies has shown interest to set up Dot Blot
- Since 1997 to June 2000 about 1622 samples were tested by using
- Feed back information from the farmers shows that PLs screened
using Dot Blot kit before stocking results better production and less
disease outbreak

Cont..
- Some of the government and private agencies such as TASPARI
have set up PCR labs for screening of brood stock, larvae and post larv.
of shrimp alongwith financial assistance of MPEDA.
- The existing diagnostic lab facilities of MPEDA were upgraded f
PCR in later half ofthe year 2000.
- MPEDA donated PCR equipments to the Dept. of Fisheries, Go1
of Andhra Pradesh to establish PCR diagnostic laboratory for the bene
of farmers at Kakinada in the name of SIFT.
- To implement the basic standards and a uniform code of practic
in hatcheries under the surveillance of MPEDA a system of registratic
has been introduced.
- As part of registration of hatcheries to maintain minimum physic
facilities at the hatcheries, setting up of PCR laboratory is one oft
prerequisite for screening of seed in order to supply quality seed to t
farmers.

PCR DIAGNOSIS RESIILTS ANAI.YZED DURING 2WI-02
Flrltlvr
Negnlne
- By seeing the encouraging results from the farmers, motivate the
hatcheries to set up PCR lab facilities for screening of brooders,
nauplii and I'Ls, juveniles from grow out ponds with the financial
assistance of MPEDA.
- Also encouraged the private laboratories to set up Inore PCR
- Motivated some of' the private R & D laboratories to come up w~th
new kit to have better sensit~vity such as M/s.Mangalore Biotech
Limited. MIS Bangalore Gene1 etd

aining programme:
Conducted a training programme to the hatchery technical personnel
acquaint more knowledge on PCR operation and importance on
tinuous programme for all h4PEDA staff is arranged in CLBA.
of Fisheries, Mangalore to acquaint latest in PCR
Hatchery operators also continuously trained who have installed an
n house PCR diagnostic lab
Trained private hatcheries operators by using all the PCR kit
vailable in the market.
Hatchery registered and subsidized:
As on date about 45 hatcheries have been registered with
MPEDA to follow code of practices.
22 hatcheries in India have been subsidized by MPEDA for
setting up of PCR laboratories as an in house facility

--
LTI-*I'*~
MPEDA - NACA Technical Assistance
Programme:
- As pan of techntcal asslstance programme MPEDA started a
project on "Shr~mp D~sease Control and Coastal Management" w~th
the tnvolvement of Network of Aquaculture Centres tn Asla-Pac~fic
(NACA) an ~ntergovernmental organlzatlon
- MPEDA - NACA techn~cal asslstance programme gtve.;
emphas~ze on
Study of hor~zontal I vert~cal transmlsston of d~sease In selected
fa~mlng areas ~nclud~ng lnvesllgatlon of hatcher~es
Development of practtcal measures for contdtntng I preventtng
shr~mp d~sease outbreak whtch should bpec~fically covers
~dent~ficat~on ot shrtmp d~sease r~sk factor, d~agnosts of problems
atid management strategies to control d~sease in farms

Conducting training and demonstration of appropriate shrimp
disease control measures, which should especially include
demonstration of efficient farm management practices for
containing viral and other diseases in selected farms.
Examining opportunities for cooperation of self help among
shrimp farmers to regulate water quality management and shrimp
disease management.
Findings from PCR test:
- One single method is not the ultimate answer for diagnostic
- Seasonal prevalence needs to be done
- Hatchery operators need scientific approach for catches of
brooders (depends on weather and depth of shore)
- Scientific farming is necessary
- Checking hatchery operators subsidized by MPEDA for PCR
labs whether following measures to be taken while analyzing the
PCR test, if not strict action to be initiated.

Standardization of PCR technology:
- Advance preparation of master mixes, without increasing
background
- Rather than one primer many In number should be lniplied as ~t is
made PCR highly specific
- Therefore prcmer design IS the most critical aspect in developing
PCR
- Standard protocal should be developed for DNA extraction in
order to get pathogen DNA out of host tissue components
- Multiplex PCR for existing shrimp viral diseases is required
- Rapid diagnostic test kits has to be desrgned to detect the presence
of virus at farmrng level
Conclusions:
PCR is found to be an imponant diagnostic tool for detection of
pathogen
- The use of d~fferent protocols affected the sensitlv~ty of results.
therefore
- Needs more staridard~ration ofthe methodologies
- Farmer friendly kits (shosi trme. high sensitrve, reliable and cost
effective) may be developed
- More R & D efforts should go In for product~on of hlgh seris~tive
PCR klts
- More competition should be developed In market on supply of
k~ts

Issues and Implications of PCR Results to Management of
WSSV :Lessons from the DFID shrimp Epidemiology
Pro-j ect
C V.Mohan, Fish Pathology Laboratory
Dqarlrnenl of Aquncullure, Colhp oFI:~sha~es, Mmgak,m, India
DFID Study
Longitudinal observational study
- September 1999 to April 2000
To identlfy risk factors associated with WSS
- 100 ponds randomly selected
- 70 enrolled
lnforn~at~on on large number of var~ables (about 900) collected from pond preparation
stage till harvest
samples of I'L, shrimp at 6 wk, dead shrimp, shrlmp at harvest, feed, plankton, wild
crustaceans In the pond and estuary collected
Details of Samples Collected and PCR used
Samples
PL at stock~ng (500ipond) from 70 ponds
After 6 weeks by cast net (100lpond) from 68 ponds
Dead duriny the production cycle from 44 ponds
At harvest (400ipond) from 62 ponds
Feed samples froin 70 ponds
Wild shrlmp and plankton In the pond and estuary
PCR
DNA extraction by alkaline cell lysis method (Kiatpathomcha~ et al 2001)
PCR by following method descr~bed by Lo et al (1996)
PCR Results of PL
500 PLIPond from 70 ponds
divided into 8 sub-samples of 50 PL each and 1 sub-sample of 100
a lnaxrlnum of 300 PL testedipond (6 sub-samples)-to detect a prevalence
of 1 % and above
a pond was considered + ve when at least one sub-sample tested + ve
either by I step or 2 step PCR

3170 ponds positive by I step PCR
32170 ponds positive only by 2 step nested PCR
35/70 (50%) of the ponds stocked WSSV +ve sced
PL Results-for sub samples
3 ponds + 1 step PCR (414 sub-samples +)
32 ponds + by 2 step PCR
- in 3 ponds 314 sub-samples + ( I -ve)
- tn 5 ponds 214 sub-samples t (2 -ve)
- In 14 ponds 114 sub-samples + (3 -ve)
- In 6 ponds 115 sub-satnples + (4 -\re)
- tn 5 ponds I16 sub-samples + (5 -ve)
35 ponds ncgatlvc by 3 step PCR
Proportion of infected PL within positive
batches
Sample used front the I00 I'L sub-sample
I0 cnd~v~dual PI. tested from dtfferent batches
- from one 2 step negat~ve batch - 011 0
- from one 314 2 step + batch - 0110
- from one 214 2 step i batch - 0110
- from one 114 2 step i batch - Oil0
- rrom the 3 one step + batches - l0110+ve
I0 sub-samplcs of 5 I'L each teted for all the 3 one step + batches
- l Oil 0 sub-samples from all the 3 one step posltcve batches were pos~tcve
PL results: issues and implications
Quantcficat~on of the proportton of c~tfected I'L
- ~uev:$lcncc \r,il.: \,cry blah m I \Iq, hnlchc,
p~rvitleocc \rii.: r,eiv low n 2 t(cli hittchcr
- var~abll~ty In the propofiron of sub-samples posctlve by 2 step IS very scsn~ficant
- scgnlficant risk of obtacn~ng a falsely negat~ve result for tlic populat~on be~ny tested
- may he because of very few, unevenly dtstr~buted, tnfected PL In the populat~on
smaller sample stze can give false negattve results
smaller sample scze glvlng +ve result (crcter~a to reject)
- Increascng the sample s~ze is the only solutcon
PL results and associations with PL quality
. assoccat~on w~th WSSV In PL
- more batches + at the beglnn~ng of the season (seasonalcty ~ssue)

- PL batches stocked before 31st Oct were 2 % (p=O 016) tlmes more l~kely to be
Infected than batches stocked In the second half of Nov-Dec
- l~ght colored PL more l~kely to test + (p=O 040)
- longer transponatlon 1s a r~sk factor (p=O 015)
no assoclatlon
- w~th PL qual~ty assessed goodlaveragelbad by the RAI farmer
- hatchery source, length of PL, number of PWbag, etc
PL results and associations with outcomes
WSSV ~nfect~on of PL (2 step nested)
- not assoc~ated w~th fa~lure outcomes (lower length of product~on cycle, low
productlon, d~sease outbreak)
- not assoc~ated w~th WSSV status of 6 wk shr~mp
- not assoc~ated w~th WSSV status of dead shr~rnp
- not assoc~ated wlth WSSV status of harvested shr~mp
h~gh load (I step +) and h~gh prevalence appear to be potentla1 r~sk factors
[.ow load (2 step +)and low prevalence may not he s~gn~ficant r~sk factors
PCR results of 6 wk sail~ples
I00 zhrnnp trom each pond for 68 ponds by cast net
d~v~ded Into 10 sub-samples of 10 pleopods eqch (680 sub-san~ples). to detect a
prevalence of 3% and above
2 ponds + ve by 1 step
10/10 \uh \smpltl\ + (2 pmd, I.11lm9
- 26 ponds + ve only by 2 step nested PCR
- 1 pond 9110 sub-samples + ve (fa~lure)
4 ponds 2/10 sub-samples + ve (2 fatlures 2 success)
- 21 ponds I11 0 sub-samples t ve ( I l fallures 10 success)
40 ponds negatlve (I8 fa~lurec 22 success)
Issues and implications of 6wk results
C'ons~der~ng the results for all the 68 ponds
No assoclatlon w~th fa~lure outcomes (lower length of product~on cycle p=O 45'
low product~on, d~sease
. cons~der~ng the results on lndlv~dual pond bass
- pondz posltlve by 1 step and ponds where large proportion of sub samples tested
posltlve always faded
- h~gh vvlral load and h~yh prevalence appeal to be Important r~sk factors
-- low load and low prevalence 1s not a slgnlficant r~sk factor
PCR results for dead samples
Dead samples collected by the farmers from 44 ponds
- ( 1-20 pleopodslsample)

19 ponds +ve by I step
10 ponds +ve only 2 step nested PCR
15 ponds -ve by 2 step nested PCR
assoclatlons
- assoctated w~th lower average wtat harvest (p=O 012)and lower length ol
product~on cycle ( pa 052)
- has prognosttc value
effect of post-mortem changes on recovery of DNA
PCR results for harvest samples
I-larvest sample from 62 ponds
400 slulmp/pond (1 pleopod each)
- d~v~ded Into 8 sub-samples of 50 pleopods eacll
a Inaxlmuln of 300 legs (6 sub-samples) tested to detect a prevalence of
l "/o and above
a pond was cons~dered + when at least one sub-sample tested + e~ther by I
step or 2 step PCR
3') ponds + by 1 step PCR
20 ponds + only by 2 step nested PCR
3 ponds nepatlve
Results for harvest sub-samples
Xfll \,lcopiKi\ 111 h wh \umples of50 leg, cuclr
- I!) \I wh \.anplc tcsled lo1 62 pond\
- :I, pond, tc.iled + hv onc step
- l i pond, tc\trd + hg 2 step
xcond 5uh-\anlplc toued tot 10 pmd,
2 )"""I\ lcrlcd + I>\ 2 stq1
I111cd 'ill) \~IIII/II~ ksted lul K pr>nd\
- pmd\ tcrted + h\ 2 tep
li,i~nh wlr wnplc Ic\!ed lor 6 pond,
-- I pimd tc\tcd + Ir! 2 5tr~
- IIIIII \uh-ulmpl~ teslcd lor 5 ponds - no publbve
.1\1h vll, mnplc leqcd for 5 pcmd.;
pvtd, tc\ted + hv 2 stql
Issues and implications of harvest sample results
I'onds poaltlve by 1 step at harvest slgn~ficantly assoc~ated w~th fallure outcomes
(shorter rearlng pertod (p=0 0016), low survtval, dtsease outbreak)
Ifnested 2 step result ~scons~dered, then there IS no statlst~cal assoclatton w~th
successlfa~lure outcomes
1 step +ve at harvest slynlficantly assoc~ated w~th cl~ntcal wh~te spots (p

- high vrral load and hrgh prevalence associated wrth chnlcal signs and drsease
outbreaks
Other samples
Pooled feed from 30170 ponds +ve by 2 step
- srgnificantly associated with decreased productton, but not wrth dlsease
6170 pond plankton samples at 6 wk t by 2 step- not analysed
samples of w~ld crustaceans (shrimp, crabs) from the pond and estuary collected at
d~fferentimes durrng the study were + by 2 step
- before stocklny, during the culture period, at harvest (assocratrons not analysed)
issues of diverse carrrer hosts, endemic nature of the vrms, low prevalence in natural
wlld populattons
- 10 hrwd steel; morrhund hcfore spawning
- 10110 plcopuds+ hv I stcp
ii eye 4alh + hy I stcp
- 30 compound eye + by I step
numplcs fiam 7 hnwd stocl. whxh spa~vrxd
- 717 cye\i~k.. negall!.c by I slcp
- 217 cac~illhs + hy 2 dcp nested
I-.."C\
Brood stock screening
- ~llhd~~tlcn, uf PCII hv a~n~pound eve7
- I 5tep + hrn

