MANAGEMENT STRATEGIES FOR CONTROLLING DISEASES IN

SHRIMP AQUACULTURE
Hassan Rostamian
fidep@qfpco.com
Preface
Shrimp aquaculture has provided opportunity for economic and social upliftment of
rural communities in the southern coastal area of our country.
Recent spread of highly Pathogenic viral diseases of shrimp, have accompanied
movements of shrimp aquaculture development.
More recently outbreaks of white spot disease (WSD) which caused by the white spot
syndrome virus (WSSV) in several part of Bushehr province (Helleh site) is a direct
result of the ill-considered of infected shrimp in our country.
Therefore, adoption of proper shrimp health management practices, based of the
principles of good aquaculture practice, is the first step towards this direction.
Abstract
This document presents the report of the management strategies for controlling
diseases in shrimp aquaculture. Also included the summery of the discussion and
recommendations for national and regional policies, legislation, and regulatory
frameworks for reducing the risks of disease outbreaks in shrimp aquaculture.
The most important step of this document is adoption of proper shrimp health
management, based on the principles of good aquaculture practice.
1- History of global disease outbreaks
At the same time as environmental concerns have increased, many diseases have
proliferated to threaten the shrimp farming industry. In 1989 there existed only six
world-wide shrimp viruses. By 1996 the number rose to over twenty.
The first outbreak of viruses on a large scale hit Taiwan 1987, when production was at
37 million pounds. By 1998, less than 1 million pounds were producted in Taiwan due
to continued disease outbreak.
WSD outbreaks were first reported from farmed Penaeus Japonicus in Japan in 1993
and the causative agent was named penaeid rod-shaped DNA virus (PRDV) or rodshaped nuclear virus of P.Japonicus (RV-PJ) later, outbreaks of viral disease with
similar gross signs and caused by similar rod-shaped viruses were reported from
elsewhere in Asia and other names were applied: hypodermal and haematopoietic
necrosis baculovirus (HHNBV) in the people s Republic of Chain; white spot
baculovirus (WSBV) and PmNOBIII in Taipei China; and systemic ecotodermal and
mesodermal baculovirus (SEMBV) or PmNOBIII in Thailand.
The virus from the people s Republic of Chain has also been called Chinese
baculovirus (CBV). Shrimp exhibiting the gross signs and histopathology of WSD
have also been reported from Korea, India, The Philippines and the USA. During
1999, WSD also had a severe impact on the shrimp industries of both Central and
South America.
Because of similar gross signs, ultrastructure and molecular biology, Lightner has
included these virus (WSSV) complex, which is refrred to have simply as white spot
virus or WSSV. In Iran the first official confirmation date of white spot disease event
is on 23th June 2005 in Bushehr Province (Helleh site), but the previous outbreaks of

white spot disease in Iran reported to OIE in 2003.

Shilna-Iran fishery organization (Shilat) in an interview with Mr.M.Ghodsi (The
president of shrimp farms union) reported that the the first event of WSD has been
happened 4 years ago in Choybdeh site (Abadan) in Khuzestan province, and the
other shrimp farms in Bushehr Province already has been affected by WSD in August
2005, such as Boyrat, Mond,

2- What Is White Spot Disease (WSD)

The WSD is caused by the white spot syndrome virus (WSSV) and affect shrimps of
all age groups. The WSD outbreaks are often characterized by high and rapid
mortality of infected populations, shortly after the first appearance of the clinical
signs. Diseased shrimp develop anorexia, lethargy and characteristic white spots on
the inside surface of the carapace. Moribund shrimp may also show a pink to red
discoloration. There may be reduction in feeding levels.
White spot syndrome virus (WSSV) has a wide host range among crustaceans and is
potentially lethal to most of the commercially cultivated penaeid shrimp species.
Virions are rod-shaped to elliptical with a trilaminar envelope and they are large (80120 250-380 nm). Negatively stained virions purified from shrimp haemolymph show
unique, tail-like appendages. The virions are occlusion bodies. In initial reports,
WSSD was described as a non-occluded baculovirus, but even while the molecular
data were still limited, the preliminary WSSD DNA sequence analysis, the
morphological characteristics and the general biological properties of the virus
already highlighted its uniqueness. Recent data, including the genome sequence and
phylogenies based on DNA Polymerase and Protein Kinase, suggest that WSSV is a
member of a new virus family. The status of work on WSSV has been reviewed by
Lightner, Flegel et al. Lob et al, Lo & Kou and Lo et al.
The size of the WSSV genome has been differently reported for different isolates.
305107 bp,292967 bp, and 307287 bp for viruses isolated from the people s Republic
of China, Thailand and Taipei China, respectively. The sequences of these three
isolates are almost identical, with the size differences being due mostly to several
small insertions and one large deletion.
For two of these isolates, the analysis of the complete WSSV genome has been
published. The following descriptions are based mostly on the WSSV isolate from the
people s Republic of China. The WSSV genome has a total G+C content of 41%.
About 3% of the WSSV genome is made up of nine homologous regions containing
47 repeated mini-fragments that include direct repeats, a typical inverted repeat
sequence is unique.
Total of 531 putative Open Reading Frames (ORFs) were identified by sequence
analysis, among which 181 ORFs are likely to encode functional proteins.
Thirty-six of these 181 ORFs have been identified by screening and sequencing to
encode functional proteins. Transcription of another 52 ORFs was confirmed by
reverse-transcription polymerase chain reaction (RT-PCR).
For 80% of the putative 181 ORFs there is a potential polydenylation site (AAT-AAA)
downstream of the ORF. Confirmation of WSD requires more detailed analysis by
PCR, antibody-based assays, in-situ DNA hybridization, or transmission electron
microscopy (TEM).

