Historic emergence, impact and current status of shrimp pathogens in Asia

It is estimated that approximately 60% of disease losses in shrimp aquaculture have been caused by viral pathogens and 20% by bacterial pathogens. By comparison, losses to fungi and parasites have been relatively small. For bacterial pathogens, Vibrio species are the most important while for viral pathogens importance has changed since 2003 when domesticated and genetically selected stocks of the American whiteleg shrimp Penaeus (Litopenaeus) vannamei (Boone 1931) replaced the formerly dominant giant tiger or black tiger shrimp Penaeus (Penaeus) monodon (Fabricius 1798) as the dominant cultivated species. For both species, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal. Next most important for P. vannamei is infectious myonecrosis virus (IMNV), originally reported from Brazil, but since 2006 from Indonesia where it was probably introduced by careless importation of shrimp aquaculture stocks. So far, IMNV has not been reported from other countries in Asia. Former impacts of Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) on this species have dramatically declined due to the introduction of tolerant stocks and to implementation of good biosecurity practices. Another problem recently reported for P. vannamei in Asia is abdominal segment deformity disease (ASDD), possibly caused by a previously unknown retrovirus-like agent. Next most important after WSSV and YHV for P. monodon is monodon slow growth syndrome (MSGS) for which component causes appear to be Laem Singh virus (LSNV) and a cryptic integrase containing element (ICE). Hepatopancreatic parvovirus (HPV) and monodon baculovirus (MBV) may be problematic when captured P. monodon are used to produce larvae, but only in the absence of proper preventative measures. Since 2009 increasing losses with P. vannamei in China, Vietnam and now Thailand are associated with acute hepatopancreatic necrosis syndrome (AHPNS) of presently unknown cause. Despite these problems, total production of cultivated penaeid shrimp from Asia will probably continue to rise as transient disease problems are solved and use of post larvae originating from domesticated SPF shrimp stocks in more biosecure settings expands.     

The role of selective breeding and biosecurity in the prevention of disease in penaeid shrimp aquaculture

About 3.5 million metric tons of farmed shrimp were produced globally in 2009 with an estimated value greater than USD$14.6 billion. Despite the economic importance of farmed shrimp, the global shrimp farming industry continues to be plagued by disease. There are a number of strategies a shrimp farmer can employ to mitigate crop loss from disease, including the use of Specific Pathogen Free (SPF), selectively bred shrimp and the adoption of on-farm biosecurity practices. Selective breeding for disease resistance began in the mid 1990s in response to outbreaks of Taura syndrome, caused by Taura syndrome virus (TSV), which devastated populations of farmed shrimp (Litopenaeus vannamei) throughout the Americas. Breeding programs designed to enhance TSV survival have generated valuable information about the quantitative genetics of disease resistance in shrimp and have produced shrimp families which exhibit high survival after TSV exposure. The commercial availability of these selected shrimp has benefitted the shrimp farming industry and TSV is no longer considered a major threat in many shrimp farming regions. Although selective breeding has been valuable in combating TSV, this approach has not been effective for other viral pathogens and selective breeding may not be the most effective strategy for the long-term viability of the industry. Cost-effective, on-farm biosecurity protocols can be more practical and less expensive than breeding programs designed to enhance disease resistance. Of particular importance is the use of SPF shrimp stocked in biosecure environments where physical barriers are in place to mitigate the introduction and spread of virulent pathogens.     

Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): a review

Penaeid shrimp aquaculture is an important industry in the Americas, and the industry is based almost entirely on the culture of the Pacific White Shrimp, Litopenaeus vannamei. Western Hemisphere shrimp farmers in 14 countries in 2004 produced more than 200,000 metric tons of shrimp, generated more than $2 billion in revenue, and employed more than 500,000 people. Disease has had a major impact on shrimp aquaculture in the Americas since it became a significant commercial entity in the 1970s. Diseases due to viruses, rickettsial-like bacteria, true bacteria, protozoa, and fungi have emerged as major diseases of farmed shrimp in the region. Many of the bacterial, fungal and protozoan caused diseases are managed using improved culture practices, routine sanitation, and the use of chemotherapeutics. However, the virus diseases have been far more problematic to manage and they have been responsible for the most costly epizootics. Examples include the Taura syndrome pandemic that began in 1991-1992 when the disease emerged in Ecuador, and the subsequent White Spot Disease pandemic that followed its introduction to Central America from Asia in 1999. Because of their socioeconomic significance to shrimp farming, seven of the nine crustacean diseases listed by the World Animal Organization (OIE) are virus diseases of shrimp. Of the seven virus diseases of penaeid shrimp, five are native to the Americas or have become enzootic following their introduction. The shrimp virus diseases in the Americas are increasingly being managed by exclusion using a combination of biosecurity and the practice of culturing domesticated specific pathogen-free (SPF) stocks or specific pathogen-resistant (SPR) stocks. Despite the significant challenges posed by disease, the shrimp farming industry of the Americas has responded to the challenges posed by disease and it has developed methods to manage its diseases and mature into a sustainable industry.     

