Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).     

Morphologic description of infectious salmon anaemia virus (ISAV)-induced lesions in rainbow trout Oncorhynchus mykiss compared to Atlantic salmon Salmo salar

In the present study the pathogenesis of experimental infectious salmon anaemia virus (ISAV) infection in rainbow trout Oncorhynchus mykiss (Walbaum, 1972) and Atlantic salmon Salmo salar was compared. The virus infection in the 2 species demonstrated different mortality patterns and pathology characteristics. Atlantic salmon showed a typical acute mortality pattern peaking at 8 to 16 d post-infection (dpi) depending on virus dose, whereas in rainbow trout, only the highest virus dose (10(7.13-7.8) TCID50/200 microl) showed a similar pattern. The middle (10(4.13) TCID50/200 microl) and lowest virus doses (10(2.13) TCID50/200 microl) in rainbow trout induced only sporadic protracted mortality, lasting up to 46 dpi. Infected rainbow trout that were live-sampled and those that died demonstrated increased erythrophagia, clusters of cellular degeneration in the haematopoietic portion of the kidney, and occasionally epicarditis, endocarditis and myocarditis. These lesions are very different from the typical necrosis in liver and kidney that occur in infected Atlantic salmon, and some of them may be indicative of an antiviral response by a resistant host to the ISAV infection. Virus was detected in the endothelium of the rainbow trout tissues using in situ hybridization, supporting our conclusions of the ISAV-induced lesions in this report.     

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the 'wild-type' agent

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.     

A salmonid cell line (TO) for production of infectious salmon anaemia virus (ISAV)

A new cell line designated TO which provides a high yield of infectious salmon anaemia virus (ISAV) has been established. The cells originate from head kidney leukocytes isolated from Atlantic salmon and grow well at 20 degrees C in EMEM with 5% CO2 and without CO2 supplement in HMEM. The cells have at present been passed more than 150 times and no changes in morphology, growth or virus production have been observed. The virus infection results in cytopathic effects (CPE) within 9 d, and the virus titre obtained from centrifuged and filtrated cell lysates, measured as TCID50, was about 10(9.1) ml(-1). The virus isolated from lysates of infected cells by a sucrose gradient provided purified ISAV when examined by silver stained SDS-PAGE. Salmon injected with diluted virus supernatant showed mortalities, hematocrit values and clinical signs in accordance with infectious salmon anaemia.     

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 microm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20 degrees C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13 degrees C, up to 7 d at 20 degrees C, but it was inactivated at 37 and 56 degrees C within 24 and 1 h, respectively. Freezing at -20 degrees C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20 degrees C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10(2.5)-50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.     

Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes,using an indirect enzyme-linked immunosorbent assay (ELISA)

Antibody detection tests are rarely used for diagnostic purposes in fish diseases. Infectious salmon anaemia (ISA) caused by ISA virus (ISAV) is an emerging disease of Atlantic salmon Salmo salar L. The virus has also been isolated from diseased coho salmon Oncorhynchus kisutch in Chile. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate serodiagnosis of ISAV infection, the study of epidemiology, and the control of ISA in farmed fishes has been developed using purified ISAV as the coating antigen, and monoclonal antibodies that detect fish immunoglobulins bound to the antigen on the plate. Application of the test to a random sample of farmed Atlantic salmon from the Bay of Fundy, New Brunswick, Canada, positively identified 5 of the 7 ISAV RT-PCR-positive fish, and all 10 RT-PCR-negative fish were also negative in the ELISA. Some RT-PCR-negative fish had an elevated non-specific antibody reactivity suggestive of chronic infection or resistance to ISAV. This test was also able to detect 11 of the 14 coho salmon pooled serum samples from a clinically affected farm in Chile that were positive by the virus neutralization (VN) test, and 2 of the 4 VN-negative samples. We conclude that this ELISA would be suitable as a routine test for ISAV infection or for assessing ISAV vaccine efficacy before placing smolts in sea cages, and for testing fishes in sea cages to detect level of resistance to ISA. The assay enables vaccination in combination with depopulation control methods.     

Identification of infectious salmon anaemia virus in Atlantic salmon from Nova Scotia (Canada): evidence for functional strain differences

Infectious salmon anaemia (ISA) is a serious disease responsible for high morbidity in farmed Atlantic salmon Salmo salar in Norway, Scotland and New Brunswick, Canada. Recent attempts to identify different strains of ISA virus (ISAV) based on nucleotide sequence variation have shown that the Norwegian and Scottish samples are similar to one another but markedly different from New Brunswick samples. These data may suggest the presence of different strains on each side of the Atlantic but no functional difference has been found with either strain. We describe the first identification and characterisation of ISAV in Atlantic salmon from Nova Scotia, Canada. Further, salmon infected with the Nova Scotia ISAV do not show typical ISAV pathology or mortality. Sequencing of this new strain showed it to possess greater similarity to ISAV from Norway and Scotland than to ISAV from New Brunswick. These findings are discussed in terms of a possible origin of the Nova Scotia ISAV strain and the existence of an avirulent ISAV strain. The impact of current strain variation studies on our knowledge of ISAV is also discussed.     

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission

Wild-caught saithe Pollachius virens were experimentally exposed to an isolate of infectious salmon anaemia virus (ISAV) of Norwegian origin. Mortality attributable to ISAV did not occur following exposure by intra-peritoneal (i.p.) injection of virus or by cohabitation with ISAV-infected Atlantic salmon Salmo salar. Despite the individual testing of 120 ISAV-exposed saithe, ISAV was not detectable using RT-PCR, the most sensitive ISAV diagnostic tool demonstrated to date. Furthermore, saithe exposed to ISAV-infected salmon were not capable of transmitting virus when transferred to tanks containing na?ve salmon. Thus saithe appear to be resistant to this Norwegian isolate of ISAV and incapable of supporting its replication. Saithe which co-exist with salmon in and around aqua-culture facilities are considered unlikely to have a significant impact on the epizootiology of ISAV.     

Fate of infectious salmon anaemia virus (ISAV) in experimentally challenged blue mussels Mytilus edulis

In order to investigate the potential role of blue mussels Mytilus edulis as a vector of the fish pathogenic infectious salmon anaemia virus (ISAV), we developed an experimental bioaccumulation system in which mussels can accumulate virus during normal filtration. Detection of virus in mussels was performed by means of real-time RT-PCR. ISAV-RNA was detected in the mussels until 72 h post-challenge. Hepatopancreas homogenate from experimentally challenged mussels was injected into salmon. All the fish injected with homogenate prepared immediately after accumulations were strongly ISAV positive 4 wk post-challenge. In the group injected with homogenate prepared 24 h after the challenge, 1 fish out of 25 was weakly ISAV positive. All of the fish that were challenged with mussel homogenate prepared 96 h after accumulation were ISAV negative. Mussels sampled from a tank with experimentally infected salmon demonstrating clinical signs consistent with ISA (infectious salmon anaemia) and mussels collected on net pen cages during ISA outbreaks in Atlantic salmon were all ISAV negative. The results indicate that the ISAV is rapidly inactivated in mussels and that mussels are not a likely reservoir host or vector for ISAV.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.