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.     

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.     

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.     

Epidemiological observations of pancreas disease of farmed Atlantic salmon, Salmo salar L., in Ireland

Epidemiological investigations into the pancreas disease (PD) of farmed salmon were conducted on populations of Atlantic salmon reared in Ireland during 2003 and 2004. The investigations surveyed all marine salmon farms operating in Ireland through a detailed questionnaire with follow-up farm visits. Information was gathered on 21 populations of fish in 2003 and 14 populations in 2004. Thirteen of the 21 populations suffered PD in 2003 and 12 of the 14 in 2004. The mean mortality due to PD on affected farms was 18.8% in 2003 and 14.8% in 2004 and the loss of growth due to PD was estimated at 11.4% over the 2-year period. The highest risk periods for outbreaks of PD were early summer and early autumn and the farms most seriously affected by PD mortality were in the western counties of Ireland. Factors which showed an indication of association with a PD outbreak or high mortality during a PD outbreak were: livestock movement to another sea site, high feeding rate prior to any PD outbreak, the presence of another PD positive farm in the same water body, greater than 250000 fish on a site, a previous history of PD on a site, a high sea lice burden, and sites located in the western regions of Ireland which reared a specific strain of salmon.     

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.     

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.     

Genetic characterization of salmonid alphavirus in Norway

Pancreas disease (PD), caused by salmonid alphavirus subtype 3 (SAV3), emerged in Norwegian aquaculture in the 1980s and is now endemic along the south‐western coast. In 2011, the first cases of PD caused by marine salmonid alphavirus subtype 2 (SAV2) were reported. This subtype has spread rapidly among the fish farms outside the PD‐endemic zone and is responsible for disease outbreaks at an increasing numbers of sites. To describe the geographical distribution of salmonid alphavirus (SAV), and to assess the time and site of introduction of marine SAV2 to Norway, an extensive genetic characterization including more than 200 SAV‐positive samples from 157 Norwegian marine production sites collected from May 2007 to December 2012 was executed. The first samples positive for marine SAV2 originated from Romsdal, in June 2010. Sequence analysis of the E2 gene revealed that all marine SAV2 included in this study were nearly identical, suggesting a single introduction into Norwegian aquaculture. Further, this study provides evidence of a separate geographical distribution of two subtypes in Norway. SAV3 is present in south‐western Norway, and marine SAV2 circulates in north‐western and Mid‐Norway, a geographical area which since 2010 constitutes the endemic zone for marine SAV2.     

Incidence of hybridization between Atlantic salmon, Salmo salar L., and brown trout, Salmo trutta L., in Ireland

The substantial growth of the farmed salmon industry in Europe since the 1970s has highlighted concerns regarding the genetic impact of escaped farmed salmon on wild salmonid stocks. High incidences of salmon × trout hybrids have been recorded in rivers situated near intensive salmon farming in Norway and Scotland, which may be indicative of a breakdown in reproductive isolation between salmon, Salmo salar L., and brown trout, Salmo trutta L. In the present study, salmonid fry and 0+ parr were collected from rivers in western Ireland. Allozyme and minisatellite DNA analysis were carried out on fry to determine the frequency of F1 hybrids from 10 rivers located within 38 km of salmon farms and three rivers at least 80 km from salmon farms. A total of 49 hybrids were recorded from 4135 salmonid fry (frequency = 1.2%). Mitochondrial DNA analysis showed that all hybrids arose from Atlantic salmon female × brown trout male crosses. Hybrid parr were recorded from one of the low-risk rivers (1.0%), but were present in seven out of the 10 catchments located within 38 km of salmon farms, with frequencies ranging from 0.7% to 3.1%. The results of the present survey, which represents the first extensive record of the levels of salmon-trout hybridization in Ireland, are discussed in relation to the continued growth of salmon farming in this country.     

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).     