Using such techniques, it has been shown that WSSV from captured juvenile and
brood stock shrimp or other carriers are identical or closely related. These molecular
techniques have also been used to confirm infection of more than 40 penarid and nonpenaeid crustacean carriers and, tentatively, an aquatic insect larval carrier. Some of
these carriers have been shown to transmit WSSV to shrimp in laboratory tests.
Detection of WSSV in carrier shrimp or other crustaceans cannot be reliably
accomplished by histological methods, and more sophisticated techniques are
required.
3- What is the Source and Causation of White Spot Disease.
The WSSV has been established as the necessary cause of the WSD.
However, presence of the necessary cause alone will not lead to a WSD outbreak in a
pond. In a farm situation, a number of component cause (risk factors) along with the
necessary cause might become sufficient cause to produce WSD outbreaks.
The recent studies clearly show that WSD is not caused by any one factor Rather a
number of risk factors influence the occurrence of WSD in the farm. These risk
factors occur throughout the shrimp cropping cycle and in general terms full into the
following categories during the different stages of the corp cycle:
Pond preparation
Pond filling and water preparation
Seed quality and screening
Water management
Pond bottom management
Feed management
Disease treatment
The risk factors at each stage of the cropping cycle and their relationship to WSD
outbreaks are illustrated in chart source and causation of white spot disease
(Please refer to Annex to see the Chart No. 1)
4- Guide lines for Controlling White Spot Disease In Shrimp Farms
4-1 Pond Preparation
Pond preparation is essential to reduce risks of shrimp disease outbreaks. The
following risk factors that can significantly reduce the risk of disease outbreaks and
improve shrimp production.
Removal of bottom sludge, Particularly in ponds stocking higher densities (up to 8
numbers Per m .
Ploughing of soil when wet (Particularly at higher stocking density farms).
Use of lime in pond preparation.
Shrimp ponds with a history of disease outbreaks have a greater likelihood of future
disease outbreaks, therefore special attention is required during pond preparation in
such farms.
Farms with poor bottom soil quality, Particularly the presence of a black soil layer,
will suffer crop failures.
4-1-1 Sludge removal
The sludge must be disposed away from the pond site. So that it dose not seep back
into waterways, ponds, or cause other environmental problems. In farms the sludge,

unless there was disease outbreak during the last crop. In such a situation extra
precaution should be taken.
Sludge removal should pay attention to areas of the pond where there is a high
accumulation of organic matter from pervious crop, such as feeding areas, and the
side ditch in extensive farms.
If the sludge is removed properly then management of the pond becomes easier
during high pH periods, due to low salinity, and plankton growth.
4-1-2 Ploughing
The main purpose of Ploughing is to expose the black soil layer underneath bottom
soil to sunlight and atmospheric oxygen. By this process, the organic waste (sludge)
will be oxidized. Ploughing on wet soil is particularly recommended for ponds if the
planned stocking density is between 6 and 10 PL/ m and when the sludge cannot be
removed properly by manual or mechanical methods.
After ploughing, dry the pond bottom for 5 to 7 days and report the procedure till no
more black soil is seen. In case a heavy tractor is used for ploughing, then plogh the
dry soil and then fill the pond with water to wet the soil and then again dry.
Ploughed pond bottom leads to turbid water conditions during culture period.
Therefore, compaction of the bottom using heavy rollers after the whole process of
pond preparation, i.e., before water intake, can avoid the turbid water condition.
4-1-3 Liming
Liming during pond preparation optimizes pH and alkalinity conditions of soil and
water. The type and amount of lime to be added depends mainly on the soil pH and
also on pond waters pH, which ideally should be checked before lime application.
Quick lime or should be used only if the soil pH is low i.e. pH <5. If it is applied on
soils of pH>5, then it may increase the water pH after filling and this high water pH
condition may remain for a prolonged period even after stocking, which is not
desirable. If the soil pH is more than 5, then shell lime or agricultural lime dolomite
should be applied.
(Please refer to Annex to see Table no. 1)
4-2. Pond filling and water preparation
There are management practices that can be adopted to reduce risk factors associated
with pond filling and preparation of water before stocking. These include:
Water filtration (mesh of 60 holes/sq inch) reduces the risk of disease outbreak
through reduced introduction of carriers to the pond.
Disinfection of pond water can also reduce the risk of disease outbreaks in farms
using higher stocking density.
Fertilization reduces the risk of disease outbreak in lower stocking density farms.
The use of water reservoir appeared to have no significant benefit to farmers in either
reducing risk of disease outbreaks or improving production. As reservoirs are
normally good to improve water supply quality in shrimp farming, the findings
suggest that reservoirs are not being used properly.
The proper use of reservoirs is therefore strongly recommended as disease control

measure and to make water management more effective during the crop cycle.
4-2-1 Reservoir maintenance and pond filling
Reservoir pond for at last 14 days before pumping to the shrimp culture ponds to
facilitate the growth of plankton in the reservoirs. This water can even be used to fill
grow-out ponds just one to two days before stoking with seeds. If the water reservoir
is not maintained then the grow-out pond should be filled directly with source water at
least 14 days before stocking.
During water intake the cultured pond, water inlet point to avoid entry virus carriers
such as crabs, wild shrimps and zooplankton and also to avoid entry of fish or
crustacean, which may be predator or competitor for shrimp. The suction line should
be placed at the deeper side of the reservoir pond and the foot valve of the pump
should be kept at least half a foot above the pond bottom to avoid turbidity during the
pumping process.
4-2-2 Fertilization of water
Inorganic fertilizers like urea or superphosphate at 30-50 Kg/ha in 3-4 dosages must
be used to boost the plankton production.
Fertilizer should be first applied at least 10 days before the planned stocking date so
as to obtain a good plankton bloom with green water color for stocking. It the water
color remains unstable, fertilizer application should be continued even after stocking.
This would avoid a risk of plankton bloom collapsing suddenly.
4-3 Seed Selection and Stocking Process
There are several important risk factor associated with shrimp seed, which include:
Stocking of poor quality of seed (less active, more mortality during transportation
and size of less than 16 mm in case of nursery reared juveniles) increases the risk of
shrimp disease outbreak.
Higher prevalence (>5% of seed population) of WSSV as determined by two-step
PCR in stocked post-larvae leads to increased risk of an outbreak and lower pond
production.
Longer transport time (>6 hours) of the seed from hatchery or nursery to the pond
also increases the likelihood of a subsequent disease outbreak.
On farm nursing of shrimp post-larvae helps to reduce disease outbreak.
Several management can be used to reduce the risk as follow:
A- Before purchasing, shrimp post larvae should be checked for their general
condition at the hatchery. Observations should be made o n activity, color, size, etc
from the selected tanks in the hatchery. The post larvae should be uniform is size with
relatively uniform body color and should be actively swimming against the swirling
water current produced in a round tab. If there are any dead and abnormal colored PL
in the tank, the entire batch should be rejected.
B- Once the post larvae pass these gross visual examination tests, a PCR test should
be conducted on 59 randomly selected post larvae from that tank. Using 59 post
larvae will allow detection of WSSV at 5% or more prevalence level. If the sample
shows negative result by following 2 step PCR then the seed is ready for
transportation to the farm for stocking.

