Timothy W. Flegel

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.

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection in Shrimp

ABSTRACT Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.

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

Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

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 (>

Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

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.

Shrimp Molecular Responses to Viral Pathogens

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

Statistical correlation between severity of hepatopancreatic parvovirus infection and stunting of farmed black tiger shrimp (Penaeus monodon)

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide.

The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in southern Thailand

From almost negligible amounts in 1970, the quantity of cultivated shrimp (∼3 million metric tons in 2007) has risen to approach that of the capture fishery and it constitutes a vital source of export income for many countries. Despite this success, viral diseases along the way have caused billions of dollars of losses for shrimp farmers. Desire to reduce the losses to white spot syndrome virus in particular, has stimulated much research since 2000 on the shrimp response to viral pathogens at the molecular level. The objective of the work is to develop novel, practical methods for improved disease control. This review covers the background and limitations of the current work, baseline studies and studies on humoral responses, on binding between shrimp and viral structural proteins and on intracellular responses. It also includes discussion of several important phenomena (i.e., the quasi immune response, viral co-infections, viral sequences in the shrimp genome and persistent viral infections) for which little or no molecular information is currently available, but is much needed.

Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

Abstract In response to complaints from shrimp farmers, samples were taken to determine whether stunting in cultured Penaeus monodon was related to the presence of known infectious agents. Shrimp were arbitrarily defined as normal at >8 cm length and stunted at Agmasoma penaei . Infections were found for only HPV, IHHNV and MBV. For all 80 specimens, prevalences were HPV 49% (39), MBV 50% (40) and IHHNV 4% (3). Mean prevalences for HPV in the normal and stunted groups (28±8% and 70±15%, respectively) were not significantly different ( P =0.057). Nor were the mean prevalences of MBV infection (45±9% and 58±15%, respectively; P =0.496). For HPV and MBV, severity of infection was estimated by an ocular grid severity index. Pearson product moment correlation tests on a subgroup of 71 pooled samples showed a negative correlation between HPV severity index and length ( P P P

Shrimp hepatopancreatic parvovirus detection by combining loop-mediated isothermal amplification with a lateral flow dipstick

Abstract The occurrence, development and histopathology of monodon baculovirus (MBV) in larvae, postlarvae (PL) and broodstock of Penaeus monodon was studied over a period of 1 year in southern Thailand. In histological samples of captured male and female broodstock obtained from the Andaman Sea, the incidence of individuals with MBV occlusion bodies (OBs) was approximately 5.7% during the period. Absence of MBV virions in electron micrographs of oocytes of MBV-infected females, mature eggs and nauplii indicated that the virus may not be transmitted transovarially. Together, these facts suggest that elimination of MBV from hatcheries by screening of quarantined wild broodstock would be feasible. In the hatchery, baculovirus OBs began to be clearly identifiable in the hepatopancreas of larvae at the third zoeal stage. During all subsequent larval and PL developmental stages, there was a high incidence of infected individuals, some with extremely large numbers of OBs. The average incidence of PL positive for OBs in histological specimens approached 100% during the period of the study in a survey that included eight commercial PL producers on both the Gulf of Thailand and the Andaman Sea. However, the incidence of OB-positive animals gradually fell as the shrimp grew older and OBs were often undetectable in healthy juveniles derived from infected PL within approximately 2 weeks of stocking in grow-out ponds. In some instances, it was possible to follow groups of PL from the nursery stage through to harvest of market-size shrimp. Average survival data for such shrimp followed in a large number of ponds over a period of 1 year indicated that MBV was well tolerated by Penaeus monodon if other rearing conditions were optimal. However, in the event of environmental or other stress, the incidence of animals showing large numbers of MBV particles increased dramatically.