Donald V. Lightner

Shrimp diseases and current diagnostic methods

Abstract In less than 30 yr, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of U.S. dollars in revenue, and augmentation of the worlds food supply with a high value crop. Concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. Major epizootics have plagued the worlds shrimp culture industries. The most important diseases of cultured penaeid shrimp have had viral or bacterial etiologies, but a few important diseases have fungal and protozoan agents as their cause. Diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. While tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. The need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. The industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like IHHNV, HPV, TSV, WSSV, MBV, and BP. Additional genomic probes for viruses, for bacterial pathogens like NHP and certain Vibrio spp., and Microsporidia have also been developed. Highly sensitive detection methods for some pathogens that employ DNA amplification methods based on the polymerase chain reaction (PCR) now exist, and more PCR methods are being developed for additional agents. These advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease.

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.

A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Kochs Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.

Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp.

Abstract Populations of the Pacific blue shrimp, Penaeus stylirostris, reared at the University of Arizonas experimental shrimp culture facility on Oahu in Hawaii from late 1980 through 1981, were severely affected by a highly acute and lethal disease of viral etiology. Also found to be susceptible to the disease were P. vannamei and P. monodon. The disease was named infectious hypodermal and hematopoietic necrosis (IHHN) disease to describe the principal lesions observed. The histopathology of acute and subacute IHHN disease in these species was dominated by the presence of conspicuous eosinophilic intranuclear-inclusion bodies of the Cowdry type A variety in ectodermally (especially the cuticular hypodermis) and mesodermally (especially the hematopoietic tissues) derived tissues that were undergoing necrosis. Electron microscopy of affected tissues demonstrated the presence of two or three types of virus-like particles with cubic morphology and diameters of 17 to 27 nm that suggest IHHN virus to be either a parvo- or picornavirus.

Experimental Infection of Western Hemisphere Penaeid Shrimp with Asian White Spot Syndrome Virus and Asian Yellow Head Virus

Abstract Postlarval and juvenile stages of four species of western hemisphere penaeid shrimp (Penaeus aztecus, P. duorarum, P. setiferus, and P. vannamei) were experimentally challenged with white spot syndrome virus (WSSV) and yellow head virus (YHV) isolates originating from Asia. Challenge exposures were accomplished by feeding minced tissue from WSSV- or YHV-infected shrimp tissues. The WSSV challenge of postlarval shrimp resulted in severe infections in P. setiferus and P. vannamei and less severe infections in P. aztecus and P. duorarum. The WSSV challenge of juvenile shrimp (∼1 g) resulted in severe infections and 100% cumulative mortality in P. setiferus and P. vannamei, moderate infections and 27% cumulative mortality in P. aztecus, and no signs of infection and 0% cumulative mortality in P. duorarum. The YHV challenge caused serious disease and mortality in juveniles of all four species, but postlarval shrimp appeared resistant to YHV because no virus-related signs of infection, mortality, or di...

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

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.

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 (>

The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp

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.

IHHN virus: Infectivity and pathogenicity studies in Penaeus stylirostris and Penaeus vannamei

Significance Since 2009, an emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing global losses to the shrimp farming industry. The causative agent of AHPND is a specific strain of Vibrio parahaemolyticus. We present evidence here that the opportunistic V. parahaemolyticus becomes highly virulent by acquiring a unique AHPND-associated plasmid. This virulence plasmid, which encodes a binary toxin [V. parahaemolyticus Photorhabdus insect-related toxins (PirAvp and PirBvp)] that induces cell death, is stably inherited via a postsegregational killing system and disseminated by conjugative transfer. The cytotoxicity of the PirAvp/PirBvp system is analogous to the structurally similar insecticidal pore-forming Cry toxin. These findings will significantly increase our understanding of this emerging disease, which is essential for developing anti-AHPND measures. Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirAvp and PirBvp) proteins and found that the overall structural topology of PirAvp/PirBvp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirABvp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirABvp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus

Transmission of exotic pathogens occurs through a variety of means, including migration with humans and animals, rapid transit by land, sea or air or through the shipment of infected frozen food products. White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) have caused mass mortalities of cultured shrimp in Asia beginning in 1992. In 1995, these viruses appeared for the first time in the Western Hemisphere causing high mortalities in farm reared shrimp in Texas, USA. The purpose of this study was to determine if WSSV and YHV are present in frozen shrimp products imported into the United States from Asia. Infectivity assays, transmission electron microscopy (TEM), and polymerase chain reaction (PCR) showed these viruses were detectable and infectious in frozen shrimp imports. Frozen shrimp were used to infect indicator shrimp (Penaeus stylirostris) which resulted in mortalities. The cause of these mortalities was determined by histology and TEM to be by YHV. PCR confirmed the presence of WSSV in the frozen, purchased products. The results from this study indicate that exotic shrimp pathogens can be transmitted via imported frozen products.

A parvo-like virus disease of penaeid shrimp

Abstract Infectivity studies of infectious hypodermal and hematopoietic necrosis virus (IHHNV) were conducted on Penaeus stylirostris and P. vannamei (representing species highly susceptible and highly refractory to the disease) via intramuscular injections of the virus. Distinctive histological lesion patterns were observed between species. Six target organ systems were compared interspecifically. The gills and nerve cord/ganglia were shown to undergo significantly higher degrees of tissue damage in infected P. stylirostris compared with infected P. vannamei . Pathogenetic differences correlated well with observed differences in epizootiology between the two species. The infectivity study confirmed that IHHN disease is virus-caused. Cell-free extracts from IHHNV-infected shrimp, when injected into healthy, susceptible shrimp, produced mortalities and Cowdry type A intranuclear eosinophilic inclusion bodies presently considered pathognomonic for the disease.