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Posted by on in Biological Products

Probiotic bacterial cultures added to shrimp ponds typically are composed primarily of heterotrophic bacteria or a mixture of heterotrophic bacteria and autotrophic nitrifiers. Heterotrophic bacteria are those bacteria that primarily obtain their nutrition from organic sources.  The primary source of carbon for these bacteria is carbohydrates.  Nitrogen is typically obtained from the proteins in the organic material consumed by the bacteria.  Just like the shrimp, heterotrophic bacteria excrete ammonia as a by-product of the metabolism of the proteins they consume.  Some heterotrophic bacteria, however, are able to utilize ammonia directly as an alternative source of nitrogen.

 

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What does this all have to do with C: N ratios?  Shrimp feeds used in intensive shrimp ponds typically have at least 35% protein.  These feeds do not contain a lot of carbohydrates.  C: N ratios in these feeds typically run around 9:1.  The bacteria require about 20 units of carbon per unit of nitrogen assimilated.  With such a low C:N ratio in the feed, carbon is the limiting nutrient for heterotrophic bacteria populations.  The bacterial population will not expand beyond a certain point due to the limited availability of carbon.  The protein in the organic detritus supplies most of the nitrogen requirement for the heterotrophic bacteria under these circumstances, and inorganic ammonia is not utilized as a nitrogen source to any great extent.

 

If the C: N ratio is increased, either by feeding lower protein feeds with a higher percentage of carbohydrate, or by adding a carbohydrate source such as molasses in addition to the regular feed, the increased availability of carbon allows the heterotrophic bacterial population to consume a higher percentage of the protein in the organic material.  This results in a complete digestion of the organic material in the pond by the heterotrophic bacteria.  As the C: N ratio increases, the heterotrophic bacteria resort increasingly to ammonia metabolism to meet their nitrogen requirements.  As C: N ratios are increased even further, a point is reached where nitrogen, rather than carbon, becomes the limiting nutrient.  At this point, ammonia concentrations should be close to 0 mg/L in the pond.

 

It should be pointed out that holding the feed protein constant and supplementing with pure carbohydrate will result in much higher bacterial counts in the pond.  The oxygen required to support this additional bacterial biomass will increase proportionally with the increase in bacterial population.  Likewise, CO2 production will increase, driving pH down.  If you are contemplating carbohydrate supplementation to increase C: N ratios, make sure that your pond is well-aerated and circulated to keep the organic detritus suspended in the water column where there is sufficient oxygen for the heterotrophs.  Also, once you develop a dense population of heterotrophs through carbohydrate supplementation, don’t discontinue the carbohydrate supplementation suddenly.  This will starve the bacteria of carbon, a die-off will occur and you will get an ammonia spike.

 

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Another point that should be considered before enhancing C: N ratios in P. monodon ponds.  P. monodon does not utilize the organic detritus and associated bacterial protein as effectively as a food source as does P. vannamei.  With vannamei, C: N ratios can be enhanced by lowering the overall feed protein levels and utilizing feeds that are high in carbohydrate.  Because vannamei feeds on the organic flocs and utilizes bacterial protein efficiently, growth rates don’t suffer and protein utilization efficiencies improve dramatically.  With monodon, feeding low-protein, high-carbohydrate diets will likely result in lower growth rates.  Therefore it might be necessary to rely more on supplementation with pure carbohydrates to boost C: N ratios.  But this will result in more bacterial biomass, more BOD, and higher CO2.  This makes it somewhat questionable, in my mind, whether it is worth the risk to manage a monodon pond with high C: N ratios.

 

Most common genera of heterotrophic bacteria used in probiotic formulations are Bacillus and Lactobacillus, both of which are gram-positive.  It is not necessary, however, to inoculate a pond with commercial probiotics in order to manage a heterotrophic production system.  This can be accomplished simply by maintaining a C:N ratio greater than 12:1, and supplying adequate aeration.  The bacteria are already present in every pond.  By removing the carbon (and perhaps oxygen) limitation, they will proliferate.

 

The counts of naturally occurring bacteria are several thousand per milliliter, so a one-hectare pond contains astronomical amounts of bacteria.  It would be very difficult to add enough bacteria to a pond to significantly change its bacterial composition.  

 

Also, one might expect that the naturally occurring bacteria species are the best adapted to the conditions in the pond.  There is no guarantee that the bacteria in the probiotic culture will be well adapted to the conditions in the pond, let alone that they will out-compete the naturally occurring bacteria species.  Even if enough bacteria were added to have an effect on bacterial composition at one point in time, it would likely be necessary to re-inoculate the bacteria periodically to maintain the predominance of the probiotic species.  I admit that there have been studies which appear to show benefits in terms of survival in probiotic-treated ponds.  But there are also a lot of studies that fail to find any measurable impact on bacterial species composition.  Perhaps there is something going on that enables the probiotic bacteria to positively influence survival even when they are not the predominant species.

 

Bacillus and Lactobacillus are common genera of heterotrophic bacteria used in probiotic formulas.  What genera of the heterotrophic bacteria are already in the ponds, but not in the commercial probiotic products?

 

Marine soil sediments contain naturally occurring beneficial bacteria such as Bacillus subtillis,B. circulans, B. megaterium, B. polymyxa, and B. licheniformis.  They are purified and multiplied in fermenters and then further processed as liquids or spray-dried powders for marketing in vegetative or spore forms).

 

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Also, what’s the best way to measure the C: N ratio in a pond? 

 

Measurement of C/N is only part of the story.  If you measure TOC (total organic carbon), some of that carbon can be refractory and not help grow bacteria and soak up the ammonia.  Measuring TOC and BOD (biological oxygen demand, with and without ammonia oxidation inhibition) along with TKN (total Kjeldahl nitrogen) will provide some useful management information.  To make these systems work, you should also be rearing a species that can use the single-cell protein being produced in the pond.  If not, all you are doing is converting ammonia into an unusable biomass using a significant amount of carbohydrate and oxygen.  You either have to discharge that biomass or oxidize it in the pond bottom when drained.  If it stays in the system, it will metabolize itself back into ammonia and CO2. 

 

The only difference between a photosynthetic system (algae in a pond) and a heterotrophic system (carbohydrate and oxygen) is the energy supply for the waste treatment function.  Sunlight limits your energy density per unit area in algae-based systems, which limits your feed/area.  With heterotrophic systems the energy density is not limited; it’s volumetric. 

 

The real trick is to get the biomass from these waste systems into a usable animal as fast and efficiently as possible so you don’t waste energy redoing the ammonia again and again as the biomass (or algae) you produced with your energy input decays.  

 

Remember: all closed aquaculture is polyculture.  The only question is how many sellable species do you have and what are your energy flows.  The job of an aquaculturist is to control that microbiological ecology to get the energy flows and treatment biomass to go where you want.

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Posted by on in Biological Products

Yellow Head Virus (YHV)

 

Yellow Head Virus was the first major viral disease problem to affect Asian shrimp farms when it was diagnosed as causing extensive losses for shrimp farming. YHV and its close relatives GAV and LOVV are single stand RNA viruses, similar to TSV.

 

 

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The first records of this virus were from P. monodon ponds in Eastern Thailand, it had moved to Southern Thailand and was causing substantial mortality. YHV is prevalent wherever P. monodon are cultured, including Thailand, Taiwan Province of China, Indonesia, Malaysia, Mainland China, the Philippines and Viet Nam. It may also have been responsible for the first major crashes in Taiwan. Losses due to YHV continued, although the severity and frequency of outbreaks declined sharply when WSSV became the prime cause of mortality in cultured P. monodon. Although research has shown that YHV is still present in culture ponds, the shrimp now rarely show gross symptoms and are latently infected. There thus appears to be a currently unknown mechanism for rapid tolerance or resistance to RNA-type viruses (such as YHV in Asia, and TSV in Latin America) in Penaeid shrimp. It is known that YHV occurs in wild shrimp, but there is no data on the extent or effects of YHV on populations of wild shrimp in Asia and its impacts are thus currently unknown.

 

The primary mechanism of spread of YHV in pond culture appears to be from water and mechanical means or from infected crustacean carriers. Some infected carriers appear to have latent infections (i.e. P.merguiensis, Metapenaeus ensis, Palaemon styliferus and Acetes spp.), while others may die from it (i.e. Euphausia superba). Other crustaceans, such as Macrobrachium rosenbergii and many crab species and Artemia appear unsusceptible. Since, like most viruses, the viability of the free virus in seawater is not more than a couple of hours, the most serious threat to farmers is latent or asymptomatic carriers, from which the virus can be spread either by ingestion or cohabitation. In addition, infected broodstock can pass on the virus to larvae in the maturation/hatchery facilities if thorough disinfection protocols are not strictly adhered.

 

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Although a distinct possibility, YHV has not yet been reported from Latin America apart from some probably spurious results from Texas. However, from work in Hawaii, YHV is known to cause high mortality in P. vannamei, P. stylirostris, P. setiferus, P. aztecus and P. duorarum when it is injected as viral extracts. Despite this, there are still no reports of “natural” infections in shrimp farms of P. vannamei and P.stylirostris with YHV in Asia. There is a strong possibility, however, that YHV may cause problems for the new culture industries for P. vannamei and P. stylirostris in Asia. This will probably be true at least until these species can gain some degree of tolerance or resistance to the virus as P. monodon appears to have done. In the meantime, the large number of latent infected hosts (including P.monodon) will serve as a potential reservoir of infection and should not be permitted to come into contact with cultures of P. vannamei or P. stylirostris.

