Historic emergence, impact and current status of shrimp pathogens in the Americas.

Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the eastern USA via frozen commodity products and it reached the main shrimp farming countries of the Americas located on the Pacific side of the continents by the same mechanism in 1999. As is the case in Asia, WSD is the dominant disease problem of farmed shrimp in the Americas. The most recent disease to emerge in the Americas was infectious myonecrosis caused by IMN virus. As had happened before, within 3years of its discovery, the disease had been transferred to SE Asia with live P. vannamei, and because of its impact on the industry and potential for further spread in was listed by the OIE in 2005. Despite the huge negative impact of disease on the shrimp farming industry in the Americas, the industry has continued to grow and mature into a more sustainable industry. In marked contrast to 15-20years ago when PLs produced from wild adults and wild PLs were used to stock farms in the Americas, the industry now relies on domesticated lines of broodstock that have undergone selection for desirable characteristics including disease resistance.

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.

Global transboundry disease politics: the OIE perspective.

Reviewed in this paper are the steps for listing or de-listing of an aquatic animal disease, the current list of OIE listed aquatic animal diseases, and the reporting requirements for listed diseases by member countries. The current OIE listed aquatic animal diseases includes two diseases of amphibians, nine of fish, seven of mollusks, and eight of crustaceans. Of interest is the difference in importance of the listed diseases in each of the four groups of aquatic animals. In mollusks, parasitic diseases dominate the list, while in fish and crustaceans virus diseases are dominant. Whether a listed disease is due to a virus, fungus, bacterium or a parasite, the occurrence of the disease may adversely affect international trade among trading partners that have, or do not have, the listed disease. By its very nature, the international trade in terrestrial animals and aquatic animals, and their products, is influenced by national and international politics. When the occurrence of an OIE listed or emerging disease becomes an issue between trading partners, trade restrictions may be put in place and disputes are often a consequence. The World Trade Organization named the OIE as the reference body for animal health as it relates to international trade. This action recognized the 88 year history of the work by the OIE in disease control, listing of diseases, the development of the terrestrial and aquatic codes and the diagnostic manuals, and the prompt notification of members by the OIE of the occurrence of listed diseases. The intent of the WTO with this action was likely to minimize disease related trade disputes brought before the WTO.

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.

A real-time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) causes a common shrimp disease that occurs in many shrimp farming regions, especially in the Indo Pacific, and infects most of the cultured penaeid species. There are seven geographic HPV isolates known, so a method to detect different HPV types is needed. We developed a sensitive and generic real-time PCR assay for the detection of HPV. A pair of primers and TaqMan probe based on an HPV sequence obtained from samples of Fenneropenaeus chinensis from Korea were selected, and they were used to amplify a 92 bp DNA fragment. This real-time PCR was found to be specific to HPV and did not react with other shrimp viruses. A plasmid (pHPV-2) containing the target HPV sequence was constructed and used for determination of the sensitivity of this assay. The assay could detect a single copy of plasmid DNA, and it was used successfully in finding HPV in shrimp samples from the China-Yellow Sea region, Taiwan, Korea, Thailand, Madagascar, New Caledonia and Tanzania.

Development and characterization of a monoclonal antibody against Taura syndrome virus.

We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes.

Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay.

We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 x 10(6) to 2.2 x 10(8) IMNV copy numbers microL(-1) RNA, and it was similar with the standard RNA extraction (from 1.2 x 10(6) to 6.3 x 10(7) IMNV copy numbers microL(-1) RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.

Comparison of Taura syndrome virus (TSV) detection methods during chronic-phase infection in Penaeus vannamei.

Methods to detect Taura syndrome virus (TSV) were assessed for their ability to detect the virus during chronic phase infection in the Pacific white shrimp Penaeus vannamei. In situ hybridization (ISH), immunohistochemistry (IHC) using monoclonal antibody 1A1, conventional RT-PCR and real-time quantitative (q)RT-PCR were compared using shrimp sampled over 60 wk following experimental TSV infection. Between Weeks 7 and 60, hematoxylin-eosin histology confirmed the presence of lymphoid organ spheroids (LOS) and an absence of lesions in the cuticular epithelium. ISH detected TSV in LOS over the duration of the study. IHC was generally less sensitive than ISH, and after Week 24, was often unable to confirm TSV infection. Detection of TSV by RT-PCR was highly dependent on sample source after Week 43, where viral RNA was detected in 12 of 14 hemolymph samples but only 5 of 16 pleopod samples. qRT-PCR detected TSV over the 60 wk in both hemolymph and pleopods, although RNA copy numbers in pleopods were consistently lower throughout the study. This study demonstrates that ISH and qRT-PCR are the most reliable methods for detecting TSV during late chronic phase infection. RT-PCR was also reliable if hemolymph was used as the sample source.