C- The post larvae should be transported to the farm site within the shortest travel
time, which should not exceed 6 hours. Bags should be packed with relative low post
larvae densities to minimize stress.
The recommended densities of seed in transportation bags are as follows:
PL 15= 1000 to 2000 PL/Liter
PL 20= 500 to 1000 PL/Liter
D- Weak and dead PL should be removed before stocking at the pond site. A
separation method should be employed. To do this PL from transport bags should be
carefully transferred into a plastic/fibre-glass tank of around 500 liter water holding
capacity. Then, formalin should be mixed at the rate of 100ml/1000 liters of water to
get a formalin concentration of 100 ppm and the PL should be treated for above 30
minutes. It is essential that water in the tank is well aerated; ideally using oxygen
cylinders. After this treatment the water is stirred to concentrated all the dead and
weak seeds at the center-bottom of the tank. There after the good/strong Pl should be
siphoned off using a plastic pipe from the upper portion of the tank, suitably from the
sides. This process should be continued till around 3/4 of the water volume gets
pumped out so that the bottom settled PL could be seen clearly. The post larvae
concentrated in the center should then be siphoned out using a thinner plastic tube.
The farmers are also advised to use the following precautions:
Formalin should be used only when aeration is available if no aeration is available,
then follow the above process but without formalin.
In case molting is observed during transportation or many of the post larvae have
died, formalin should not be used.
If the seed comes from a nursery then the juveniles should be treated with 150ppm
formalin (150ml/ton of water) for 15 minute. The weak juveniles cannot be separated
easily as they are quite big and can withstand the above dosage. Any further increase
in formalin concentration may stress healthy shrimp. This treatment is helpful in
reducing the external parasites and fouling only.
E- During the above process of weak seed elimination, PLs should also have to be
acclimatized to pond water conditions like salinity with gradual addition of pond
water to the tank.
The whole process of seed selection (hatchery visit, PCR testing, transportation and
screening should be done at least 2-3 days prior to stocking.
4-4 Water Quality management
A number of risk factors that are significantly related to shrimp disease outbreaks and
shrimp production and can be addressed through routine water quality management.
These include:
Water exchange practices pond exchanging water to maintain water quality
appeared to yield better shrimp production, particularly in low stocking density farms.
Water filtration-ponds using water filter nets of fine mesh have better production.
Aeration-ponds using aeration tend to have higher shrimp production.
High salinity and pH (>8.5) have an affect on risk of disease outbreaks, but the study
found that salinity had less influence on shrimp production. In high saline waters it is

difficult to maintain water quality, especially a stable bloom compared with that in
low saline waters. Therefore, the stress on shrimp due to changes in bloom conditions
may make it more susceptible to viral infection and subsequent disease outbreak. In
case where pH exceeds 8.5, the toxicity of ammonia increases leading to higher stress
condition for shrimps. The studies showed significant relation between shrimp disease
outbreaks and algal populations within ponds:
Ponds with clear water at stocking and during the culture cycle are at risk from
lower production and shrimp disease outbreaks.
Green water (pond colour) ponds have better production and lower risk of disease
outbreak.
Clear water with benthic and filamentous algae lead to lower production.
Ponds with dead benthic algae observed during culture are risk of disease and poor
production.
The management recommendations are directed towards maintaining a healthy algal
bloom in the pond. Sufficient water must be kept in the pond to reduce risks of the
growth of benthic algae. The water depth in the shallowest part of the pond should
beat least 80 cm.
Ideally, water quality should be checked on daily basis for the following parameters
and records kept in a recording sheet as below.
(please refer to Table no. 2)
4-5 Pond Bottom Management
The studies did not find any direct relationship between the condition of pond
sediment and risk of disease outbreaks or production. However, in order studies the
occurrence of black and toxic bottom sediments has been shown to adversely affect
shrimp health and lead to disease outbreak or poor production.
There is a relationship between ponds with higher stocking densities and feeding
rates, and poor pond bottom conditions. Therefore, as farmers stock with higher
density, more attention must be given to pond bottom management.
The pond bottom soil should be checked on weekly basis, especially at the feeding
area or trench.
The occurrence of black soil, benthic algae and bad smell should be recorded. If the
soil is black and smelly, water exchange should be carried out and feed reduced (using
a feed tray to monitor requirements). During water exchange, the feeding area and
where black soil occurs should be midly and carefully agitated to dislodge the soil
from the pond bottom. This will facilitate its drainage from the pond. Special attention
has to be given to management of feed to reduce wastage and avoid deterioration of
pond bottoms.