 

YHV principally affects pond reared P. monodon in juvenile stages from 5-15 g. Shrimp typically feed voraciously for two to three days and then stop feeding abruptly and are seen swimming near the pond banks. YHV infections can cause swollen and light yellow coloured hepatopancreas in infected shrimp, and a general pale appearance, before dying within a few hours. Total mortality of the crop is then typically seen within three days. Experimentally infected shrimp develop the same signs as those naturally infected, indications of the disease are noted after two days and 100 percent mortality results after three to nine days. Yellow head virus can be detected by RT-PCR or with a new probe for dot-blot and in situ hybridisation tests. It can also be diagnosed histologically in moribund shrimp by the presence of intensely basophilic inclusions, most easily in H&E stained sectioned stomach or gill tissue, or simply by rapid fixation and staining of gill tissue and microscopic examination. Exact protocols for all of these techniques are given in the OIE website and by Flegel et al

 

Eradication methods in ponds are much the same as for other viruses and involve a package including: pond preparation by disinfection and elimination of carriers, storage and/or disinfection of water for exchange with chlorine (30ppm active ingredient), filtering water inlet to ponds with fine screens, avoidance of fresh feeds, maintenance of stable environmental conditions, disinfection of YHV infected ponds before discharge, and monitoring (by PCR) and production of virus free broodstock and PL for pond stocking. Various immunostimulants, nutrient supplements and probiotics have been tried, but there remains a paucity of conclusive evidence of the benefits of such treatments.

 

The rapid tolerance gained by P. monodon to YHV provoked theories as to its mechanism. Whether this theory is correct or not, field data has indicated that shrimp surviving a YHV epidemic are already infected and thus are not killed by subsequent infections, suggesting that some type of “vaccination” with a dead or attenuated virus might provide some resistance. Some commercial products are already being marketed and trials have been partially successful. YHV is not causing much loss at present in Asia, but general management practices as described above (to maintain optimal environmental conditions and minimize viral loadings) are still required to help prevent infections.

 

Lymphoid Organ Vacuolization Virus (LOVV)

 

Lymphoid Organ Vacuolization Virus was first noted in P. vannamei farms in the Americas in the early 1990. In P. vannamei, LOVV has been shown to result in limited localized necrosis of lymphoid organ cells, but has never been shown to impact production. It was later discovered in Australia, along with the other TSV-like virus GAV.

 

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Due to the coincidence in dates, it is possible that the main cause of the problems with P. monodon, was a result of the introduction of viral pathogens carried by P. vannamei. A RNA viral pathogen very similar to LOVV in P. vannamei has recently been discovered in Thailand in the lymphoid organ of P. monodon. This new type of LOVV might be the causative agent of this slow growth phenomenon. Evidence for this was provided by Timothy Flegel (per. com.), who found that juvenile P. monodon injected with this virus grew to only 4g after two months, whilst those injected with a placebo reached 8g in the same time. Injections of the same virus into P. vannamei caused no obvious effects, suggesting that it probably originated from this species.

 

Other viruses

 

There are a number of other viruses in the Asia-Pacific region. Penaeus monodon from Australia have been found to be hosts for a number of viruses not yet present in other Asian countries. These include two viruses closely related to YHV: GAV (only 20 percent genetically different to YHV) and MOV (only 10 percent genetically different from GAV), which are quite recently discovered viruses that are already prevalent in 100 percent of P. monodon from Queensland. MOV was only discovered in 1996, but has already been found in P. japonicus and is associated with disease episodes in P. monodon farms in Australia and elsewhere in Asia. The strong possibility for the introduction of these viruses into Asia exists due to frequent shipments of P. monodon broodstock from Australia into Thailand, Viet Nam and other Southeast Asian countries.

 

Many of the viruses infecting shrimp are hidden or cryptic and, although present in their host, may produce no gross signs of disease or notable mortality. Many of these viruses, without methods of diagnosis, are probably being harboured unknown within the wild and cultured populations of shrimp throughout the world. It may not be until shrimp species from one location are moved to another and their viral flora comes into contact with new and/or naive or intolerant hosts that disease epidemics begin. Crustaceans may be particularly problematic since they tend to have persistent, often multiple, viral infections without gross or even histological signs of disease.

 

 

Examples of this problem include the transfer of IHHNV from the tolerant P. monodon in Asia to the susceptible white shrimp P. vannamei and P. stylirostris in Latin America. Another possibility in this category is the LOVV virus thought to be causing the slow growth phenomenon in P. monodon around Asia. This virus may have been imported with live P vannamei broodstock and PL brought to Asia from the Americas in the mid 1990s. For this reason, extreme caution should be placed on the transboundary movements of live shrimp.

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White Spot Syndrome Virus (WSSV) is now and has for some time been the most serious threat facing the shrimp farming industry in Asia. It is an extremely virulent pathogen with a large number of host species.

This disease is probably the major cause of direct losses of shrimp farming in Asia. Similarly, in Latin America, losses due to WSSV have been substantial. In addition, indirect losses in hatchery, feed and packing plant capacities and so on resulted in lost earnings. Similar problems have occurred throughout Central and South America, with the exception of Brazil and Venezuela, which remain WSSV-free due to the prompt and effective closure of their borders to all crustacean imports.The United States also managed to eradicate WSSV from its shrimp culture industry after initial losses through implementation of biosecurity measures, including the use of all SPF broodstock, although there are reports of its recent re-emergence in Hawaii.

 

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WSSV is a large double-stranded DNA baculovirus. Other names for probably the same viral complex include Chinese baculovirus (CBV), White spot syndrome baculovirus complex (WSBV), Mainland China’s Hypodermal Hepatopoietic Necrosis Baculovirus (HHNBV), Shrimp Explosive Epidermic Disease (SEED), Penaeid Rod-shaped DNA Virus (PRDV), Japan’s Rod-shaped Nuclear Virus (RV-PJ) of P. japonicus, Thailand’s Systemic Ectodermal and Mesodermal.

Baculovirus (SEMBV) of P. monodon, red disease and white spot virus or disease. WSSV was first reported in farmed P. japonicus from Japan, but was thought to have been imported with live infected PL from Mainland China. At roughly the same time, it was discovered in cultured P. monodon, P. japonicus and P. penicillatus in Taiwan Province of China and then in P. monodon in southern Thailand. WSSV then spread rapidly throughout most of the shrimp growing regions of Asia, probably through infected broodstock and PL P. monodon. Then, in 1995, it was detected for the first time in farmed P. setiferus in Texas. It was also shown to be infective experimentally to both P. vannamei and P. stylirostris. WSSV did not reach the Philippines, which had an effective government ban on live imports, until an illegal introduction of Chinese PL P. monodon.

Other susceptible host species include the shrimp species P. merguiensis, Metapenaeus ensis, Metapenaeus monoceros and various crab species, whilst Palaemon setiferus, Euphausia superba, Metapenaeus dobsoni, Parapenaeopsis stylifera, Solenocera indica, Squilla mantis, Macrobrachium rosenbergii and a range of crab species can act as latent carriers, although Artemia appear unsusceptible. Later, in 1999, WSSV began affecting Latin America from Honduras, Guatemala, Nicaragua and Panama in Central America to Ecuador and Peru in the south and later to Mexico. The only shrimp farming countries to remain free of WSSV in Latin America are Brazil and Venezuela, who (like the Philippines) both placed immediate and effective bans on the importation of live crustaceans and developed their domestication programmes for producing virus-free seedstock.

 

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The mode of transmission of WSSV around Asia was believed to be through exports of live PL and broodstock. The outbreaks in Texas and then Honduras followed by Spain and Australia, were thought to be due to the virus escaping from processing plants which were importing and processing frozen shrimp from infected parts of Asia, although this has never been proven. Regardless of their origin, isolates of WSSV have shown little genetic or biological variation, suggesting that the virus emerged and was spread from a single source. WSSV, as with most viral diseases, is not thought to be truly vertically transmitted, because disinfection of water supplies and the washing and/or disinfection of the eggs and nauplius is successful in preventing its transmission from positive broodstock to their larvae. Instead, it is generally believed that the virus sticks to the outside of the egg, since, if it gains entry to the egg, it is rendered infertile and will not hatch. Thus, using proper testing and disinfection protocols, vertical transmission can be prevented in the hatchery, as proven by the Japanese who to date have successfully eliminated WSSV from captive stocks in the country through disinfection and PCR checking of broodstock and nauplii)

Using mathematical epidemiology modelling, Soto and Lotz (2001) showed that WSSV was more easily transmitted through ingestion of infected tissues than through cohabitation with infected hosts, and that P. setiferus was much more susceptible than P. vannamei to infection. Although it is clear that live Penaeids can carry the virus and infect new hosts through reproduction (transmission from broodstock to larvae), consumption or cohabitation with diseased or latent carriers, and that it is possible for frozen shrimp to be infective, other modes of transmission are also possible. For example, Australia is considered WSSV (and YHV)-free, although WSSV was detected in the Northern Territories in 2000 associated with imported bait shrimp, before being eradicated.

Data regarding the presence and effects of WSSV in wild shrimp populations in infected countries is scarce, but it is known to be present in wild shrimp in both Asia and Latin America. WSSV infects many types of ectodermal and mesodermal tissues, including the cuticular epithelium, connective, nervous, muscle, lymphoid and haematopoietic tissues. The virus also severely damages the stomach, gills, antennal gland, heart, and eyes. During later stages of infection, these organs are destroyed and many cells are lysed. The shrimp then show reddish colouration of the hepatopancreas and the characteristic 1-2mm diameter white spots (inclusions) on their carapace, appendages and inside surfaces of the body. They also show lethargic behavior and cumulative mortality typically reaches 100 percent within two to seven days of infection.

