MANAGEMENT STRATEGIES FOR CONTROLLING DISEASES IN SHRIMP AQUACULTURE

Hassan Rostamian fidep@qfpco.com

PrefaceShrimp 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 rod-shaped 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 peopleۥs 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 peopleۥs 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 (80-120 250-380 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 peopleۥs 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 peopleۥs 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 non-penaeid 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 treatmentThe 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 PreparationPond 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 removalThe 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 PloughingThe 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 LimingLiming 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 preparationThere 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 fillingReservoir 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 ProcessThere 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 managementA 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 ManagementThe 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 ManagementThe 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 chemicalThe 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. Chloramphenicol2. Nitro furans including Furazolidone, Nitrofurazone, Furaltadone, Nitrofurantoin, Furylfuramide, Nifuratel, Nifuroxime, Nifurprazine and all their derivatives3. Neomycin4. Nalidixic Acid5. Sulphamethoxazola6. Aristolochia spp. And preparations thereof 7. Chloroform8. Chloropromazine9. Colchicine10. Dapsone11. Dimetridazole12. Metronidazole

13. Ronidazole14. Ipronidazole15. Other nitromidazoles16. Clenbuterol17. Diethystilbestrol (DES)18. Sulfonamide (except approved Sulfadimethoxine, Sulfabromomethazine and Sulfaethoxypyridazine)19. Fluoroquinolones20. 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 it’s 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 countries’networks.• 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.

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Table 1– Recommended lime for pond preparation

Solid pH Quantity of CaCo lime (Kg/hectare)

Quantity of Ca(OH) lime (Kg/hectare)

<6 >1000 >5005 to 6 >2000 >1000

>5 >3000 >1500

Table 2- Water quality parameters checking

Parameter Checking time Recommended values1 .pH Morning (8-10 am) and

Evening (3-5 pm)7.5 to 8.5

2 .Water transparency Morning (8-10 am) )30 to 40 cm(3 .Water color Morning Green water

4 .Water temperature Morning (8-10 am) andEvening (3-5 pm)

28 to 32 c

5 .Alkalinity Only once in a weak during first month of crop.

80 to 120 ppm

Thereafter to be continued based on requirements.