Dr.Hao, R1A No 2, Vietnam

Background in PCR Application
m
2000 One step PCR - KII fro111 Mal~~dol Unlvcrs~t\ and nlrll a 2 s~cp
Nested PCR k11 from a commerc~al sl~onller fro~ii USA
20Olh 2002 S~ngle tube nested QPCR \v~lll;I co~n~~~crc~ill k~t fro111
Tawan
m Tnal wllll a dual deteci~on klt from Frdncc
PCR Scrcening'for YHVIGAV. IHHNV and MBV do~ic on an
exper~mcntl basis. commercial screcnlng 1101 donc

m DNA T.\trnct~o~>

DNA Amplification
The amplification protocol
is as specified by the kit
supplier
Documented results are
provided to the farmer to
make the process of
testing transparent and to
give the farmer confidence
in testing

False Negatives and False Positive PCR
Results
Steps taken to avoid false negatives include a larger sample
size and periodic verification by spiking a duplicate or
known negative sample with a 20 copies positive control
or DNA of a known positive tissue. Invariably the results
had come positive validating the test and sensitivity of the
reaction
False posit~ves are prevented by isolation. hygiene, and
other measures that prevent contaminat~on
Correlation between PCR Results and
Crop performance
rkre.5 a negdln carrrlafa,n Iwlwen PrR
m
parm\c rr~alti a) PI and llx mop
perfunnm~c .IIIC spprurmmtr mop wveear
TL \ were rclnu\d\
.
lllgll
l.'~' ll,o\,~l, ,I,< IICI< pu\,,,rc pcrc.nmp< ill '=
PI 'A ,r Ion 11,. ~nildrncl 01 \\'SS\' I!, t1,r
tncllir lhnvc hasn ~rnrc pnt~ottnard, tllr XR%OI~
c4d hc
.
$0 Iho~$zot~t~t IW~II~~~IISSIOI~
!."or 0, hlrr In the obrn\.rti rs5a1,r muldnnsl:
CO,,, \elea,rr. nl~powl of PCR possvc u"ks .
and rr.pcn!cd tnx ol PCK srsrt\r Is"ka

Some Questions on WSSV Spread
m h m g Jan-March ZOO2 random scrcelung olBd stoch and Nauplai showed
60% lws!tlvc for WSSV
m TJI lndla only n 1mcuo11 of lhctlalchenti meen Gmv~dslBrd StwL nnd or
Nnuplrt. Ulmfore irrcq~cttve olwhellm Lhc Nauplit were ~nlected or not a
mn,or~ty afhc Ilutchenes lhad stocked and ptducd Pl.'s
m WSSV posltlver I PL's wcrc s~gnlfiuntly lower at aboul 15% (Altholigh Ibc
~nfect~on 111 I'L's 1s hgh cornpared to previous yam)
conceulratcons reduce to unddectnhle levels once Lhey ellla lllc Hnlcllrr~
A Case Study in a Hatchery
rills Halcllcq produces ~t's o\vn Naoplil from gm\,1d5
Mass Sp~rvnlng and Hatcb~ng IS dor~c Inn large tank 51nd tile N;~upl~l
are I~anested. u~asl~cd tl~oroughly and transferred to lllc Lansl qectlon
m When screcncd Br WSSV b~, QPCR. Ihe spcnt an~nuls and thc
Natlpl~~ from the SpawnlndH;llcLng tank gave sevcrc pos~tnc resulls
B PL samples Inkcr~ mndon~l) lrolll different tanks gave ncgatlvc results
Q W!th Inass spn~vnsg and a1111 lllc levcl of tllc ~nlecllon III Brwd sloch
and Naupll~ observed 11's q111lc llkely that the \.~lus would Ila\c
entered Ule Larval and PL tanks Dws washlng 01 Nauplr~ bnng do11 11
tlle ural load to undcleclable l~rn~ls In PL's7

Fate of WSSV in Hatchery
m
m
m
Allhou$~ the the WSSV prevalence from Jan-April 2002 has been lligh
in bmod stock. thc Hatcheries performance has been good Good
convenlons and survival rates have ken reported.
PL production lus been all trnlc higli and tl~e prices qu~tc lotv.
On most occasions WSSV positive tanks Iuvc done very well in the
Q. How do (he WSSV ~nlccled Larvae and PL survive well In a lank
which is crowded. has Iugll orgaluc and bacterial loads and a relativel\
smssed env~ronmenll and when the m e PL IS stocked in ponds even
at low densltles de witliln a vcr). shon (!me
Is low Tempcruturc 11 Lriggcr for fhc replicution und virulence of WSSI'

Priorities
Commercial Labs are the first link in the field with the
FarmerMatchery operator, they could provlde valuable
information regarding disease outbreaks It would be in the
interest of the industry in general that there's an
association between the Labs and Research community
Urgent need for Assessment of Lab procedures and skills.
Accredltion of Labs and Harmonisation of PCR and other
Lab procedures
In India too mt(ch importance is given for MBV and Vibrio
screening in PL's Quite a few farmers screen for MBV
and not for WSSV Such misleading technical
dissemination should be avoided

PCR plays a cruc~al role In SPF and Selective breedlng
programs. the servlces of the labs could be made use by the
Industry In such programs
m MF'EDA should fac~l~tate free Import of PCR and other
rap~d d~agnost~c klts that are recognlsed worldw~de
m If the Ind~an Shrlmp Industry has to benefit from the global
technolog~cal advances In shrlmp farmlng ~ntellectual
property rlghts should be respected
Thank you

Rapid methods for diagnosis of
shrimp diseases in Thailand
T.W. Flu~cl
CeMu Shrimp. Oldvn Pmld Building, Fowh of
Scirncr. Mmhldol Unhvaiy. F!ma 6 R d , hghk
/ Acknowledgements . I
*lhmkr to ACIAR. NACA. FAOord the
Indim bpt. Expart Pmmatim for thr
opportunity to porticipdc in this rnctiq
+W to m ~ collcogur y in Thilmd whose
work1 will nfu
to
+W to BIOTEC for contilwauw$prt
for ow nrcarch in shrimp biotcchwlogy
I The future is domestication 1 I Two types of pathogen detection /
+The key too susto~nublt indwtry is
donwsticated, gmeticdly impmved Rock
*These rill bc urtified fnr of dl mojw vim1
path-. so m PUI w m s q
+ 5m&iq by PCR will then be used only for
monitorlq ~ c kmd r pmdc &iq w!q
+We w in a tmmawn p-bd plqing him
mukttc with apmd hxktock
*Detection of rnlwtive agents dwirg d~swse
outhk
- Pmhoprnr pwnt 8n hogh wti*
- Simple mrthodsof i&ntificationlauolly ruftcr
- HIsioIqi~d trrkniguu but ~iird for the*
+htcction of covert infcctiow
- Pdhopw w n t in wy lw qmnttiy
- Molrcubr trchniguu Nadd tor drtution
- PC11 and imnwnochonry hi tulkd for this
* Fww on Pa( md immvochunutry Should mt
lend to ncglcct of histdogirnl tcchnqw

Organs/tissues vital to shrimp
*Similar to othw ~imnls
- CNS. gillr, hmrt. kdptopacnu. ontcnml
plmd, hmmntrlc tinuu
*All located in the uphdothomx ngion
*Thus, the mqior focus of diagnostic rmrk is
on the cepholothomx
* KMwlcdgc of thuc orgmrr and ti$= is
nuwury fw d-stic wwk
I Shrimp diagm
I
All gross signs are unreliable
*No gmss signs CM bc talun as conclusive
for any disease
*A minimum of micposwpic Lxmirntion is
alqs q i n d
*Micmswpic exnmimtim may Imd to other
required tests
At brrt, the gposs signr moy sugg~ct what
to Iwk out for in the microscopic
Lxmimtion
Example of brown gills
*Brown gills may result in scvml wayr
- LM OVW kvl m r pond
- Fouling by dwt. hctcria, alpc. pmtozmw
- Infection by bacttno or fungl
- Nvtritionol dcficiencin
Chcmial or baotic toxtm
*A gross cxarnirntion would rat allow my
distinction amongst these possibilitiu
Must preserve living shrimp
I Post mortem chawes ~
*Fa histology. dad shrimp are gmmlly
wdw. even if prescrvd on ice
*Frozen mplu may bc used fa PCR
rmalpii ~d emuction of some v i ~ u
*Shrimp nuDt be alive when fixed with
Mdaon's fixative
*If not, postmortem changa ocar wy
*dly ad pmnt proper &is
*This ism utwmly imprtont pint

Aquarium test
+Bring moribmd hbnp to the lobomtwy live
+Pkc sane in a well d r d ogwriun with
elm watm of sane mliniiy ar sarrrc pond
+Lcovetheshrimpforafa hwm
*If they revive fulty and my discolorotion
di-:- pObDbly M undrrlyirq diimrc
*#ajar focus would then be on pnd
rmimmwvl and nmmguncnt
Monitoring and screening
+A little ua in sampling is o big help
+Conrult~vetmi~ry sompllng tobls for
target pnvalcnce
*Pnwlaue tabla sha* # ndcd for 95%
confiduvcto obtain ot last me infrctcd
indivihl in the mple
*Hatchery, ~ r wand y pond popuhtiotn w
uswlly in ucas of lOO,OW
*Suprisigly small sompls arc d c d to
detect pnvalrncr as low as 2%
mw.
1 Sampling probability
Sendsrd \ caw sbh eves Ihc suoplc slzc odd
for 91% prchbllll) of ~OCCM& 8 ~#A%LB
No gua~ntees, only probabilities
* Tuting md wling are bxed on proprobity
r Trrt pmccdunr ore limited in rwitivhy
*Also atmot tut cvvy shrimp. only sa@u
* hmpks Nbject to mrr -no gwmtcu
+For cmprim: m m con giy~yw 100%
wumrrr thot a plow will M crmh: pu arrpt
th law risk whm p get on
* W is the mcptoblr risk fa oulbmkr bya
prticular pi* blia-w-4 hr nr*php.l.)
Major diseuse agents
I
Virus groups affecting shrimp
+5omcpmsitu h m specific infection
mcchmha and moy h man pnalrmotic
+Havcw, the d pmblem is vim1 dimu
VIM- done ore ~ ~pmnble for me
mq)or#ty of the ha Asnon quocultur 1
+SI trmimcnts w mt miwihbk,
pmrntion is Hu only effective @+proah
am,

Parvoviridae
Parvoviridae
IHHNV. HPV
*Size of genome is 4 to 6 kb (vety dl)
*They ore resistant to k t imctiwtim
+Two reported fmm Thniiand
*They do not c w mwlolity
*One appears to be inwcuarr while the
other cnmu retarded g d h
IHHNV
Histopathology of IHHNV
IHHNV summary
+Still much work ncdcd to arras its impact
on P. monwbnd other Mi
sh~itnp
+DNA pmbcr mihbie but mq be nmin
spcific
*Could k w d to s m bmodstock carriers
*Should also k urd to M far other
cwstou~ corrirn
*This vould be tk on)y effective control
m r e
Hepatopancreatic parvovirus (HPV)
We first sow it in Thailand in arty 1990's
aft* begimirg of high intuuity raring
*Not found in brwdrtockor hatchery lorwe
tut in runcly and growout pnds
*Extent of louts unknown then and MY
Ow doto indimits that it stunts pwth
but dar not wse mortdity
*R&edvimts npartd dsewhvr md.-a.
h *dm. hum**. Mm+d
$Ii*k mokcular work ~QIM
mm,

hphilr, inimnwhc inrlulim in hnvtmlhird
nude8 of the hrptop-
HPV in HP smears
I Commercial PCR primers with HPVmoa I 7

DNA mmdhm
Cmlrlfe~ SWO rpn
DNA
Sttprutmt
I/ i
PCR ud Dot Molhyb~Uo~ 1
Sensitivity of HPV-PCR
in NS boiled f- or PI.
Dot blot sensitivity with
NS boiled foeces and PL
Still needed for HPV
t Nud to identify viw nsrrvoir
+Nud to develop a hbamtoy infection
madd for further st*
*Should use PCR to soun Pi since DNA
alrccldy utmctd for WSSV orsay
*Acceptable prevalence in PL sharld be
uiabliskd
t The cumt best m of control is to
r m n Pi by PCR and nject r batches
Ww,
I
Rod shaped, enveloped dsDNA viruses
+This group includesthe baculonnrw
- W o n hulanrw (Ma9
- Dorvlon",, rn,dgut*lal -u viw @MNl
- Tlpd hulonruur
t Als included is white spot spdrnmc vim
(WSSV) which is cvrtntly ~clmifid
* W5sV is c mtly the mort serious shnmp
vim1 diseuse in the world