A comparison of epidemiological patterns of salmon lice, Lepeophtheirus salmonis, infections on farmed Atlantic salmon, Salmo salar L., in Norway and Scotland

This paper examines two large national data sets collected over several years and contrasts the patterns of sea lice, Lepeophtheirus salmonis (Krøyer, 1837), infections, the use of treatments and the occurrence of chalimus peaks between Norwegian and Scottish farms. Infection levels in Scotland were significantly higher in general over the period under study. For the chalimus stage group in the first quarter of the year, Norwegian mean abundance stayed below 10 lice per m² while Scottish means reached 45 lice per m² of fish skin per m³ of water. Both countries had more chalimus in summer than at other times of year, but in the last 3 months of the year Scottish fish had, on average, two to four times as many chalimus as Norwegian fish. Peaks of chalimus abundance were more frequent in Scotland, particularly in winter, but the most prominent peaks occurred in summer in both countries. In Scotland a marked mid-year build-up of mobile pre-adult and adult stages was seen, and both countries showed a tendency for mobile counts on the second year fish to increase towards the end of the year. Scottish fish carried, on average, three times as many mobile lice per m² of skin as Norwegian fish in the last 3 months of the year. The difference in lice loads was reflected in the greater use of veterinary medicines on Scottish farms. The higher infection levels in Scotland may be due to shallower and more enclosed water bodies used for farming, smaller and shallower pens, differences in sea water temperatures or in access to appropriate medication. The results highlight the importance of ensuring that effective veterinary medicines are available in the UK for the control of infection.     

Changes in epidemiological patterns of sea lice infestation on farmed Atlantic salmon, Salmo salar L., in Scotland between 1996 and 2006

Analyses of a unique database containing sea lice records over an 11 year period provide evidence of changing infestation patterns in Scotland. The data, collected from more than 50 commercial Atlantic salmon farms, indicate that both species of sea lice commonly found in Scotland, Lepeophtheirus salmonis and Caligus elongatus, have declined on farms over the past decade. Reductions for both species have been particularly marked since 2001 when more effective veterinary medicines became available. Treatment data were also available in the database and these show a growing trend towards the use of the in-feed medication emamectin benzoate (Slice), particularly in the first year of the salmon production cycle. However, this trend towards single product use has not been sustained in 2006, the latest year for which data are available. There is some evidence of region to region variation within Scotland with the Western Isles experiencing higher levels of infestation. However, compared to the levels observed between 1996 and 2000, all regions have benefited from reduced lice infestation, with the overall pattern showing a particular reduction in the second and third quarters of the second year of production.     

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.     

The epidemiology of the sea lice, Caligus elongatus Nordmann, in marine aquaculture of Atlantic salmon, Salmo salar L., in Scotland

Although Caligus elongatus is one of two major species of sea lice that parasitize farmed salmon, its epidemiology has not been extensively studied. In this communication, the abundances of the adult stage of C. elongatus in salmon populations from 33 farms in the West of Scotland between 1997 and 2000 have been analysed for evidence of seasonal and annual patterns. The findings indicate that the pattern of C. elongatus is remarkably consistent from year to year, and directly opposed to that reported for Lepeophtheirus salmonis, the other major caligid species. In particular, adult infestations rapidly increase from the start of July each year, and are more prevalent on salmon in the first year of production than the second year. Treatment is seen to have an effect on levels of infestation but it is not clear why this species should have significantly lower levels of abundance in the second year of production. There is also evidence that fallowing has no effect on abundance. Strategic management programmes for the control of sea lice on salmon farms, which are increasingly effective in controlling L. salmonis , may also have to give greater consideration to C. elongatus .     

Potential disease interaction reinforced: double-virus-infected escaped farmed Atlantic salmon,Salmo salar L., recaptured in a nearby river

The role of escaped farmed salmon in spreading infectious agents from aquaculture to wild salmonid populations is largely unknown. This is a case study of potential disease interaction between escaped farmed and wild fish populations. In summer 2012, significant numbers of farmed Atlantic salmon were captured in the Hardangerfjord and in a local river. Genetic analyses of 59 of the escaped salmon and samples collected from six local salmon farms pointed out the most likely source farm, but two other farms had an overlapping genetic profile. The escapees were also analysed for three viruses that are prevalent in fish farming in Norway. Almost all the escaped salmon were infected with salmon alphavirus (SAV) and piscine reovirus (PRV). To use the infection profile to assist genetic methods in identifying the likely farm of origin, samples from the farms were also tested for these viruses. However, in the current case, all the three farms had an infection profile that was similar to that of the escapees. We have shown that double-virus-infected escaped salmon ascend a river close to the likely source farms, reinforcing the potential for spread of viruses to wild salmonids.     

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.     

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.