Opportunities for training in shrimp diseases.

Opportunities for formal training in shrimp diseases were not available 30 years ago. This was because the shrimp farming industry was in its infancy with few significant disease issues and even fewer shrimp disease specialists investigating the causes of production losses. In 2006, more than two million metric tons of the marine penaeid shrimp were farmed, accounting for more than half of the world's supply. With most of the world's shrimp fisheries at maximum sustainable yields, the ratio of farmed to fished shrimp appears likely to continue to increase. The remarkable growth of sustainable shrimp farming was made possible through the development of methods to diagnose and manage disease in the world's shrimp farms. This occurred as the result of the development of training opportunities in shrimp disease diagnosis and control methods and the application of that knowledge, by an ever increasing number of shrimp diseases specialists, to disease management at shrimp farms. The first type of formal training to become generally available to the industry was in the form of special short courses and workshops. The first of these, which was open to international participants, was given at the University of Arizona in 1989. Since that first course several dozen more special short courses and workshops on shrimp diseases have been given by the University of Arizona. Dozens more special courses and workshops on shrimp diseases have been given by other groups, including other universities, industry cooperatives, governments and international aid agencies, in a wide range of countries (and languages) where shrimp farming constitutes an important industry. In parallel, graduate study programs leading to post graduate degrees, with shrimp disease as the research topic, have developed while formal courses in shrimp diseases have not become widely available in veterinary or fisheries college curricula in the USA and Europe, such courses are appearing in university programs located in some of the shrimp farming countries of SE Asia. The trend towards more formal training programs in shrimp diseases and disease management is likely to continue as the industry continues to mature and become increasingly sustainable.

Application of molecular diagnostic methods to penaeid shrimp diseases: advances of the past 10 years for control of viral diseases in farmed shrimp.

The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.

Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.

Traceability of aquatic animals.

Effective methods of traceability are urgently required for use in research as well as in different types of aquaculture operations and to control trade in aquatic animals and products. In regard to the marking of fish, many different tagging methods have been described and the method to be used depends on the purpose and need for tagging. In contrast, for molluscs and crustaceans, only a few methods of marking such animals have been described, due to the practical difficulties. The authors first describe the different methods for tracing fish and fishery products, by means of external tags, such as Floy tags, Carlin tags and passive integrated transponder tags; chemical marking using inorganic substances such as silver nitrate or potassium nitrate, pigments, oxytetracycline, etc.; and several different types of electronic devices in which basic information such as the strain of fish, farm of origin or weight can be stored. Genetic traceability using deoxyribonucleic acid profiling is developing quite rapidly for cultured brood stocks and wild populations. This technique may be used with very high degrees of confidence to assign to or exclude animals or products from their claimed origin, paternity or strain, and may be used as evidence in court proceedings. The second section of this paper describes the traceability of live molluscs for restocking and for human consumption. In these applications, genetic markers have been demonstrated to be suitable. Mechanical tagging on a small scale for research purposes has also been used. Otherwise, the only means of tracing live molluscs are the movement documents and the labelling on boxes that certifies the origin of the commodity. The third section describes the methods available for tracing live and dead crustaceans. A large variety of physical tagging methods for decapod crustaceans is described, such as the injection of biological stains (fast green, Niagara sky blue, trypan red and blue) and external tags such as coloured streamer tags, wire tags and a variety of anchor tags. Furthermore, a number of different internal coding methods, such as the coded micro-wire tags and injected elastomer tags are discussed in detail. As is the case for fish, genetic molecular techniques are also applied in population studies of crustaceans; some of the molecular genetic methods are described. Prawns for human consumption are most frequently packed whole or as tails after the necessary sorting, washing and freezing and the only way of performing a traceback is through documents relating to movement, invoices, health certificates and labelling of the boxes. The minimum requirements for labelling would be the content of the packages, i.e. species, quantity, identification of the manufacturer (name and address), packing place, importer/exporter or vendor of the product, in addition to the loading bill number.

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR.

A real-time PCR method using a fluorogenic 5' nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 x 10(7) copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and contained up to 10(9) copies of IHHNV microg(-1) of DNA. Similar quantities of IHHNV were detected in hatchery-raised, small juvenile P. stylirostris collected from Guam in 1995 and in farm-raised, post-larval P. monodon from the Philippines in 1996. Laboratory-infected P. stylirostris contained approximately 10(8) copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissue. In contrast, individuals of Super Shrimp, a line of P. stylirostris selected for IHHNV resistance, showed no signs of infection 32 d after ingesting IHHNV-infected shrimp tissue. Laboratory-infected P. vannamei also contained approximately 10(8) copies of IHHNV 30 d after being fed infected shrimp tissue. A time-course study of IHHNV replication in juvenile P. vannamei showed that the doubling time in the exponential growth phase was approximately 22 h.

