Mortality and weight loss of Atlantic salmon,Salmon salar L., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from Norway

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD‐endemic zone. This subtype has spread rapidly among fish farms in mid‐Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific‐based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post‐smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3‐infected fish showed higher mortality than SAV 2‐infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV‐infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection.     

Longitudinal serological surveys of Atlantic salmon, Salmo salar L., using a rapid immunoperoxidase-based neutralization assay for salmonid alphavirus

Longitudinal serological surveys for salmon pancreas disease virus (SPDV), the causal agent of pancreas disease (PD), were conducted on multiple caged populations of Atlantic salmon, Salmo salar L., on two farms over a 77-week period (farm 1, freshwater and marine stages) and a 36-week period (farm 2, marine stage only), using a microtitre-based virus neutralization (VN) assay. Collected sera were also screened for viraemia with SPDV, and pancreas, heart and muscle tissues were examined for lesions consistent with PD. Outbreaks of PD occurred during the marine phase on both farms, as demonstrated by seroconversion, the isolation of virus and progressive histopathological changes consistent with a PD outbreak. All populations monitored showed a progressive increase in seroprevalence of 90-100%, typically accompanied by rises in geometric mean antibody titres. With the exception of one caged population, which showed a marked biphasic seroprevalence pattern, the seroprevalence figures in the remaining four monitored populations remained high (> or =70%) until the end of the study period. Peak VN titres of > or =1/1280 were detected on both farms. The results provide essential baseline information for the interpretation of SPDV VN serology results, and indicate that this methodology is suited to both the diagnosis and seroepidemiology of SPDV infections.     

Salmonid alphavirus (SAV) and pancreas disease (PD) in Atlantic salmon,Salmo salar L., in freshwater and seawater sites in Norway from 2006 to 2008

A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD‐affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real‐time RT‐PCR from samples collected in the freshwater phase, nor were any SAV‐neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (±7.06) (range 0.7–26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (±1.11) (range 1–6).     

Ultrastructural morphogenesis of salmonid alphavirus 1

Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.     

Prevalence of piscine orthoreovirus and salmonid alphavirus in sea‐caught returning adult Atlantic salmon (Salmo salar L.) in northern Norway

Heart and skeletal muscle inflammation (HSMI) caused by piscine orthoreovirus (PRV) and pancreas disease (PD) caused by salmonid alphavirus (SAV) are among the most prevalent viral diseases of Atlantic salmon farmed in Norway. There are limited data about the impact of disease in farmed salmon on wild salmon populations. Therefore, the prevalence of PRV and SAV in returning salmon caught in six sea sites was determined using real‐time RT‐PCR analyses. Of 419 salmon tested, 15.8% tested positive for PRV, while none were positive for SAV. However, scale reading revealed that 10% of the salmon had escaped from farms. The prevalence of PRV in wild salmon (8%) was significantly lower than in farm escapees (86%), and increased with fish length (proxy for age). Sequencing of the S1 gene of PRV from 39 infected fish revealed a mix of genotypes. The observed increase in PRV prevalence with fish age and the lack of phylogeographic structure of the virus could be explained by virus transmission in the feeding areas. Our results highlight the need for studies about the prevalence of PRV and other pathogens in Atlantic salmon in its oceanic phase.     

Geographical distribution of salmonid alphavirus subtypes in marine farmed Atlantic salmon, Salmo salar L., in Scotland and Ireland

