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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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