4-6 Feed Management

The studies showed no significant relationship between amount of feeding patterns
and shrimp disease outbreaks. However, good feeding practice is essential to maintain
water and soil quality and a healthy environment within the ponds.
Peelt feed should be given according to a fixed schedule. This schedule will depend
on the body weight of the shrimp and the feed tray result during previous meal. On

the basis of body weight, the feed amount should be calculated. For this purpose the
feed tables given on the feed bag by the manufacture can be used. If fresh (cooked)
feed such as snail or trash fish (but not crustacean based fresh feed) is used during the
later stage of cycle to improve the growth, it should be only under close monitoring of
water quality and a careful feeding strategy to avoid wastage.
Feed trays should be introduced after one week of stocking and should be used to
check general condition of the shrimp of the shrimps during the first month. From 30
days of culture (DOC) onwards, feed size should be changed according to the actual
size of the shrimp. A mix of two feed pellet sizes should be used for at least 7 days if
there is any size variation during the regular check-up. Feeding area should be
changed at least once in 10 days depending on the bottom condition along feeding
area. This allows shrimps to feed in a clean area.
4-7 Treatments and use of chemical
The study showed that many chemicals were used against shrimp disease without any
beneficial effect. Farms with higher stocking density tend to use more chemicals, both
in terms of numbers and the quantities applied. The study showed that antibiotics and
a range of probiotics had no significant effect on the risk of shrimp disease outbreaks.
Use of lime, fertilizers in low input systems, and disinfectants in farms using higher
stocking densities had some protective effect against shrimp disease.
Feed additive including vitamin and mineral premix as well as some bacterial
products had some beneficial effect on shrimp production in higher stocking density
farms.
There is a concern that in low input farms, pesticides maybe used because they lead to
residues in harvested shrimps which are hazardous for human health. Only calcium
hypochlorite (bleach) should be used as disinfectant. Other chemicals such as lime
compounds, fertilizer, zeolite and related compounds that do not lead to residues in
the harvested shrimps can be used to manage the pond condition and shrimp health.
It has been clearly shown that shrimps of good quality can be grown with very
minimal chemical usage and without the usage of antibiotics. Minimal usage of
chemicals and no usage of banned chemicals lead to reduced cost of production and
easy marketing of the harvested shrimp in domestic and export markets.
There is a serious concern on the use of antibiotics, and their use in shrimp farming
should be avoided.
The following antibiotics and pharmacologically active substances, which are banned
for use in aquaculture should not be used in any circumstances:
1. Chloramphenicol
2. Nitro furans including Furazolidone, Nitrofurazone, Furaltadone, Nitrofurantoin,
Furylfuramide, Nifuratel, Nifuroxime, Nifurprazine and all their derivatives
3. Neomycin
4. Nalidixic Acid
5. Sulphamethoxazola
6. Aristolochia spp. And preparations thereof
7. Chloroform
8. Chloropromazine
9. Colchicine
10. Dapsone
11. Dimetridazole
12. Metronidazole

13. Ronidazole
14. Ipronidazole
15. Other nitromidazoles
16. Clenbuterol
17. Diethystilbestrol (DES)
18. Sulfonamide (except approved Sulfadimethoxine, Sulfabromomethazine and
Sulfaethoxypyridazine)
19. Fluoroquinolones
20. Glycopeptides

5- Conclusion
Closed-cycle breeding programs to produce PL that are certified free of specified
endemic and exotic prawn viruses will improve disease security. This, combined with
treatment of pond inlet water and good control of water quality on farms, should
minimize the risk of disease. Further work is necessary to identify all potential viral
pathogens and to develop sensitive and specific tools for their detection.
Ultimately, intensive semi-closed farming systems using high quality SPF,
domesticated and genetically selected stock will be a forward way.
Planners, policy markers and the industry must site face to face to discuss strategies
and options and come up with a pragmatic and practical approach to sustainable
shrimp farming. Traditional shrimp farming is a case in point.
The Shilat (I.R.IRAN Fishery Organization), with the help of state and district
authorities, has to regulate shrimp farming through licensing of farms meeting the
basic environmental and social criteria laid down by the experts.
On the other hand, shilat and its fisheries research institutions are addressing various
relating to technical and commercial aspects of sustainable shrimp farming and a plan
for sustainable development of coastal aquaculture should be developed, focusing on
a comprehensive health management strategy for shrimp farming.
Enforcement of coastal area management regulations that are relevant to shrimp
culture is needed, including increased monitoring and law enforcement to maintain
environmental quality, development of law authority capacity, extensive
dissemination of information on laws and regulations to local communities; increased
effectiveness, co-ordination and integration of law enforcement; improvement and
completion of regulations that have not been defined in detail, and encouragement of
community capability and awareness in implementing regulations.
Assessment of shrimp culture production, including small-and large-scale hatcheries,
grow-out pond, production toward year 2006. THE following research and
development activities cover priority elements to be included:
Identification of a shrimp health management program, including aspects to pond
construction and irrigation systems, water quality management, feed and nutrition,
disease and mortalities, disease control and health management, drugs and chemical
treatment, and genetics.
Assessment of shrimp health management services, including facilities at individual
farms and hatcheries, for disease diagnosis and control, provision of guidance for

disease control, national and regional facilities for diagnosis and control, a net work
system for disease control services, and research and field-testing facilities for disease
control.
Allocation of necessary supplies, equipment, and other required assessing the shrimp
health management status.
Overseas and local training in shrimp disease diagnostics and health management
and training for extension specialists, farmers and hatchery operators.
Some actions needed to obtain reliable information on identified shrimp health
management problems in order to establish substantial shrimp culture follow:
Establishment of national diagnostics and disease control systems, to be liked with
other countriesnetworks.
Establishment of specific laboratory and research activities for shrimp health
management.
Strengthening of training and research capabilities.
Supporting information systems for research and training via a regional Shrimp
Health Management (SHM) unit in NACA.
Establishment of a website for recent shrimp disease diagnostics and control
measures, so that member countries can immediately access needed information.
Organization of regional annual meeting and workshops on shrimp health
management.
Conducting a collaborative project among member countries on various aspects of
disease diagnosis and control.
6- Acknowledgements
I would like to give my special thanks to Mr Mohammad Sahari for his participation
and cooperation in this paper.
7- References
1. Bairoch, A., Bucher, P., and Hofmann, K. (1997) The PROSITE database, its status
in 1997. Nucleic Acids Res. 25, 217 221
2. Bell T.A. & Lightner D.V. (1988). A Handbook of Normal Penaeid Shrimp
Histology. World Aquaculture Society, Baton Rouge, USA, 114 pp
3. Chang P.S., Lo C.F., Wang Y.C. & Kou G.H. (1996). Identification of white spot
syndrome associated baculovirus (WSBV) target organs in the shrimp Penaeus
monodon by in-situ hydridization. Dis. Aquat. Org., 27, 131-139.
4. Chen L.L., Chen H.C., Huang C.J., Peng S.E., Chen Y.G., Lin S.J., Chen W.Y., Dai
C.F., Yu H.T., Wang C.H., Lo C.F. & Kou G.H. (2002). Transcriptional analysis of the
DNA polymerase gene of shrimp white spot syndrome virus (WSSV). Virology, 301,
136-147.
5. Chen L.L., Leu J.H., Huang C.J., Chou C.M., Chen S.M., Wang C.H., Lo C.F. &
Kou G.H. (2002). Identification of a nucleocapsid protein (VP35) gene of shrimp
white spot syndrome virus and characterization of the motif important for targeting
VP35 to the nuclei of transfected insect cells. Virology, 293, 44-53.
6. Chen, X. F., Chen, P., and Wu, D. H. (1997) Study on a new bacilliform virus in
cultured shrimps. Sci. China Ser. C 27, 415 420

