Lucas M. Hart

Factors controlling the early stages of viral haemorrhagic septicaemia epizootics: low exposure levels, virus amplification and fish‐to‐fish transmission

Viral haemorrhagic septicaemia virus, Genogroup IVa (VHSV), was highly infectious to Pacific herring, Clupea pallasii (Valenciennes), even at exposure doses occurring below the threshold of sensitivity for a standard viral plaque assay; however, further progression of the disease to a population-level epizootic required viral amplification and effective fish-to-fish transmission. Among groups of herring injected with VHSV, the prevalence of infection was dose-dependent, ranging from 100%, 75% and 38% after exposure to 19, 0.7 and 0.07 plaque-forming units (PFU)/fish, respectively. Among Pacific herring exposed to waterborne VHSV (140 PFU mL(-1) ), the prevalence of infection, geometric mean viral tissue titre and cumulative mortality were greater among cohabitated herring than among cohorts that were held in individual aquaria, where fish-to-fish transmission was prevented. Fish-to-fish transmission among cohabitated herring probably occurred via exposure to shed virus which peaked at 680 PFU mL(-1) ; shed virus was not detected in the tank water from any isolated individuals. The results provide insights into mechanisms that initiate epizootic cascades in populations of wild herring and have implications for the design of VHSV surveys in wild fish populations.

Efficacy of a glycoprotein DNA vaccine against viral haemorrhagic septicaemia (VHS) in Pacific herring, Clupea pallasii Valenciennes.

Viral haemorrhagic septicaemia virus (VHSV) and its associated disease state, viral haemorrhagic septicaemia (VHS), is hypothesized to be a proximate factor accounting for the decline and failed recovery of Pacific herring populations in Prince William Sound, AK (Marty et al. 1998, 2003, 2010). Survivors of laboratory-induced VHSV epizootics develop resistance to subsequent viral exposure (Kocan et al. 2001; Hershberger et al. 2007, 2010), which is likely the result of immune system recognition of the viral glycoprotein (G) (Lecocq-Xhonneux et al. 1994), a surface antigen that contains neutralizing epitopes (Lorenzen, Olesen & Jorgensen 1990; Jørgensen et al. 1995) and cell attachment domains (Lecocq-Xhonneux et al. 1994; Estepa & Coll 1996). These properties have proven useful in the development of G-gene-based DNA vaccines for VHSV and a related rhabdovirus, infectious haematopoietic necrosis virus (IHNV) (Anderson et al. 1996; Heppell et al. 1998; Corbeil et al. 1999; Einer-Jensen et al. 2009). Rainbow trout fingerlings, Oncorhynchus mykiss (Walbaum), vaccinated with 1 lg of either the VHS or IHN vaccine are protected from VHS when exposed to virus as early as 4 days (44 degree days) post-vaccination (p.v.) (Lorenzen et al. 2002). At later time points (80 days p.v.; 880 degree days), the level of cross-protection against VHS by IHN vaccination is either completely lost (60 days p.v.; 660 degree days) (3 g rainbow trout; 1 lg vaccine dose) (Lorenzen et al. 2002) or present at intermediate levels (6.5 g rainbow trout; 1 lg vaccine dose) (Einer-Jensen et al. 2009). Comparatively, VHS vaccination remains effective as long as 9 months (2520 degree days) p.v. (100 g rainbow trout; 0.5 lg vaccine dose) (McLauchlan et al. 2003). These results suggest that IHN and VHS vaccination activate a rapid transitory innate immune response against VHSV that is followed by longterm adaptive immunity in VHS-vaccinated trout (Lorenzen et al. 2002). To determine whether DNA vaccines are protective in Pacific herring, we injected age 10 months (approximately 3020 degree days) specific pathogenfree (SPF) herring (9.3 g mean weight) (Hershberger et al. 2010) into the left epaxial muscle with 20 lL of saline containing 4 lg pcDNA3-vhsG (VHS vaccine, derived from VHSV isolate DK3592B; genogroup Ia) (Heppell et al. 1998), pcDNA3-ihnG (IHN vaccine, derived from IHNV isolate IT 217/A; genogroup M) (Einer-Jensen et al. 2009), pcDNA3 plasmid (plasmid control) or 0.9% saline alone (negative control) and placed them into separate holding tanks supplied with single-pass, ambient Journal of Fish Diseases 2012 doi:10.1111/j.1365-2761.2012.01364.x

