BackgroundCardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.ResultsUsing high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.ConclusionsAlthough causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Piscirickettsia salmonis, a serious bacterial pathogen of farmed marine fish, previously considered culturable only in eukaryotic cell-culture systems, was grown for the first time on agar and broth containing enhanced levels of cysteine, thus greatly increasing the potential for isolation, in vitro culture and study of this organism. Virulence towards Atlantic salmon following passage on agar media was retained in a controlled laboratory trial. Of the studied temperatures, optimal growth on agar was observed at 22 degrees C.

Summary1. Indigenous European freshwater crayfish (ICS) are threatened due to invasive North American freshwater crayfish that are natural carriers of Aphanomyces astaci which causes crayfish plague. Infectious A. astaci zoospores are released from carrier crayfish, but little is known about the spore abundance in water systems that either host non-indigenous crayfish species (NICS) or experience crayfish plague outbreaks. We tested two large-scale filtering approaches to generate new insight about the abundance and dynamics of A. astaci spores in natural freshwater systems. 2. Depth filtration (DF) and dead-end ultrafiltration (DEUF) followed by A. astaci-specific quantitative real-time PCR was used to monitor A. astaci spores in large Nordic lakes hosting A. astaci-positive Pacifastacus leniusculus, the dominating NICS in Northern Europe. Crayfish and water were sampled together to compare the A. astaci pathogen load in tissues, A. astaci prevalence in the population and the corresponding spore density in water. Samples were also obtained from a river where indigenous noble crayfish suffered from acute crayfish plague. The sensitivity of the filtering techniques was evaluated using simulation of random events. 3. We detected A. astaci spores in lakes hosting NICS with both filtering methods but predominantly at concentrations below c. 1 spore L À1 . We found a significant positive association between A. astaci spore density in water, the A. astaci prevalence in the corresponding NICS population and the tissue pathogen load. Water from the river with the ongoing crayfish plague outbreak contained overall c. 43 times more spores L À1 than water hosting NICS. Both filtering techniques proved suitable and equally sensitive, but simulations suggest that an optimization of the spore recovery could yield a 10-fold increase in the DEUF-method sensitivity. 4. Synthesis and application. Our study demonstrates a low amount of pathogen spores are present in aquatic environments with non-indigenous crayfish species, emphasizing the need for large-volume filtering techniques for successful detection. The approach can be used for risk assessments and to improve conservation and management strategies of crayfish in Europe. Applications of this method include targeted disease surveillance, habitat evaluation prior to crayfish re-stockings and water monitoring that can minimize disease transmission and spread, for example in crayfish farms and prior to fish movements for stocking purposes.

This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid Ectopleura larynx frequently colonises cage nets at high densities (thousands of colonies per m2) and is released into the water during regular in-situ net cleaning. Contact with the hydroids’ nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba Paramoeba perurans, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections–using P. perurans as an example. Salmon in tanks were exposed either to freshly ‘shredded’ hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living P. perurans, or first to ‘shredded’ hydroids and then to P. perurans. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group.Nematocysts of E. larynx contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of P. perurans or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning.

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.KEY WORDS: Heart and skeletal muscle inflammation · HSMI · Transmission · Reovirus · Wild marine fish species · Farmed fish · PCR Resale or republication not permitted without written consent of the publisherDis Aquat Org 97: [255][256][257][258] 2012 Research, Bergen. Sampling was financed and performed by the project 'Viral haemorrhagic septicaemia virus (VHSV) in wild and farmed fish in Norway' (NFR-190245). Briefly, the fish were captured by trawling and 1 g samples of spleen, kidney and brain were pooled and directly frozen in 9 ml Leibovitz (L-15) culture medium supplemented with 4 mM L-glutamine and 100 ng ml -1 gentamicin (all from SigmaAldrich). Each pool consisted of material from 2 to 5 individuals. Samples were immediately transferred to −80°C for storage. RNA was extracted from 100 µl of mixed tissue homogenate with the NucliSENS easyMAG nucleic acid extraction system (bioMérieux) according to the manufacturer's recommendations. A total of 1627 fish (379 pools) from 37 different species were sampled and screened by means of a PRV-specific RT-qPCR assay (Palacios et al. 2010). RESULTSThe majority of the 379 pools of samples tested negative for the PRV-specific real-time PCR, but pools from 4 different fish species yielded positive results (Table 1) that had relatively high threshold cycle number (C t )-values (great silver smelt Argentina silus, C t = 37; capelin Mallotus villosus, C t = 38; Atlantic horse mackerel Trachurus trachurus, C t = 37; and Atlantic herring Clupea harengus, C t = 31). DISCUSSIONThe fish species that tested positive with the PRV-specific real-time PCR are common species found along the Norwegian coast at different periods of the year. Argentina silus is a smelt of the Argentinidae family. It is a pelagic species that is common along the Norwegian coastline. The positive pool originated from A. silus that were caught by trawl in northern Trøndelag. Trachurus trachurus is a pelagic species that is common in west and northern parts of Norway during late summer and autumn. Only one T. trachurus was caught and it was in the Andøyfjord. Mallotus villosus is a small fish of the smelt family Osmeridae. This is an arctic species, but local stocks are also found along the coast of the northern part of Norway. The positive pool of M. villosus was from the Altafjord. Clupea harengu...

BackgroundFish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds.ResultsMorphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes.ConclusionsShortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietary fish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is a severe cardiac disease characterized by a necrotizing myocarditis involving the atrium and the spongious part of the ventricle. The disease is caused by piscine myocarditis virus (PMCV), a double-stranded RNA virus likely belonging to the family Totiviridae. The objective of this study was to evaluate the genetic variation in Norwegian PMCV isolates focusing on the putative structural proteins encoded by open reading frames (ORFs) 1 and 3. The virus isolates were sampled from a total of 36 farms along the Norwegian coastline. This study represents the first investigation of PMCV genome variation and shows that Norwegian isolates are highly similar, with the most divergent isolates sharing 98.6% nucleotide identity. Interestingly, amino acid sequence diversity within ORF3 is approximately threefold higher than for ORF1. While phylogenetic analysis based on concatenated nucleotide data covering ORF1 and ORF3 revealed four main clusters, the maximum sequence variation of 1.4% at the nucleotide level suggests that all Norwegian isolates belong to a single genogroup. Substantial sequence variation within farms was also observed, which may complicate future molecular epidemiological investigations. The genetic homogeneity among the Norwegian isolates might facilitate development of both diagnostic tools and an efficient vaccine against CMS in the future.

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.