7. Chou H.Y., Huang C.Y., Wang C.H. Chiang H.C. & Lo C.F. (1995). Pathogenicity
of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in
Taiwan. Dis. Aquat. Org., 23, 165-173.
8. Claros, M. G., and von Heijne, G. (1994) TopPred II: an improved software for
membrane protein structure predictions. Comput. Appl. Biosci. 10, 685 68.
9. Durand S., Lightner D.V., Nunan L.M., Redman R.M., Mari J. & Bonami J.R.
(1996). Application of gene probes as a diagnostic tool for the white spot baculovirus
(WSBV) of penaeid shrimp. Dis. Aquat. Org., 27, 59-66.
10. Durand S., Lightner D.V., Redman R.M. & Bonami J.R. (1997). Ultrastructure
and morphogenesis of white spot syndrome baculovirus. Dis. Aquat. Org., 29, 205211.
11. Durand S.V., Tang K.F.J. & Lightner D.V. (2000). Frozen commodity shrimp:
potential avenue for introduction of white spot syndrome virus and yellow head virus.
J. Aquat. Anim. Health, 12, 128-135.
12. Flegel T.W. (1997). Special topic review: Major viral diseases of the black tiger
prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol., 13, 433-442.
13. Flegel T.W., Boonyaratpalin S. & Withyachumnarnkul B. (1997). Current status of
research on yellow-head virus and white-spot virus in Thailand. In: Diseases in Asian
Aquaculture III, Flegel T.W. & MacRae I.H., eds. Fish Health Section, Asian Fisheries
Society, Manila, the Philippines, 285-296.
14. Global Aquaculture Alliance (GAA) (1999). Shrimp white spot virus confirmed in
Central America. GAA Newsletter, Vol. 2, Issue 2.
15. Global Aquaculture Alliance (GAA) (1999). Shrimp white spot disease in Latin
America - an update. GAA Newsletter, Vol. 2, Issue 3.
16. Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L., and Brunak,
S. (1998) NetOglyc: prediction of mucin type O-glycosylation sites based on
sequence context and surface accessibility. Glycoconj. J. 15, 115 130.
17. Hofmann, K., Bucher, P., Falquet, L., and Bairoch, A. (1999) The PROSITE
database, its status in 1999. Nucleic Acids Res. 27, 215 219.
18. Hong, T., Summers, M. D., and Braunagel, S. C. (1997) N-terminal sequences
from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66
and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope,
intranuclear microvesicles and the envelope of occlusion derived virus. Proc. Natl.
Acad. Sci. U. S. A. 94, 4050 405.
19. Huang, C., Shi, Z., Zhang, J., Zhang, L., Chen, D., and Bonami, J. R. (1999)
Establishment of a model for proliferating white spot syndrome virus in vivo. Virol.
Sin. 14, 358 364
20. Huang, C., Zhang, L., Zhang, J., Xiao, L., Wu, Q., Chen, D., and Li, J. K. (2001)
Purification and characterization of white spot syndrome virus (WSSV) produced in
an alternate host: crayfish, Cambarus clarkii. Virus Res. 76, 115 125
21. Huang J., Song X.L., Yu J. & Yang C.H. (1995). Baculoviral hypodermal and
hematopoietic necrosis - study on the pathogen and pathology of the explosive

epidemic disease of shrimp. Mar. Fish Res., 16, 1-10.