 

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Increasingly, since the late 1990s, it has become clear that the presence of WSSV in a pond does not always lead to disaster. Work in Thailand has shown that outbreaks are usually triggered from latent P. monodon carriers by some environmental changes, probably related to osmotic stress through changes in salinity or hardness or rapid temperature changes. Similarly in Latin and North America, fluctuations in temperature have been shown to induce mortalities of infected P. vannamei. However, there have been conflicting reports about constant temperatures which have been reported to: limit mortality due to WSSV at 18 ºC or 22 oC and induce 100 percent mortality at 32 oC in the US, yet induce mortality at less than 30 oC and protect from it at greater than 30 oC in Ecuador

Additionally, three to four years of genetic selection work (selection of shrimp surviving WSSV outbreaks) on the domesticated stocks of P. vannamei appear to have resulted in enhanced resistance to WSSV in Ecuador. Thus the culture industries for P. vannamei in Central and South America have been slowly recuperating since the start of the WSSV epidemic in 1999. For example, Ecuador was exporting 115 000 metric tonnes in 1998, which dropped to only 38 000 metric tonnes in 2000 after the arrival of WSSV in 1999. Subsequently, Ecuador has recovered to export an estimated 50000 metric tonnes in 2003.

Prevention methods are similar to those with TSV. All live and frozen shrimp should be checked by PCR prior to importation from infected areas to those currently disease-free. Broodstock should be PCR screened before breeding. PL should also be PCR screened before stocking into ponds, as this has been proven to result in a higher percentage of good harvests. PCR is not an infallible method for detection of WSSV, but it is the best diagnostic procedure currently available. Washing and disinfection of eggs and nauplii have also been shown to prevent vertical transmission of WSSV from infected broodstock to larval stages. Feeding with fresh crab and other crustaceans to broodstock should be avoided. Polyculture techniques with mildly carnivorous fish species (such as Tilapia spp.) have also proven effective at limiting the virulence of WSSV in ponds, as the fish can eat infected carriers before they become available to the live shrimp. 

The white spot virus only remains viable in water for 3-4 days, so disinfection of water used for changes and fine screening is effective in preventing transmission. Dose rates of 70ppm formalin have been shown to prevent transmission and not cause any harm to shrimp. In addition, all effluent from farming or processing operations with the possibility of WSSV infections should be disinfected (i.e. with formalin or chlorine) prior to discharge. 

WSSV can be detected by using PCR, or with probes for dot-blot and in situ hybridisation tests. It can also be visually diagnosed through the presence of the characteristic white spots (although these are not always present in infected animals). WSSV can be confirmed histologically (particularly for asymptomatic carriers) by the presence of large numbers of Cowdrey A-type nuclear inclusions and hypertrophied nuclei in H&E-stained sectioned tissues, or simply by rapid fixation and staining of gill tissue and microscopic examination.

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This virus was first discovered in P. vannamei and P. stylirostris in the America, starting in Hawaii. However, it was probably not an indigenous virus, but was thought to have been introduced along with live P.monodon from Asia. IHHNV has probably existed for some time in Asia without detection due to its insignificant effects on P. monodon, the major cultured species in Asia, meaning that nobody was looking for it. Recent studies have revealed geographic variations in IHHNV isolates, which suggested that the Philippines were the source of the original infection in Hawaii, and subsequently in most shrimp farming areas of Latin America.

 

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IHHNV is a small single-stranded DNA-containing parvovirus, which is only known to infect only Penaeid shrimp. “Natural” infections are known to have occurred with P. stylirostris, P. vannamei, P. occidentalis and P. schmitti, while P.californiensis, P. setiferus, P. aztecus and P. duorarum were proven susceptible experimentally in Latin America. Penaeus monodon, P. semisulcatus, P. japonicas and P. chinensis and others are known to be susceptible in Asia. Catastrophic epidemics and multi- million dollar losses in shrimp culture have been attributed to IHHNV and it has had significant negative consequences for cultured P. vannamei in the America. Some indication of its impact may be gauged from work done in intensive culture systems in Hawaii, which improved yields by 162 percent through the stocking of shrimp bred specifically to be IHHNV resistant.

 

IHHNV was also largely responsible for the temporary cessation of Mexican commercial shrimp fishing for several years once it escaped from farms into the wild shrimp populations. IHHNV is now commonly found in cultured and wild Penaeid on the Pacific coast of Latin America from Mexico to Peru, but not yet from the eastern coast of Latin America. It has also caused problems for the Hawaiian broodstock and farm- based culture industries. IHHNV has also been reported from both cultured and wild Penaeid from throughout the Indo-Pacific region. IHHNV is fatal to P. stylirostris (unlike P. vannamei), which, although highly resistant to TSV are extremely sensitive to IHHNV , especially in the juvenile stages. However, IHHNV has not been associated with mass mortalities of P. stylirostris in recent years, probably due to the selection of IHHNV-resistant strains (i.e. the so-called “supershrimp” P. stylirostris. This emphasises the potential benefits offered from the domestication and genetic selection of cultured shrimp.

 

Penaeus vannamei are fairly resistant to this disease with certain modifications in management practices. In P. vannamei, IHHNV can cause runt deformity syndrome (RDS), which typically results in cuticular deformities (partic ularly bent rostrums), slow growth, poor feed conversion and a greater spread of sizes on harvest, all combining to substantially reduce profitability. These effects are typically more pronounced where the shrimp are infected at an early age, so strict hatchery biosecurity including checking of broodstock by PCR, or the use of SPF broodstock, washing and disinfecting of eggs and nauplii is essential in combating this disease. Even if IHHNV subsequently infects the shrimp in the grow-out ponds, it has little effect on P. vannamei if the PL stocked can be maintained virus free.

 

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Some strains of IHHNV, however, have recently been found to be infectious for P. vannamei, including a putative strain collected from Madagascan P. monodon and a putative attenuated strain in an American laboratory. In addition, recent laboratory studies with P. stylirostris has shown that juveniles that are highly infected with IHHNV (by feeding them with IHHNV-infected tissue) were able to show 28-91 percent survival three weeks after subsequent infection with WSSV (by feeding them with WSSV infected tissue), whilst control animals suffered 100 percent mortality within five days. Surviving shrimp were found to be heavily infected by IHHNV, but had at most only light infection with WSSV which was not enough to kill all of them. Similar trials showed that neither IHHNV pre-infected P. vannamei nor IHHNV-resistant P. stylirostris (SPR “Supershrimp”) were able to tolerate subsequent WSSV infections. Nonetheless, these results raise the question whether exposing shrimp to putative strains of IHHNV may prevent them from getting infected by an infectious strain of IHHNV or possibly WSSV.

 

IHHNV typically causes no problems for P. monodon since they have developed a tolerance to it over a long period of time, but they may suffer from runt deformity syndrome (RDS). Penaeus merguiensis and P. indicus meanwhile appear refractory to the disease. They are, however, life-long carriers of the disease and so could easily pass it onto P. vannamei, which typically suffer from slow growth (RDS) when exposed to IHHNV. This presents a potential problem if the two species are cultured in close proximity at any phase of their life cycle. This should be a cause for great concern for P. vannamei farms that are currently being established throughout Asia.

 

As with most important shrimp viruses, transmission of IHHNV is known to be rapid and efficient by cannibalism of weak or moribund shrimp, although waterborne transfer due to cohabitation is less efficient. Vertical transmission from broodstock to larvae is common and has been shown to originate from the ovaries of infected females (whilst sperm from infected males was generally virus-free). Although the embryos of heavily infected females may abort, this is not always true and selection of IHHNV-free broodstock (by nested PCR) and disinfection of eggs and nauplii would help ensure production of virusfree PL.

 

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As with TSV, IHHNV may be transmitted through vectors such as insects, which have been shown to act as carriers for the disease. However, their mode of action is thought to be mechanical rather than real, as insect extracts do not react to in situ hybridisation tests for IHHNV. The probability that IHHNV in frozen shrimp can cause problems is suggested from OIE data that IHHNV remains infectious for more than 5 years of storage at minus 20oC. Gross signs of disease are not specific to IHHNV, but may include: reduced feeding, elevated morbidity and mortality rates, fouling by epicommensals, bluish coloration, whilst larvae PL and broodstock rarely show symptoms.

 

Diagnosis and detection methods include DNA probes for dot blot and in hybridisation and PCR techniques as well as histological analysis of H&E-stained sections looking for intracellular, Cowdrey type A inclusion bodies in ectodermal and mesodermal tissues. One of the big problems with IHHNV is its eradication in facilities once they have been infected. The virus has been shown to be highly resistant to all the common methods of disinfection including chlorine, lime, formalin and others in both ponds and hatcheries. Complete eradication of all stocks, complete disinfection of the culture facility and avoidance of restocking with IHHNV-positive animals.

 

White Spot Syndrome Virus (WSSV) will continue in Part 3

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In Shrimp Disease control, they are Six viruses were known to affect Penaeid shrimp, but there are more than 20 viruses were identified as having affected wild stocks and commercial production. The OIE now lists seven viral diseases of shrimp in the Aquatic Animal Health Code, which are considered to be transmissible and of significant socio-economic and/or public health importance.

