Phylogenetic analyses and molecular epidemiology of European salmonid alphaviruses (SAV) based on partial E2 and nsP3 gene nucleotide sequences

Sequence data were generated for portions of the E2 and nsP3 genes of 48 salmonid alphaviruses from farmed Atlantic salmon (AS), Salmo salar L., and rainbow trout (RT), Oncorhynchus mykiss (Walbaum), in marine and freshwater environments, respectively, from the Republic of Ireland, Northern Ireland, England, Scotland, Norway, France, Italy and Spain between 1991 and 2007. Based on these sequences, and those of six previously published reference strains, phylogenetic trees were constructed using the parsimony method. Trees generated with both gene segments were similar. Clades corresponding to the three previously recognized subtypes were generated and in addition, two further new clades of viruses were identified. A single further strain (F96-1045) was found to be distinct from all of the other strains in the study. The percentage of nucleotide divergence within clades was generally low (0-4.8% for E2, 0-6.6% for nsP3). Interclade divergence tended to be higher (3.4-19.7% for E2, 6.5-28.1% for nsP3). Based on these results and using current SAV terminology, the two new clades and F96-1045 were termed SAV subtypes 4, 5 and 6, respectively. SAV4 contained AS strains from Ireland and Scotland, while SAV5 contained only Scottish AS strains. Recently identified SAV strains from RT in Italy and Spain were shown to belong to SAV2. In addition, marine AS strains belonging to SAV2 were identified for the first time. Analysis of the origin of several clusters of strains with identical E2 and nsP3 sequences strongly support horizontal transmission of virus between farms and aquaculture companies. Evidence in support of vertical transmission was not found.     

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.     

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.     

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.     

Observational study of erythrocytic inclusion bodies in farmed Atlantic salmon, Salmo salar L., in the British Isles

An observational study was conducted of farmed Atlantic salmon, Salmo salar L., in the British Isles with regard to intraerythrocytic inclusions and disease prevalence. Intraerythrocytic inclusions were observed to be widespread in Atlantic salmon from farms in Ireland and Scotland. The percentage of fish infected with these inclusions and the intensity of infection were greater in sea stage salmon than those in fresh water. This corresponded with reduced values for haematological parameters (haematocrit, erythrocyte count, haemoglobin, MCV, MCH, MCHC). There appeared to be a significant association between the presence of the intraerythrocytic inclusions and focal cardiomyopathy, although there was no established association with overt clinical disease and the presence of the inclusions. There was also a significant association between the presence of raised splenic haemosiderin and multiple intraerythrocytic inclusions.     

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.     

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).     

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.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

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.     

Cultural characteristics of salmonid alphaviruses – influence of cell line and temperature

Laboratory studies were carried out to investigate the cultural characteristics of salmonid alphaviruses (SAV) from Atlantic salmon (AS, Salmo salar) and rainbow trout (RT, Oncorhynchus mykiss), particularly in relation to cell line and temperature. In an initial study, SAV was isolated from 12 viraemic sera and passaged in Chinook salmon embryo (CHSE‐214) cells at 15 °C. Geometric mean titres (GMT) after initial isolation were found to be significantly higher (P < 0.05) relative to those after two or four passages. Primary isolation of SAV was conducted from 12 viraemic sera (six AS and six RT) in seven different cell lines at 15 °C: CHSE‐214, rainbow trout gonad (RTG‐2), TO (derived from Atlantic salmon head kidney leucocytes), salmon head kidney (SHK‐1), blue fin‐2 (BF‐2), fat head minnow (FHM) and Epithelioma papulosum cyprini (EPC). Overall, significant differences were found between cell lines in both the numbers of strains where growth was detected and in the GMT obtained. For both AS and RT strains, GMT values were significantly (P < 0.01) higher in both TO and BF‐2 cells relative to the others, including CHSE‐214 and RTG‐2, the cell lines conventionally used for SAV. The effects of temperature of incubation (4, 10, 15 and 20 °C) on growth in TO, CHSE‐214 and RTG‐2 were investigated. In TO and RTG‐2 growth was optimal at 15 °C, whereas in CHSE‐214 results at 10 and 15 °C were more similar. Little or no growth was detected at 4 or 20 °C.     

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.     

Genetic diversity of piscine myocarditis virus in Atlantic salmon Salmo salar L. in Ireland

Piscine myocarditis virus (PMCV) is a double‐stranded RNA virus which has been linked to cardiomyopathy syndrome (CMS) in Atlantic salmon (Salmo salar L.). The first recorded outbreak of CMS in Ireland occurred in 2012. Heart tissue samples were collected in the current study from farmed Atlantic salmon from various marine sites around Ireland, and the open reading frames (ORFs) 1 and 3 were amplified and sequenced in order to examine the genetic diversity of PMCV. Results showed PMCV to be largely homogenous in Irish samples, showing little genetic diversity. However, several amino acid positions within both ORF1 and ORF3 showed consistent variations unique to the Irish PMCV strains when compared with previously published Norwegian strains. The phylogeny generated in the present study suggests that PMCV may have been introduced into Ireland in two waves, both coming from the southern part of PMCV's range in Norway. In addition, over three‐quarters of the PMCV strains which were sequenced came from fish not exhibiting any clinical signs of CMS, suggesting that either PMCV is evolving to become less virulent in Ireland or Irish Atlantic salmon are developing immunity to the disease.     

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.     

A historical review of the key bacterial and viral pathogens of Scottish wild fish

Thousands of Scottish wild fish were screened for pathogens by Marine Scotland Science. A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken. Despite many reported cases in farmed fish, there was a limited number of positive samples from Scottish wild fish, however, there was evidence for interactions between wild and farmed fish. A slightly elevated IPNV prevalence was reported in wild marine fish caught close to Atlantic salmon, Salmo salar L., farms that had undergone clinical IPN. Salmonid alphavirus was isolated from wild marine fish caught near Atlantic salmon farms with a SAV infection history. Isolations of VHSV were made from cleaner wrasse (Labridae) used on Scottish Atlantic salmon farms and VHSV was detected in local wild marine fish. However, these pathogens have been detected in wild marine fish caught remotely from aquaculture sites. These data suggest that despite the large number of samples taken, there is limited evidence for clinical disease in wild fish due to these pathogens (although BKD and furunculosis historically occurred) and they are likely to have had a minimal impact on Scottish wild fish.     

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.     

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.     

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.