Sequence data from salmonid alphavirus (SAV) strains obtained from farmed marine Atlantic salmon, Salmo salar L. , over a 20-year period between 1991 and 2011 was reviewed to examine the geographical distribution of the genetically defined SAV subtypes in twelve regions across Ireland and Scotland. Of 160 different Atlantic salmon SAV strains examined, 62 belonged to subtype 1, 28 to subtype 2, 34 to subtype 4, 35 to subtype 5 and 1 to subtype 6. SAV subtypes 1, 4 and 6 were found in Ireland, while subtypes 1, 2, 4 and 5 were found in Scotland. In the majority of regions, there was a clear clustering of subtypes, with SAV subtype 1 being the dominant subtype in Ireland overall, as well as in Argyll and Bute in Scotland. SAV subtype 2 predominated in the Shetland and Orkney Islands. The emergence in Atlantic salmon of subtype 2 strains typically associated with sleeping disease in rainbow trout in Argyll and Bute, strongly suggesting transmission of infection between these species, was noted for the first time. SAV subtype 4 was the most common subtype found in the southern Western Isles, while SAV subtype 5 predominated in the northern Western Isles and north-west mainland Scotland. No single strain was dominant on sites in the western Highlands, with a number of sites in this region in particular having more than one subtype detected in different submissions. The significance of these results in relation to aspects of the epidemiology of infection, including transmission, biosecurity and wildlife reservoirs are discussed and knowledge gaps identified.     

Pancreas disease in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in Norway

The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples.     

Lack of evidence for vertical transmission of SAV 3 using gametes of Atlantic salmon, Salmo salar L., exposed by natural and experimental routes

Pancreas disease (PD) is an important cause of losses in farmed salmonids in Norway, the United Kingdom and Ireland. As the spread of salmonid alphavirus (SAV), the causal agent, to naïve populations is of major concern to the farming industry, it is important to uncover the transmission routes of the virus. This study was conducted to investigate the potential for vertical transmission of SAV subtype 3. Progeny of broodstock with signs of late‐stage PD and persistent RT‐PCR signals for SAV were followed from fertilization to smoltification in an experimental facility. Fertilized ova were either not disinfected or taken through one of three different disinfection regimes. Also, ova and milt from uninfected broodfish from a different population were exposed to a cell‐cultured strain of SAV 3 immediately before fertilization to simulate a viraemic phase in parent fish. A group of uninfected controls were also included in the study. Fertilized ova from bath exposed and negative control groups were double disinfected. Following fertilization, experimental fish went through a normal freshwater phase. However, fry were stressed at first feeding to enhance replication of possibly latent virus. Smoltification was induced by an artificial light regime, and experimental fish were followed to the late smoltification phase. Selected samples were investigated by real‐time RT‐PCR for SAV, by histology for evidence of PD and by serology for neutralising antibodies against SAV. All analysed samples of progeny were negative. This result shows that SAV 3 is not readily transmitted vertically from parents to offspring. Additional negative PCR results from salmon sampled in commercial hatcheries support these findings. Also, recent studies have shown that risk factors for the horizontal transmission route explain the vast majority of PD outbreaks in Norway. It is concluded that if it happens at all, vertical transmission is of minor importance in the spread of SAV 3.     

Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum)

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.     

鲑鱼甲病毒病研究进展

1疾病概述
　　冷水鲑科鱼类价格昂贵,是世界三大养殖鱼类之一,主要分布于欧洲和北美。目前我国的鲑科鱼养殖以虹鳟（Oncorhynchus mykiss）为主,年产量占世界总产量的1％以下,每年需要进口大量的鲑鱼,尤其是大西洋鲑鱼（Salmo salar ）［1-2］,鲑鱼甲病毒病对于我国的鲑科鱼养殖具有潜在的威胁。     

Susceptibility of salmonid alphavirus to a range of chemical disinfectants

A range of commercially available disinfectants were tested for efficacy against salmonid alphavirus under a range of different conditions including variations in concentration, temperature, contact time, water type and presence or absence of organic matter. Testing was based on the protocol defined in the draft European Standard prEN 14675, for which the effective standard is a 4 log(10) reduction in viral titre. All disinfectants were found to be effective under at least some of the conditions tested. However, the presence of organic matter in particular was shown to be detrimental in some cases, either through rendering some disinfectants ineffective, or by production of a visible inhomogeneity.     

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co‐varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.     