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level.

A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.

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.

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.

Identification of genomic variations among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization.

White spot syndrome virus (WSSV) is widely distributed in most of the Asian countries where penaeid shrimp are cultured, as well as in some regions of the USA. Six geographic isolates of WSSV-1 each from penaeid shrimp from China, India, Thailand, and the US states of Texas and South Carolina, and 1 isolated from crayfish at the National Zoological Park in Washington, DC-were compared by combining the methods of restriction analysis and Southern blot hybridization. DNA was extracted from purified viruses and then digested with selected endonucleases: AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI and XhoI. The blots were detected with digoxigenin-11-dUTP-labeled WSSV genomic probes: LN4, C42 and A6. No distinctive differences among the 5 WSSV isolates from penaeid shrimp were detected; however, differences in the WSSV isolate from crayfish were observed. A 2.8 kb DNA fragment originating from the crayfish isolate and encompassing the LN4 region was subcloned into pBluescript and sequenced for comparison with the LN4 fragment from the Thailand WSSV isolate. The results indicate that some genomic components of WSSV from different geographic regions share a high degree of homology. This method can be used to distinguish between the WSSV isolate from crayfish and the WSSV isolates from penaeid shrimp.

Protein analysis of geographic isolates of shrimp white spot syndrome virus.

Six geographic isolates of the white spot syndrome virus (WSSV) of penaeid shrimp, from China, India, Thailand, South Carolina, Texas, as well as from crayfish kept at the US National Zoo in Washington D. C, were compared by electron microscopy and sodium sulfate polyacrylamine gel electrophoresis (SDS-PAGE). Amino acid compositions of four of the major structural polypeptides of the South Carolina WSSV were analyzed, and three of the four polypeptides were partially sequenced from their NH2 termini. The morphologies of purified virions of the six geographic isolates of WSSV were indistinguishable by transmission electron microscopy. By SDS-PAGE, the protein profiles of the six isolates were very similar, but not identical. They all contained three major polypeptides with sizes of approximately 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the geographic isolates. The WSSV isolated from crayfish presented a slightly different structural protein profile, particularly with regard to the protein in the 19 kDa range that appeared larger in size than those of the other isolates. The NH2 terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the South Carolina WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. No significant homologous sequence was found in the GenBank. These protein sequences have been submitted to the SWISS-PROT Protein Data Bank and assigned the accession numbers P82004, P82005 and P82006.

A yellow head virus gene probe: nucleotide sequence and application for in situ hybridization.

A portion of the genome of yellow head virus (YHV) of penaeid shrimp was cloned and the cDNA fragment (1161 bp) was designated clone 3-27. The fragment was labeled with digoxigenin and hybridized in situ to tissue sections of YHV-infected Penaeus vannamei. Positively reacting tissues included those of the lymphoid organ, cuticular epithelium, and gills. In addition, connective tissue of hepatopancreas, heart, antennal gland, hematopoietic organ, nerve tract, midgut cecum and muscle reacted to the probe. The probe was highly specific since it hybridized only to tissues from YHV-infected shrimp. It did not react to those of uninfected shrimp or shrimp infected with WSSV (white spot syndrome virus), IHHNV (infectious hypodermal and hematopoietic necrosis virus), or TSV (Taura syndrome virus). The clone was sequenced, and primers were synthesized for rapid detection of YHV in hemolymph using RT-PCR (reverse transcription-polymerase chain reaction). The strand that constituted the viral sequence in the cDNA was also determined via RT-PCR and in situ hybridization with a single-stranded RNA (ssRNA) probe.

Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp.

Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this viral agent has spread throughout the shrimp growing regions of South and Central America to become established in North America in the short span of 5 yr. Diagnostic methods for TSV include histopathology, bioassay using susceptible Penaeus vannamei as the indicator species and in situ hybridization with TSV specific complimentary DNA (cDNA) gene probes. An additional method for detecting TSV is through the use of reverse transcription polymerase chain reaction (RT-PCR). Two oligonucleotide primers were selected using the sequence information from a cloned cDNA segment of the TSV genome. The primers, designated 9195 and 9992, used in the RT-PCR procedure amplify a 231 base pair (bp) fragment of the cDNA. Using the RT-PCR technique, TSV has been detected in the hemolymph of P. stylirostris and P. vannamei with experimentally induced TSV infections.