Increasing production in Korean shrimp farms with white-spot syndrome virus PCR-negative brood stock

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fry epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.     

Disease will limit future food supply from the global crustacean fishery and aquaculture sectors

Seafood is a highly traded food commodity. Farmed and captured crustaceans contribute a significant proportion with annual production exceeding 10 M metric tonnes with first sale value of $40bn. The sector is dominated by farmed tropical marine shrimp, the fastest growing sector of the global aquaculture industry. It is significant in supporting rural livelihoods and alleviating poverty in producing nations within Asia and Latin America while forming an increasing contribution to aquatic food supply in more developed countries. Nations with marine borders often also support important marine fisheries for crustaceans that are regionally traded as live animals and commodity products. A general separation of net producing and net consuming nations for crustacean seafood has created a truly globalised food industry. Projections for increasing global demand for seafood in the face of level or declining fisheries requires continued expansion and intensification of aquaculture while ensuring best utilisation of captured stocks. Furthermore, continued pressure from consuming nations to ensure safe products for human consumption are being augmented by additional legislative requirements for animals (and their products) to be of low disease status. As a consequence, increasing emphasis is being placed on enforcement of regulations and better governance of the sector; currently this is a challenge in light of a fragmented industry and less stringent regulations associated with animal disease within producer nations. Current estimates predict that up to 40% of tropical shrimp production (>$3bn) is lost annually, mainly due to viral pathogens for which standard preventative measures (e.g. such as vaccination) are not feasible. In light of this problem, new approaches are urgently required to enhance yield by improving broodstock and larval sourcing, promoting best management practices by farmer outreach and supporting cutting-edge research that aims to harness the natural abilities of invertebrates to mitigate assault from pathogens (e.g. the use of RNA interference therapeutics). In terms of fisheries losses associated with disease, key issues are centred on mortality and quality degradation in the post-capture phase, largely due to poor grading and handling by fishers and the industry chain. Occurrence of disease in wild crustaceans is also widely reported, with some indications that climatic changes may be increasing susceptibility to important pathogens (e.g. the parasite Hematodinium). However, despite improvements in field and laboratory diagnostics, defining population-level effects of disease in these fisheries remains elusive. Coordination of disease specialists with fisheries scientists will be required to understand current and future impacts of existing and emergent diseases on wild stocks. Overall, the increasing demand for crustacean seafood in light of these issues signals a clear warning for the future sustainability of this global industry. The linking together of global experts in the culture, capture and trading of crustaceans with pathologists, epidemiologists, ecologists, therapeutics specialists and policy makers in the field of food security will allow these issues to be better identified and addressed.     

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.     

Comparison of Protein Expression Profiles of the Hepatopancreas in Fenneropenaeus chinensis Challenged with Heat-inactivated Vibrio anguillarum and White Spot Syndrome Virus

Fenneropenaeus chinensis (Chinese shrimp) culture industry, like other Penaeidae culture, has been seriously affected by the shrimp diseases caused by bacteria and virus. To better understand the mechanism of immune response of shrimp to different pathogens, proteome research approach was utilized in this study. Firstly, the soluble hepatopancreas protein samples in adult Chinese shrimp among control, heat-inactivated Vibrio-challenged and white spot syndrome virus-infected groups were separated by 2-DE (pH range, 4–7; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pH range, 3–10; tricine-SDS-PAGE). Then the differentially expressed protein spots (≥1.5-fold or ≤0.67-fold averagely of controls) were analyzed by LC-ESI-MS/MS. Using Mascot online database searching algorithm and SEQUEST searching program, 48 and 49 differentially expressed protein spots were successfully identified in response to Vibrio and white spot syndrome virus infection, respectively. Based on these results, we discussed the mechanism of immune response of the shrimp and shed light on the differences between immune response of shrimp toward Vibrio and white spot syndrome virus. This study also set a basis for further analyses of some key genes in immune response of Chinese shrimp.     

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.     

Controlled bioassay systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus

In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 +/- 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27 degrees C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD50 and LD90. The LD50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 x 10(7) (Trial 1) and 1:6.667 x 10(7) (Trial 2). The LD50s of 2 different WSSV injection preparations were estimated at 1:4.444 x 10(6) and 1:4.505 x 10(6). The LD50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20 degrees C and 1:1272 at 27 degrees C. A second waterborne TSV inoculum challenge at 27 degrees C produced an LD50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens.     