 

These viral diseases as follows:

 

1.    White spot disease (WSSV).

2.    Yellowhead disease (YHV),

3.    Taura syndrome virus (TSV),

4.    Spawner-isolated mortality virus disease (SMV),

5.    Tetrahedral baculovirosis (Baculovirus penaei - BP),

6.    Spherical baculovirosis (Penaeus monodon-type baculovirus) and

7.    Infectious hypodermal and haematopoietic necrosis (IHHNV)

 

Penaeus vannamei and P. stylirostris are known to be carriers of the following viral diseases: WSSV, BP, IHHNV, REO, LOVV and TSV. These viruses can be transmitted to native wild Penaeid shrimp populations. Penaeus monodon are known carriers of: WSSV, YHV, MBV, IHHNV, BMNV, GAV, LPV, LOVV, MOV and REO.

 

Taura Syndrome Virus (TSV)

 

Perhaps the biggest concern to Asian countries already or currently wanting to import P. vannamei is the possibility of introducing TSV. Despite original work suggesting Taura syndrome (TS) was caused by a toxic pesticide, it is now known that a single or perhaps several very closely related strains (mutations) of the Taura syndrome virus (TSV) are responsible for the TS pandemic in the Americas. TSV is a single strand RNA virus and hence susceptible to mutations, causing more concern, and is closely related to other insect viruses.

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Taura Syndrome Virus was first identified from farms around the Taura river in Ecuador and subsequently spread rapidly to the whole of Latin and North America. TSV spread first throughout Ecuador and to Peru, Colombia (Pacific and Atlantic coasts), Honduras, Guatemala, El Salvador, Nicaragua, Hawaii, Florida and Brazil, Mexico, Texas, South Carolina and Belize and subsequently Asia including Mainland China and Taiwan Province of China and most recently Thailand probably through the regional and international transfer of live PL and broodstock P. vannamei.

 

Taura syndrome caused serious losses in revenue throughout Latin America. It has been suggested that TSV caused direct losses (due to shrimp mortality) and indirect losses due to loss of sales, increased seed cost and restrictions on regional trade were probably much higher. Taura syndrome so far appears to occur largely as a sub-clinical infection in populations of wild shrimp. Although P. monodon and P.japonicus appear largely unaffected, the potential impact of TSV on native stocks of P. indicus and P. merguiensis in Asia remains unknown, but a definite cause for concern.

 

The mechanism of spread of TSV is still uncertain, although initial theories concentrated on the spread through contaminated PL and broodstock between farms. Limited data have shown that TSV was introduced to Colombia and Brazil through contaminated broodstock from Hawaii. These broodstock were untested for TSV since it was not yet known that Taura syndrome had a viral cause. Such cases demonstrate once again more of the problems involved with transboundary movements of animals, even supposedly SPF ones. Recent research has shown that mechanical transfer through insect and avian vectors may be an equal or even more likely route of infection. TSV has sometimes been found in tissue bioassays of the water boatman (Trichocorixa reticulata), an estuarine insect common worldwide, and virus-containing extracts of this insect have been shown to induce infection in SPF P. vannamei under laboratory conditions. Patterns of the spread and mortality of P. vannamei in Texas have also suggested that the ingestion of infected insects is the probable mechanism of spread of TSV.

 

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Infective TSV has also been demonstrated in the faeces of shrimp-eating seagulls (Larus atricilla) collected near ponds infected with TSV in Texas, USA. Experimental results have also shown that healthy shrimp can be infected through injection of cell-free homogenates prepared from infected shrimp, and by direct feeding on infected shrimp. Taura syndrome virus has also been shown to remain infective after one or more freeze-thaw cycles, indicating the possibility of regional transmission through infected frozen shrimp. With proper disinfection procedures and controls, however, this route is currently considered to be low-risk.

 

Taura syndrome virus is highly infective for P. vannamei, P. setiferus and P. schmitti. Penaeus stylirostris can be infected by injection, but appear to be highly refractory to TSV and have demonstrated tolerance to TS in growing areas affected by this disease. Other species including P. aztecus, P. duorarum, P.monodon, P. japonicus and P. chinensis have been experimentally infected, developed the disease and remained carriers, but show some resistance. Interestingly, like P. stylirostris, P. monodon and P. japonicas appear highly refractory to TSV, and although it retards growth rates, they remain asymptomatic and the virus has not yet been demonstrated to cause mortality in these species. However, since TSV is an RNA virus, with a high propensity to mutate, there is no guarantee that it will not mutate into a more virulent form for native Asian shrimp (as it did in Central America)

 

Taura Syndrome Virus has already been detected in P. vannamei in Mainland China and Taiwan Province of China with 19 cases reported to OIE from Taiwan Province of China in 1999, ten (resulting in 700 000 cases and 200 000 deaths) and seven (resulting in 500 000 cases and 50 000 deaths). Recently, TSV has been identified in Thailand. The Taura syndrome virus tends to infect juvenile shrimp within two to four weeks of stocking ponds or tanks (0.1-1.5g body weight) and occur largely within the period of a single moult cycle. In the acute phase of the disease, during pre-moult the shrimp are weak, soft-shelled, have empty digestive tracts and diffuse expansion of the red chromatophores, particularly in the tail (hence the common name - red tail disease). Such animals will usually die during moulting (5-95 percent), although the reasons for the large variability in survival rates remains unknown; adult shrimp are known to be more resistant than juveniles. Those shrimp that survive will show signs of recovery and enter the chronic phase of the disease. Such shrimp will show multiple, randomly distributed, irregular, pitted, melanised lesions of the cuticle. These gross or microscopic lesions will persist, but may be lost during moulting, the shrimp thereafter appearing and behaving normally. However, although the shrimp may then be resistant to recurrence of the disease, they often remain chronic, asymptomatic carriers of TSV for life, as has been shown by bioassays.

 

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Standard histological and molecular methods may be used for detection, diagnosis and surveillance, although specific DNA probes applied to in situ hybridization assays with paraffin sections currently provide the greatest diagnostic certainty of this virus (OIE website). RT -PCR assays can also be used providing advantages of larger sample sizes and non-lethal sampling for broodstock. Additionally, live shrimp bioassays and serological methods with monoclonal antibodies can also be used for diagnosing infections with TSV.  Eradication methods for TSV in culture facilities are possible and depend upon total destruction of infected stocks, disinfection of the culture facility, avoidance of reintroduction of the virus (from nearby facilities, wild shrimp and carriers) and restocking with TSV-free PL produced from TSV-free broodstock. Other methods suggested for controlling the virus include: switching to the refractory P. stylirostris, and (similar to those suggested for other viruses): maintenance of optimal environmental conditions, weekly applications of hydrated lime (CaOH) at 50 kg/ha, polyculture with fish (to consume dying and dead carriers) and development of TSV resistant lines of P. vannamei. In the past few years, considerable success has been achieved in the development of families and lines of P. vannamei which are resistant to TSV.

 

Most of the SPF P. vannamei suppliers from Hawaii and Florida now offer stocks of P. vannamei which have demonstrated resistance to TSV (SPF and SPR). Genetic selection programmes run throughout the Americas have also resulted in the production of SPR lines for TSV. The use of such SPR lines enabled the Latin American industry to recuperate from the worst of the TSV pandemic within three to four years. However, importation of such lines must be done with caution, since non-SPF animals, even though resistant to TSV, may still act as carriers and can result in the introduction of TSV into areas of Asia currently free from the disease.  Aquacultural establishments, zones within countries, or countries that are considered TSV-free, are those which have been tested in an official crustacean health surveillance scheme for a minimum two years using the procedures without detection of TSV in any susceptible host species of shrimp 19. Additionally for aqua cultural establishments, they must be supplied with water that has been suitably disinfected and have barriers preventing contamination of the establishment and its water supply. New or disinfected facilities, may be declared free from TSV in under two years if all other requirements are met.

 

Whilst this degree of control may be possible in large-scale highly organized shrimp farms, the reality is that most farms are too small or disorganized to undertake such comprehensive measures. The lack of supporting infrastructure in regulation, testing and diagnosis is an additional constraint. This problem is not confined to Asia where farms are typically very small, but also occurs in Latin America where farms are far larger.

 

 

IHHNV Viral disease will continue in part 2……

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Rickettsial Infections

 

This infection is not recorded yet from Indian waters, systemic rickettsial infections were reported from cultured P. monodon from Malaysia and Singapore. In P. monodon, the rickettsia occurred within large cytoplasmic vacuoles where it formed-microcolonies of 19 to 33 f.lm in diameter. In heavy infections, cells with rickettsial inclusions were widespread in mesodermally and ectodermally derived tissues, but absent in endodermally derived tissues such as midgut, hepatopancreas and caeca. Experimental treatment using medicated feeds containing 1.5 to 2. 0 kg of oxytetracycline per 1000 kg offered was found to be successful in reducing monalities.

 

Vibriosis

 

Vibrio sp. were found to constitute the predominant normal microflora of the culturable species of shrimps. Due to their rich presence in the shrimp's microflora, researchers have found Vibrio sp. as frequent and opponunistic pathogen of the shrimps. The opponunistic pathogenic Vibrio sp. establish lethal influence as a result of other primary conditions such as other infectious diseases, nutritional disorders, extreme environmental stress and wounds. However there are a few Vibrio sp. which are true pathogens.