The baculoviruses
rid sbpd mi -I-+ d~M.l.4 vim
+First dcsrrikd s 0 pbhm wiih silhrorna
+ Inhr fwd in skimp
r smu alld ~~hpoClyhml(l viruw
v i unbrddcd in o puyhllire ptrin
&X @++cd%n) af &+dnI drip
+ spd by Iml ingution of potyhuhin
Ppticlu
Characterization of MBV
+ Typiml occludd hculwirus or wchr
potyhdmsis vim (WV)
*Va~ons armed In polyhedrin pmtein
prticlu thot ''occlude" thrvim
rl-hus, called occlusion bodies; my olro be
mlld inclusmn bdtu
Also mMy fncviriow p-t in nuch
+h+, double stronded DNA vim witha
trilmimr enwelop
1 Occlusion bodies and inclusions 1
+Vim1 pnvticlu cmbddcd in polyhdnn
particlu arc "occludd" or cowd
*Thus, the porticlu sometlmcs alld
"occlusion bodiu" for this group
+Theymay dso be nfd
to by the
g~ncrol terms "inclusion" or "inclusion
+Inclusiow moy w nur,mt bL prticuldc
and so moy not dvayr be dld "bodies"
1 MBV in HP squash mount of PL 1
MBV in PL direct
With low PL stages it
is sanctimcs possible
m sa MBV
polykdnl inclusions
diratlythmughme
cuticle using a 401
mirros~pc objective

MBV in HP smear
Histopathology by HIE
Polyhedrin granules
*Visible by light mimscape in
hrpotopanmatic celk
*Contain "occludrs' viml porticllr
MBV by TEM
Transmission and prevention
*All of the badoviruslr in shrimp are
tmmittcd fmm the b d m to h e
*Mostly this mlts
fmm z 4 i@m of
vim1 mtrrOl slwghd fmm the brooders
*The but methodof pnwntim is to wash
the eggs adfor qlii (before N61)
*Done uriq clan mwnter with w without
disinfectant

Nauplius and egg washing
Comparison of disease groups
Detection by dot blot and PCR
*Method of Wcher 6 Y wg (1998)
* Nested PCR methOd
*Bpid utmction pmtocol with force; is
pouiblc, as wiL HPV (PCR and dot blot)
*Shwld be used toscnrn PL sincc DNA
alrcody uhpctcd for WSSV as*
*Acccpthle pduw. in PL shwld tx
establishrd
A multiplu detection system needed
a3w
1 MBV detection by nested PCR
Background 1
Nimaviridae
dsbNA virus
White Spot Syndrome Virus
( wssv)
*White spot syndrome vims (WSSV) ha
1
been the most swims shrimp d i r e
problem in Ariasince 1993
*Clmulativc losses in Asio sincc I993 nt
kmt 3 billion US dollam
*Described fmm maycountric; with
different mc; (L'qhtnrr. 1996)

Gross signs in removed cuticle
Distinctive white inclusions inaitick:
1 cnnmt k scr& off with thvnbmil
Beware of bacterial white spots!
I Conduslon while spds unreliaMe chamder
I
WSSV histolopthology of stomach
Dinlnctiw hinslogy. hypertrophied rucki.
I uuh/ rtoge hat-, A-typ incluslmc (-1 I
WSSV histopathology of gills (rapid) 1
Should scc Cowdry A-typ inclusions twthcr I
Histology to confirm outbreaks
+Cmt confim o m l o fmm gmss si$w
+Histology b needed & b sufficient to
confirm outbnokr
1 Benefits f~om molecular techniques
+Pmkr for dot blot& rsituDNA
hybridizdion and for PCR developed
+Helped to detminc W55V sources and
mod- of trnn9mission
+Helping in xruniq of curriers
+HJping in epidemiological studies

I
WSSV by TEM
I I TEM and genetic material of WSSI
I --
Summary of WSSV
characterization
*A large. rod-shaped wrw, w~th 3 loyrnd envelope
*Rrst called a boculovlnu by m~stnkz
*Not a baculov!rus bewse
- Umuml~
1- gmmr (3M kbp Xu Xcn 2000)
i i wssv p rote~ns 1
No 11gnlftant homo101 to ~CU~YINI M A no 6 h,
Wdk" VWIX"W
*Probably a new v~rnlgcnw or fm~lylwhwh~~
and Mmw&ppud by Vhk)
Main m ode of transmission Kou and Lo (1999)
*Examnncd lnghtly mfccted broodstock
(I c . nrsted PCR rrqutrrd to dctcct
WSY)
*Found 38% false q t m ~f twted
kfore Splrnlng
*No folst ngot~vw ~f tested after
Spnm'ng
*They recommend PCR trstlnq ofttr

Post larvae must also be checked
+PL used to stock rewitq pds havr bun
f d to amy WSSV
+Ponds stocked with ~ c PL h hovr a hioh risk
of faikn
*scrrming by POI a.sw is ~cnmmcndcd
+It is &ended that PL positive fw
WSSV by PCR be discorded
- I
l
WSSV in many crustaceans
+Ckv 40 spccies of cwtacanc kmvn to
be potential carrim by PCl( tests
+Infections Iethd fw row, for 0 t h ~t
+All cultlnd pcnocidr susceptible with high
mortality
*Toluwt cmicrs my be a dangu to
farmers
+Tests we needed to rrr whethv they cm
t m f w the wim to shrimp
Horizontal transmission risk is low /
&mHs fm Or. tkmsim Ww,thpdxnurmhJ
Recent outbreaks in the Americas
+OngiMI report from Tuns in 1996
Wldcspnad reparts of WSSV in R
wniwmeiin Amcrim since ewly 1999
+Onglml source m hwn but processing
waste of Asian shrimp swpcted
*Bollost water also possible
+Further tmnsfer by liw PL from
hatcheries
+Molecular epidehiolqn studies have bcgvl
%ww,
Background
Picornaviridae
Taura Syndrome Virus
(TW
+ Vtw mused win mDhhtt.r in P m m e r
~n Amulca ln nud 90's
+ udopcd. icwddml vina of about 30 nm
dt~lrtV with pxitiw-swm SWA (Polio)
t Rrrt nported in &io fmn Taim with cultwe
of i m e d R WmamirTun d m9)

Gross sians of TSV 1
1 Histopathology of TSV
TSV by electron microscopy 1
I
RT-PCR
(Nunon et al. 1998)
Bunyaviridae
+Reant mlts fmm Peter Walker's lob in
Austmlio
+ NcpDtiw sense. sRNA virus on @plum
t Almst 100% wild P. momdonumy this
vim in oddltim to MV
*Tms with P.jqDOnWpvegutMd ll~m
vMroonc
+We~hwtonar~.rrWIconCcptof

Suspect from Thailand 1
I
Bunyavirus in Malaysio
Wc hve ncrnt somplcr psitive from
klclysiu
+Pa amplicon rcqunce diffuvlt from
Aurtmlion stmin by M 5%
Roniviridae
Yellow-had virus (YHV)
Background
*Ydlaw-hcod virus (YtiV) or ow of the most
senour mnmp djrcarc rODlomS on
~hm~ona (Zrn to WssJ;
*YHV f i 2 d~scovrnd in 1992 and cousd
losrw of opproxinvlhly 40 million dollars
t Subsequu~t losses difficult to asrcsr due
to monser~ous outbrrak of whitc-spot
virus
+hrety verified from other countrlcs
* L OVd 6AV fmm Aurtrdio samilor bl TEM
3ww,
-1 of Gill histopathology with YHV I
DINI!~ bmphillc indudom In H6E hid tlaw rct-
Ya mh/ in mibvd Jpcinunr. rn pdictii wlu

Rapid staining of whole gill frngments
*Normal histology toku; scvml dqs for
embeddig, cuttig, stainiq
We dmlopcd o mpid pcw for fixing
ond stainiw whokqill f m s
*Fix for 1 hr in tmvidron's&h SOX conc
HCI rrplming acetic a id
*Stain with H~E, dehydmtc ond keep in
xylenc hfon momting in pvmmt (total
time obout 3 hr).
Rapidly stained whole gill fragments
1
aim*
YHV-infrctd gill
Normal gill
Haemowes of YHV infected shrimp show
lcarvorrhectic and wcwtic nuclei
*hrr signs o n entirely unnlioble
Normal
'" ' 1 % awl
TEM of infected shrimp tissue
!

Purified virions negatively stained
Vkims bve .n mwlwc and a MO ofmmidxa
Characteristics of YHV
r Nhluc ocid (32 ko) is MNA of ps#t#vc
MI COW!^ el 01 1999)
~YHVM~~AVAOV cbscb doted (Code
Recent developments
Recent results with the MAb
t 3-28 to maop pmtrln dm no1 murroct
wnth 6AVILOV from Austmlm
+Two others to capsid ad nntrix pmtciw
do cmn reat with 6AVnOV
+None wxi with VHV-lilu hinology fmm
Ec&r or India
+ EcIDdorian rmd Mii maidol tMy
rrpnnnt nu struins fmm the gmup
An indirect viral pathogen
Baaviophogc of Wbio haytmduus ktM
toxicity to rhrimp immmnd 1994
+ Ta brangill shrimp yidd Y hmwyi
miw rot kthai on re-injection
+Bactvin&gc fmd in shrimp by TEM
*Cunbimth rquid fw &nth
+h mYnuuih lost virulence of Y
1 &;on u-trn&d hb cuhuv
+ Swh phagw pmbobly owlwkcd often in
1 irdatbiu from d i d shrimp

Siphophage in culture
I
/ Dual and multiple viral infectio;] I Dual HPVIMBV infection
/-WSSVNHV infectr--1 Triple viral infection -1

Multiple infections at Chumpol 17
Bacterial pathogens
I
1 Types of bacterial pathogens
*4 wain group in or& of imprtoncr wnth
wprct to IDUIS
+ Vibriomd nhed sprcirr of gmn Ngotivc
bacteria
*Intmccllulw bortda (Mollcutes or
Myccpbrmor, Rickettsia, etc.)
*Mycokhrio and othvs
+ Fwliq bactwio
Unculturable microbes
+lhe new frontiv
+Only 0.1-10% of micmbu an be ixlhtcd
from soil and water on dircct count plates
*Most are uncultumble on stmdord media
May nme .mprtant fwct~oh~ b.t CM no*
be laent8f1ed by ~ ILCYIO~ tecnnnques
wed on 165 rRNA gcnc sqmcu
Gram negative-oxidase positive
rods

Vibrio species
+ A W t , tk mst vinlmtgmw
- vh,v-rrrac4b.v*~~
+ fihqxcicr on grrm nqnth, motile. cUMd
mdr thot on mihe paitiw
*T~xommvof tmDimlrprirruamM
+Ow XI &-:in f& YibKioMcw
+BOX of fatal shrimp infdar cased bf
I!pm+m"@iMd Khvwi
I
Mortality from vibriosis
*Causestill unurtain but toxinr imolvcd for
%nu (esp. I! hnwyhnd Y ~ i c i & ]
*Puzzle bccousc they rn common quotic
inhabitants ~d not always p athwc
*Pathogwis nuy nsdt from virulent
stmirs or m secondary ~nfections
*Most caries opwr to be opprtunistic
infectionr nnce isolotcd stmlrs do rot kill
hdthy shrimp upn injection of high doses
8 mew,
Control of toxin production
t Potmt protein toxiinr p&ed
- l!hony,mrt, 1539)
- Vpamur,h(wrtd 2m)
Toxin production IS canditionol:
- on gmth phou ( b r t d I5391
- Dcpndl a tmprotun 1-t et d 2ml
- Other rondlttotu
+ Toxtn pmductian b/ I! pua,cidotempmturr
ww,
I Histopathology of vibriosis
Note marswe hocmocytc occurnulotion
Ofttn occompanlcd by mel~~zat~on
--
Vibriosis of HP -1
In HbE r t d hnob* m r wuld nnol the penre
of hornom d ,p* (bolophoLc) rods

-
1
IL*
Fermentation tests rcquind for species
idmt~hcctntlm
WL detection of hderia
*Pa methods prrrcntly milable for a few
spcias V p m h m E v , ~ V ~ rudvfrna ~ .
Y
pm.~d
Admntqc is speed md no nud far culture
+Boiled hounolymph samples urn be used for
allobis withwt or with (more sensit~vr)
DNA extmctlon
+~eloti.tivcl~ low sensitivity is pmbably on
advantqc in rnonitorlrg or scrrrnirg
%@w,
PCR detection of K pwahaemotyiicus
1 in shrimp hoemolyny~h
Lmel pFPhvt for 1W Wwid dl# inPCR -tic+
(2 x l@ulls/ml huno)lnph)
MI
1 Still needed for bmeria
*A rnultiplrx test for smd species ot
o m
*Application of Mw's published prmrs
for RT-PCR dctrctim~ of K puxxicirk
+Application of NtP probe
*A better unduvtonding of virulence
fnciors Md their i M t M e in tatvia
*A better undvstmdiq of shrimp drfuur
-3icgaimt kterial "hop115
mw,