The parasite Ichthyophonus sp. in Pacific herring from the coastal NE Pacific

The protistan parasite Ichthyophonus occurred in populations of Pacific herring Clupea pallasii Valenciennes throughout coastal areas of the NE Pacific, ranging from Puget Sound, WA north to the Gulf of Alaska, AK. Infection prevalence in local Pacific herring stocks varied seasonally and annually, and a general pattern of increasing prevalence with host size and/or age persisted throughout the NE Pacific. An exception to this zoographic pattern occurred among a group of juvenile, age 1+ year Pacific herring from Cordova Harbor, AK in June 2010, which demonstrated an unusually high infection prevalence of 35%. Reasons for this anomaly were hypothesized to involve anthropogenic influences that resulted in locally elevated infection pressures. Interannual declines in infection prevalence from some populations (e.g. Lower Cook Inlet, AK; from 20-32% in 2007 to 0-3% during 2009-13) or from the largest size cohorts of other populations (e.g. Sitka Sound, AK; from 62.5% in 2007 to 19.6% in 2013) were likely a reflection of selective mortality among the infected cohorts. All available information for Ichthyophonus in the NE Pacific, including broad geographic range, low host specificity and presence in archived Pacific herring tissue samples dating to the 1980s, indicate a long-standing host-pathogen relationship.

Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay to Detect Antibodies to Viral Hemorrhagic Septicemia Virus

ABSTRACT Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Influence of Temperature on the Efficacy of Homologous and Heterologous DNA Vaccines against Viral Hemorrhagic Septicemia in Pacific Herring

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species. Received August 1, 2016; accepted March 10, 2017.

Viability and Infectivity of Ichthyophonus sp. in Post-Mortem Pacific Herring, Clupea pallasii

Abstract:  Ichthyophonus-infected Pacific herring, Clupea pallasii, were allowed to decompose in ambient seawater then serially sampled for 29 days to evaluate parasite viability and infectivity for Pacific staghorn sculpin, Leptocottus armatus. Ichthyophonus sp. was viable in decomposing herring tissues for at least 29 days post-mortem and could be transmitted via ingestion to sculpin for up to 5 days. The parasite underwent morphologic changes during the first 48 hr following death of the host that were similar to those previously reported, but as host tissue decomposition progressed, several previously un-described forms of the parasite were observed. The significance of long-term survival and continued morphologic transformation in the post-mortem host is unknown, but it could represent a saprozoic phase of the parasite life cycle that has survival value for Ichthyophonus sp.

Larval and juvenile Pacific herring Clupea pallasii are not susceptible to infectious hematopoietic necrosis under laboratory conditions

Infectious hematopoietic necrosis (IHN) leads to periodic epidemics among certain wild and farmed fish species of the Northeast (NE) Pacific. The source of the IHN virus (IHNV) that initiates these outbreaks remains unknown; however, a leading hypothesis involves viral persistence in marine host species such as Pacific herring Clupea pallasii. Under laboratory conditions we exposed specific pathogen-free (SPF) larval and juvenile Pacific herring to 10(3) to 10(4) plaque-forming units (pfu) of IHNV ml(-1) by waterborne immersion. Cumulative mortalities among exposed groups were not significantly different from those of negative control groups. After waterborne exposure, IHNV was transiently recovered from the tissues of larvae but absent in tissues of juveniles. Additionally, no evidence of viral shedding was detected in the tank water containing exposed juveniles. After intraperitoneal (IP) injection of IHNV in juvenile herring with 10(3) pfu, IHNV was recovered from the tissues of sub-sampled individuals for only the first 5 d post-exposure. The lack of susceptibility to overt disease and transient levels of IHNV in the tissues of exposed fish indicate that Pacific herring do not likely serve a major epizootiological role in perpetuation of IHNV among free-ranging sockeye salmon Oncorhynchus nerka and farmed Atlantic salmon Salmo salar in the NE Pacific.

Optimization of a Plaque Neutralization Test (PNT) to Identify the Exposure History of Pacific Herring to Viral Hemorrhagic Septicemia Virus (VHSV)

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations. Received November 7, 2016; accepted January 14, 2017.