 

Luminescent Vibrio in hatcheries

 

In hatcheries, larval mortalities associated with luminescence are reported in epizootic proponions in P. monad Oil and P. merguiellsis. The causative bacteria are strains of Vibrio parahaemolyticus, Vibrio alginolyticus, V. harveyi and V.spiendidus. The affected larvae refuse to feed. Scanning Electron Microscopy (SEM) studies indicate that the vibrios colonise specifically the feeding appendages and oral cavity. Rigorous management and sanitation helps to control the infestation. The separation of mother shrimps and their faecal matters from the eggs has to be done as soon as possible after spawning. Anemia nauplii being used as live feed should be rinsed before introducing into the hatchery during feeding. Chlorination and other forms of water treatment such as ultraviolet irradiation and filtration should be done to reduce the initial load of the rearing water. The affected shrimps are treated using antibiotics such as chloramphenicol, sodium nitrostyrenate and the nitrofurans (furazolidone, nitrofurazone and prefuran)

 

Vibriosis in P. indicus and P. monodon culture ponds

 

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In India two species of Vibrio are found to be pathogenic to the shrimps. These include, Vibrio anguillarum and Vibrio alginolyticus. In shrimps moralities were reported to begin towards the end of the growout period as the shrimp reach marketable size of 25 to 35 g or 2 to 3 months after stocking PL-20. Dead shrimps can be harvested in the morning from the pond edges. It is estimated that at harvest 5 to 30 percent of the

production only could be attained. Most of the surviving shrimps also exhibit stunted growth. Indian white prawn, P.indicus cultured in brackishwater ponds are affected by Vibrio anguillarum. Blackening or whitening of the basal pan of the antenna, the oviduct and edges of the abdominal shells are the symptoms. Frequent water exchange, feeding with compounded diets containing antibiotics chloramphenicol help to control the disease. In addition to the above furacin at 1 mg per liter of water, terramycin at 40 mg per kg of biomass for 10 to 15 days through feed or feeding tylosin or tiamutin at 100 mg active ingredient per kg offced for two weeks help to contain the sickness.

 

Tiger prawn, Penaeus monodon cultured in ponds are affected by V. alginolyticus. Septicaemic conditions followed by loss of reflexes and cuticular fouling by epibionts are the symptoms. The gills are often brown in colour. Early signs include, body reddening, extended gill covers and slight melanized erosions of the uropods, pleopods and periopods. Affected shrimps reveal empty stomachs and midguts and in some cases white watery liquid oozes out. Reducing the biomass (by panial harvest) and increasing the water exchange help to contain this disease. For the subsequent production cycle, it is advisable to dry the pond bottom until the bottom soil cracks. If excessive detritus is noticed the same has to be physically removed. Quicklime (CaO) is normally applied at the rate of 0.5 kg/m' of pond bottom. The treatment pattern is much the same as that of Vibrio anguillarum infections.

 

Brown spot shell disease / burned spot disease / rust disease / shell disease / black spot disease

 

The above-referred names are synonyms of bacterial disease caused by a group of bacteria. In majority of cases, Vibrio, Pseudomonas and Beneckea have been known to cause this disease.

The disease is recorded from the freshwater prawns as well as from Penaeus sp. cultured in India. Providing better water quality, removal of infected and dead prawns, reducing the stock and adequate nutrition help to control the disease. Feeding terramycin incorporated feed at 0.45 mg per kg of feed for two weeks, bath treatment using 0.05 to 1. 0 mg of malachite green per litre of water are suggested.

 

Fungal diseases, diagnosis and management:

 

Fungal diseases have been reported to cause extensive morality ranging from 20 to 100 percent. Several fungi are known to be shrimp pathogens. Three groups of fungi commonly infect the larval stages of shrimps while another one attacks the juvenile or larger shrimps. The common fungus affecting the larval shrimps is Lagenidium. Apart from this species, Siropodium and Haliphthoros also affect the larvae. These fungi

generally require a thin cuticle which is noticed only in shrimp larvae. The most common fungi affecting the larger shrimp belongs to Fusarium sp. Environmental factor such as low salinity prevailing in the monsoon season is found to precipitate fungal infections in the hatchery as well as growout systems.

 

Fungal infections in shrimp larvae:

 

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In the case of larval fungal infections (larval mycosis), it is interesting to note that the infection starts from a fungal spore which attaches itself to the egg of the shrimp and then germinates. The germling (mycelium) then grows as the larva of shrimp grows, ramifies through the body wall of the larva and develops rapidly inside it replacing the muscles and soft tissues of the larva. Ultimately the entire body of the larva becomes a mess of mycelia of fungus. P. monodon, P. indicus and P. merguiensis (banana shrimp) are affected by the fungi Lagenidium and Fusarium sp. To prevent this disease in the hatchery, the inflow water has to be thoroughly filtered. Chemical and ultraviolet irradiation of inflow water is also effective. Application of malachite green at 0.001 to 0.006 mg per litre of water and treflan at 0.01 mg per litre are also found to be effective.

 

Mycosis of adult shrimps:

 

Although the exact extent to which the mycosis of adult shrimps caused by Fusarium sp. affect the shrimp aquaculture is not known, it is certainly considered as a potential threat. Almost all the culturable shrimps are known to be affected. The Fusarium sp. may be identified by the presence of canoe-shaped microconidia and also due to the presence of cotton wool like growth.

 

The fungus gains entry into the body through the already eroded areas or cracks on the cuticle. Preventation and treatment courses are much like that of larval mycosis.

 

Protozoan diseases, diagnosis and management:

 

Protozoan parasites and commensals of shrimp are found to occur externally or internally. The externally occurring ones are considered harmless unless they are present in large numbers. Those present internally can cause disease and are representatives of Microsporidia, Haplosporidia and Gregarina.

 

Cotton shrimp disease/milky disease of shrimps:

 

The cotton/milky shrimp disease is caused by the protozoan parasite belonging to Microsporidian group. Almost all the culturable shrimps are affected. The muscie tissue becomes milky. The microsporidians are abundant in the infected shrimp and cause the white appearance. No eggs are found in milky shrimps and it is inferred that the microsporidians infection render the shrimp incapable of reproduction. Microsporidians are present in the affected shrimp in the form of spores which are microscopic. These spores are transmitted horizontally. Providing better water quality in the hatcheries and growout ponds and following strict farm husbandry practices prevent this disease. Although no satisfactory treatment is evolved yet, experimental results indicated the usage of 0.0075 mg of malachite green per litre of water in static condition for the Post Larvae and addition of commercial bleach to the culture system are successful.

 

Ciliate infestation:

 

The Ciliates, Zoothamnium, Epistylis and Vorticella and suctoreans, Ephelota gemmipara and Acineta may invade all the life stages of the shrimps and cause respiratory and locomotory difficulties when present in large numbers on the gills and shell. In the pond grown shrimps, the ciliates may form a fuzzy mat on the shell as a result of the deterioration of the culture water. Ciliate infestation can be prevented by avoiding heavy silt, high nutrient load, turbidity, and low oxygen tension. Affected shrimps can be treated with baths of chloroquin diphosphate or formalin to remove the ciliates.

 

Gregarine disease:

 

Gregarines are common parasites of the digestive tract of shrimps. Their presence in large numbers in the gut interferes with particle filtration to the hepatopancreatic ducts or through the gut resulting in large scale mortalities. Providing better water quality conditions prevents this disease.

 

DISEASES INFLUENCED BY DIETS:

 

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Pond grown shrimps are more often subjected to nutritional disorders than their younger stages in hatchery or nursery rearing period primarily because of the culture facility. In the case of unfed shrimps, they lose their normal full and robust appearance. The shell becomes thin and flexible as it covers the underlying tissue such as tail meat that becomes greatly resorbed due to lack of nutrients. The moulting is curtailed and in

due course of time the shell and gill become darker.

 

Chronic soft shell syndrome:

 

This disease occurs among the juveniles and adults. The affected shrimp is characterized by a persistently soft and thin exoskeleton, weakness and greater susceptibility to cannibalism. Inadequate amounts of nutrients such as calcium and potassium is known to create this sickness. Dietary and environmental manipulation prevents the occurrence of this disease. This occurs among the juveniles to adults. Affected shrimps have bluish exoskeleton which is also soft and thin. The shrimps also become lethargic. Low levels of the carotenoid astaxanthin in the diet, poor soil and water qauality are the causative factors. Reducing the stocking density, feeding with high quality feed and frequent Water exchange in the culture system prevent this disease.

 

DISEASES OF UNKNOWN AETIOLOGY

 

Inflammation and melanization:

Instances of tissue darkening is observed in shrimp farming. The blood· cells congregate in particular tissue areas (inflammation) where damage has occurred and this is followed by pigment (melanin) deposition. An invasion by infective agent, injury or presence of toxins causes this defect.

 

Gills are prone to darkening due to their fragile nature and their function as collecting site for elimination of the body's waste products. Gills darken readily upon exposure to toxic metals or chemicals and also as a result of infection by fungi like Fusarium sp.

 

Cramped shrimp: Shrimps exposed to a variety of cuIture conditions develop cramped nature. The tail is drawn under the body and becomes rigid to the point where it cannot be straightened. The cause and remedy are not yet studied in detail.

 

VI. DISEASE INSPECTION AND CERTIFICATION

 

The rapid growth of shrimp farming in recent years has led to increase in the live transport ofthe shrimp young ones from one region to another. Such large movements inevitably pose a potential risk of introduction of hitherto unknown pathogens. With the improvised culture techniques, the chances of spreading of the disease is increased. The spread of viral pathogens in shrimps world over and Epizooti Ulcerative Syndrome

(EUS) in the case of the fish in Asia can be taken as the best example in this regard. Transfer and introduction of different stages of shrimps has to be controlled by suitable. regulations. This will reduce the risk of transferring pathogens from one place ot another or from an imported stock to the native stock. Regular inspections on the health status and sanitary conditions of the shrimp farms are to be carried out by trained personnel. The International Office ofEpizootics and Animal Health Problems in Aquaculture, of late has dealt with crustacean pathogens through the Fish Diseases Commission (de Kinkelin, 1992).