Mycoplasmas and Rickettsiae
+Gcnmlly non-cultumblc or vuy difficult
to culture
+Once confirmed, wwlly tded wily with
antibiotics
Parasites and other problems
+Rclat,vcly spking thuc are mimr problcms
+Best assessed by histological examimt~on
+Rapid smrors sometimes sufficient
+Embedding and sectioning moy be required in
m e msu
Conclusions
+H~stolog~cal examimt~an rill remoin os the
bockbow of dqnostic methods for
olrtbnakc and genrml mon~twing
+Rapid mounts and wears an sjmplc and
effective for many disuses
+Embedding and sectioning my also be
required
*PCR and MA0 metnoas arc llrefu. for covert
mfoct:ens of rwc.f~ wtqcnr

Prevalence of WSSV Infection in P. monodon
Broodstock and Postlarvae: A comparison Study
Between Thailand and India
Bnonsirnr Whynchuntnarnkul, M.I)., PPD.
Department of Anatomy and Centex Slirimp
Faculty of Science, Mahidol Ilniversity
Bangkok, Thailand
Number of P. ntnnndnn Wild Broodstock Determined
3.s00
3,WO
2.500
2,wo
1.sw
1,wn
500
0
Jan Peb Mar Apr May Jun Jul Aug Sfpl Oct Nor Drc
l0l99.3 B1999 a2000 m20011

Number of Batches of t nnrndon Postlarvae Determined
Jan Fcb Mnl Ap. May Jun Jul AUK bpt Or1 No,
Drc
01998 1999 m20(111 B2W1
Total Number of Wild Rroodstock and Bntches of l
monodon Postlarvae Determined in Each Year
A posihlu increarc In tbnumbcr olPCR dcterminutlunr fur wild
hrnndstocli hut not thc pr1lan.a~

20
18
16
14
12
$ in
X
6
4
2
0
Prevalence of WSSV lnfstion in the Wild Broodstock (by PCR)
$8 $ \a 8 8 $a ocL s $$ s o~L s ,&4 a 06'
1998 1999 TT
20
18
16
14
12
$ lo
8
6
4
2
0
Prevulcnce of WSSV lnlslion in the Postlarvae (I). PCR)
$8 o* \, $a o* $8 $ a 0~' @ $ \a 'y'
1998 1999 -7

Average Prevalence of WSSV Infection (by PCR) is Wild
Broodstock and Postlnwae olP. nrono[lon in Each Year
1998 I999 2000 2lNl1
A perribic dcrmnr in the pmvaluncr of WSSV infcclion in the psstirrvae, hut
1\01 the wild hroedrtect (due 10 u hctter hstchrv munnpcmcet)
I'revelesce of WSSV Infections
Between Male and Female Broodstock in 2001

Yearly Prevalence of WSSV Infection in the Postlarvne:
Comparison Between Hatcheries that Screen-Out WSSV-
Infected Broodstock and Those thnt Did Not
Brooddocli Screening
Hatchcrie~ nith
Yearly Prevalence of WSSV Infectioll of P. nwno(lnn
Broodstock in India, in 2001
100
90
80
70
60
s 50
40
30
20
I0
0
Broodstock
Postlarvae
r of Deternrinations
ji
57 Nellore, 538
HChenni~i, 73
13Ki1liinailn. 1117
@Puntl\, 1687

1
Application of PCR for control of
'..,.. shrimp disease - strategies for risk
management
I
Dr Peter Walker
CSIRO, Brisbane, Australia
Livestoch Industrics
CSllO
Diagnostic tests as disease
management tools
U
U
U
Disease diagnosis
Disease outbreak investigation and resolution
Disease prevention
Hatchery screening of broodstock/seed
Disease control
Zonale rnonitoring/surveillance
import screening
Livestock Industries

~
.
PCR Assay for WSSV
I-PCR
n-PCR
M C Samples
941 bp
/ HEAVILY-1 JCIGHTLY-I
CS8.0
Semi-quantitative n-PCR for
detection of WSSV
b0
1.100 --
526 --
M I 2 3 4 5 6 7
2 9 -
-
I43 -
WSSV DNA 10 PC
0 I ig
Act,"
-.
DNA extracted from
Gsnstlc Enpl-dnp
and
1,ivestock Industries

., .-"..,,
;@- F i
CII.0
Appropriate use of PCR for screening of
seed can greatly reduce the risk of WSS
on farm
BUT
PCR screening must be linked to
appropriate on-farm disease management
L
Livestock lndustr~es
C.llo
Key elements of WSSV biology
0 Host range and the role of carriers
Persistent infections + vertical transmission
0 The role of stress as a trigger of disease
Livestock Industries
.:.

,"-..,,. Biological cycle of major shrimp
VERTICAL
BROODSTOCK
JUVENILES
SUB-ADULTS
NAUPLl' POSTLARVAE
MYSlS
ZOEAE
[cionliphasi]
Disease
Trigger
Vlral load
cannibalization
immersion
jAcuG&sej
L~vestock Industr~ca
"....,,
($my
ll#' i
CIIIO
Viral load and WSSV detection
_ - ____ _ _ - -
,Chronic phase _ - - Acute phase]
-- -
- --- -- -
VIRAL LOAD
n-PCR
I-PCR Histology
G~~~~ signs
DETECTION
1.1vestock lndustr~es

;$"&
, . PCR test sensitiAty
CS110
U A negative PCR result provides an indication that
the level of viral infection is below the detectable
limit of the test
0 Different PCR tests have different limits of
sensitivity
0 Sensitivity will also be determined by:
- Sampling strategy
- Szunple preparation (collection, storage, extraction,
processing)
- Operator proficiency (adherence to SOPS)
As for any test, application must be linked to a
rational strategy for achieving a specific outcome
Livestock Industries -
PCR test specificity
0 Test specificity is determined by many of the same
factors as test sensitivity
0 In defining specificity, the possible existence of
multiple strains, pathotypes and related viruses must
be considered
0 For WSSV, there is evidence of significant strain
variation in shrimp - the genome comprises both
highly stable sequences and a relatively small
number of short, highly variable regions
0 Specificity is an important issue but should not be
of practical significance if the PCR test targets
stable sequences
Livestock Industries

.

CII.0
Key observations
0 Spawning stress induces viral replication
and so also sensitivity of PCR testing
0 n-PCR (-) broodstock tested after spawning
produce negative eggs
0 Lightly infected hroodstock In-PCR (+) after
spawning] produce lightly infected eggs
(Hsu ei al, 1999)
Livestock Industries
FlllO
Key observations
0 The most common source of WSSV in ponds is
through infected seed (Lo et al., 1997)
0 Lightly infected [n-PCR (+)I PLs can survive
up to 13 months without mortalities (Tsai eial.
1999)
0 1-PCR (-) PLs are more likely to survive to
harvest than 1-PCR(+) shrimp
(Withyachumnarnkul, 1999)
Livestock lndustries

Key observations
0 Ponds testing n-PCR (-) 1 ma after stocking have
low risk of crop failure -
0 Ponds in which 150% of shrimp tested n-PCR (+)
1 ma after stocking have h& risk of crop failure
Spawners testing n-PCR positive before spawning
produce nauplii with high WSSV prevalence
0 Spawners testing n-PCR positive after spawning
produce nauplii with low WSSV prevalence
0 Nauplii with low WSSV prevalence have low risk
of crop failure
(Peng a/ 2001) 1,ivestock Industries
,".,..,
;itUN$!
i [mi' :
CSIIO
PCR testing of brooders
1-PCR
, c -3
' '/d
iL,
+ Rarely suwive spawning
- Lightly or uninfected PLs
n-PCR
+ Lightly infected PLs
~3 - Highest quality PLs
1,lvestock lndustr~es

.\
i@l
P"'-"I..
r
C'I"0
PCR testing of nauplii or PLs
1 -PCR + High disease risk
~3
# - Low disease risk
n-PCR
i---",
+ Survive 13 months - no disease
- Highest quality PLs
Livestock Industries
>
'csp!
L"'"."
CIllO
-
Examples of screening strategies
Strategy 1 - Minimize risk of infection
alln for lowest possible prevalence of
infection in PLs
Strategy 2 - Minimize risk of disease
aim for lowest practicable prevalence of
infection in PLs
L~vestock Industr~es

Strategy 1
0 Screening method - n-PCR
0 Test sensitivity - 5 targetslsample
0 Screening target - broodstock postspawning
Tissue sample - epidermis
0 Exclude all positive PL batches from n-
PCR (+) spawners
Re-test PL, batches to confirm n-PCK (-)
I.ivestock lndustrics
)."""'
{cr;.
Il1 :.
CS4"O
Strategy 1 - problems
0 Cross-infection of broodstock in the
hatchery prior to spawning
0 PLs batch separation essential until test
results are available
0 Prerniu~n price demanded for negative PLs
0 What happens to PLs testing positive
R Regulationlfarmer confidence
I
Li\,estock Industries

($#
'F Z Strategy 2
511.0
0 Screening method - 1-PCR (or other less
sensitive test)
0 Test sensitivity - 1000 targetslsample
0 Screening target - PL batches
0 Tissue sample - 150 pooled PLs
O Exclude all positive PL batches
L~vestock lndustr~es
ClllO
Strategy 2 - problems
Pooled PLs can be few heavily infected or many
lightly infected - potentially different outcomes in
the pond
O PLs will have a higher risk of crop failure
more attention to farm management practices
required (training)
0 crop failures will still occur - farmer confidence
problem
Better if farmers could test PL batches themselves
( dip-stick test)
Livestock Industries

'&
- . PCR screening of ~ ~sjnau~lii
CS1"O
Viral Viral PCR Pond
prevalence load test outcome
L L n-PCR (+)I(-) ~uccaasful crop
to)
L H n.PCR (+) lncnaslngl late-cmp
mortal8ttes
,--
H L n-PCR (r) Llkcly successful crop " '
(.:.)
H H 1-PCR (+) Cnsh (: , i
L~vestock lndustr~ea
..,
1 :
CSt"0
Select PCR Screening Strategy
U
U
Which type of culture system
What is the prevalence of WSSV infection in
broodstock
What is the existing level of infrastructure
and training
0 What is the available level of investment
0 What is the acceptable level of risk

CII.0
Other issues
0 Test standardization, laboratory accreditation,
operator training and farmer education are
important issues
0 Trigger of disease remains an important
consideration
0 Farm management is an important aspect of
WSSV control
Domestication and SPF seed will be an important
element of long term disease control strategies
Livestock Industries
CSIIO
-
Good health management
Good risk management
ACTION
PCR screen shrimp in hatcheries
Treat hatchery and pond inlet water
RISK
0
a
Treat hatchery and pond effluents
Monitor and control pond water quality Eliminate carriers from ponds 0
L~vestock Industries

.\...
I., ..
Cll"0
Standardization/ barhonization of
pathogen detection tests
Aims to deliver reproducible and reliable level of test
sensitivity and specificity upon which disease
management strategies can be based
0 To be effective, the prescribed levels of sensitivity and
specificity of standardized tests must be achievable by
participating laboratories
The prescribed lwels ofsensitivity and specificity must
also be linked to a disease management strategy
Accreditation and/or regulation of testing Inboratorics
will assist effective implementation
I .~vestoch lndustr~rs
.< b."'.,
:@)'j . . PCR Standardization
CSIIO
Issue standard operating procedures (SOPS)
Train technical staff
Regulate and police adherence to SOPs
Accreditation based on adherence to SOPs
Requires that all laboratories use the same tests
Based on assumption that adherence to SOPs will
generate uniform results
Based on assumption tlfit daily adherence to SOPs
will occur and can be monitored/policed
Livestock lndustr~es

*..'..,,
{@ r
Harmonization
ClllD
Issue example standard operating procedures (SOPS)
Offer training to technical staff
Accreditation based on adequate laboratory design,
operation test performance on unknown samples
Conduct regular inter-calibrations and random spot
checks and inspections
Failure in inter-calibration or spot checks results in loss
of accreditation
Allows labs to select their own tests
Based on performance rather than adherence to process
Monitoringipolicing feasible
I
~ivktock Industries 1

--
lntegrat~on of Pathogen Detection mto Shnmp Cultur;
Pwd Ermromt

I
NACA programmes and the I
assistance I
I MPEDAINACA technical 1
I
Thanks
Melba Reantaso, NACA aquatic animal health
management specialist
m Angus Cameron, AusVet
Pornlerd Chanratchakool, AAHRl
Vishnu Bhat, MPEDA and MPEDA staff
Drs lndrani and lttya Karunasagar, Mangalore
Dr CV Mohan, Mangalore
m Arun Padiyar, NACA
TASPARC
Farmers and survey assistants

NACA programme
tAO Regional Technical Cooperation Programme FCP) Project
TCPlRAS16714 and 9605 'Assistance for the Responsible Movement of Live
Aquatic Animals"
Project was implemented by NACA from 1998-2001.
. Asia Regional Technical Guidelines on Health Management for the
Responsible Movement of Live Aquatic Animals and the Beijing Consensus
and implementation Strategy" (TGBCIS or Technical Guidelines); and
supporting
m Manual of Procedures (MOP) for the implementation of the Asia Regional
Technical Guidelines on Health Management for the Responsible
Movement of Live Aquatic Animals and the
m Asia Diagnostic Guide (ADG) to Aquatic Animal Diseases.
. National strategies to implement Technical Guidelines
m Regional reporting system in cooperation with OIE
All rely on good diagnostic tools and capacity
Objectiveslscope:
m Vertical and horizontal study
of shrimp disease (risk factor
Development of practical
measures for shrimp disease