白斑综合症病毒感染相关蛋白质相互作用的研究进展

白斑综合症病毒（white spot syndrome virus,WSSV）是危害对虾的主要病原,给全球水产养殖业带来了巨大经济损失,但至今仍未发现有效的防治方法。研究病毒与宿主的相互作用对于深入了解病毒的致病机理和宿主的免疫机制,从而寻找合适的抗病毒措施具有非常重要的理论意义和实际应用价值。该文主要介绍了蛋白质相互作用的研究方法,以及WSSV病毒蛋白之间、病毒—宿主蛋白之间和宿主蛋白之间相互作用的研究进展,为有效地防治WSSV及相关科研提供参考。     

Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28

It has been generally accepted that invertebrates such as shrimp do not have an adaptive immune response system comparable to that of vertebrates. However, in the last few years, several studies have suggested the existence of such a response in invertebrates. In one of these studies, the shrimp Penaeus monodon showed increased protection against white spot syndrome virus (WSSV) using a recombinant VP28 envelope protein of WSSV. In an effort to further investigate whether this increased protection is limited to P. monodon or can be extended to other penaeid shrimp, experiments were performed using the Pacific white shrimp Litopenaeus vannamei. As found with P. monodon, a significantly lower cumulative mortality for VP28-fed shrimp was found compared to the controls. These experiments demonstrate that there is potential to use oral application of specific proteins to protect the 2 most important cultured shrimp species, P. monodon and L. vannamei, against WSSV. Most likely, this increased protection is based on a shared and, therefore, general defence mechanism present in all shrimp species. This makes the design of intervention strategies against pathogens based on defined proteins a viable option for shrimp culture.     

Viral diseases of penaeid shrimp with particular reference to four viruses recently found in shrimp from Queensland

The culture of penaeid shrimp world-wide is primarily dependent on wild-caught broodstock which has an enormous potential to introduce new pathogens, particularly viruses, into culture systems. Of the 13 viruses described for cultured penaeid shrimp, seven have been described within the past 5 years; the most devastating viral epidemics on record for cultured penaeid shrimp have also occurred within the past 5years. During examination of local wild and cultured shrimp, four new viruses were found. Bennettae baculovirus was discovered in the digestive gland of wild Metapenaeus bennettae. It closely resembles monodon baculovirus (MBV) but has a more slender virion, does not cross-react with a DNA probe for MBV and is not infectious to Penaeus monodon. Two morphologically indistinguishable viruses, one pathogenic (gill-associated virus, GAV) and the other benign (lymphoid organ virus, LOV), were found in cultured P. monodon. LOV and GAV closely resemble yellow head virus (YHV) of Thailand. A parvo-like virus was found recently in dying post-larvae of P. japonicus. As the intensity of shrimp culture world-wide increases, researchers can expect to discover more penaeid viruses. The need to close the life cycle of P. monodon and other cultured species and develop rapid diagnostic methods for viral infections has become imperative.     

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.     

A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.     

Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.     

Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii

The concept of polymicrobial disease is well accepted in human and veterinary medicine but has received very little attention in the field of aquaculture. This study was conducted to investigate the synergistic effect of white spot syndrome virus (WSSV) and Vibrio campbellii on development of disease in specific pathogen-free (SPF) shrimp Litopenaeus vannamei. The juvenile shrimp were first injected with WSSV at a dose of 30 SID(50) shrimp(-1) (SID(50) = shrimp infectious dose with 50% endpoint) and 24 h later with 10(6) colony-forming units (cfu) of V. campbellii shrimp(-1). Controls receiving just one of the pathogens or negative inocula were included. In the treatment with WSSV only, shrimp started to die at 48-108 h post injection (hpi) and cumulative mortality reached 100% at 268-336 hpi. In the treatment with only V. campbellii injection (10(6) cfu shrimp(-1)), cumulative mortality reached 16.7%. Shrimp in the dual treatment died very quickly after V. campbellii injection and 100% cumulative mortality was obtained at 72-96 hpi. When WSSV-injected shrimp were given sonicated V. campbellii instead of live V. campbellii, no synergistic effect was observed. Density of V. campbellii in the haemolymph of co-infected moribund shrimp collected 10 h after V. campbellii injection was significantly higher than in shrimp injected with V. campbellii only (P < 0.01). However, there was no difference in WSSV replication between shrimp inoculated with WSSV only compared with dually inoculated ones. This study revealed that prior infection with WSSV enhances the multiplication and disease inducing capacity of V. campbellii in shrimp.     

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.