 

The listed shrimp pathogens include: MBV, BP, BMNV and IHHNV. The approach for health control in aquaculture involves:

 

(i)   assessment of health status of animals in a production site based upon inspections and standardised sampling procedures followed by laboratory examinations conducted according to the OlE codes.

(ii)   constraints of restocking open water and farming facilities only with products having a health status higher than or equal to that of animals already living in the considered areas,

(iii) eradication of disease when possible, by slaughtering of infected stocks, disinfection and restocking with pathogen free animals, and

(iv) notification by every member country of its particular requirements, besides those provided by the code, for importation of aquaculture animals and animal products.

 

CONCLUSION

 

A number of diseases of shrimps cultured has been enumerated in the preceding discussion. The diseases pose threat to obtain maximum production. In many instances the poor water quality conditions of the culture system only predispose the candidate species towards diseases. Maintenance ofbest water quality could therefore be described as 'health maintenance' and be given top priority in shrimp farming. This will reduce the harmful effects of chemoprophylaxis as well as chemotherapy in farming activities.

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Litopenaeus vannamei is the most commonly cultured shrimp in Latin America and Southeast Asia, representing over 90 % of total shrimp production. India with its 8,118 km of coastline and 1.24 million Ha of brackish water area is the second shrimp producer in the world, with Andhra Pradesh being India’s largest vannamei farming area. Andra Pradesh, situated on the southern coast of the country, has 974 km of coastline and 175,000 Ha of brackish water. Andhra Pradesh has gradually increased its share in total marine exports of the country, with the United States and Vietnam as the main export markets.

Currently, the state’s L. vannamei aquaculture is facing different issues and challenges to achieve sustainability related to diseases outbreaks, lack of availability of quality seed, high feed costs, unauthorized farming, international price fluctuations, less demand in the domestic market, and others. If farmers implement Better Management Practices (BMP) and biosecurity in L. vannamei culture supported by the Government policy measures then sustainability can be achieved. This article discusses the present culture practices, major problems, future perspectives and suggestive measures for sustainable L. vannamei farming in Andhra Pradesh.

A brief history of L. vannamei:

 

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Shrimp farming started as an initiative of the Government of India (GoI) with a study of brackish water fish farming in the late 1970’s. Due to the economic benefits from shrimp farming, the culture of Penaeus monodon developed rapidly during the early 1990’s. The intensification of culture systems and the lack of biosecurity led to disease outbreaks of White Spot Syndrome Virus (WSSV) in 1994. The P. monodon culture almost collapsed in the late 1990’s so in 1999 the fresh water prawn, ‘scampi’ Macrobrachium rosenbergii was introduced as an alternative to P. monodon. The 1990’s are well known as the “era of virus disease” and Andhra Pradesh’s shrimp aquaculture was not the exception.

In 2001- 2002, fresh water prawns faced severe disease outbreaks that affected the state’s production significantly. This is when the Litopenaeus vannamei was proposed as an alternative species due to their disease resistance and tolerance to high stocking densities, low salinity and temperature, as well as their high growth rate. At the same time a risk analysis was carried out by the Central Institute of Brackishwater Aquaculture (CIBA) and National Bureau of Fish Genetics and Resources (NBFGR) with the aim of evaluating the feasibility of the introduction of this new species.

After the experimental studies and due to the constant pressure of growers and traders for the introduction of L. vannamei due its potential in the export market, in 2009 the Coastal Aquaculture Authority (CAA) approved vannamei culture through import of Specific Pathogen Free (SPF) brood stock and strict regulatory guidelines. In order to reduce the risk of adverse effects of the introduction of this exotic shrimp, the Rajiv Gandhi Centre for Aquaculture (RGCA) created the “Aquatic Quarantine Facility of L. vannamei” (AQF) at the behest of Ministry of Agriculture, which is a state-of-the-art facility located in Chennai, Tamil Nadu for quarantine of L. vannamei broodstock imported to India.

L. vannamei in Andhra Pradesh 

For over 25 years, the P. monodon was the mainstay of Indian aquaculture but since L. vannamei’s introduction in 2009, its production and culture area has gradually decreased while L. vannamei has increased.

Potential for development of L. vannamei culture

The production of L. vannamei shrimp is concentrated in East Godavari, West Godavari, Krishna, Prakasam and Nellore state districts. Andhra Pradesh produces more than half of the country’s farmed shrimp and still has a lot of potential to exploit this resource by expanding culture to low salinity waters and through the rehabilitation of abandoned farms in Krishna district. Currently, Srikakulam (the northernmost district of the Andhra Pradesh Coastline) is considered as the ‘sunrise’ of the state’s shrimp farming.

Nursery, culture and feeding practices

 

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The CCA recommends a density of 60 shrimp/m2 but depending on the pond and soil conditions as well as the farmers’ experience the culture densities vary, occasionally reaching 2,000,000 to 6,000,000 Post Larvae (PL) per hectare. Prior to stocking, the pond is tested in order to maintain a pH of 6.5-7. The PL 10 -12 is regularly stocked in high salinities with more than 10 ppt, while PL 15 is stocked when the salinity is low. During the production cycle the water temperatures are maintained between 24-32ºC and the Dissolved Oxygen at 4-5 ppm. The culture cycles in the region range from 90 – 120 days and producers regularly have 2 cycles per year, with stocking in February-March and later in September-October. Shrimp of 17 – 19 grams are considered as a marketable size for the species. Currently the farmers are practicing partial harvests after 60 – 70 days of culture to overcome the slower growth rates of L. vannamei after reaching a size of 19 grams and the increase of operational cost as the days of culture increase. According to the Department of Fisheries of Andhra Pradesh the average production per hectare in the state is 3,000 to 4,000 kg.

As a consequence of the intensification of L. vannamei culture systems in recent years, higher Feed Conversion Ratios (FCR) have been registered, ranging from 1.5:1 to 1.8:1. The feeding frequency in the state is typically 2-4 times per day.

Challenges for sustainable L. vannamei farming

 

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The growth of L. vannamei in the state has been impressive but for further expansion and sustainability the main issue is the lack of availability of quality seed from Specific Pathogen Free brood stock. By 2015, in Andhra Pradesh the CAA has given permission to 192 L. vannamei hatcheries and the Government of India permitted 17 hatcheries for nauplii rearing in facilities outside the jurisdiction of the CAA. For the last couple of years, L. vannamei farms started to develop their own brood stocks from grow out ponds and began producing seed; these seed are sold in the market as SPF and due to the lack of proper testing facilities is impossible for farmers to known the real quality of the seeds. Disease outbreaks are another issue that L. vannamei farming is facing nowadays; they have increased the economic risks and slowed the industry’s development. The White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) resulted in catastrophic losses in Asian and Latin American shrimp farms. However, no major disease outbreaks have been registered in Andhra Pradesh.

WSSV, White Faeces Syndrome (WFS), Loose Shell Syndrome, Black Gill Disease (BGD), Running Mortality Syndrome (RMS) and White Muscle Disease (WMD) are the most common diseases that have affected L. vannamei in Andhra Pradesh. And most recently, Enterocytozoon hepatopenaei (EHP) which does not cause mass mortalities but has been shown to reduce growth. Globally, the feed prices are gradually increasing as a consequence of the rise of raw materials and fishmeal price hikes and Andhra Pradesh shrimp producers are resenting this situation, reflected in the increment of their operational costs.

In Andhra Pradesh, small farm holdings are the most common. Price fluctuations and the lack of information on international prices and demand have generated economic losses for small-scale producers. The uncertainty of market prices has made farmers unable to buy high quality feed, which is very costly. In addition, the quality of more economical feed is often unknown and has to be tested but there is a dearth of technical manpower and laboratories.

Suggestions for achieving sustainability

The shrimp farming industry in the region has been consolidated over the years, but to achieve sustainability it is necessary to increase the Aquatic Quarantine Facilities (AQF) and create more SPF brood stock and nauplii rearing centers. At the same time, it is important to prevent the operation of unauthorized hatcheries and nauplii rearing centers. It is also fundamental to generate protocols and guidelines for probiotic use in soil, water and feed; as well as promote the implementation of best management practices and biosecurity in shrimp culture. The installation of reservoir ponds in L. vannamei farms should be a must, as well as effluent treatment. The government should incentivize the rehabilitation of abandoned shrimp farms and expansion of culture areas as well as promote the development of alternate species with culture systems and hatcheries for mud crabs, sea bass and cobia. Techniques for reducing bacterial loads in shrimp culture systems should be addressed, among other topics.

The Andhra Pradesh L. vannamei aquaculture sector is characterized by small-scale farms, therefore it is important to organize shrimp producers into Farmer Producer Organizations to provide technical support and training in Best Management Practices and Biosecurity, as well as information about the national and international market.

