Nucleotide sequence variation in isolates of infectious salmon anaemia virus (ISAV) from Atlantic salmon Salmo salar in Scotland and Norway

The nucleotide sequences of segments 2 and 8 of infectious salmon anaemia virus (ISAV) isolates from Scotland and Norway were determined and compared. Sequence variations were found in both segments, with segment 2 being more variable. These variations were of the same degree as those found in previous comparisons of the first recorded strain of ISAV in Scotland and Norwegian strains for which sequence information was available. The sequences again demonstrate the separation of European and Canadian strains of ISAV but, as is expected for an RNA virus, reveal that the European ISAV is not homogeneous. These results demonstrate the value of nucleotide sequences as a tool for strain identification and epidemiological investigation of ISAV.     

Genetic analysis of infectious salmon anaemia virus (ISAV) from Scotland

The nucleotide sequences of segments 2 and 8 of infectious salmon anaemia virus (ISAV) isolates from Scotland were determined. These were used to predict amino acid sequences of the products of these segments. The sequences were compared with those previously published for ISAV from Norway and North America. Nucleotide and amino acid variations were found in the Scottish isolate, indicating that it is not identical to those Norwegian or North American strains. Although phylogenetic analysis of the sequences was not possible, it was clear that the Scottish isolate is more closely related to the Norwegian strain than the North American. Sequencing further isolates that are geographically or temporally separated will be important in advancing understanding of the epidemiology of this important disease.     

Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.     

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.     

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.     

Absence of vertical transmission of infectious salmon anemia virus (ISAV) from individually infected Atlantic salmon Salmo salar

Atlantic salmon Salmo salar L. eggs were collected from grilse that were individually identified as ISAV-positive based on the detection of pathogen in ovarian fluid by RT-PCR. The eggs were fertilised, disinfected and reared under quarantine conditions. To address the possibility of vertical transmission, fertilised eggs, alevins and parr were screened for the virus by SHK-1 cell culture and RT-PCR. In addition, ISAV-negative parr were injected with homogenates of potentially infected eyed eggs. ISAV was not detected in eyed eggs, alevins or parr. No mortalities occurred among fish injected with the egg homogenates. These observations suggest the absence of a vertical transmission route for ISAV infection.     

Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway

Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.).

Infectious salmon anemia (ISA) virus is the cause of infectious salmon anemia in farmed Atlantic salmon. The virus has been shown to contain RNA with structural characteristics similar to those of accepted members of the Orthomyxoviridae. Further biochemical, physiochemical, and morphological characterization of ISA virus was undertaken to clarify its taxonomic position. The virus was found to be sensitive to chloroform, heat, and low pH and agglutinated erythrocytes from fish. Erythrocytes from mammals or birds were not agglutinated. Receptor-destroying enzyme activity was detected, and the nature of this enzyme was suggested to be an acetylesterase. The buoyant density of the virus was 1.18 g/ml in sucrose and CsCl gradients. The maximum rate of virus replication was observed at 15 degrees C, while no virus was produced at 25 degrees C. Actinomycin D inhibited viral replication, and viral antigen was detected in nuclei by immunofluorescence. The addition of trypsin to the culture medium during virus replication had a beneficial effect on virus replication. ISA virus contains four major polypeptides with estimated molecular sizes of 71, 53, 43, and 24 kDa. Electron microscopy revealed structures closely resembling the nucleocapsids of influenza virus. Mushroom-shaped surface projections were a distinctive morphological feature, which differed from the rod-shaped hemagglutinin projections of the influenza viruses. The data reported here support the relationship of ISA virus to the Orthomyxoviridae, although ISA virus differs from influenza viruses in some morphological characteristics and in showing restricted hemagglutination, in different specificity of the receptor-destroying enzyme, in different polypeptide profile, in being unable to replicate at temperatures above 25 degrees C, and in host range.     

Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis

The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.     

Incidence of Mentum Deformities in Midge Larvae (Diptera:Chironomidae) from Northern Nova Scotia, Canada

Deformities in the mouthparts of larval Chironomidae, particularly of the teeth on the mentum, have been proposed as a bioindicator of sediment quality and environmental stress. Most work to date has concentrated on relatively few abundant, responsive genera common in soft-bottom lakes. We examined mentum deformities in 25 genera of Chironominae, Orthocladiinae and Diamesinae (one genus) from streams and a lake in rural Nova Scotia where farming and forestry are the principal land uses. Incidence of deformity at similar stream sites varied across genera from zero to >10%. Average frequencies of deformity across all three subfamilies at sites with no known sources of contamination ranged from <4% to 8%, and increased to nearly 15% at a site receiving treated municipal sewage effluent. Differences in chironomid community structure and rates of leaf litter decomposition above and below the sewage effluent outfall were congruent with the difference in mentum deformities. Frequencies of deformity observed here are an order of magnitude greater than in similar studies of rural areas. Low-level stress from agriculture or forest harvesting may be widespread in rural regions even in aquatic ecosystems that are seemingly free of industrial discharges or sediment contamination.     

Experimental infection models and susceptibility of Atlantic salmon Salmo salar to a Scottish isolate of infectious salmon anaemia virus.

Infection models for both fresh and seawater salmon were established using a Scottish isolate of infectious salmon anaemia virus (ISAV). Modes of infection were intra-peritoneal injection, cohabitation and immersion exposure, and a range of doses was tested. Development of these models using a Scottish isolate of ISAV provided an approximation of the minimum infective dose leading to mortality under different infection regimens. The models also allow prediction of the time to first mortality and an estimation of expected total mortality following the various routes of infection. Such knowledge is important to the development of anti-ISAV vaccines and to future studies aimed at understanding the biology of ISAV in general.     

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.