James R. Winton

Emerging Viral Diseases of Fish and Shrimp

The rise of aquaculture has been one of the most profound changes in global food production of the past 100 years. Driven by population growth, rising demand for seafood and a levelling of production from capture fisheries, the practice of farming aquatic animals has expanded rapidly to become a major global industry. Aquaculture is now integral to the economies of many countries. It has provided employment and been a major driver of socio-economic development in poor rural and coastal communities, particularly in Asia, and has relieved pressure on the sustainability of the natural harvest from our rivers, lakes and oceans. However, the rapid growth of aquaculture has also been the source of anthropogenic change on a massive scale. Aquatic animals have been displaced from their natural environment, cultured in high density, exposed to environmental stress, provided artificial or unnatural feeds, and a prolific global trade has developed in both live aquatic animals and their products. At the same time, over-exploitation of fisheries and anthropogenic stress on aquatic ecosystems has placed pressure on wild fish populations. Not surprisingly, the consequence has been the emergence and spread of an increasing array of new diseases. This review examines the rise and characteristics of aquaculture, the major viral pathogens of fish and shrimp and their impacts, and the particular characteristics of disease emergence in an aquatic, rather than terrestrial, context. It also considers the potential for future disease emergence in aquatic animals as aquaculture continues to expand and faces the challenges presented by climate change.

Status and opportunities for genomics research with rainbow trout

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

Viral hemorrhagic septicemia virus in North America

Abstract The first detections of viral hemorrhagic septicemia virus (VHSV) in North America were in Washington State from adult coho (Oncorhynchus kisutch) and chinook (O. tshawytscha) salmon in 1988. Subsequently, VHSV was isolated from adult coho salmon returning to hatcheries in the Pacific Northwest in 1989, 1991 and 1994. These isolates represented a strain of VHSV that was genetically different from European VHSV as determined by DNA sequence analysis and T1 ribonuclease fingerprinting. The North American strain of VHSV was also isolated from skin lesions of Pacific cod (Gadus macrocephalus) taken from Prince William Sound (PWS), Alaska in 1990, 1991 and 1993. In 1993 and 1994, the virus was isolated from Pacific herring (Clupea harengus pallasi) in Alaskan waters of PWS, Kodiak Island, Auke Bay and Port Frederick. During 1993 and 1994 the herring fishery in PWS failed from a probable complex of environmental stressors but VHSV isolates were associated with hemorrhages of the skin and fins in fish that returned to spawn. Also in 1993 and 1994, VHSV was isolated from apparently healthy stocks of herring in British Columbia, Canada and Puget Sound, Washington. Thus, the North American strain of VHSV is enzootic in the Northeastern Pacific Ocean among Pacific herring stocks with Pacific cod serving as a secondary reservoir. Although the North American strain of the virus appears to be moderately pathogenic for herring, causing occasional self-limiting epizootics, it was shown to be relatively avirulent for several species of salmonids. Pacific herring are common prey for cod and salmon and were most probably the source of the VHSV isolates from the adult salmon returning to spawn in rivers or at hatcheries in Washington State. Compelling circumstances involving the European isolates of VHSV suggest that this strain of the virus also is enzootic among marine fish in the Atlantic Oean. The highly pathogenic nature of the European strain of VHSV for salmonid fish may be the result of the exposure of rainbow trout (O. mykiss), an introduced species, in a stressful environment of intensive culture and the high rate of mutation inherent in all rhabdoviruses. Consequently, we recommend that efforts be made to eradicate the North American strain of VHSV when detected in live salmonids to reduce the possibility of its evolution into a more virulent salmonid virus.

A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

BackgroundA large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression.ResultsThree distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues.ConclusionOur findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish.

North American Strain of Viral Hemorrhagic Septicemia Virus is Highly Pathogenic for Laboratory-Reared Pacific Herring

Abstract Specific-pathogen-free Pacific herring Clupea pallasi were reared in the laboratory from eggs and then challenged at 5, 9, and 13 months of age by waterborne exposure to low (101.5–2.5 plaque-forming units [PFU] per milliliter), medium (103.5–4.5 PFU/mL), or high (105.5–6.5 PFU/mL) levels of a North American isolate of viral hemorrhagic septicemia virus (VHSV). The fish were extremely susceptible to the virus, showing clinical disease, mortality approaching 100%, and only a limited increase in resistance with age. Mortality began 4–6 d after exposure and peaked at approximately day 7 in fish exposed to high levels of virus. Whereas the mean time to death showed a significant dose response (P < 0.001), the percent mortality and virus titers in dead fish were generally high in all groups regardless of initial challenge dose. External signs of disease were usually limited to 1–2-mm hemorrhagic areas on the lower jaw and isthmus and around the eye, but 2 of 130 infected fish exhibited extensive cutan...

