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.     

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon,Salmo salar,infected with salmonid alphavirus (SAV3)

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on‐farm qualitative test and an in‐laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ₃₄₀). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ₃₄₀ in infected fish rose from undetectable to a maximum at 6 weeks post‐infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.     

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.     

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).     

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.     

Pancreas disease (PD) in sea‐reared Atlantic salmon,Salmo salar L., in Norway; a prospective,longitudinal study of disease development and agreement between diagnostic test results

A prospective longitudinal study was performed on three cages at each of three Norwegian Atlantic salmon seawater sites that experienced outbreaks of pancreas disease (PD). Once salmonid alphavirus (SAV) ribonucleic acid (RNA) was detected by real‐time RT‐PCR (Rt RT‐PCR) at a site, it became detected in all studied cages and was persistently found until the end of the study period up to 19 months after first detection. SAV‐specific antibodies were detected at all sites until the end of the study period and were also found at a high prevalence in broodfish at the time of stripping. No evidence of increased viral activity was detected in these broodfish. One site tested negative over several months prior to the first detection of SAV by Rt RT‐PCR and SAV‐specific antibody, which occurred 1 month prior to clinical manifestations of PD. Moribund fish or thin fish/runts that were sampled after the first PD diagnosis had almost twice the risk of testing positive by one or more diagnostic tests compared to that of randomly selected apparently healthy individuals. This paper describes the first detailed investigation of the disease development of PD at site and cage level in Norway, as well as an assessment of the performance and agreement of the commonly used diagnostic tests.     

Molecular cloning of MDA5, phylogenetic analysis of RIG‐I‐like receptors (RLRs) and differential gene expression of RLRs,interferons and proinflammatory cytokines after in vitro challenge with IPNV,ISAV and SAV in the salmonid cell line TO

The RIG‐I receptors RIG‐I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG‐I‐like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full‐length MDA5 sequence in Atlantic salmon, and compared it with RIG‐I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa‐d) and proinflammatory cytokines (TNF‐α1, TNF‐α2, IL‐1β, IL‐6, IL‐12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV‐infected cells. In the two aforementioned, TNF‐α1 and TNF‐α2 were highly upregulated, while in SAV‐infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.     

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.     

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.     

Effect of low salinity on the expression profile of Na+/K+‐ATPase and the growth of juvenile chub mackerel (Scomber japonicus Houttuyn)

Chub mackerel (Scomber japonicus Houttuyn) is one of the most commercially important scombroid fish used as a food resource. Recently, there has been a demand for efficient rearing methods of this fish for a full‐life cycle aquaculture. In the present study, we evaluated the physiological responses in the juvenile S. japonicus to different ambient salinities. A significantly higher gain of the body mass was observed in the juveniles reared in 24 g/L and 13 g/L seawater than in those reared in natural seawater (34 g/L) within 40 days of the experimental period without affecting mortality. A principal enzyme for osmoregulation, Na⁺/K⁺‐ATPase, was expressed in the ionocytes located in the gill filaments of the juveniles. The number and the cell size of ionocytes and the enzymatic activity of Na⁺/K⁺‐ATPase in the gills decreased within 10 days after the low‐salinity challenge, which implies the reduction of the energy‐consuming active ion secretion under the low‐salinity environment. The physiological capacity for adaptation to low‐salinity seawater in chub mackerel could be basic knowledge to carry out culturing of these fish in coastal sea pens where ambient salinity fluctuates. The improvement of the growth performance by rearing in low‐salinity seawater will contribute to the efficient production of the seed juveniles for aquaculture.     

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.     