22. Huang J., Yu J., Song X.L., Kong J. & Yang C.H. (1995) Studies on fine structure,
nucleic acid, polypeptide and serology of hypodermal and hematopoietic necrosis
baculovirus of penaeid shrimp. Mar. Fish Res., 16, 11-23.
23. Inouye K., Miwa S., Oseko N., Nakano H., Kimura T., Momoyama K. & Hiraoka
M. (1994). Mass mortality of cultured kuruma shrimp Penaeus japonicus in Japan in
1993: Electron microscopic evidence of the causative virus. Fish Pathol., 29, 149-158.
24. Inouye K., Yamano K., Ikeda N., Kimura T., Nakano H., Momoyama K.,
Kobayashi J. & Miyajima S. (1996). The penaeid rod-shaped DNA virus (PRDV)
which causes penaeid acute viremia (PAV). Fish Pathol., 31, 39-45.
25. Kanchanaphum P., Wongteerasupaya C., Sitidilokratana N., Boonsaeng V.,
Panyim S., Tassanakajon A., Withyachumnarnkul B. & Flegel T.W. (1998).
Experimental transmission of White-Spot Syndrome Virus (WSSV) from crabs to
shrimp Penaeus monodon. Dis. Aquat. Org., 34, 1-7.
26. Karunasagar I., Otta S.K. & Karunasagar I. (1997). Histopathological and
bacteriological study of white spot syndrome of Penaeus monodon along the west
coast of India. Aquaculture, 153, 9-13.
27. Karunasagar I., Otta S.K. & Karunasagar I. (1998). Disease problems affecting
cultured penaeid shrimp in India. Fish Pathol., 33, 413-419.
28. Kasornchandra J. & Boonyaratpalin S. (1998). Primary shrimp cell culture:
Applications for studying white spot syndrome virus (WSSV). In: Advances in
Shrimp Biotechnology, Flegel T.W., ed. National Center for Genetic Engineering and
Biotechnology, Bangkok, Thailand, 273-276.
29. Kasornchandra J., Boonyaratpalin S. & Itami T. (1998). Detection of white-spot
syndrome in cultured penaeid shrimp in Asia: Microscopic observation and
polymerase chain reaction. Aquaculture, 164, 243-251.
30. Kim C.K., Kim P.K., Sohn S.G., Sim D.S., Park M.A., Heo M.S., Lee T.H., Lee
J.D., Jun H.K. & Jang K.L. (1998). Development of a polymerase chain reaction
(PCR) procedure for the detection of baculovirus associated with white spot syndrome
(WSBV) in penaeid shrimp. J. Fish Dis., 21, 11-17.
31. Kimura T., Nakano H., Momoyma K., Yamano K. & Inouye K. (1995).
Purification of the rod-shaped nuclear virus (RV-PJ) from kuruma shrimp, Penaeus
japonicus. Fish Pathol., 30, 287-288.
32. Kimura T., Yamano K., Nakano H., Momoyama K., Hiraoka M. & Inouye K.
(1996). Detection of penaeid rod-shaped DNA virus (PRDV) by PCR. Fish Pathol.,
31, 93-98.
33. Kou G.H., Peng S.E., Chiu Y.L. & Lo C.F. (1998). Tissue distribution of white
spot syndrome virus (WSSV) in shrimp and crabs. In: Advances in Shrimp

Biotechnology, Flegel T.W., ed. National Center for Genetic Engineering and
Biotechnology, Bangkok, Thailand, 267-271.
34. Kozak, M. (1987) An analysis of 5'-noncoding sequences from 699 vertebrate
messenger RNAs. Nucleic Acids Res. 15, 8125 8148.
35. Lightner D.V. (Ed.) (1996). Handbook of Pathology and Diagnostic Procedures
for Diseases of Penaeid Shrimp. World Aquaculture Society, Baton Rouge, USA.
36. Liu W.J., Yu H.T., Peng S.E., Chang Y.S., Pien H.W., Lin C.J., Huang C.J., Tsai
M.F., Huang C.J., Wang C.H., Lin J.Y., Lo C.F. & Kou G.H. (2001). Cloning,
characterization and phylogenetic analysis of a shrimp white spot syndrome virus
(WSSV) gene that encodes a protein kinase. Virology, 289, 362-77.
37. Lo C.F., Ho C.H., Chen C.H., Liu K.F., Chiu Y.L., Yeh P.Y., Peng S.E., Hsu H.E.,
Liu H.C., Chang C.F., Su M.S., Wang C.H. & Kou G.H. (1997). Detection and tissue
tropism of white spot syndrome baculovirus (WSBV) in captured brooders of Penaeus
monodon with a special emphasis on reproductive organs. Dis. Aquat. Org., 30, 53-72.
38. Lo C.F., Ho C.H., Peng S.E., Chen C.H., Hsu H.C., Chiu Y.L., Chang C.F., Liu
K.F., Su M.S., Wang C.H. & Kou G.H. (1996). White spot syndrome baculovirus
(WSBV) detected in cultured and captured shrimps, crabs and other arthropods. Dis.
Aquat. Org., 27, 215-225.
39. Lo C.F., Hsu H.C., Tsai M.F., Ho C.H., Peng S.E., Kou G.H. & Lightner D.V.
(1999). Specific genomic DNA fragment analysis of different geographical clinical
samples of shrimp white spot syndrome virus. Dis. Aquat. Org., 35, 175-185.
40. Lo C.F. & Kou G.H. (1998). Virus-associated white spot syndrome of shrimp in
Taiwan: a review. Fish Pathol., 33, 365-371.
41. Lo C.F., Leu J.H., Ho C.H., Chen C.H., Peng S.E., Chen Y.T., Chou C.M., Yeh
P.Y., Huang C.J., Chou H.Y., Wang C.H. & Kou G.H. (1996). Detection of
baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using
polymerase chain reaction. Dis. Aquat. Org., 25, 133-141.
42. Lo C.F., Peng S.E. & Kou G.H. (2001). PCR screening for white spot syndrome
virus (WSSV) in Penaeus monodon brooders: a general effort to combat shrimp WSS.
In: Aquaculture and Fisheries Resources Management: Proceedings of the Joint
Taiwan-Australia Aquaculture and Fisheries and Management Forum - TFRI
Conference Proceedings 4, Liao I.C. & Baker J., eds. Taiwan Fisheries Research
Institute, Keelung, Taipei China, 63-69.
43. Loh P.C., Cesar E., Nadala B. Jr, Tapay L.M. & Lu Y. (1998). Recent
developments in immunologically-based and cell culture protocols for the specific
detection of shrimp viral pathogens. In: Advances in Shrimp Biotechnology, Flegel
T.W., ed. National Center for Genetic Engineering and Biotechnology, Bangkok,
Thailand, 255-259.
44. Loh P.C., Tapay L.M., Lu Y. & Nadala E.C. (1997). Viral pathogens of the penaeid
shrimp. Adv. Virus Res., 48, 263-312.

