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.     

Alphavirus infections in salmonids--a review

The first alphavirus to be isolated from fish was recorded in 1995 with the isolation of salmon pancreas disease virus from Atlantic salmon, Salmo salar L., in Ireland. Subsequently, the closely related sleeping disease virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), in France. More recently Norwegian salmonid alphavirus (SAV) has been isolated from marine phase production of Atlantic salmon and rainbow trout in Norway. These three viruses are closely related and are now considered to represent three subtypes of SAV, a new member of the genus Alphavirus within the family Togaviridae. SAVs are recognized as serious pathogens of farmed Atlantic salmon and rainbow trout in Europe. This paper aims to draw together both historical and current knowledge of the diseases caused by SAVs, the viruses, their diagnosis and control, and to discuss the differential diagnosis of similar pathologies seen in cardiomyopathy syndrome and heart and skeletal muscle inflammation of Atlantic salmon.     

Heritability of mortality in response to a natural pancreas disease (SPDV) challenge in Atlantic salmon,Salmo salar L., post‐smolts on a West of Ireland sea site

Pancreas disease (PD) is an economically important disease of European farmed Atlantic salmon. It can cause significant losses because of morbidity, mortality and reduced production. The disease is caused by an alphavirus, known as salmon PD virus (SPDV) or salmonid alphavirus subtype 1 in Ireland. To examine whether it is possible to improve the natural resistance of Atlantic salmon to SPDV by selective breeding, 6000 genotyped, tagged, pedigreed fish from 150 full‐sib families were exposed to a natural challenge during 2005 in a sea cage on a commercial salmon farm in the West of Ireland. Histopathological and serological examination was performed weekly on a proportion of all moribund fish to determine the onset of the infection and the likely cause of death. Heritabilities and genetic correlations are presented for resistance to a natural PD challenge and smolt input weight. The results indicate that the susceptibility of salmon to SPDV could be reduced by selective breeding based on the survival in a natural challenge to the virus.     

Isolation of salmon pancreas disease virus (SPDV) from farmed Atlantic salmon, Salmo salar L., in Scotland

A toga-like virus was recently isolated in the present authors' laboratory from Atlantic salmon during an outbreak of PD in Ireland (Nelson, McLoughlin, Rowley, Platten & McCormick 1995). When inoculated into Atlantic salmon post-smolts, this virus, known as salmon pancreas disease virus (SPDV), caused pathological changes in the pancreas, heart and skeletal muscle which were indistinguishable from those observed in natural outbreaks of PD (McLoughlin, Nelson, Rowley, Cox & Grant 1996). In 1996 a serological survey was carried out to determine the presence and distribution of antibodies to SPDV in Irish, Scottish and Norwegian farm-reared Atlantic salmon (McLoughlin, Rowley & Doherty 1998). The results of this survey indicated that SPDV is present in the major European salmon-producing countries. The present paper describes the first isolation of SPDV from farm-reared, PD-affected Atlantic salmon in Scotland.     

Transmission dynamics of pancreas disease (PD) in a Norwegian fjord: aspects of water transport,contact networks and infection pressure among salmon farms

Pancreas disease (PD) in marine salmon farming is caused by salmon pancreas disease virus (SPDV). Virus survival, infection pressure and contact networks among farms influence the potential of PD to spread. The present study aims to explore contact networks and infection pressure and their ability to explain transmission dynamics of PD in a Norwegian fjord. In this study, we included all records of PD by subtype 3 (SPDV3) in the study population from the first reported in August 2006 to the last reported in November 2009. Using logistic regression analyses, we found that contact network by water transport explained better transmission of PD than contact networks defined by ownership or close distance to infected farms. Hydrodynamic modelling can be a valuable tool to forecast the spread of PD and thereby take actions to reduce the transmission. Knowing the risk of getting infected, it is important to avoid water transport from infected farms when new cohorts are transferred to sea water, and to have conscious control regarding management operations between farms.     

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.     

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.     

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).     