Quality of raw and smoked fillets from clinically healthy Atlantic salmon,Salmo salar L., following an outbreak of pancreas disease (PD)

Pancreas disease (PD) is a viral disease of farmed salmonid fish, which causes huge economic losses. Pathological changes in skeletal muscle, pancreas and heart are hallmarks of PD. Stakeholders in the fish‐smoking industry have claimed that fillets from PD‐affected Atlantic salmon, Salmo salar L., are of poor quality. We therefore examined harvest‐ready, clinically healthy Atlantic salmon from a population of fish previously affected by PD. Histopathological changes in skeletal muscle tissues ranged from minor to severe. Fillet quality measurements showed that fish with severe skeletal muscle changes provided a paler raw fillet and a yellowish and harder cold‐smoked fillet than normal. PD had no significant effect on fillet gaping, bacteriological quality or off‐odour development during storage. An unexpected finding was a significant subendocardial fibrosis in 23% of the PD‐affected fish. The latter may indicate susceptibility to stress‐related heart failure.     

Identification of a novel cyprinid herpesvirus 3 genotype detected in koi from the East Asian and South‐East Asian Regions

Cyprinid herpesvirus 3 (CyHV‐3) is a highly contagious virus that causes significant morbidity and mortality in common carp Cyprinus carpio L. and considered to be one of the most important pathogens of koi and common carp worldwide. Cyprinid herpesvirus 3 infected consignments imported from East Asian and South‐East Asian regions were identified during quarantine period in Singapore, and virus from a 2005 consignment was successfully isolated in koi fin cells. A combination of sequence analyses and duplex PCR were used to characterize 15 CyHV‐3 isolates detected in koi consignments between 2005 and 2011. Sequence analyses of the enlarged 9/5, SphI‐5 and TK gene regions identified both the Asian 1 (n = 11) and European 4 (n = 4) genotypes. Duplex PCR analysis of two variable marker regions between ORF29 and ORF30 (marker I) as well as ORF133 and its upstream region (marker II) revealed viruses of genotypes J (I⁺⁺II⁺), U/I (I⁻⁻II⁻), an intermediate genotype (I⁺⁺II⁻) and a novel genotype, I⁺⁺II^+Δ, which was identified in viruses from seven different consignments. This novel genotype has a 13‐bp deletion in marker II, while maintaining the I⁺⁺ allele of marker I. The I⁺⁺II^+Δ genotype may have emerged from East Asian and South‐East Asian regions in recent years.     

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.     

Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua)

The objective of this study was to examine whether digestive capacity correlates with growth rate in Atlantic cod (Gadus morhua). Ninety fish (1035±240 g; 478±36 mm) were assigned to one of three treatments: deprived of food for 5 weeks and then re-fed for 24 days, deprived for 10 weeks and then re-fed for 24 days, or fed for 24 days without deprivation (controls). Three times a week during feeding, the fish were provided with meals of capelin (Mallotus villosus) in excess. Within each treatment, ten fish were sacrificed before and twenty after the feeding period. The relative masses of the pyloric caeca, intestine, and white muscle were determined. Cytochrome c oxidase (CCO), citrate synthase (CS), and nucleoside diphosphate kinase (NDPK) activities were assayed in the pyloric caeca, intestine, and muscle as was trypsin activity in the pyloric caeca. During the re-feeding period, fish that had been deprived of food for 10 weeks showed compensatory growth, growing 1.9 times faster than the controls (0.94±0.26 versus 0.50±0.31% body mass ⋅ day⁻¹). Fish that displayed compensatory growth had a lower relative white muscle mass than controls (66.5±4.1% vs. 69.6±7.4%) while the relative masses of the pyloric caeca (2.21±0.49% vs. 1.78±0.31%) and intestine (0.95±0.20% vs. 0.77±0.15%) were greater than the controls, suggesting that the sizes of these digestive tissues might correlate with compensatory growth capacity. Citrate synthase in the pyloric caeca was the only enzyme that showed higher activity during compensatory growth (8.57±0.94 U ⋅ g tissue⁻¹ compared to 7.13±1.03 U ⋅ g tissue⁻¹ in the control group). This suggests that aerobic catabolic capacity of pyloric caeca could be related to growth capacity during recovery, possibly via the energetic cost of digestive enzyme synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.