45. Lu Y., Tapay L.M., Gose R.B., Brock J.A. & Loh P.C. (1997). Infectivity of
yellow head virus (YHV) and the Chinese baculo-like virus (CBV) in two species of
penaeid shrimp Penaeus stylirostris (Stimpson) and Penaeus vannamei (Boone). In:
Diseases in Asian aquaculture III, Flegel T.W. & MacRae I.H., eds. Asian Fisheries
Society, Manila, the Philippines, 297-304.
46. Lu Y., Tapay L.M., Loh P.C., Brock J.A. & Gose R. (1997). The pathogenicity of a
baculo-like virus isolate from diseased penaeid shrimp obtained from China for
cultured penaeid species in Hawaii. Aquaculture Int., 5, 277-282.
47. Magbanua F.O., Natividad K.T., Migo V.P., Alfafara C.G., de la Pena F.O.,
Miranda R.O., Albaladejo J.D., Nadala E.C. Jr, Loh P.C. & Mahilum-Tapay L. (2000).
White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines
Dis. Aquat. Org., 42, 77-82.
48. Mann, M., and Pandey, A. (2001) Use of mass spectrometry-derived data to
annotate nucleotide and protein sequence databases. Trends Biochem. Sci. 26, 54
61..
49. Mayo M.A. (2002). (International Committee on Taxonomy of Viruses [ICTV]
Secretary), virology division news: ICTV at the Paris ICV: Results of the Plenary
Session and the Binomial Ballot. Arch. Virol., 147 (11), 2254-2260.
50. Mayo M.A. (2002). A summary of taxonomic changes recently approved by
ICTV. Arch. Virol., 147 (8), 1655-1656.
51. Mohan C.V., Shankar K.M., Kulkarni S. & Sudha P.M. (1998). Histopathology of
cultured shrimp showing gross signs of yellow head syndrome and white spot
syndrome during 1994 Indian epizootics. Dis. Aquat. Org., 34, 9-12.
52. Momoyama K., Hiraoka M., Inouye K., Kimura T. & Nakano H. (1995).
Diagnostic techniques of the rod-shaped nuclear virus infection in the kuruma shrimp,
Penaeus japonicus. Fish Pathol., 30, 263-269.
53. Momoyama K., Hiraoka M., Nakano H., Koube H., Inouye K. & Oseko N. (1994).
Mass mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993:
histopathological study. Fish Pathol., 29, 141-148.
54. Naaby-Hansen, S., Waterfield, M. D., and Cramer, R. (2001) Proteomics-postgenomic cartography to understand gene function. Trends Pharmacol. Sci. 22, 376
384.
55. Nadala E.C.B., Tapay L.M., Cao S. & Loh P.C. (1997). Detection of yellow-head
virus and Chinese baculovirus in penaeid shrimp by the Western blot technique. J.
Virol. Methods, 69, 39-44.
56. Nakano H., Koube H., Umezawa S., Momoyama K., Hiraoka M., Inouye K. &
Oseko N. (1994). Mass mortalities of cultured kuruma shrimp, Penaeus japonicus, in
Japan in 1993: Epizootiological survey and infection trails. Fish Pathol., 29, 135-139.
57. Nunan L.M. & Lightner D.V. (1997). Development of a non-radioactive gene

probe by PCR for detection of white spot syndrome virus (WSSV). J. Virol. Methods,
63, 193-201.
58. Nunan L.M., Poulos B.T. & Lightner D.V. (1998). The detection of white spot
syndrome virus (WSSV) and yellow head virus (YHV) in imported commodity
shrimp. Aquaculture, 160, 19-30.
59. OReilly, D. R. (1997) in The Baculoviruses (Miller, Lois K., ed) 1st Ed., pp.267
295, Plenum Press, New York
60. Park J.H., Lee Y.S., Lee S. & Lee Y. (1998). An infectious viral disease of penaeid
shrimp newly found in Korea. Dis. Aquat. Org., 34, 71-75.
61. Poulos B.T., Pantoja C.R., Bradley-Dunlop D., Aguilar J. & Lightner D.V. (2001).
Development and application of monoclonal antibodies for the detection of white spot
syndrome virus of penaeid shrimp. Dis. Aquat. Org., 47, 13-23.
62. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric
sequencing of protein silver-stained polyacrylamide gels. Anal. Chem. 68, 850 858.
63. Shih H.H., Wang C.S., Tan L.F. & Chen S.N. (2001). Characterization and
application of monoclonal antibodies against white spot syndrome virus. J. Fis. Dis.,
24, 143-150.
64. Supamattaya K., Hoffmann R.W., Boonyaratpalin S. & Kanchanaphum P. (1998).
Experimental transmission of white spot syndrome virus (WSSV) from black tiger
shrimp Penaeus monodon to the sand crab Portunus pelagicus, mud crab Scylla
serrata and krill Acetes sp. Dis. Aquat. Org., 32, 79-85.
65. 64. Wang C.S., Tang K.F.J., Kou G.H. & Chen S.N. (1997). Light and electron
microscopic evidence of white spot disease in the giant tiger shrimp, Penaeus
monodon (Fabricius), and the kuruma shrimp, Penaeus japonicus (Bate), cultured in
Taiwan. J. Fish Dis., 20, 323-331.
66. Takahashi Y., Itami T., Kondom M., Maeda M., Fujii R., Tomonaga S.,
Supamattaya K. & Boonyaratpalin S. (1994). Electron microscopic evidence of
bacilliform virus infection in Kuruma shrimp (Penaeus japonicus). Fish Pathol., 29,
121-125.
67. Takahashi Y., Itami T., Maeda M., Suzuki N., Kasornchandra J., Supamattaya K.,
Khongpradit R., Boonyaratpalin S., Kondo M., Kawai K., Kusuda R., Hirono I. &
Aoki T. (1996). Polymerase chain reaction (PCR) amplification of bacilliform virus
(RV-PJ) DNA in Penaeus japonicus Bate and systemic ectodermal and mesodermal
baculovirus (SEMBV) DNA in Penaeus monodon Fabricius. J. Fish Dis., 19, 399-403.
68. Tapay L.M., Lu Y., Gose R.B., Nadala E.C.B. Jr, Brock J.A. & Loh P.C. (1997).
Development of an in vitro quantal assay in primary cell cultures for a non-occluded
baculo-like virus of penaeid shrimp. J. Virol. Methods, 64, 37-41.
69. Tsai M.F., Lo C.F., Van Hulten M.C.W., Tzeng H.F., Chou C.M., Huang C.J.,
Wang C.H., Lin J.Y., Valk J.M. & Kou G.H. (2000). Transcriptional analysis of the
ribonucleotide reductase genes of shrimp white spot syndrome virus. Virology, 277,
92-99.