Phase 2 - Risk factor study
m Objective:
. To identify major risk factors for key shrimp
diseases and practices for management of risks
of these diseases
Study sites:
Kandaleru creek - an enclosed creek area with
heavy clustering
Bhimavaram - an area with heavy concentration
of small-scale farms
. Small number of sea based fans at Naaai
Knndleru Creek, AP, India: FCC a2 ms-IC ~lssrll (mcmul)
1
I

Factors studied
Pond ownership and history
Pond preparation
Stocking, source of PL, quality of PL
. Management practices
Water and soil
Feed
. Chemicals, prob~ol~cs
Pond environment
. Environmenffclimate

Diagnostic methods
. PLs and juveniles
. PCR (WSSV) (n=238)
Single step
. Nested PCR
. Histopathology
Shrimp disease outbreak samples (n=250)
. Histopathology
Key points from study
. The study identifies several significant factors
related to outbreaks of White Spot Syndrome
. Several causes act indirectly through other
causes
. Alone, each cause may have a limited effect
. Removal of a single cause cannot stop the
disease
. An integrated approach to disease control is
essential

Data analysis
Risk factors for disease outbreak
Risk factors for poor production
. Nellore: < 1000 kglha
. Bhimavaram < 200 kglha

Time of outbreaks (calendar)
Bhimavaram
k]
n
O o B B
!om-
.
Time of outbreaks (DOC)
Bhimavaram
8)
0
- 0

PL WSSV prevalence by time
0
0
P-emm
Stocking and seed factors
High prevalence of WSSV in seed batches by
nested PCR
However, single step PCR tests were negative
This result is interpreted as a high prevalence in
the population but low viral load in anlmals
Significant relations between risk of an outbreak
and lower pond production at >5% prevalence.
MBV and white spot are significantly related in
shrimp nurseries - multiple infections
Increased risk of outbreak related to seed
quality factors

.- -
pp -- -
60
50
40
Percentagesof WSSV pevalencc groupsln Mllore
(87 samples) and Bhlmavaram (151 samples)
30
20
la
0
Tt% 12% 5-1L% 20JWb M% >BW6
L
m% In ~e~
m% In West
r

- - -- - -- - - - . -
Carpansn tetveen the percentagwns, batchesd seeds
mth lo/.pe\a!%nce &lusdW-ama and
n s ,

Implications of PCR findings
Farms stocking:
PL populations have widespread prevalence
of WSSV but low viral load
PCR has an important role in management
PCR combined with quality screening of PLs
before stocking is recommended
Risk can be reduced through a combination of
PCRlscreening and better farm management
practice

Bmodstock \Prevalence ~ ~ ~ l ~ h ~
Transport
in led
Introduced
Dlslnfeclian
Rerervalrs
Predators
Cannlbal,sm-.,
Not remavmng/
dead shrimp
Multiplication
in pond
Disease
outbreak
Benlhlc algae
Clear water
/'
Lack of fertilirer
I Shrimp seed is

Standardisation/harmonisation
m Use of PCR and shrimp seed screening important part
of health (risk) management
However, a harmonised approach to PCR test to define
the virus load (within the shrimp) and prevalence (w~thin
the population) should be useful.
Can we find an acceptable level of risk for stocked PLs
Thankyou

16 tstlmla Ihe prevalenc~ 01 WSSV ~n 1s brood rmck
of Penahis mvwdon
BroM %lock&mp
wrrl roll~cl~6fmmndour
hatch.rlts sihl.1.d abnp MI *anand*tr( rand d
*dl*
A tout 0149 brmd,, sampl#s w m rollrcl*d
Twemymur nmplnr mn .nalyud
Nim nr. found pollbvl in fil sZp PCR
Thlneln mn p~sltivo In %.ccndnlp PCR
To sWy lhs axpcnnlen su3c.pllblllty ddli(w.d
life sbgo of liarh$nler piam. Macmbrwh~m
mrenhrpn 10 WSSV
I
F U ~ N cmflrmlllon war dona uslng PCR
I

6
Obpnortic arvb
To dao- WSW hllCua hrmmshNn~Irm
vMws pat# Itma In mbmnhm
Issues and Suggestions
Kll cmlro!ars) IS nu1 ncw
' Ovcreslilllulc Ills Inlpdnnu: illplhogcni
Il81nnon1wuon wrscs slwdurdtwlliln
Pnonn!l/ill~t,~~ olohj~~llrcr
Accmlllaltuu

Monoclonal antibody based immunodot for rapid
and sensitive detection of
white spot syndrome virus of shrimp
K. M. Shankar
Fish .ihology and Biotechnology Laboratory
Department of Aquaculture, C~llege of Fisheries
Mangalorc-575 002, INDIA
Monoclonal antibodies (MAbs)
- Highly specific- P-tact at epitope !eve1
- No background -eaction
- Application - Diagnosis
- Laboratory
- Field level

MAbs to WSSV- Review

Production of MAbs to WSV
Gills. epithelial layer from cephalothorax from WSV dated shrimp
I
3-
WSV purified by ultracentnfugation on sucrose gradient.
Electron microscopy and SDS-PAGE
Balb C mice immunised with purified WSV, Spleen cells fused with SP2 0
myeloma cells
Development of Immunodot assay, Hybridoma screened by imm1111odot assay
Hybridoma charactensed by Westemblot and Isotyping
No of clones produced: 500
No of immuonodot positive: 83
No of immunodot positive with SDS treated Ag: 35

Electron Microscope
Negative staining

SCREENING OF HYBRIDOMAS

Westernblot analysis of WSV antigens by MAbs
Lane 1 - molecular weight marker
2 - purified virus
3 - MAb C-5,4 - MAb C-33,s - MAb C-38,6 - MAb C-56

Characteristics of the MAbs to WSSV
-No reaction, - Weak reaction, -+ strong reaction, +++ Very strong reactlon

Development of MAb based lmmunodot test
Homogenate of Infected gill tissue (3 ul ) wtth appropnate Ag controls
on to Nimocellulose (NC) discs
dotted
4
NC blocked with 3 % BSA for 30 mln
1.
Reacted with a MAb for 90 min
i
Reacted with rabb~t anti-mouse I&-HRP for 30 m
Reacted with substrate (4 chloro I naphthol)
i
Purple blue dots recorded
Positive control: Punfied WSV,
Negahve control: Normal shnmp hssue. hepatopancreas of MBV ~nfected shnmp

Reaction of MAb C38 with WSV infected and uninfected
shrimp tissue in the immunodot test.
A1 - Positive control
B1 - Negative control with normal shrimp

Epitope analysis of WSV by Irnmunodot
Gill tissue homogenate of WSV infected shnrnp from east and west mast
1
J
Centrifuged at 8000 rprn,
3 ul of each of the six isolates dotted on NC in one colurnnilane
Each column of dots treated with one MAb for 90 min
1
Reacted with rabbit antimouse IgG -HRP
Reacted wth Substrate (4chloro I naphthol)
i
Purple blue dots recorded

CS6
Kundapur 1
Kundapur2
Q Kundapur 3
Kakinada
Nellore
Madras
Uninfected shrimp

Determination of Sensitiviw of lmmunodot Test
WSV purified twice on sucrose gradient and protein estimated
Senal dilution of the virus preparation
2 ul from each dilution dotted on NC
Reacted with MAb( 4 day old cell culture supernatant)
1
Reacted with rabbit antimouse IgG -HRP
I
J
Reacted with substrate (1 chloro I naphthol)
End point recorded

Vim1 protein concentration (in ng)
Fig. 5 Sensitivity of immunodot test

Limit of detection of \\ SV by lmmunodot compared with PCR
Gills from a WSV ~nfectsd shnmp
I
J
Ground to paste and,di\lded Into two equal parts
1-
Diluted with TNE 1 .lo.
Cfg 8000 rpm
1
Log dilut~on
i
3ul from each dl1 dotted :n NC
1
hrnunodot PCR
I
DNA extraction
I
J
Log dilution
1,
1
End point recorded
3 ul from each dil used for PCR
End point recorded

LIMIT OF DETECTION OF IMMUNODOT IN
COMPARISON TO PCR

Table 2. Lit of detatlon of Immunodot In cornpanson with I step PCR for
detection of WSSV
1 Shrirno Test Dilut~on Nenat~ve I
sampk No
control
I I 0 I I lod
A1 PCR+ + + . - .
Idot + + + . . -
I
PCR+ + + - - -
Idot + + + - - -
I
- I

Comprritive Performance of immunodot ~ C for R detection of WSV in [
shrimp from WSS outbreak
S h p samples collected, washed and packed individually from WSS outbreak
from different farms in kundapur
J.
'Gills from each shrimp colle~ted, ground to paste and divided into two equal parts
l
TNE Ill0 wlv
I
Fixed ."methml
\1 J.
Cfg 8000 rpm
DNA extracted
1 I
,1
1
3ul used for immunodot
jul used for PCR
1
1
Immunodot
PCR
I
End points recorded

Preliminary evaluation of irnmunodot for detection of WSSV in a
sequential study in grow out ponds-
12 ponds - Kundapur, West coast of India
13 8 samples
1 step PCR + ve 11 samples
Immunodot dark positive 9
Immunodot light positive 2
Small sample study
Further sequential study:
- Experimental infection
Screening of seed, brood, grow out

Conclusion
Mab based Immunodot Test
-Sensitive( 500 pg, 1 step PCR)
-Rapid(3 hrs)
-Simple (Farmer Level)
-Cheap
Development of Immunodot Kit
Screening for - Brood
-Seed
-Growout Pond
-Water

PATHOGEN
1 GUT-AND-I

An overview of PCR techniques for shrimp disease
diagnosis in Asia, with emphasis on Thailand
T.W. Flegel
Centex Shrimp,Chalm Prakiat Bld., Fac. Science, Mahidol University
Rama 6 Road, Bangkok 10400, Thailand
ABSTRACT
Asia leads the world in cultivated shrimp production with export earnings in the order
of billions of US dollars per year. In spite of this success, annual production decreased in the
late nineties because of widespread epimtics caused by new viral pathogeos. Although,
these viruses were no cause for alarm to human health authorities, they were economically
crippling for Asian shrimp farmers. In Thailand, shrimp production trends have mirrored
those in the rest of Asia, except that recovery born the viral epizootics has been somewhat
better than it has for most of its close neighbors. Our work in Thailand has focused on the
characterization of the causative viruses and on the development of rapid diagnostic probes
for them. Similar work has been done elsewhere. The aim of the work has been to develop
effective control measures to help shrimp farmers. We are engaged in similar work on
bacteria and parasites. The major viruses of concern (in our estimated order of economic
impact for Thailand) are white-spot syndrome virus (WSSV), yellow-head virus (YHV),
hepatopancreatic pawovirus (HPV), monodon baculovirus (MBV) and infectious hypodermal
and haematopoeitic virus (IHHNV). We have also prepared probes for Vihrio parahaemolyricus
and for a microsporidian parasite, Agmmoma penaei. These highly specific and
sensitive tools for detection are already helping shrimp farmers and we hope that new
technological advances will make them practicable in the field. At the moment, however, the
most rapid test is the polymerase chain reaction (PCR) test, which takes approximately 3
hours to complete. This review covers important Asian shrimp diseases for which PCR tests
are currently available
lntroductlon
Asia has always led world production of cultivated shrimp with a market value of
billions of US dollars per year. Thailand alone has been the world's leading producer since
I992 with ns export earnings alone reaching more than one billion US dollars per year.
However, in Thailand in 1995, largely due to yellow-head virus (YHV), production decreased
by about 5,000 metric tons (equal to approximately 40 million US dollars in lost export
revenue) (Flegel el a1 1995b). In 1996 and 1997, another virus called white-spot syndrome
virus (WSSV) was even more d~sashvus, with cumulative lost expoti revenue estimated at
approximately 1 billion US dollars (Flegel & Alday-Sanz 1998; Flegel 1997). After 1997,
Thai production began to recover, reaching the previous highest production of 250,000 metric
tons again in 1999. The rest of Asia did not fare so well. For example, WSSV outbreaks in
China began in 1993, reducing export production bom the 1992 high of 115,000 metric tons
to 35,000 metric tons. Recovery has been slow, with production reaching only 70,000 metric
tons by 1999.
These examples serve to illustrate how serious disease losses can be m the shrimp
aquacultwe industry. The pilous position of the shrimp farmer and the shnmp industry can
be greatly improved by the implementation of relevant skategies which include programs for

improved farmer coopemtion and technological changes. These strategies could lead to a
long term, stable shrimp industry with little negative environmental impact. Biotechnological
research can make substantial contributions towards maintaining achieving this goal but it is
csscntial that government and indwhy provide continuous support for the infrastructure and
training required to maintain the relevant capability.
This review covers steps in the development of DNA probes and PCR technology for
detection of shrimp pathogens. Much of the work described bas been done in Thailand and it
has been reviewed in a broader context elsewhere (Flegel 1997). Where appropriate, similar
work done elsewhere will be included. While focusing on these probes, one should not forget
that the probes play only one smaU part in the overall strategy to fight against disease. l'bey
are not an answer in themselves hut must be used properly in tbe overall context of a shrimp
health program involving such topics as environmental safety, nutrition, and genetics, to name
only three.
This review will cover the development of DNA diagnostic probes for white-spot
syndrome virus (WSSV), yellow-head virus (YHV), hepatopancreatic parvovims (HPV),
monodon baculovirus (MBV) and infectious hypodermal and haematopoeitic virus (IHHNV).
Also briefly discussed will he probes dpeloped in Thailand for Vibrio puruhuemolylicus and
for the microsporidian parasite, Agmasoma penoei, and probes of others for Taura syndrome
virus (TSV), spawner mortality virus (SMV) and a mycoplasma. In terms of losses to the
Asian shrimp indushy WSSV, YIIV, HFV and MBV are undoubtedly the most important (in
decreasing order). Losses from the virus IHHNV and from bacteria and microsporidians are
less clearly evident.