Conclusions

The potential of shrimp culture in Andhra Pradesh is extraordinary; it generates a great number of direct and indirect jobs in the region, represents a great opportunity for rural development and brings a significant economic impact. Thus, it is important for all shrimp farmers to practice responsible aquaculture by only purchasing seed from authorized hatcheries, implementing strict biosecurity protocols and following strict quarantine measures and best management practices in culture systems. This way crop losses will be reduced, as well as the risk of disease outbreaks. Andhra Pradesh has the possibility to become an aquaculture hub in India, that’s the reason why the State government has considered incentives and subsidies to foment aquaculture and its sustainability.

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In light of the devastating disease problems currently plaguing the global shrimp farming industry, water exchange has apparently become a risky management option for maintaining acceptable water quality. The biosecure shrimp farming system is an evolving culture practice which provides means to achieve a higher degree of biosecurity. Biosecurity in aquaculture is the sum of all procedures in place to protect living organisms from contracting, carrying, and spreading diseases and other non-desirable health conditions, with biotherapeutic agents like probiotics.

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The Central Institute of Brackish water Aquaculture (CIBA) has developed a Bio secure Shrimp Farming Technology (BSFT) based on three years of study, which includes several yard experiments and two pond trials, involving investigations on utilization of bio therapeutic agents, water and sediment quality parameters in relation to modifications in culture practices. It differs from conventional farming with regard to practices on water utilization, biosecurity measures followed and in addressing disease concerns without the use of antibiotics and chemotherapeutics. In a way, this is a modification of the widely practiced zero water exchange system relying more on increased provisions of biosecured environment even in the absence  of reservoir ponds. It is also advantageous over the existing zero water exchange system due to reduction in disease risks and input costs. The technology has been successfully tested in on-station field trials with improved productivity and feed conversion ratio (FCR) achieved by using defined biotherapeutic agents. This technology is more suitable for states like West Bengal, Orissa, Kerala, Karnataka, Goa and Maharashtra, since it takes advantage of good monsoon precipitation.

The biosecure shrimp farming technology can be applied to extensive, semi-intensive or intensive shrimp aquaculture. The salient features of this technology are:

• High scoring for biosecurity compared to conventional farming and zero water exchange systems

• Reduced operational costs (no use of antibiotics, chemicals, water exchange and related feed costs)

• Use of efficient biotherapeutic agents

• Reduced risk of disease outbreaks

• Growth and production performance at par or better than conventional farming and zero water exchange systems

• Better feed conversion efficiency as there is no loss of in situ nutrients

• Efficient water budgeting and utilization of  harvested rainwater

• Better profitability and rate of return

Farming technology

The following gives the various steps to be followed with regard to site selection and pond design, biosecurity features, pond preparation, stocking, water and soil quality monitoring, feeding strategy, health management, harvesting and post harvest measures. These are steps unique for BSFT along with standard procedures followed in conventional farming.

Site selection and pond designing

➢ Good monsoon precipitation is one of the prerequisites for this farming to compensate evaporation or seepage loss throughout the culture period.

➢ Site must be free from pollution by industrial effluents and domestic discharge.

➢ Good quality brackishwater (salinity 10- 25 ppt, temperature 25-31o C) and optimum soil quality parameters are essential.

➢ Electricity and communication accessibility  is indispensable  as this technology requires heavy aeration throughout the production cycle.

➢ Rectangular pond with strong non porous high dykes is recommended.

➢ Sluice gate of the simple type with minimum cost compared to the heavy structural investment required in conventional system can be used.

Biosecurity features

➢ With minimal or zero water exchange a high degree of pathogen exclusion is maintained.

➢ Biosecurity barriers or fences around the pond, prevention of the carrier/vectors including birds, disinfection of intake water, avoidance of cross contamination, use of certified healthy seeds, quality feed, use of allowable chemotherapeutics, strict feeding management, water quality monitoring and overall hygiene including that of the equipment and personnel are some of the in-built features of this farming system.

➢ A key step in BSFT is the introduction of a defined microbial community in to the  culture environment which can work synergistically to enhance the overall productivity of the shrimp ponds without resorting to commercial probiotics.

Pond preparation

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➢ Pond preparation is to be started with the drying of the pond bottom till it cracks and surface soil scraped to remove the black soil accumulated from the previous crop since it results in the deposition of considerable load of organic matter.

➢ Soil amendment measures like lime application must be practiced (depending on soil  pH) similar to any other conventional shrimp farms.

➢ Water intake is to be done after proper screening and a higher depth of at least 1.5 m is maintained unlike conventional systems.

➢ Disinfection is recommended with application of Calcium hypochlorite (Ca (OCl) @ 60 ppm.

➢ Optimum natural productivity should be maintained using inorganic fertilizers (Urea: SSP: 2:1 @ 3 ppm) in frequent doses, if necessary; yeast based extract or some good biotherapeutic agents like Bacillus spp. can also  be used for start up a good bloom.

Stocking

➢ Ponds should be stocked with healthy seeds (postlarvae) from a certified hatchery.

➢ 12 nos./m2 postlarve is considered ideal for the BSFT system

➢ Proper acclimatization is to be done to avoid any kind of stress during stocking.

➢ The crop should be scheduled to take advantage of the monsoon rain.

Water and soil quality monitoring

➢ Maintain a stable environment with effective recycling of nutrients and other metabolites through the use of farm made probiotics.

➢ Good aeration is required for supporting the detritus food chain and effective mineralization of the higher level of organic matter in this closed system. .

➢ Parameters influencing productivity like alkalinity, pH and dissolved oxygen are to be maintained within optimum range.

➢ Nutrients like phosphate and nitrate are maintained at higher levels in the BSFT system throughout the culture, resulting in optimum levels of natural productivity.

Feeding strategy

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➢ Feed requirement is to be appropriately estimated through regular sampling (growth and survival) and check tray observation.

➢ Over feeding should be avoided at any cost to prevent eutrophication and associated additional operational costs.

➢ During molting or any other stressful conditions, restricted feeding should be adopted.

➢ The pond bottom in the feeding area should be monitored periodically and if necessary bottom treatment including application of lime mixed with sand is to be adopted.

Health management

➢ Periodical health monitoring with due consideration to proper biosecurity measures is required.

➢ Beneficial microorganisms (such as Lactobacillus spp, Bacillus spp, Pseudomonas spp. and probiotic yeast Saccharomyces spp.) are to be applied as either in single/multi-strain for effectively controlling the pathogenic microorganisms and maintaining the water quality parameters in the optimum range.

➢ Best management practices (BMPs) like care for preventing pathogen carriers, pond bottom

managements, certified healthy seeds, quality feeds and non use of antibiotics /chemicals must be incorporated in the practices followed.

Harvesting and Post harvest measures

➢ There should be minimum stress during harvesting.

➢ After chill killing, the shrimps must be packed in good quality ice and must be transported to processing factory which adopts Hazard Analysis Critical Control Point (HACCP) principles.

Conclusion

This biosecure shrimp farming system is a better farming practice for the coastal ecosystem for its high scoring on biosecurity measures and avoidance of antibiotics and banned chemicals. Effective recycling of nutrients and other metabolites which results in a stable environment  are features of this system. A reduced level of nitrogenous metabolites and better water quality could be maintained even with no water exchange. This BSFT system is amenable for control of disease through best management practices. This evolving farming practice with all its biosecurity features and effective utilisation of biotherapeutic agents can pay rich dividends with reduction in input cost coupled with higher level of production, besides its environment friendly features of utilising harvested rainwater.

 

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Disease challenge by viruses, bacteria, fungi and toxic algae presents a major threat to profitable aquaculture production. Biosecurity, in other words reducing the number of infectious organisms in the aquaculture environment, is the most effective form of protection. Biosecurity is a set of management practices, which reduce the potential for the introduction, and spread of disease-causing organisms onto and between sites. Bio-security procedures, particularly disinfection and sanitation, should be combined with selection of pathogen-free seed and strategic treatments to either eradicate or reduce these pathogens to non-infectious levels.

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The Neospark Biosecurity Programme has been developed over many years with leading aquaculture producers around the nation. Neospark products and procedures have proven effective in practical farm conditions against a broad spectrum of pathogens. These include persistent and difficult to destroy immunosuppressive viruses causing WSSV, MBV and Vibriosis, which make the shrimps and other aquaculture organisms more susceptible to additional disease challenge. Neospark disinfectants are also proven effective against bacteria casing a threat to food safety such as E. coli, Pseudomonas, Aeromonas, Salmonella, Shigella, Edwardsiella etc.

HACCP (Hazard Analysis and Critical Control Points) principles are increasingly being applied on all stages of aquaculture sector to control such threats. Neospark Bio-security Programmes are entirely consistent with HACCP principles.

Disease transmission

The mode of disease transmission between shrimps/prawns/fishes or between ponds or even between sites may or may not differ depending on the type of infection. For example, the occurrence of WSSV in shrimps depends on several factors. The shrimps that carry WSSV may not show any prominent symptoms or mortalities at all times. This may be due to the number of physico-chemical and microbiological factors, which triggers the stress factors causing severe mass mortalities. Therefore the management aspects should be considered for occurrence or non-occurrence of diseases in aquaculture ponds. Subsequently the virus particles through drained water spread the disease by waterborne transmission. On the other hand, the secondary infections caused my bacteria or other microbes, which are native flora, also causing the diseases and heavy mortalities. Infectious agents spread through droppings is also a major threat to the aquaculture sector.

Other diseases persist on sites through the contamination of equipment and organic matter by stubborn virus particles. Many organisms will persist outside the host, and Vibrio, Zoothamnium, Aspergillus and many viruses can survive in this way for a considerable time, especially in organic material.