70. Tsai M.F., Yu H.T., Tzeng H.F., Leu J.H., Chou C.M., Huang C.J., Wang C.H., Lin
J.Y., Kou G.H. & Lo C.F. (2000). Identification and characterization of a shrimp white
spot syndrome virus (WSSV) gene that encodes a novel chimeric polypeptide of
cellular-type thymidine kinase and thymidylate kinase. Virology, 277, 100-110.
71. 57.Tzeng H.F., Chang Z.F., Peng S.E., Wang C.H., Lin J.Y., Kou G.H. & Lo C.F.
(2002). Chimeric polypeptide of thymidine kinase and thymidylate kinase of shrimp
white spot syndrome virus (WSSV): thymidine kinase activity of the recombinant
protein expressed in baculovirus/insect cell system. Virology, 299, 248-255.
72. Van Hulten M.C.W., Westenberg M., Goodall S.D. & Vlak J.M. (2000).
Identification of two major virion protein genes of white spot syndrome virus of
shrimp. Virology, 266, 227-236.
73. Van Hulten M.C.W., Witteveldt J., Peters S., Kloosterboer N., Tarchini R., Fiers
M., Sandbrink H., Lankhorst R.K. & Vlak J.M. (2001). The white spot syndrome
virus DNA genome sequence. Virology, 286, 7-22.
74. Van Hulten M.C.W., Witteveldt J., Snippe M. & Vlak J.M. (2001). White spot
syndrome virus envelope protein VP28 is involved in the systemic infection of
shrimp. Virology, 285, 228-233.
75. Wang Y.G., Lee K.L., Najiah M., Shariff M. & Hassan M.D. (2002). A new
bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and
its comparison with white spot syndrome (WSS) caused by virus. Dis. Aquat. Org., 41
(1), 9-18.
76. Wang C.H., Lo C.F., Leu J.H., Chou C.M., Yeh P.Y., Chou H.Y., Tung M.C.,
Chang C.F., Su M.S. & Kou G.H. (1995). Purification and genomic analysis of
baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis.
Aquat. Org., 23, 239-242.
77. Wang C.H., Yang H.N., Tang C.Y., Lu C.H., Kou G.H. & Lo C.F. (2000).
Ultrastructure of white spot syndrome virus development in primary lymphoid organ
cell cultures. Dis. Aquat. Org., 41, 91-104.
65. Wang C.S., Tsai Y.J., Kou G.H. & Chen S.N. (1997). Detection of white spot
disease virus infection in wild-caught greasy back shrimp, Metapenaeus ensis (de
Haan) in Taiwan. Fish Pathol., 32, 35-41.
79 Wongteerasupaya C., Vickers J.E., Sriurairatana S., Nash G.L., Akarajamorn A.,
Boonsaeng V.,
Panyim S., Tassanakajon A., Withyachumnarnkul B. & Flegel T.W. (1995). A nonoccluded,
systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and
causes high
mortality in the black tiger prawn Penaeus monodon. Dis. Aquat. Org., 21, 69-77
.
80 Wongteerasupaya C., Vickers J.E., Sriurairatana S., Nash G.L., Akarajamorn A.,

Boonsaeng V., Panyim S., Tassanakajon A., Withyachumnarnkul B. & Flegel T.W.
(1995). A non-occluded, systemic baculovirus that occurs in cells of ectodermal and
mesodermal origin and causes high mortality in the black tiger prawn Penaeus
monodon. Dis. Aquat. Org., 21, 69-77.
81. Wongteerasupaya C., Wongwisansri S., Boonsaeng V., Panyim S., Pratanpipat P.,
Nash G.L., Withyachumnarnkul B. & Flegel T.W. (1996). DNA fragment of Penaeus
monodon baculovirus PmNOBII gives positive in situ hybridization with viral
infections in 6 penaeid shrimp species. Aquaculture, 143, 23-32.
82. Wongteerasupaya C., Wongwisansri S., Boonsaeng V., Panyim S., Pratanpipat P.,
Nash G.L., Withyachumnarnkul B. & Flegel T.W. (1996). DNA fragment of Penaeus
monodon baculovirus PmNOBII gives positive in situ hybridization with viral
infections in 6 penaeid shrimp species.
Aquaculture, 143, 23-32.
83. Yang F., He J., Lin X., Li Q., Pan D., Zhang X. & Xu X. (2001). Complete
genome sequence of the shrimp white spot bacilliform virus. J. Virol., 75, 1181111820.
84. Zhang, X., Huang, C., Xu, X., and Hew, C.-L. (2002) The transcription and
identification of an envelope protein gene (p22) from shrimp white spot syndrome
virus (WSSV). J. Gen. Virol. 83, 471 477.
85. Zhang X., Xu X. & Hew C.L. (2001). The structure and function of a gene
encoding a basic peptide from prawn white spot syndrome virus

Table 1 Recommended lime for pond preparation

Solid pH

Quantity of CaCo 3 lime

(Kg/hectare)
1000>
2000>
3000>

6<
to 6 5
5>

Quantity of Ca(OH) 2 lime

(Kg/hectare)
500>
1000>
1500>

Table 2- Water quality parameters checking

Parameter

Checking time

Recommended values

pH .1

Morning (8-10 am) and

Evening (3-5 pm)
Morning (8-10 am)
Morning
Morning (8-10 am) and
Evening (3-5 pm)
Only once in a weak during first
.month of crop

to 8.5 7.5

Water transparency .2
Water color .3
Water temperature .4
Alkalinity .5

(to 40 cm 30)
Green water
to 32 0 c 28
to 120 ppm 80

Thereafter to be continued
.based on requirements