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.

The serum proteome of Atlantic salmon, Salmo salar, during pancreas disease (PD) following infection with salmonid alphavirus subtype 3 (SAV3).

Salmonid alphavirus is the aetological agent of pancreas disease (PD) in marine Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, with most outbreaks in Norway caused by SAV subtype 3 (SAV3). This atypical alphavirus is transmitted horizontally causing a significant economic impact on the aquaculture industry. This histopathological and proteomic study, using an established cohabitational experimental model, investigated the correlation between tissue damage during PD and a number of serum proteins associated with these pathologies in Atlantic salmon. The proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting. A number of humoral components of immunity which may act as biomarkers of the disease were also identified. For example, creatine kinase, enolase and malate dehydrogenase serum concentrations were shown to correlate with pathology during PD. In contrast, hemopexin, transferrin, and apolipoprotein, amongst others, altered during later stages of the disease and did not correlate with tissue pathologies. This approach has given new insight into not only PD but also fish disease as a whole, by characterisation of the protein response to infection, through pathological processes to tissue recovery. BIOLOGICAL SIGNIFICANCE: Salmonid alphavirus causes pancreas disease (PD) in Atlantic salmon, Salmo salar, and has a major economic impact on the aquaculture industry. A proteomic investigation of the change to the serum proteome during PD has been made with an established experimental model of the disease. Serum proteins were identified by two-dimensional electrophoresis, trypsin digest and peptide MS/MS fingerprinting with 72 protein spots being shown to alter significantly over the 12week period of the infection. The concentrations of certain proteins in serum such as creatine kinase, enolase and malate dehydrogenase were shown to correlate with tissue pathology while other proteins such as hemopexin, transferrin, and apolipoprotein, altered in concentration during later stages of the disease and did not correlate with tissue pathologies. The protein response to infection may be used to monitor disease progression and enhance understanding of the pathology of PD.

Dietary synbiotic application modulates Atlantic salmon (Salmo salar) intestinal microbial communities and intestinal immunity.

A feeding trial was conducted to determine the effect of dietary administration of Pediococcus acidilactici MA18/5M and short chain fructooligosaccharides (scFOS) on Atlantic salmon (Salmo salar L.) intestinal health. Salmon (initial average weight 250 g) were allocated into triplicate sea pens and were fed either a control diet (commercial diet: 45% protein, 20% lipid) or a synbiotic treatment diet (control diet + P. acidilactici at 3.5 g kg(-1) and 7 g kg(-1) scFOS) for 63 days. At the end of this period, fish were sampled for intestinal microbiology, intestinal histology and the expression of selected immune-related genes (IL1ß, TNFα, IL8, TLR3 and MX-1) in the intestine. Compared to the control fish, the total bacterial levels were significantly lower in the anterior mucosa, posterior mucosa and posterior digesta of the synbiotic fed fish. qPCR revealed good recovery (log 6 bacteria g(-1)) of the probiotic in the intestinal digesta of the synbiotic fed fish and PCR-DGGE revealed that the number of OTUs, as well as the microbial community diversity and richness were significantly higher in the anterior digesta of the synbiotic fed fish than the control. Compared to the control fed fish, the mucosal fold (villi) length and the infiltration of epithelial leucocytes were significantly higher in the anterior and posterior intestine, respectively, in the synbiotic group. Real-time PCR demonstrated that all of the genes investigated were significantly up-regulated in the anterior and posterior intestine of the synbiotic fed salmon, compared to the control group. At the systemic level, serum lysozyme activity was significantly higher in the synbiotic fed fish and growth performance, feed utilisation and biometric measurements (condition factor, gutted weight and gut loss) were not affected. Together these results suggest that the synbiotic modulation of the gut microbiota has a protective action on the intestinal mucosal cells, improving morphology and stimulating the innate immune response without negatively affecting growth performance or feed utilization of farmed Atlantic salmon.