Factors influencing biosecurity

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Infection may be harboured and spread in a variety of ways. In relation to aquaculture, these may include by crustaceans, in feed, in feacal matter, by birds, by inadvertent human intervention and on equipment. These factors all influence the planning of a bio-security programme.

However, disease avoidance measures can be undertaken elsewhere. For example, use a heavy-duty broad-spectrum virucidal and bactericidal disinfectant (eg KloSant or ViraNil, Neospark), which will be capable of dealing with gross organic challenge.

People are the most important animate factor – including employees, servicemen, vehicle drivers, fishermen (cast net sampling). Staff movements should be as limited as possible, particularly where the disease situation on a particular site has deteriorated.

Control site traffic:

Keep to a minimum and exclude all unauthorized persons. All visitors should enter on foot. Use regularly refilled foot dips, charged with a suitable disinfectant (eg. SparkDin, Bionex, ViraNil - Neospark).

All possible vehicles should be excluded from the site. Vehicles, which must enter, should be subject at the site entrance to spray disinfection of wheels and wheel arches. All visitors should observe standard operating procedures on vehicle cleansing and protective clothing used by vehicle crew.

All site visitors should be provided with adequate protective clothing, and should wash their hands prior to visiting ponds. Use an effective hand hygiene system, which is equally effective even when there is no available water supply (KloSant or ViraNil, Neospark). A shower in, shower out facility should also be seriously considered.

The fish or prawn or shrimp themselves can also be a cause of disease spread. Incoming seed should therefore be from high health status sources and there should be a well-defined health monitoring and audit procedure for bravids/broodrs/nauplei supply flocks. This should extend to hatchery hygiene procedures with regular microbiological/PCR monitoring. Avoid the potential spread of infection by diseased carcasses and dead once by on-site incineration.

Effective cleaning and disinfection reduces pathogen numbers and the weight of disease challenge, and enhances any biosecurity programme. It can only be achieved with sufficient turnaround/down time to allow removal of all organic matter or litter, and to satisfy required contact times for the disinfection products used prior to stocking or restocking. Cleaning and disinfection should include ponds, equipment and surroundings.

 

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Use water with a low total viable count for better growth of fish, prawn and shrimp. It is always better to maintain a reservoir for ideal disinfection of total volume of water. At turnaround, clean and disinfect the water system with a non-tainting product (eg. KloSant or ViraNil, Neospark) to remove the greasy biofilm that will harbour and protect pathogens.

Treat feed trays and feed delivery systems. Feed delivered to the site must be of high health status and vermin protected. Finished feed and stored raw materials should be sampled regularly for its quality. “High risk” feed or raw materials or sources should not be used.

Check biosecurity procedures regularly. Use only biosecurity products with proven broadspectrum efficacy against all viral and bacterial pathogens and use them according to manufacturers’ instructions. Maintain an effective, audited biosecurity programme and prevent entry of pathogens through good farm/pond design and repair.

Biosecurity Checklist:

01. Properly implemented biosecurity measures will limit the spread of disease causing organisms.

02. When these are combined with disinfection and sanitation, vaccination and strategic treatments, many pathogens can be reduced to non-infectious levels.

03. Remember – different infectious agents spread by different methods, so use appropriate measures against each type.

04. Site location and design, and density of fish/prawn/shrimp in a given geographical area, are vital. When planning a new site, there is the opportunity for very effective biosecurity to be implemented at the design stage. However, biosecurity practices must concern themselves with practicalities, rather than a theoretically deal set-up.

05. All sites must have traffic – in personnel, feed, stock and equipment – but this should be kept to an absolute minimum.

06. Only essential vehicles should have access to a site, and these should be sanitized where possible on arrival.

07. Use protective clothing to prevent pathogen spread.

08. Priority should be given to biosecurity measures on breeding and hatching sites since errors here are magnified greatly at the commercial level.

09. Site decontamination, turnaround times and a well audited and structured cleansing and disinfection procedure should be in place for all sites.

10. Effective disease/pathogen carrier control must be maintained.

11. Only disinfectants with proven broad-spectrum efficacy against all viral and bacterial pathogens should be used and then at manufacturers’ stated dilutions and directions.

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Aquaculture is one of the fastest growing food-production sectors but the economic impact that parasites and bacterial, fungal and viral diseases have on the industry is highly significant for the many countries that rely heavily on this industry. Research into the diseases that affect penaeid shrimps that are grown in aquaculture systems is therefore vital, writes Bob Carling for TheFishSite.

 

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Two diseases that are currently being actively in Shrimp farming are: 

1. The bacterial infection, Acute Hepatopancreatic Necrosis Disease (AHPND) – also called Early Mortality Syndrome (EMS) 

2. The fungal infection, hepatopancreatic microsporidiosis caused by Enterocytozoon hepatopenaei (EHP).

AHPND 

Acute Hepatopancreatic Necrosis Disease (AHPND) is a problem of the main countries that farm shrimps – China, Thailand, Vietnam and Malaysia. AHPND can occur in the first 30 days after stocking shrimp into ‘grow-out’ ponds, which is why AHPND is commonly, but incorrectly, called early mortality syndrome (EMS).

The disease is caused by a bacterium that colonizes the shrimp gastrointestinal tract and produces a toxin that causes tissue destruction and dysfunction of the the hepatopancreas, the shrimp digestive organ. The culprit is the bacterium Vibrio parahaemolyticus (Vp), a common enough bacterium in brackish saltwater which, when ingested, can cause gastrointestinal illness in humans.

The species of shrimp affected are:

  • Giant tiger shrimp (Penaeus monodon)
  • Whiteleg (or Pacific white) shrimp (Litopenaeus vannamei, formerly Penaeus vannamei)
  • Chinese white shrimp (Penaeus chinensis)
  • Mortality of infected shrimp stock can exceed 70%.

 

Prevention, control and mitigation measures include:

 

  • Improved biosecurity at the farm, zone, national, and regional levels
  • Zonal management of production units
  • Disease risk assessments, and
  • Development and implementation of aquatic veterinary health plans.

Unless they are certified by independent third parties as being free from AHPND, the use of live feeds for shrimp broodstock should be discouraged – or, where live feeds are used, these should be sterilised or frozen to reduce the likely transfer of AHPND.

Stocking density can have an effect, as does the use of probiotics. The use of Biofloc technology – enhancing water quality by balancing carbon and nitrogen in the system – has also been reported as helping in reducing the impact of AHPND 

EHP

In comparison to AHPND, very little is known about the effects on aquaculture systems by the fungal infection hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP).

Hepatopancreatic microsporidiosis was first detected in P. monodon and then the infection is also reported as being an increasing problem in India, exacerbated by flooding 

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What is known so far is that EHP infects only the tubule epithelial cells of the hepatopancreas of the shrimp. The spores are very small (1.1 × 0.60–0.07μm) and have a polar filament with 4–5 coils.

Although previously EHP was only reported as being found in the shrimp hepatopancreas tubule epithelial cells.

In 2010, EHP was reported as being associated with ‘white faeces syndrome’ (WFS), but further experiments have however failed to show the association with WFS, although transmission has been demonstrated. EHP spores are extremely hardy and EHP can be transmitted horizontally between shrimp. As a result EHP infection can spread progressively and is believed to intensify with successive shrimp crops over time.

Prevention, control and mitigation

Detection and screening is done by using nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) tests. 

Histological analysis for the spores is possible, but is difficult unless performed by expert histopathologists. Live feeds should be avoided and/or freezing/sterilization should be carried out.

Research is currently being prioritised on:

  • A better understanding the lifecycle of EHP
  • The EHP transmission pathway
  • Development of real-time PCR testing
  • Easier histopathological identification of spores <liDevelopment and registration of surface disinfectants to control EHP
  • Development and registration of drug treatment for the treatment of live shrimp infected with EHP
  • iDevelopment of cell lines to culture microsporidia

Shrimp farmers have tried a variety of measures to try and eradicate EHP from hatcheries, nurseries and grow-out farms but with little success. There has been some experimentation with alternative treatments, including coccidiostats used to treat poultry like Monensin, but no research has yet been conducted to ensure the product is safe to use in shrimp production systems.

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Shrimp Probiotics

Biosecurity involves following strict management protocols to prevent specific pathogens from entering a system or reducing the numbers. A good understanding of pathogen reservoirs is important. Quarantine, sanitation and disinfection are all important components of biosecurity.

Quarantine, defined as the isolation of an organism or group of organisms to prevent the introduction or spread of infectious disease, is a standard procedure that is extremely important in aquaculture. In practical terms, quarantine is a standard set of procedures that should be observed to prevent the introduction of pathogens or diseases into a population of fish, prawn and shrimp in aquaculture. The quarantine protocols should be strictly adhered and should follow as many of the following protocols as are practical:

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Shrimp Probiotics

In order L. Vannamei can grow optimally, it needs a place to live that can provide state physics, chemistry, and biology is optimal. Physical environmental conditions are including temperature and salinity. While the chemical conditions is including pH, dissolved oxygen (DO), nitrate, orthophosphoric, and the presence of plankton as natural feed. Should be noted that environmental conditions can inhibit the growth of shrimp, shrimp can be deadly, such as the emergence of toxic gases and pathogenic microorganisms.

Temperature is one factor controlling the speed of biochemical reactions. This is because the temperature can determine the metabolic rate of shrimp and other aquatic organisms. Low temperatures will result in a lower metabolic system in contrast to the high temperatures will spur a more rapid metabolism. In order for the cultivation of L. Vannamei to work well, pond waters temperature range suggested is between 28 - 32o C.

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