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.     

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.     

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno‐histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post‐seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II⁺ cells in heart, pancreas and pyloric caeca, IgM⁺ cells in heart and pancreas, and CD3⁺ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD‐affected tissues were MHC II⁺ and CD3⁺ cells suggesting that antigen‐presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.     

Serum enolase: a non‐destructive biomarker of white skeletal myopathy during pancreas disease (PD) in Atlantic salmon Salmo salar L.

Diseases which cause skeletal muscle myopathy are some of the most economically damaging diseases in Atlantic salmon, Salmo salar L., aquaculture. Despite this, there are limited means of assessing fish health non‐destructively. Previous investigation of the serum proteome of Atlantic salmon, Salmo salar L., during pancreas disease (PD) has identified proteins in serum that have potential as biomarkers of the disease. Amongst these proteins, the enzyme enolase was selected as the most viable for use as a biomarker of muscle myopathy associated with PD. Western blot and immunoassay (ELISA) validated enolase as a biomarker for PD, whilst immunohistochemistry identified white muscle as the source of enolase. Enolase was shown to be a specific marker for white muscle myopathy in salmon, rising in serum concentration significantly correlating with pathological damage to the tissue.     

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.     

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.     

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.     

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.     

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.     

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).     

Liquid fat,a potential abiotic vector for horizontal transmission of salmonid alphavirus?

Viral diseases represent serious challenge in marine farming of Atlantic salmon (Salmo salar L). Pancreas disease (PD) caused by a salmonid alphavirus (SAV) is by far the most serious in northern Europe. To control PD, it is necessary to identify virus transmission routes. One aspect to consider is whether the virus is transported as free particles or associated with potential vectors. Farmed salmonids have high lipid content in their tissue which may be released into the environment from decomposing dead fish. At the seawater surface, the effects of wind and ocean currents are most prominent. The aim of this study was primarily to identify whether the lipid fraction leaking from dead infected salmon contains SAV. Adipose tissue from dead SAV‐infected fish from three farming sites was submerged in beakers with sea water in the laboratory and stored at different temperature and time conditions. SAV was identified by real‐time RT‐PCR in the lipid fractions accumulating at the water surface in the beakers. SAV‐RNA was also present in the sea water. Lipid fractions were transferred to cell culture, and viable SAV was identified. Due to its hydrophobic nature, fat with infective pathogenic virus at the surface may contribute to long‐distance transmission of SAV.     

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.‐approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA‐positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.     

Multiple passage of infectious salmon anaemia virus in rainbow trout,Oncorhynchus mykiss (Walbaum), did not induce increased virus load

The infectious salmon anaemia virus (ISAV) has not been observed to cause natural disease in farmed rainbow trout, Onchorhynchus mykiss (Walbaum), but may cause high mortality in farmed Atlantic salmon, Salmo salar L. In this study, ISAV was passaged 10 times in succession by intraperitoneal injections of serum from previous passage into naïve rainbow trout. The serum viraemia was monitored by real‐time qPCR. The rainbow trout in this study became infected but did not develop ISA. No clinical signs were observed in the rainbow trout in any passage, but replication of ISAV was detected from Day 4 post‐infection (p.i.). Neither increased relative virus loads nor histopathological and immunohistochemical findings consistent with ISA were observed. However, the expression of interferon type I and Mx genes were slightly up‐regulated in the hearts of some individual fish at day 17 p.i. Sequencing of all open reading frames in the ISAV genome of the 10th passage revealed two nucleotide mutations, one in segment 6 coding for the haemagglutinin–esterase (HE) and one in segment 1 coding for the basic polymerase 2 (PB2). The mutation in HE resulted in an amino acid substitution T/K₃₁₂.     

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.     

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.     

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.