Cross‐neutralization studies with salmonid alphavirus subtype 1–6 strains: results with sera from experimental studies and natural infections

The serological reactivity between strains of each of the six currently genetically defined subtypes of salmonid alphavirus (SAV) was examined by comparison of homologous and heterologous virus neutralization titres on sera from experimentally infected fish. With the exception of the level of SAV subtype 6 neutralization by heterologous sera, good cross‐neutralization was detected between all subtypes, albeit with variation in geometric mean titres when each subtype‐specific serum set was tested against the panel of virus subtypes. A similar pattern was evident with field sera, except that heterologous neutralization of the SAV6 strain was more evident. In only 23% of available pairwise comparisons was the homologous titre recorded with an experimentally derived serum fourfold or greater than the heterologous titre, and in only two instances was this difference demonstrated in both directions. No virus strains consistently met the old serology‐based criteria (Sub‐committee on Inter‐relationships Among Catalogued Alphaviruses) to be considered separate subtypes within an alphavirus species. Only when testing with an SAV subtype‐2‐specific monoclonal antibody was a major difference between homologous and heterologous neutralization capacity evident. These results provide new direct or indirect information in terms of SAV classification, vaccine efficacy and the selection and validation of reagents for serological and immunological diagnostic purposes.     

A comparative study of marine salmonid alphavirus subtypes 1-6 using an experimental cohabitation challenge model

A comparative challenge study of six marine isolates representing subtypes 1-6 of salmonid alphavirus (salmon pancreas disease virus, Genus Alphavirus, Family Togaviridae) was conducted in Atlantic salmon in a fresh water cohabitation trial. Histopathological lesions typical of pancreas disease were observed with all subtypes, and virus was re-isolated from serum of cohabitant fish in each case. Using a virus neutralization (VN) test neutralizing salmonid alphavirus (SAV) subtype 1 strain F93-125, VN antibodies were detected in all challenge groups, consistent with serological cross-reactivity between these subtypes. Using real-time RT-PCR, SAV RNA was detected in heart tissue from 2 to 3 weeks post-challenge (wpc) in all cohabitant groups excluding controls. The results obtained suggested differences in the dynamics of infection between strains of SAV and potentially between subtypes. Results for SAV subtypes 1 and 3 suggested essentially synchronous infection of cohabitant fish. These two study groups also had the highest virus load in heart tissue as measured by quantitative RT-PCR and also had the most extensive histopathological changes. In contrast, results for SAV subtypes 2 and 6 strains were consistent with asynchronous infection in the cohabitant fish and were characterized by slow spread, low virus loads and mild histopathological changes. The SAV subtype 4 and 5 strains occupied an intermediate position in this regard. Despite the use of concentration procedures, it was not possible to detect SAV RNA in water samples from selected study tanks. However, testing of faeces from the SAV subtypes 1, 3 and 6 challenge groups found positive signals in each beginning at 1-3 wpc and remaining detectable for a further 2-3 weeks. Parallel testing of mucus samples found these became positive at 2-3 wpc and remained positive for a further 1-3 weeks. These results demonstrate for the first time that shedding and transmission of virus may occur by both these routes and suggest that dispersal in these matrices should be included in any disease transmission models.     

A rapid immunoperoxidase-based virus neutralization assay for salmonid alphavirus used for a serological survey in Northern Ireland

A modified virus neutralization (VN) assay was developed to replace an existing assay read on the presence or absence of virus-induced cytopathic effect (CPE). The modified assay used a monoclonal antibody to salmon pancreas disease virus as the first layer of an immunoperoxidase (IPX)-based immunostaining technique to detect viral growth. The IPX-based VN assay required only 3 days to perform, and the adoption of a 96-well microtitre format facilitated a high throughput of samples requiring small volumes of serum, cells and virus. When 352 sera from farmed salmon and 302 sera from farmed trout were tested by both the modified and the original CPE-based assays, overall correlations of 97.72 and 96.03% were, respectively, obtained (96.94% combined). When the modified assay was used to test 188 sera collected from wild salmonids in freshwater river systems in Northern Ireland, no positive results were recorded.