Major histocompatibility complex loci are associated with susceptibility of Atlantic salmon to infectious hematopoietic necrosis virus

Infectious hematopoietic necrosis virus (IHNV) is one of the most significant viral pathogens of salmonids and is a leading cause of death among cultured juvenile fish. Although several vaccine strategies have been developed, some of which are highly protective, the delivery systems are still too costly for general use by the aquaculture industry. More cost effective methods could come from the identification of genes associated with IHNV resistance for use in selective breeding. Further, identification of susceptibility genes may lead to an improved understanding of viral pathogenesis and may therefore aid in the development of preventive and therapeutic measures. Genes of the major histocompatibility complex (MHC), involved in the primary recognition of foreign pathogens in the acquired immune response, are associated with resistance to a variety of diseases in vertebrate organisms. We conducted a preliminary analysis of MHC disease association in which an aquaculture strain of Atlantic salmon was challenged with IHNV at three different doses and individual fish were genotyped at three MHC loci using denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of all differentiated alleles. Nine to fourteen alleles per exon-locus were resolved, and alleles potentially associated with resistance or susceptibility were identified. One allele (Sasa-B-04) from a potentially non-classical class I locus was highly associated with resistance to infectious hematopoietic necrosis (p < 0.01). This information can be used to design crosses of specific haplotypes for family analysis of disease associations.

Distribution of an Invasive Aquatic Pathogen (Viral Hemorrhagic Septicemia Virus) in the Great Lakes and Its Relationship to Shipping

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus found in fish from oceans of the northern hemisphere and freshwaters of Europe. It has caused extensive losses of cultured and wild fish and has become established in the North American Great Lakes. Large die-offs of wild fish in the Great Lakes due to VHSV have alarmed the public and provoked government attention on the introduction and spread of aquatic animal pathogens in freshwaters. We investigated the relations between VHSV dispersion and shipping and boating activity in the Great Lakes by sampling fish and water at sites that were commercial shipping harbors, recreational boating centers, and open shorelines. Fish and water samples were individually analyzed for VHSV using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell culture assays. Of 1,221 fish of 17 species, 55 were VHSV positive with highly varied qRT-PCR titers (1 to 5,950,000 N gene copies). The detections of VHSV in fish and water samples were closely associated and the virus was detected in 21 of 30 sites sampled. The occurrence of VHSV was not related to type of site or shipping related invasion hotspots. Our results indicate that VHSV is widely dispersed in the Great Lakes and is both an enzootic and epizootic pathogen. We demonstrate that pathogen distribution information could be developed quickly and is clearly needed for aquatic ecosystem conservation, management of affected populations, and informed regulation of the worldwide trade of aquatic organisms.

Tissue-Specific Induction of Hsp90 mRNA and Plasma Cortisol Response in Chinook Salmon following Heat Shock, Seawater Challenge, and Handling Challenge.

Abstract: In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6°C; + 10.6°C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold. Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight.

Molecular epizootiology and evolution of the glycoprotein and non-virion protein genes of infectious hematopoietic necrosis virus, a fish rhabdovirus

Infectious hematopoietic necrosis virus (IHNV) causes a highly lethal, economically important disease of salmon and trout. The virus is enzootic throughout western North America, and has been spread to Asia and Europe. The nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes of 12 diverse IHNV isolates were determined in order to examine the molecular epizootiology of IHN, the primary structure and conservation of NV, and the evolution of the virus. The G and NV genes and their encoded proteins were highly conserved, with a maximum pairwise nucleotide divergence of 3.6 and 4.4%, and amino acid divergence of 3.7 and 6.2%, respectively. Conservation of NV protein sequence (111 amino acids in length) confirms that the protein is functional and plays an important role in virus replication. The phylogenetic relationship of viruses was found to correlate with the geographic origin of virus isolates rather than with host species or time of isolation. These data are consistent with stable maintenance of virus in enzootic foci. Two main IHNV genetic lineages were identified; one in the Columbia River Basin (Oregon, Washington and Idaho), the other in the Sacramento River Basin (California). The first major IHNV outbreak in chinook salmon in 1973 in the Columbia River was genetically linked to importation of virus-infected fish eggs from the Sacramento River where outbreaks in chinook salmon are common. However, the introduced virus apparently did not persist, subsequent virus outbreaks in Columbia River chinook salmon being associated with Columbia River genetic lineages. In general, virus monoclonal antibody reactivity profiles and phylogenetic relationships correlated well.

Ichthyophoniasis: An Emerging Disease of Chinook Salmon in the Yukon River

Abstract Before 1985, Ichthyophonus was unreported among Pacific salmon Oncorhynchus spp. from the Yukon River; now it infects more than 40% of returning adult Chinook salmon O. tshawytscha. Overall infection prevalence reached about 45% in the Yukon River and about 30% in the Tanana River between 1999 and 2003. Mean infection prevalence was greater in females than males in the main-stem Yukon River during each of the 5 years of the study, but the infection prevalence in males increased each year until the difference was no longer significant. Clinical signs of ichthyophoniasis (presence of visible punctate white lesions in internal organs) were least at the mouth of the Yukon River (∼10%) but increased to 29% when fish reached the middle Yukon River and was 22% at the upper Tanana River. However, clinical signs increased each year from 7% in 1999 to 27% in 2003 at the mouth of the river. As fish approached the upper reaches of the Yukon River (Canada) and the spawning areas of the Chena and Salcha rivers...