Assessment of infectious salmon anaemia virus prevalence for different groups of farmed Atlantic salmon, Salmo salar L., in New Brunswick

Abstract Infectious salmon anaemia (ISA) virus (ISAV) has been causing disease in New Brunswick since 1996. As a control measure, all fish in an outbreak cage are killed. The objective of this study was to compare ISAV prevalence in cages experiencing an outbreak with healthy cages from the same farm, neighbouring farms and distant farms. Atlantic salmon from five different groups were tested using an RT-PCR test. Groups included moribund fish from a cage experiencing an outbreak (A), healthy fish from an outbreak cage (B), healthy fish from a negative cage from a farm experiencing an outbreak in a different cage (C), healthy fish from a negative farm near an outbreak farm (D), and healthy fish sampled at a negative farm located in an area with only negative farms (E). Apparent prevalences (standard error) for the different groups (A-E) were 0.94 (+/-0.026), 0.41 (+/-0.062), 0.29 (+/-0.040), 0.08 (+/-0.037) and 0.08 (+/-0.037), respectively. All groups were significantly different (P < 0.002) from each other except for groups B and C and groups D and E. Because the prevalence of the virus was significantly higher in the outbreak cage (B) compared with other sites, early harvest of outbreak cages will remove one source of virus. However, ISA negative cages (C) that remain on the positive farm may potentially act as a viral reservoir.     

In vivo virulence and genomic comparison of infectious Salmon Anaemia Virus isolates from Atlantic Canada

The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo‐controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high‐virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA‐HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid‐virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT‐qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence.     

Viral co‐infections in farmed Atlantic salmon,Salmo salar L., displaying myocarditis

Several different viruses have been associated with myocarditis‐related diseases in the Atlantic salmon aquaculture industry. In this study, we investigated the presence of PMCV, SAV, PRV and the recently identified Atlantic salmon calicivirus (ASCV), alone and as co‐infections in farmed Atlantic salmon displaying myocarditis. The analyses were performed at the individual level and comprised qPCR and histopathological examination of 397 salmon from 25 farms along the Norwegian coast. The samples were collected in 2009 and 2010, 5–22 months post‐sea transfer. The study documented multiple causes of myocarditis and revealed co‐infections including individual fish infected with all four viruses. There was an overall correlation between lesions characteristic of CMS and PD and the presence of PMCV and SAV, respectively. Although PRV was ubiquitously present, high viral loads were with a few exceptions, correlated with lesions characteristic of HSMI. ASCV did not seem to have any impact on myocardial infection by PMCV, SAV or PRV. qPCR indicated a negative correlation between PMCV and SAV viral loads. Co‐infections result in mixed and atypical pathological changes which pose a challenge for disease diagnostic work.     

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.     

Seasonal increase in sea temperature triggers pancreas disease outbreaks in Norwegian salmon farms

Pancreas disease (PD) is a viral disease causing negative impacts on economy of salmon farms and fish welfare. Its transmission route is horizontal, and water transport by ocean currents is an important factor for transmission. In this study, the effect of temperature changes on PD dynamics in the field has been analysed for the first time. To identify the potential time of exposure to the virus causing PD, a hydrodynamic current model was used. A cohort of salmon was assumed to be infected the month it was exposed to virus from other infective cohorts by estimated water contact. The number of months from exposure to outbreak defined the incubation period, which was used in this investigation to explore the relationship between temperature changes and PD dynamics. The time of outbreak was identified by peak in mortality based on monthly records from active sites. Survival analysis demonstrated that cohorts exposed to virus at decreasing sea temperature had a significantly longer incubation period than cohorts infected when the sea temperature was increasing. Hydrodynamic models can provide information on the risk of being exposed to pathogens from neighbouring farms. With the knowledge of temperature‐dependent outbreak probability, the farmers can emphasize prophylactic management, avoid stressful operations until the sea temperature is decreasing and consider removal of cohorts at risk, if possible.     

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon,Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real‐time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post‐infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post‐infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).     

Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.