Moondon bnculovirus (MBV)
We were quite alarmed when we saw this vinw (Figs. 1-3) in Thailand for the first
time in 1990 (Fegan er 01. 1991), because it had been implicatedin the collnpse of the shrimp
industry in Taiwan in the mid 1980's (Lio 1989). However, we soon found out that it did not
cause shrimp mortality so long as rearing conditions were good. This was lo spite of the fact
that some of the infected shrimp larvae had very large numbers of viral inclusion bodies. In
other words, they survived high levels of virus production with no ill effccb and no visible
resistance response. The opinion that MBV did not cause mortality was later expressed in
Taiwan as well (Liao el a/. 1992).
Figure I. Squash mount of a larval hepatopancreatic cell showing
'# several polygonal viral inclusion bodies in the enlarged nucleus. The
inclusions are composed of a protein matrix called polyhedrin which
contains embedded viral panicles. Because these protein particles
enclose or protect viral particles, they are sometimes called occlusion
bodies. They are released in the shrimp feces and ingested by other
larvae so that the infection is spread horizontally.
Figure 2. Sca~ing electron micrograph of MBV inclusion bodies
purified from shrimp larvae by differential centrifugation of tissue
homogenates. Such particles can be used for subsequent viral
purification and nucleic acid extraction.
Figure 3. Transmission electron micrographs
of MBV. The upper left photograph
shows two virions with intact envelopes on
the left and right and one with a fractured
envelope revealing the inner nucleocapsid.
The lower right photograph shows a section
of an inclusion body with rod shaped virions
embedded in the polyhedrin matrix.
Even though we knew that MBV was not a serious pathogen for the black t~ger prawn,
we still wanted to eliminate it from the farming system because we did not believe that the
shrimp could carry such heavy viral infections without paying some price. Indeed, we have
done furlher work showing that the mean length of MBV infected shrimp is significantly
shorter than uninfected shrimp from the same pond (Fig. 4) (Flegel et al. 2001). although this
difference was not easily detected until late in the cultivation cycle. These results support
findings from studies in the Philippines indicated that MBV infections could slow growth in
intensive cultures. We also suspect that poor rearing wnditions could lead to a flare-up of the
virus followed by secondary bacterial infcctions resulting in shrimp death. In fact, it turned

out the that the virus could be eliminated from the rearing system by a combination of
washing eggs and/or early naupliar stages with clean sea water, separate rearing of singlespawn
larval btcbcs and discard of occasionally infected batches of larvae or post larvae
(PL).
The experience with MBV had important spin-offs in terms of cooperation with other
scientists working on shrimp diseases. It started our contact with Australian scientists through
cooperation with Dr. Joan Vickers at the University of Queensland in what comprised our
start on viral gene cloning work and DNA diagnostic probe development. It also started our
interactoos with Dr. S.N. Chen in Taiwan, Dr. D.V. Lightner in Arizona and Dr. J.A. Bmck in
Hawaii.
Figure 4. Comparison of infection status
with length for uninfected shrimp and
shrimp infected with MBV, HPV or both
MBV and HPV (dual). The shortest shrimp
where those with HPV or dual MBVmPV
infections and the longest where those with
no infection. Bars marked with the different
letters (a,b,c) indicate means that are
significantly different (P

lot DNA hybridization and PCR amplification. This would allow for nondestructive testing
of broodstock and pond reared shrimp. More work is needed on the MBV genome
particularly with respect to comparative analysis with the insect NPV for which much more
information is cumntly available. Such studies should give some insight into the interaction
between MBV and its shrimp host
Since MBV is a DNA virus like white spot syndmme virus (WSSV) and
hepatopancreatic parvovirus (HPV) (see below for details on these two viruses). It should be
possible to devise a multiplex PCR method that would be capable of detecting any
combination of these viruses in DNA exhacts from PL. Sinee PL in Thailand are already
regularly tested for WSSV by PCR, it would seem a worthwhile goal to assay for all of these
viruses in a single PCR reaction.
Additional figure. Photomicrograph on the left shows MBV occlusion bodies (arrows) as
viewed directly through thc cuticle of an early PL specimen using the light microscope with a
40x objective. The photomicrograph on the right shows early to late stages of MBV infection
in an HBE stained tissue section of the hepatopancreas (HP). Nuclei become enlarged with an
acidophilic (pink) center with nucleoli and chromatin condensed along the nuclear membrane.
At the final stage the very enlarged nuclei contain acidophilic, paracrystalling protein
inclusions (occlusion bodies). In this photomicrograph. 4 occlusion bodies can be seen free in
the lumen of the HP where they have been discharged from a lysed cell.

Yellow-head virus (YW)
Our work on MBV was intempted by the amval of the first really serious viral
pathogen of shrimp in Thailand in 1992. In retrospect, we know that this vim first began to
cause problems in Thailand in 1990 (Limsuwan 1991), but it was not discovend as a new
pathogen until 1992 (Boonyaraplin era/. 1993; Chantanachookin elal. 1993). The virus was
named from the gross signs of disease which included a yellowish cephalothorax and very
pale overall coloration of moribund, infected shrimp (Fig. 5). Histologically, it can be
recognized by densely basophilic inclusions, particularly in the gills by rapid staining (Flegel
eta/. 1997a) (Fig. 6) and the haexnolymph (Nash el a1 1995) (Fig. 7). Research on YHV in
Thailand ha8 ban reviewed (Flegcl et a/. 1997a; Flegel el a/. 199513) along with current
practices for diagnosis, prevention and conf~ol.
YHV was first thought to be a baculovims but we discovered during purification and
characterization that it had curious morphology (Figs. 8 & 9) and that it was an RNA virus
(Wongtc~asupaya et 01. 1995a). DNA diagnostic probes were prepared by cDNA preparation
and cloning, although these cumntly work best in RT-PCR assays (Wongteerasupaya el a/.
1997) rather than in situ hybridintion (Fig. X), probably because of the instability of the viral
RNA. Even with the PCR ansay, samples must be processed quickly, since storage at -XO°C
does not prevent deterioration of the RNA.
Figure 5. Gross signs of yellow-head infection
are seen here in the 3 shrimp on the right. They
are generally bleached in color with a yellowish
discoloration of the cephalothorax ("head")
region when compared to shrimp of normal
appearance on the left.
Figure 6. Gills of YHV infated shrimp
stained with H&E in normal paraffi
sections (u~mr date) and in ra~idlv fured

The PCR amplicon sequence and the primm for YHV detection in infected shrimp by RT-
PCR are as follows (Wongtcemupaya el al. 1997).
CCG CTA ATT TCA AAA ACT ACG
I
ACA GAR ACA CCG GCA TGT CCT GTT CTC TCA
CTG RAT TCC AGC TCT CTC TCT CTC ACA TCC TCT ACC GTT CTG AAG CAC AGC
GTA CTC CTG ACG ACT TCC TCG ACA TAA CAC CTT
Figure 7 Haemolymph from normal and YHV
%*"< mfected shnmp The dlslntegrabon of the nuclel IS
clearly evldent m the YHV rnfected shnmp These
can be seen m early stages of mfecuon, but not later
when the haemocyte populahon has been depleted by
the vuus In addltron, such d~srntegratlon can be
found wtth some bacterral lnfcchons
Figure 8. Transmission electron micrograph of
YHV-infected shrmp tissue showing the unusual
filamentous nucleocapsid precursors (on the left) and
mature, rod-shaped, enveloped vki0u.s (on the right).
I
Figure 9. Transmission electron micrograph of
negatively stained purified virions of YHV. Note that
the virus particles are enveloped and that the envelope
bas a halo of appendages characteristic of some RNA
This fragment can also be labeled and used for dot blot or in sihr hybridization assays.
The problem with these techniques is that the viral RNA is very labile and must be protected
during specimen preparation. For in sifu hybridization using fued tissues, it is important not
to use Davidson's fixative which is acidic and will destroy the RNA. More successful in sihr
hybridiition results using RNA-friendly fuation of shrimp tissues has been reported by Tang
and Lighhler (Tang Lightner 1999). Briefly this method uses a formula where the acetic acid
in Davidson's fixative is replaced with additional formalin. AAer overnight fixation, the
tissues (without cuticle, which cannot be cut unless decalcified) arc quickly embedded in
paraffm atter which they can be stored indefhitely before in situ hybridiition assays are
carried out. However, once rehydratcd, the specimen sections will be extremely vulnerable to
attack by RNase and all precautions must be taken to protect the specimens. We have used
neutral buffered fonnalin and Davidson's fixative with the acetic acid replaced by distilled
water as alternative fixatives to Davidson's and these appear to work equally well. It has also
been proposed that normal Davidson's fixative may be used, so long as the fixation is not
longer than overnight and followed immediately by dehydration and embedding in paraffm.

The Tang and Lightner (1999) probe used for in rilu hybridization was a 1051 bp
digoxygenin labeled probe derived from a 1061 bp YHV cDNA clone and produced from that
clone template using PCR with the following primers:
Sense: 5'-ACA TCT GTC CAG AAG GCG TC- 3'
Antisense 5'-GGG GGT GTA GAG GGA GAG AG- 3'
They also gave primen derived from the same cDNA fragment for detectionof a 273 bp
YHV specific mplicon by RT-PCR:
Sense:
Antisense:
5'-CAA GAT CTC ACG GCA ACT CA-3'
5'-CGA CGA GAG TGT TAG GAG G-3'
In addition to these reagents, Cowley et al. (Cowley et al. 2000a) have published primer
sequences that can be used for detection of both YHV and the related Australian lymphoid
organ virus (LOV) (Spann et al. 1995) and gill associated virus (GAV) (Spann et a/. 1998).
These primers were designed fran a 781 bp GAV cDNA clone to give a 618 bp RT-PCR
product and are:
Sense:
Antisense:
5'-AAC TTT GCC ATC CTC GTC AC-3'
5'-TGG ATG TTG TGT GTT CTC AAC-3'
Together with Dr. Walker's group from CSIRO Australia, sequencing and comparison
ofthe 618 bp RT-PCR kagments obtained using these primers with YHV, GAV and LOV has
shown that all are closely related single stranded, positive sense RNA viruses that will likely
be the fust invetebrate representatives from the Order Nidovirales (Cowley et al. 20Wb).
LOV and GAV shared approximately 95% DNA sequence homology and 100% amino acid
homology establishing that they are the same virus shin, while GAV and YHV shared
approximately 85% DNA sequence homology and 96% amino acid homology indicating that
they are diffe~nt strains (Cowley et al. 1999). An excellent commercial kit is available from
lntelligene of Taiwan that gives differential and graded RT-PCR detection for GAV and
YHV.
Dr. Walker's (CSIRO, Brisbane) has recently found a third, apparently nonpathogenic
YHV variant in Viet Nam and Thailand that is closer in sequence to GAV than the
original Thai YHV. Using the Intelliene kit, the new YHV shin gives the same PCR band
as Australian GAV. These RT-PCR probes are also useful for examining suspected carriers of
YHV and testing whether they can transfa the virus to cultivated shrimp. The results of such
studies will have an important impact on disease control programs for shrimp fanners. Dr.
Walker's has developed an excellent preservative solution for field samples. It contains 80%
ethanol, 20% glycerol and 0.25% mercapto-ethanol (prepared by mixing 80 ml absolute
ethanol, 20 ml pure glycerol and 0.25 rnl of mercapto-ethanol). Samples are crushed or
homognized in this at 1 part sample to 10 parts preservative and survive storage for a
reasonable length of time at mom temperature. At the laboratory, supernatant preservative is
removed before nucleic acid extraction (RNA or DNA) tom tissues in the usual manner for
us as a PCR template.

