Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch''s postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.     

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.     

Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.     

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.     

Effects of functional feeds on the lipid composition,transcriptomic responses and pathology in heart of Atlantic salmon (Salmo salar L.) before and after experimental challenge with Piscine Myocarditis Virus (PMCV)

Background

Cardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon (Salmo salar) recently associated with a double-stranded RNA virus, Piscine Myocarditis Virus (PMCV). The disease has been diagnosed in 75-85 farms in Norway each year over the last decade resulting in annual economic losses estimated at up to €9 million. Recently, we demonstrated that functional feeds led to a milder inflammatory response and reduced severity of heart lesions in salmon experimentally infected with Atlantic salmon reovirus, the causal agent of heart and skeletal muscle inflammation (HSMI). In the present study we employed a similar strategy to investigate the effects of functional feeds, with reduced lipid content and increased eicosapentaenoic acid levels, in controlling CMS in salmon after experimental infection with PMCV.

Results

Hepatic steatosis associated with CMS was significantly reduced over the time course of the infection in fish fed the functional feeds. Significant differences in immune and inflammatory responses and pathology in heart tissue were found in fish fed the different dietary treatments over the course of the infection. Specifically, fish fed the functional feeds showed a milder and delayed inflammatory response and, consequently, less severity of heart lesions at earlier and later stages after infection with PMCV. Decreasing levels of phosphatidylinositol in cell membranes combined with the increased expression of genes related with T-cell signalling pathways revealed new interactions between dietary lipid composition and the immune response in fish during viral infection. Dietary histidine supplementation did not significantly affect immune responses or levels of heart lesions.

Conclusions

Combined with the previous findings on HSMI, the results of the present study highlight the potential role of clinical nutrition in controlling inflammatory diseases in Atlantic salmon. In particular, dietary lipid content and fatty acid composition may have important immune-modulatory effects in Atlantic salmon that could be potentially beneficial in fish balancing the immune and tissue responses to viral infections.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-462) contains supplementary material, which is available to authorized users.     

Phylogenetic Evidence of Long Distance Dispersal and Transmission of Piscine Reovirus (PRV) between Farmed and Wild Atlantic Salmon

The extent and effect of disease interaction and pathogen exchange between wild and farmed fish populations is an ongoing debate and an area of research that is difficult to explore. The objective of this study was to investigate pathogen transmission between farmed and wild Atlantic salmon (Salmo salar L.) populations in Norway by means of molecular epidemiology. Piscine reovirus (PRV) was selected as the model organism as it is widely distributed in both farmed and wild Atlantic salmon in Norway, and because infection not necessarily will lead to mortality through development of disease. A matrix comprised of PRV protein coding sequences S1, S2 and S4 from wild, hatchery-reared and farmed Atlantic salmon in addition to one sea-trout (Salmo trutta L.) was examined. Phylogenetic analyses based on maximum likelihood and Bayesian inference indicate long distance transport of PRV and exchange of virus between populations. The results are discussed in the context of Atlantic salmon ecology and the structure of the Norwegian salmon industry. We conclude that the lack of a geographical pattern in the phylogenetic trees is caused by extensive exchange of PRV. In addition, the detailed topography of the trees indicates long distance transportation of PRV. Through its size, structure and infection status, the Atlantic salmon farming industry has the capacity to play a central role in both long distance transportation and transmission of pathogens. Despite extensive migration, wild salmon probably play a minor role as they are fewer in numbers, appear at lower densities and are less likely to be infected. An open question is the relationship between the PRV sequences found in marine fish and those originating from salmon.     

Effect of functional feeds on fatty acid and eicosanoid metabolism in liver and head kidney of Atlantic salmon (Salmo salar L.) with experimentally induced Heart and Skeletal Muscle Inflammation

Heart and Skeletal Muscle Inflammation (HSMI) is an emerging viral disease caused by a novel Atlantic salmon reovirus (ASRV) affecting farmed fish. Primary symptoms associated with HSMI include myocardial and skeletal muscle necrosis indicating a severe inflammatory process. Recently, we applied the concept of clinical nutrition to moderate the long-term inflammatory process associated with HSMI in salmon subjected to experimental ASRV challenge. The use of functional feeds with lower lipid (hence energy) content reduced the inflammatory response to ASRV infection and the severity of associated heart lesions. The aim of the present study was to elucidate possible mechanisms underpinning the observed effects of the functional feeds, focussing on eicosanoid and fatty acid metabolism in liver and head kidney. Here we show that liver was also a site for histopathological lesions in HSMI showing steatosis reflecting impaired lipid metabolism. This study is also the first to evaluate the expression of a suite of key genes involved in pathways relating diet and membrane phospholipid fatty acid compositions, and the inflammatory response after ASRV infection. The expression of hepatic Δ6 and Δ5 desaturases was higher in fish fed the functional feeds, potentially increasing their capacity for endogenous production and availability of anti-inflammatory EPA. Effects on mobilization of lipids and changes in the LC-PUFA composition of membrane phospholipids, along with significant changes in the expression of the genes related to eicosanoid pathways, showed the important role of the head kidney in inflammatory diseases caused by viral infections. The results from the present study suggest that clinical nutrition through functional feeding could be an effective complementary therapy for emerging salmon viral diseases associated with long-term inflammation.     

Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.     

Virus-like particles associated with heart and skeletal muscle inflammation (HSMI)

The first cases of heart and skeletal muscle inflammation (HSMI), in Atlantic salmon Salmo salar were registered in 1999 in the Hitra/Fr?ya area of Norway. The disease has since spread south to Rogaland, i.e. the southernmost county with salmon farming in Norway. The disease outbreaks usually start 5 to 9 mo after release into seawater but may occur as early as 2 wk after sea release. The present study focuses on possible pathogens associated with HSMI. It was not possible to find any parasites or bacteria that could explain HSMI, and none of the well-known viruses (infectious salmon anaemia virus, Norwegian salmonid alphavirus, infectious pancreatic necrosis virus, Atlantic salmonid paramyxovirus) were consistently present. Use of transmission electron microscopy showed the presence of epitheliocystis agent in 3 of 4 farms included in this study, and several virus-like particles. Type I and Type II virus particles, previously described for salmon suffering from haemorrhagic smolt syndrome (HSS), and erythrocytic inclusion body syndrome (EIBS) virus were consistently present in salmon suffering from HSMI in all 4 farms included in this study. The 2 HSS viruses (Type I and Type II) were also cultured in Atlantic salmon kidney (ASK) cells from salmon suffering from HSMI. However, a causal relationship between the observed virus particles and HSMI remains to be demonstrated.     

Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

Pancreas disease (PD) of farmed Atlantic salmon Salmo salar L., which is caused by an alphavirus known as salmon pancreas disease virus (SPDV), can have serious economic consequences. An epidemiological survey carried out in Ireland in 2003 indicated that within individual farms there were significant differences in the susceptibility of different strains of farmed Atlantic salmon to infection with SPDV, as measured by levels of clinical disease and mortality. The aim of this preliminary study was to investigate this field observation by comparing lesion development, viraemia and serological responses of 3 commercial strains of Atlantic salmon (A, B and C) experimentally infected with SPDV. Highly significant differences in the severity of lesions in the pancreas at Day 21 post-infection (pi) were detected (p < 0.01), with Group B being more severely affected. There were also significant differences in the prevalence and severity of lesions in heart and skeletal muscle at Day 21 and 35 pi respectively, with Group B results again significantly higher than those from both Groups A and C (p < 0.05). There was no overlap between viraemia and the presence of specific SPDV antibody. Some fish in all groups had no viraemia, lesions or evidence of seroconversion. There were no significant differences seen between the challenged groups in relation to the percentage of viraemic fish at each time point. Viral loads were not determined. Differences between the number of antibody-positive fish in each challenge group were found at Days 28 and 35 pi (p < 0.1). Highly significant differences (p < 0.01) in the geometric mean titres of seropositive fish were detected at Day 28. These results, obtained using a challenge model, confirm that there are strain differences in the susceptibility to experimental SPDV infection in commercial farmed Atlantic salmon.     

Genetic Stock Identification of Chum Salmon in the Bering Sea and North Pacific Ocean Using Mitochondrial DNA Microarray

A newly developed DNA microarray was applied to identify mitochondrial (mt) DNA haplotypes of more than 2200 chum salmon in the Bering Sea and North Pacific Ocean in September 2002 and also 2003, when the majority of maturing fish were migrating toward their natal river. The distribution of haplotypes occurring in Asian and North American fish in the surveyed area was similar in the 2 years. A conditional maximum likelihood method for estimation of stock compositions indicated that the Japanese stocks were distributed mainly in the north central Bering Sea, whereas the Russian stocks were mainly in the western Bering Sea. The North American stocks were abundant in the North Pacific Ocean around the Aleutian Islands. These results indicate that the Asian and North American stocks of chum salmon are nonrandomly distributed in the Bering Sea and the North Pacific Ocean, and further the oligonuleotide DNA microarray developed by us has a high potential for identification of stocks among mixed ocean aggregates of high-seas chum salmon.     

A review of genetic variation in Atlantic salmon, Salmo salar L., and its importance for stock identification, enhancement programmes and aquaculture

The increasing exploitation of Atlantic salmon as a food source and sport fish demands a better understanding of salmon genetics and the dynamics of Atlantic salmon populations. Surveys of salmon populations for protein electrophoretic variation reveal that the average heterozygosity in Salmo salar is low and that four gene loci account for more than 95% of the total electrophoretically detectable variation. Populations that have been studied by this means fall into one of three groups: Western Atlantic, Eastern Atlantic or Baltic. However, biochemical genetics involving starch gel electrophoresis cannot be used routinely to identify the continent of origin of an Atlantic salmon, let alone its native river. The mitochondrial genome can be used to identify North American or European salmon with the aid of restriction endonucleases that have six base pair recognition sites. Restriction endonucleases that recognize four base pairs appear to be able to identify salmon from a particular river system. There has been a move from protein variation to mitochondrial DNA variation and this will inevitably lead to more extensive studies on the nuclear genome. Chromosomal studies suggest differences between salmon from Europe and North America but these have been hampered by lack of good banding procedures. Preliminary studies using cloned segments of salmonid genomes suggest that repeated sequences such as the genes for ribosomal RNA will be most useful for identifying specific stocks of Atlantic salmon. The need for continued genetic studies on the Atlantic salmon and the relevance and importance of the results of this research for stock identification, enhancement programmes, aquaculture and basic science are discussed.     

Cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI)

Heart and skeletal muscle inflammation (HSMI) is a disease of marine farmed Atlantic salmon where the pathological changes associated with the disease involve necrosis and an infiltration of inflammatory cells into different regions of the heart and skeletal muscle. The aim of this work was to characterize cardiac changes and inflammatory cell types associated with a clinical HSMI outbreak in Atlantic salmon using immunohistochemistry. Different immune cells and cardiac tissue responses associated with the disease were identified using different markers. The spectrum of inflammatory cells associated with the cardiac pathology consisted of mainly CD3(+) T lymphocytes, moderate numbers of macrophages and eosinophilic granulocytes. Proliferative cell nuclear antigen (PCNA) immuno-reaction identified significantly increased nuclear and cytoplasmic staining as well as identifying hypertrophic nuclei. Strong immunostaining was observed for major histocompatibility complex (MHC) class II in HSMI hearts. Although low in number, a few positive cells in diseased hearts were detected using the mature myeloid cell line granulocytes/monocytes antibody indicating more positive cells in diseased than non-diseased hearts. The recombinant tumor necrosis factor-α (TNFα) antibody identified stained macrophage-like cells and endothelial cells around lesions in addition to eosinophilic granular cells (EGCs). These findings suggested that the inflammatory response in diseased hearts comprised of mostly CD3(+) T lymphocytes and eosinophilic granular cells and hearts exhibited high cell turnover where DNA damage/repair might be the case (as identified by PCNA, caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) reactivity).     

Microsatellite Variation in Populations of Atlantic Salmon from North Europe

Our aim was to investigate the level of genetic differentiation in northern European populations of Atlantic salmon, to establish the genetic relationship among major salmon populations in Russia and North Norway, and to compare these to populations from the western Atlantic lineage. Samples were collected along an east—west axis, from Pechora River in Russia to Restigouche River in Quebec, Canada. A total of 439 individual salmon were collected from seven rivers (sample sizes from 50 to 84 individuals). The samples were analysed for variation at four microsatellite loci; Ssa13.37, Ssa14, Ssa171 and Ssa171. Significant differences were found between most of the European populations, and the populations from the Tana and Pechora Rivers were most distinct. The samples from the Rivers Mezenskaya Pizhma and Emtsa in Arkhangelsk oblast in Russia were not significantly different from each other in an exact test of population differences. All other river pairs were significantly different. These results confirmed the deep genetic divergence between American and European salmon populations demonstrated in earlier studies, with alleles specific to continent found in three of the microsatellites.     

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.     

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV^901_09 and rISAVr^S6-NotI-HPR containing a NotI restriction site and rISAV^S6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 10⁵ PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.     

Pathogenesis and immune response in Atlantic salmon (Salmo salar L.) parr experimentally infected with salmon pancreas disease virus (SPDV)

Atlantic salmon parr were injected intraperitoneally with salmon pancreas disease virus (SPDV) grown on CHSE-214 cells. The viraemia, the histopathological changes in target organs and some immune parameters were taken at intervals up to 30 days post-infection (dpi). The earliest kind of lesion was necrosis of exocrine pancreas, appearing as soon as 2 dpi. It progressed towards complete tissue breakdown at 9 dpi before resolving gradually. Concurrent to this necrosis, a strong inflammatory response was in evidence from 9 dpi in the pancreatic area for a majority of fish. A necrosis of the myocardial cells of the ventricle occurred in infected fish mainly at 16 dpi and it faded thereafter. The monitoring of the plasma viral load showed a rapid haematogenous spreading of SPDV, peaking at 4 dpi, but also the absence of a secondary viraemia. No interferon (IFN) was detected following the infection of parr with SPDV, probably owing to an IFN activity in Atlantic salmon below the detection level of the technique. Neutralising antibodies against SPDV were in evidence from 16 dpi and they showed a time-related increasing titre and prevalence. The phagocytic activity in head-kidney leucocytes was always significantly higher in the infected fish than in the control fish, being particularly high by 9 dpi. Lysozyme and complement levels were both increased and they peaked significantly in the infected fish at 9 and 16 dpi respectively. These results demonstrated that an experimental infection of Atlantic salmon parr with SPDV provoked a stimulation of both specific and non-specific immunity with regards to the viraemia and the histopathology.