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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) in carrier sea trout Salmo trutta after experimental infection

This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting.     

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.     

Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase

This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process.The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His₆-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 °C and not at 28 °C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE’s own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart.In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.