Whitespot syndrome virus (WSSV)
Historically, this was the second viral infection to seriously affect Thai shrimp
farmers. We have recently reviewed the studies and control methods for this virus in Thailand
(Flegel er a/. 1997a). Infections with it usually give gross signs of white inclusions of various
sizes embedded in the carapace at the late stages of infection (Fig. 10). These characteristic
gross signs of infection were first reported from an outbreak which occumd in P. japonicw
in Japan in 1993. The causative agent was a new hacillifm vim (Takahashi el al. 1994). In
the same interval viral infections with similar gross signs w m seen in P. japonim. P.
monodon and P. penicillatus in Taiwan and C hi (Chou er 01. 1995). With hindsight, we
now know that in Thailand the virus was first seen in laboratory reared P. monodon m late
1993 (Wongteerasupaya er a/. 1995b). Now, the virus is called white spot syndrome virus
(WSSV) by genml consensus (Lightner 1996; Lightoer & Redman 1998). However, it was
not found in Thai farmpd shrimp until late 1994, when mass mortalities began to be reported
with characteristic gross signs of WSSV infection (Wongteerasupaya er al. 1996).
We originally called WSSV a baculovirus based on its cylindrical morphology and
histological lesions that resembled those of "non-occluded" baculoviruses (Wougteerasupaya
era/. 1995b) and in one publication, we were actually requested by the reviewers to call it
PmNOBII for "Penaeus monodon non-occluded baculovirus U" (Wongteerasupaya el a/.
1996). In the end, this turned out to he a mistake. We now how that WSSV is a tailed, rod
shaped, double stranded DNA virus with a very large circular genome in the order of 300 kbp
that is available at GenBank (Lightner 1996; van I-iulten er a/ 2001; Wongteerasupaya er a/.
1995b). Since the genome has no significant homology to any hnown virus, a new viral
family (Nimaviridae) and genus (Whirpoviius) have been proposed for it (van Hulten er a1
2001).
Figure 10. Gross signs of WSSV infection.
White inclusions in the cuticle of moribund
shrimp are indication of infection with this
Fhre 11. Hislooatholom of WSSV. The low mamifica-tion micromaoh on the lee shows manv
characteristic inciusions ZWSSV under the cuticle ofthc gul epithelium. 'The high magni-fication on
the riaht clearlv shows the hmcrtronhied ,. . nuclei. Note the nucleus in the lower rinht comer wtth a
densly red stained center (acidophilic) surrounded by a clear space and then a ring of purple
(basophilic) chromatin. This is a Cowdry A-type inclusion characteristic of nuclei in the curly stages
of infection.

On the basis of gross signs of disease, histopathology with the light (Figs. 11 and 12)
and electron microscopes (Fig. 13). and DNA characteristics (Fig 14). it soon became obvious
that these infections wuld be ascribed to the same virus or closely related fonns of it (Chon el
01. 1995; Durand er a/. 1996; Kimura er al. 1996; Wang era/. 1995; Wongteerasupaya el a/
1995b). This contention was further supported by in siru DNA hybridization tests with whitespot
syndrome, cultivated shrimp of various species from several Asian countries
(Wongteerasupaya et a/. 1996) (Fig. 15).
Flgure 13. Transmission electron microscopy of WSSV. On the left is a low magnification
view of a WSSV infected nucleus fom gill tissue showing large numben of rod sbaped
virions. On the right is a view of negatively stained enveloped virions showing unusual
appendages and somewhat variable morphology.
Figure 14. Polyacrylamide gel elecrtophoresis of nucleic acid
from WSSV. Lane M contains a DNA marker and Lane 3
, contains the undigested nucleic acid. The nucleic acid in Lane I
, was digested with BamHI while that in Lane 2 was digested with
EcoRI. The EcoRl digest was cloned and screened for fragments
that was specific for WSSV. The selected fragments were used for
in sihr hybridization assays and for development of PCR detection
assays.

It is extremely important to understand that diagoosis for WSSV infection cannot be
based on the pss signs of wbite inclusions in the cuticle. A recent report by Wang n al.
(Wang el a/. 2000) has shown that bacterial infections of the cuticle can also be associated
with the formation of white inclusions, in the absence of WSSV infection (Fig. 15). Since the
management response to bacterial and viral infections is fundamentally different, it is always
necessary to confirm whether shrimp with such gross signs also show the histolopathology
characteristic of WSSV. Thrs can be done by microscopic examination of whole gill
fragments that have ban rapidly fixed and stained in a simple, inexpensive process that takes
only 3 hours.
Figure 15.. Gross appearance of the carapace of shrimp
showing bacterial wbite spot syndrome. These spots cannot be
easily distinguished from those caused by WSSV, so histological
examination is always required in the confmtion of WSSV
infections from (Wang el al. 2000).
Figure 15b. Sanning electron micrograph of bacteria
colonizing the white spots in bacterial white spot syndrome.
Examination of the epithel~al tissues oi the shrimp showed
absence of any WSSV histopathology. The shrimp were also
negative for WSSV by nested PCR assay (Wnng era/. 2000).
We developed DNA hybridization probes for WSSV (Wongtecmupaya er a/. 1996)
(Fig. 16) and soon thereafter the primers for PCR detection WSSV by PCR, although the
primer sequeoce was not published (Kanchauaphum ef a/. 1998) but they are available
commercially from the Shrimp Biotechnology Business Unit, National Center for Genetic
Engineering and Biotechnology, Bangkok. These primers are widely used in Thailand for
screening broodstock and PL in an attempt to stem the spread of the WSSV and restore
production to former levels. The rapid implementation of this PCR screening system together
with other appropriate management (Chanratchakool & Limsuwan 1998; Flegel er a/. 1997a;
Withyachumnarnkul 1999) has probably rescued the Thai shrimp industry from a disaster
similar to that which occurred in China in 1993. By ow reckoning, the preventative measures
have probably saved the countq in the order of 1 billion US$ per year in export earnings
since 1995. Again we are cooperating with Dr. Walker's gmup in Australia and with
scientists in Taiwan and Japan in the analysis of viral DNA sequences from various sources,
in order to understand the relationship amongst the epizootics that are occurring throughout
Asia and now-the Americas.
Another use of the PCR test has been to Identify and monitor the transfer of WSSV
from rese~ou hosts to shrimp. In these studid, the most surprising feahue has been the wide
range of potmtial hosts. WSSV infects not only several spies of penaeid shrimp including
those cultivated in the westem hemisphere (Lu el a/. 1997). but apparently also a wide range
of other decapods, including crabs and more distantly related cmstaceans such as copepods
and p h p even aquatic insect larvae (Lo et a/. 1996a; Lo el a/. 1996b). For shrimp farmers,
it is extremely important to establish whether these non-cultivated cmtacuw are bona fde
reservoirs of the virus that can transmit it to cultivated shrimp. Studies in Thailand

(Supamatlsya el al. 1998) have pmvm th.1 the swimming crab, Pomnis pelagicw, and the
mud crab, Scylla semara. can be infected with WSSV by injection or feeding, and we have
shown by time-course PCR ansay, histopathology, in situ -DNA hyhridizntion that P.
pelagims, and the mangmve m b can transmit the virus hack to shrimp within a few days via
water (Kanchanaphum el a/. 1998) (Fig. 17).
Figure 16. In situ hybridization detection of WSSV. The panel on the left shows H&E
staining of WSSV infected tissue and that on the right shows a serial section of the same
tissue wyed by in silu hybridization. The dark cells indicate positive hybridization.
Figure 17. Examples of PCR amplification tests for WSSV in a reservoir host. Here on the
left is seen an agarose gel with PCR products derived from samples of the crab Sesarma with
a strong product hand sea in lane 4 at 36 h after injection of the virus. The gel on the left
shows the appearance of positive PCR bands for the presence of WSSV in the haemolymph of
shrimp cohabitants with the infectad crabs. The virus was transferred and became evident in
the haemolymph by 24 h. Lane 1 =molecular marker; lanes 1-9 =products 6um barnolymph
at 0 h (lane I) and every 12 hours thaeafm.

Hepatopaocrc~tlc parvovirus (HPV)
We have been interested in this virus for some years (Flegel el a/. 1992b; Flegel el a[.
1995a) but it has been hard to get information. Our preliminary data (Flegel er al. 1995a)
suggested to us that the virus was I& to shrimp larvae during the interval of the first month
after stocking. However. our most rcccnt results (Flegel el al. 1999) suggest that there is a
strong statistical comlation belween HPV infection and small size, suggesting that most
HPV-infected shrimp simply p w very slowly and stop growing at around 6 cm in length
(Figs. 4 & 19). Thus, when the shrimp are sampled by cast net. the% non-growers escape and
are usually counted as non-survivors. Functionally speaking for the farmer, lhis is almost
equivalent to death since shrimp 6 cm in length weigh only about 5 g (200 pcices per kg) and
have so little market value that they do not wver the wst of rearing. If HPV infected shrimp
constitute a substantial part of a pond population, the resulting crop mry be a financial loss.
On the basis of our recent results, we are recommending that PL batches with moderate to
high prevalence of HPV be rejected for stocking by shrimp farmers. However, there is no
indication that HPV infections spread horizontally in growout ponds, so it may be acceptable
to use PL hatches with a low prevalence of HPV, if no uninfected batches are available and if
the projected loss would be economically acceptable.
01.- --
-- 7-T-j
0 2 4 8 8 1 0
HPV ssvamy Index
Figure 19. Severity of HPV infection
versus length for shrimp from a
farming system in southern Thailand
(Flegel er a/. 1999). Please also refer
back to Fig. 4, where shrimp size is
shown according to infection group.
There, HPV infected shrimp comprise
the groups with the smallest sizes as
either a single or dual infection with
MBV.

DiagXotics primers
DiagXotics probe
Figure 21. PCR assay of Thai HPV using primers from DiagXotics Co. Ltd. The gel on the
left shows the conlrol PCR product at 350 bp and the Thai product at 732 bp. The southern
blot membrane on the right was prepared from the gel on the left and it shows that Thai HPV
does not react so strongly with the DiagXotics labeleled probe as does the DiagXotics control
HPV product.
Detection of HPV is not easy because there are no unique and distinctive gross slgns
associated with infection. Since 11 occurs only in the hepatopancreas (Fig. 20), the shrtmp
must be killed to be examined histologically. This is not problematic for larval samples, hut it
is for broodstock and pond reared shrimp. We need diagnostic reagents that can preferably be
used with the shrimp feces, and DNA probes are ideal for such applications. Commercial
DNA diagnostic probes and PCR primers for HPV are available (DiagXotics Co. Ltd.,
Wheaton, Conn.) based on an HPV isolate in P, chinensir (HPV-chin) ffom Korea, but ~t
turned out that these were not ideal for HPV from Thailand. HPV m P. monodon (HPV-man)
from Thailand is quite different (Sukhumsirichan e/ a/. 1999) with a.larger genome of 6 kb
single stranded DNA compared to 4 kb oTHPV-chin. The DiagXotics primen designed from
HPV-chin DNA give a 732 hp fragment with HPV-man rather than the predicted one of 350
hp. When we compared the 732 hp fragment from HPV-man to that of HPV-chin at
GenBank, we discovered that HPV-mon had only 70% homology to HPV-chi (Phmmjai er
a/. 2001) and this explained the weak DNA hybridization in Southern blots (Fig. 21). To
improve sensitivity with HPV-mon, the primers of Sukhumsirichari el a1 (Sukhumsiricharl c/
a/. 1999) may be usd instead of those of DiagXotics to yield an HPV specific fragment of
156 bp. This fragment is shown below with the primer sequences underlined.
5'-GCA
3'-CGT
CTT ATC ACT GTC TCT ACC CAA GTC ATG AGC TGT CTG
GAA TAG TGA CAG AGA TGG GTT CAG TAC TCG ACA GAC
RRA GCC TTG TAT ATA TGG CAA CCA GAC TTT GCT CAR GRR
TTT CGG AAC ATA TAT ACC GTT GGT CTG AAA CGA GTT CTT
ATC CTC CTT CAT GGT TAG CAT TTT CAC AGC TAT ACT AAT
TAG GAG GAA GTA CCA ATC GTA A M GTG TCG ATA TGA TTA
CTT ATG ACA GAG CA& GGT ATT TAC AAU GTT CAC-3'
GAA TAC TGT CTC GTT CCA TAA ATG TTT CAR GTG-5'
One drawback of these primers is that they yield a very small PCR fragment that may
be confused with primerd'imcr pairs. As a result, we have designed a sccond set of primers

for PCR amplification of a 441 bp spific DNA fragment from HPV-moo (submitted to
DAO). These primers have also been used to produce a 441 bp digoxygenin labeled specific
probe for HPV by PCR using the 732 bp cloned fragment as a template. These methods are
currently being applied in Thailand for detection of HPV DNA in extracts derived from PL
and faeces (Fig. 22) by PCR (Fig. 23) and dot blot hybridization (Fig. 24) and they are
available commercially corn the Shrimp Biotechnology Business Uoit, National Center for
Genetic Engineering and Biotechnology. Bangkok The primer squcnces are:
primer HPV441F: 5' ACA CTC AGC CTC TAC CTT GT 3'
primer HPV441R: 5' GCA TTA CAA GAG CCA AGC AG 3'
and the PCR protocol is: 95°C for 5 min followed by 40 cycles of 95°C for I min. 60°C for I
min and 7Z°C for I min with final extension at 7Z°C for 7 min. The expected amplicon is
441bp. Other primer sequences and in sihr probes have been described as well (Pantoja &
Lightner 2000,2001).
One major mystery that remains unsolved is the source of ELPV in the farming system.
We have never seen it in hatchery larvae (unpublished) and then are, as yet, no published
reports of it there. Nor did we fmd it in captive broodstock (Flegel eta/. 1997b) although the
sample was small and we used histology only since we did not have PCR techniques at the
time. The fust place we have seen it with certainty is in nursery tanks to which the post
larvae are transferred from the hatchery for outdoor acclimatization before stocking in shrimp
ponds. This suggests that there may be existence of an unknown reservoir carrier(s) of the
virus. DNA probes would be useful in identifying this carrier(s) as a prelude to excluding it
from the cultivation system.
Figure 22. Pracccurcs for
PL AND FAECES SAMPLE PREPARATION preparation of DNA cxtracts
FOR HPV DETECTION
from PL and faeces for PCR
detection and dot blot detection
of HPV and other DNA viruses.
in 0.05%NaOH+
Sunple in Lysis buffer
The lys~s solution on the nghl
o.ozs%sDs can be used with fresh material
4 4 only but has the advantage that
no DNA extraction step is
Phenol-chloroform boil for 10 min necessary. Normal lysis buffer
DNA extraction t
(50 mM Tris-HCI pH 9. 100
centrifuge St3OO rpm mM EDTA, 50 mM NaCl, 2%
1
l0dn 4 SDS and Protcinasc K 1 uglml
added immediately before wc)
DNA
supernatant
has the advantage that samples
can be stored at room
I 4
temoerature for several years
PCR and Dot blot hybridhation
without degradation, but DNA
extraction is necessary.

Sensitivity of HPV-PCR
in NS boiled faeces or PL
Pigore 23. Example of PCR
detection results for boiled PL or
faeces spiked with HPV DNA at
Figure 24. Example of a dot blot
assay for HPV using ffesh faeces or
PL samples boiled in sodium
hydroxide1 SDS solution.
Dot blot sensitivity with
NS boiled faeces nnd PL
Figure 24% Negatively sta~ned trausmlsslon
electron micrograph of purified virus part~cles
from HPV-mon. Bar = 50nm. Like other typical
parvovimses, the virions are unenveloped,
icosahedral and very small (around 24 nm in
diameter).

Infeeitous hypodermal and h.ematopoeitie necrosis vim6 (IHHNV)
There is little hard data available for this virus in Thailand (Flegel el 01. 1995a).
However there is some circumstantial evidence for its presence. First of all, IHHNV is
probably endemic in Asian P. monodon (Ligbtner 1996; Lighiner & Rcdman 1991).
Secondly, we have found histopathology typical of the vim on one occasion in apparently
healthy shrimp (Flegel er d 1992b) (Fig. 25) and the relevant specimens were confmed for
WiMr by in situ hybridization (D.V. Lightner. unpublished). Third, we have frequently
cncountercd paracrystalline arrays of 23 nm virus-like particles, which might be those of
IHHNV, during examination of lymphoid organ specimens by electron microscopy (Flegel er
a/. 1997b) (Fig 25). Fourth, Thai shrimp fanners have complained about the increasing
tendency for wide size variation in harvested crops, a feature of IHHNV infection ~n P.
vannornei in which IHKNV infection causes runt deformity syndrome rather than massive
mortality (Lightner 1993, 1996). We still do not know whether MHNV has any impact on
the Thai shrimp culture industry. The situation requires further study, and commercial DNA
diagnostic probes developed for IHHNV in Dr. Lightnet's laboratory are available for rapid
detection by in situ hybridization or by PCR assay using shrimp haemolynph @iagXotics.
Wilton CT). We have hied these reagenls and they do work with Thai material (Fig. 26), so
there are no tcchoical obstacles to the work.
Figure 25. MHNV from Thai shrimp. The photomicrograph on the
left shows one nucleus of the anlennal gland with a lypical IHHNV
Cowdry-A type inclusion (red, acidophilic, central inclusion
surrounded by a clear wne and then a ring of marginated purple,
hasophiic, chromatin). The electron micrograph on the right shows
what may be MHNV viral particle arrays from lymphoid organ tissue.
The viral particles in the array are approximately 23 nm in diameter.
One curious feature of this virus is what appears to be P. monodon's high tolerance to
it. The Thai specimens in which we found typical IHHNV histopathology came from a
feeding test group that exhibited normal growth aod survival (Flegel and Sriurairatana 1994).
Yet in situ hybridization tests gave very strong positive reactions, indicating a heavy viral
infection (D.V. Lightncr, personal communication). Unfortunately, no b~oassay could be
performed using P. stylirostris with these specimens. There are also anecdotal reports of
LHHNV tolerant or "mistant" P. stylimstris from Tahiti which were found to transmlt
virulent IHHNV to naive P. stylimfris in bioassay trials (D.V. Lightner, personal
communication). This scenario seems lo have common features with the accommodation to
WSSV and YHV in Thailand, and in the case of IHHNV, it is clear that it the shrimp have
changed, not the virus.

Primers for the PCR detection of MHNV appear in the OIE Diagnostic Manual for Aquatic
Animal Diseases (2000). These give a 356 bp MHNV specific fragment. The sequence of the
pruners is as follows:
Sense :
Antisense:
5'-ATC GGT GCA CTA CTC GGA
5' TCG TAC TGG CTG TTC ATC
It is possible that PCR labeling of the 356 bp 6agment would yield a probe suitable
for either dot blot DNA hybridization or in sifu hybridization, hut this would have to be
ascertamed by appropriate testing.
The full sequence of MHNV is now available at GenBank and we have done a
comparison of our HPV-mon sequence and the full sequence oTHPV-chin at GenGanL with
that of LHHNV. Although HPV and IHHNV are both parvovimses, rt was luteresting to
discover that there was no significant homology between the sequences of the HPV strains
and that of I HW. We have also done comparative dot blols with HPV and l H W probes
and targets and we have found no cross-hybridiition.
Figure 26. Example of a positive in situ hybridization reaction of DiagXotics commercial
probe for IIiHNV with shrimp 'om Thailand. The photomictograph on the left sllows a
negative reaction with normal uninfected nerve tissue, wh~le that on the right shows a positive
reaction (blackened areas).

Taura syndrome virus VSV)
The newest viral pathogen to arrive on the Asian scene is Taura syndromc virus (TSV)
(Lightner 1996; Tu el al. 1999). Taura syndrome was fmt recognized as a new disease in the
Americas in 1992 but its viral etiology was not established until 1994 (Brock et a/. 1995;
Brock era/. 1997; Hasson el a/ 1995). The causative agent has been tentatively classified as
a member of the Picomiviridae because it is an unenveloped. 32 nm icosahedral virus
containing a 10.2 kb ssRNA genome of positive sense (Bonami er 01. 1997). TSV infectious
present characteristic gross pathology io P. vannamei that can serve for presumptive
diagnosis (Fig. 27). These include reddening of the tail fan and visible necrosis of the
epithelial tissue there in the acute phase of the disease. In the recovery or chronic phase of
survivors, black lesions in the cuticle are often found However, histological examination is
required for confirmation of the disease (Fig 28). By TEM icosahedral virions can be seen in
the cytoplasm of infected cells (Fig. 29).
Figure 27. Cross signs of Taura syndrome. On the left is a tail fm of P. vannaplei with
reddish necrotic areas (arrow). The right photo shows black lesions in the cuticle
characteristic of the chronic stage of TSV infection (Lightner 1996).
Figure 28. Histopathology of TSV i nfed P. vannemei in the acute phase of infection. Note
the large masses of spherical, cytoplasmic inclusions that begin as eos~nophilic to light
basophilic bodies and later become intensely basophilic.

Figure 29. TSV by
- TEM. The electron
micrograph on the left
shows TSV in the
cytoplasm by notmal
TEM using thin tissue
sections from TSV
infected P. vannamei.
The micrograph on the
right shows purified TSV
virions by negative
staining. (From (Nunan
et aL 1998).
TSV outbreaks were frst reported in Aisa from Taiwan where P. vannemei had been
imported live as fry and brooders for use in commercial aquaculture ponds (Tu ef a1 1999) 11
was probably introduced with the imported stocks but molecular epidemiological tests would
be needed to confirm this. Although TSV is not highly lethal to P. monodon, ~ts effect on
other species of Asian shrimp is not known.
Molecular DNA methods for the detection of TSV have been reported (Mari ef a/.
1998; Nunan ef a/. 1998). Primers for an RT-PCR method that yields a 231 bp TSV specific
fragment (Fig. 30) have been reported (Nunan eta/. 1998). The primers are:
Sense: 5' TCA-ATG-AGA-GCT-TGG-TCC 3'
Antisense: 5' AAG-TAG-ACA-GCC-GCG-CTT 3'
Figure 30. Agarose gel of PCR products
I from shrimp infected with TSV. Lane 1.
100 bp ladder; lanes 2-8, samples from
shrimp injected with TSV and infection
confumed by histology; lane 9 posctlve
TSV control; lane 10, negatlve control
sample from prc-injection shrimp (Nunan et
a/. 1998).

~briopar~haemoIyticus and Y. penaecida
Species of Vibrio are often the cause of shrimp death. although we believe that they
are usually opportunistic pathogens that overcome shrimp defenses when they are weakened
by some sort of predisposing shes (Flegel et al. 1995a). The way to solve the problem is to
remove the cause of the underlying stnss. The main Vibrio species responsible for shrimp
mortality are Y. parahaernolyticus, V. harveyi, V. wlnificur, and perhaps Y. panaeicida. We
fust worked on V. parahaemolyticus because it is also a serious human pathogen and a target
for screening of frozen shrimp for export from Thailand A probe was developed by random
cloning and selection procedures and it was subsequently utilized for a rapid and sensitive
PCR detection that can be used directly with shrimp haemolymph samples (Rojlorsakul el al
1998) (Fig. 31). This system cnn be used to nondestructivtly check shrimp haemolymph
samples without the necessity of traditional steps for bacterial isolat~on, purification and
nutribonal testing to ob:ain an identification. Like the other PCR assays described, the
process takes only a few hours and contrasts sharply with the traditional methods which
require several days and may risk misidentification of atypical strains. It is hoped that a cheap
multiplex system will eventually be developed which would allow for the simultaneous PCR
assay for all of the major Yi6rio pathogeas, but this will probably take a few years lo reach
fruition.
Figure 31. Agarose gel of
PCR producb horn shrimp
haemolympb samples containing
cells of V. parahoemolyticus.
The highest
sensitivity seen in the gel is
for lane 8 using a sample
containing 2 x 10%acterial
cells per ml of haemolymph
and 100 cells per
PCR reaction vial.
The PCR primers that yield the 285 bp fragment are as follows:
Sense:
Antisense:
5'-GTT ACG CAC AGA TGC GAC AT-3'
5'-CTT GTG GAT TGG ATT CTC GC-3'
Another species of bacterium that appears to cause high mortality in P. monodon is V.
penaeicida (Ishimam et a/. 1995). Tbis bacterium does not grow well on TCBS agar and may
sometimes be overlooked in examining shrimp for bacterial infections. An RT-PCR method
bas been published for V. penaeicida 6om Japan (Gonmoto ef a/. 1996) and a PCR method
has been published for V. penaecida !om New Caledonia (Saulnier et a/. 2000).

The microrporldian Agm~~ontrr pm~ci
This intracellular parasite infects both P. monodon and P. merguiemis, but apparently
does not usually cause very high mortality (Flegel er al. 1992a). - It is most damaging because
it disfigures the shnmp with white discoloration of the musculature ("white-back" or
"cotton" shrimp) and reduces the selling price considerably. The parssite scuns to be a bigger
problem with P. merguiemir and it is probably the major reason that it cmot be used as an
alternate species to P. monodon, even when higher prices for it or its resistance to yellowhead
virus infection, for example, would make its cultivation advantageous.
To address this problem, we have hed to fmd the source of the pathogen in the shrimp
cultivation system. When diict infection tests between shrimp failed, we began to look for an
intermediate host. To do this. DNA diagnostic reagents were developed and used to screen
potential reservoir (i.e., alternate) hosts (Pasharawipas & Flegel 1994; Pasharawipas er a/.
1994; Pasharawips et a/. 1997). By this process, two fish species (Scatophagus argus and
Priacanrhus fayenus) were identified as potential hosts (Fig. 32). However, bioassay tests
with one of these (Scaiophaguc argus) and shrimp have not yet been successful in closing the
life cycle of the pamite, so the issue of the allernate host is still open.
Figure 32. Agarose gel of PCR products using
Agmasoma specific primers with DNA extracts
from Scatophagus orgus and Priacanthus rayenus
as the template. Lane M = molecular marker;
lane 1 = 600 bp product from Agmasoma positive
control tcmplatc; lanes 2-5, no product from
various negative controls including bacterial,
protozoan and noml shrimp DNA templates;
lanes 6 and 7, positive PCR product from the
respective fd DNA templates.
Additional Qure. The photograph on the left shows the gross appearance of white shrimp
tissue infected with the microsporidian Agmasoma penaei. The photomicrograph on the right
shows a fresh mount from infected tissue with immahlre sporoblasts stained with malachite
green while mature sporoblasts with 8 spores each are refractory to staining.

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