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Disease outbreaks in fish aquaculture are often of concern due to the possibility of pathogen transmission to fish in neighbouring farms and to wild fish populations. To be able to assess the risk and manage transmission and outbreak scenarios, it is of great importance to understand the impact of various biological and physical conditions affecting the survival of the pathogen in the environment. In this study, we examined the effect of temperature on the viability of salmonid alphavirus subtype 3 (SAV3) in three separate experiments using SAV3 in culture medium, SAV3 in seawater or SAV3 shed by infected fish mimicking the virus produced at marine sites during outbreaks. SAV cultured on CHH-1 cells and cultured SAV mixed with seawater were incubated at different temperatures over a period of 4 weeks and 2 weeks, respectively. The surviving virus was quantified by two in vitro methods, RT-qPCR and a 50% tissue culture infectious dose (TCID50) assay. In addition, seawater containing SAV shed by Atlantic salmon post-smolts was incubated at different temperatures over 4 weeks. The viability of SAV shed by SAV-infected fish was examined both by in vitro methods and assayed in vivo using recently seawater-transferred post-smolts. The survival of SAV decreased with increasing temperature in all three experiments. Generally, the titre of SAV propagated in cell culture was more stable in culture medium compared to SAV diluted in seawater or shed by infected fish. Cells could be infected with a low titre of SAV, 24 TCID50 L−1 of seawater, whereas a SAV concentration above 448 TCID50 L−1 of seawater was required for successful SAV transmission to the post-smolts in the in vivo assay. SAV3 shed by infected fish at a starting titre of 882 TCID50 L−1 of seawater was completely inactivated after three and four weeks of incubation at 16 °C and 12 °C, respectively, as analysed by the TCID50 in vitro assay. In addition, the TCID50 resulted in a higher sensitivity than RT-qPCR, especially at very low virus titres, probably due to the multiplication of the virus in cells and the possibility of analysing a greater volume of sample in the assay compared to RT-qPCR. The information acquired in the present study may be incorporated into pathogen dispersal models in order to aid understanding and management of SAV outbreaks. Calculation of the risk of transmission would provide new prospects for the control of pancreas disease.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection. acceptedVersion

Salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) and adversely affects salmonid aquaculture in Europe. A better understanding of disease transmission is currently needed in order to manage PD outbreaks. Here, we demonstrate the relationship between viral dose and the outcome of SAV3 infection in Atlantic salmon post-smolts using a bath challenge model. Fish were challenged at 12 °C with 3 different SAV3 doses; 139, 27 and 7 TCID50 L−1 of seawater. A dose of as little as 7 TCID50 L−1 of seawater was able to induce SAV3 infection in the challenged population with a substantial level of variation between replicate tanks and, therefore, likely represents a dose close to the minimum dose required to establish an infection in a population. These data also confirm the highly infectious nature of SAV through horizontal transmission. The outcome of SAV3 infection, evaluated by the prevalence of viraemic fish, SAV3-positive hearts, and the virus shedding rate, was positively correlated to the original SAV3 dose. A maximal shedding rate of 2.4 × 104 TCID50 L−1 of seawater h−1 kg−1 was recorded 10 days post-exposure (dpe) from the highest dose group. The method reported here, for the quantification of infectious SAV3 in seawater, could be useful to monitor PD status or obtain data from SAV3 outbreaks at field locations. This information could be incorporated into pathogen dispersal models to improve risk assessment and to better understand how SAV3 spreads between farms during outbreaks. This information may also provide new insights into the control and mitigation of PD. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0385-2) contains supplementary material, which is available to authorized users.

Background Pancreas disease (PD), caused by salmonid alphavirus (SAV), is an important disease affecting salmonid aquaculture. It has been speculated that Atlantic salmon post-smolts are more prone to infections in the first few weeks following seawater- transfer. After this period of seawater acclimatization, the post-smolts are more robust and better able to resist infection by pathogens. Here we describe how we established a bath immersion (BI) model for SAV subtype 3 (SAV3) in seawater. We also report how this challenge model was used to study the susceptibility of post-smolts to SAV3 infection in two groups of post-smolts two weeks or nine weeks after seawater - transfer. Methods Post-smolts, two weeks (Phase-A) or nine weeks (Phase-B) after seawater- transfer, were infected with SAV3 by BI or intramuscular injection (IM) to evaluate their susceptibility to infection. A RT-qPCR assay targeting the non-structural protein (nsP1) gene was performed to detect SAV3-RNA in blood, heart tissue and electropositive-filtered tank-water. Histopathological changes were examined by light microscope, and the presence of SAV3 antigen in pancreas tissue was confirmed using immuno-histochemistry. Results Virus shedding from the Phase-B fish injected with SAV3 (IM Phase-B) was markedly lower than that from IM Phase-A fish. A lower percentage of viraemia in Phase-B fish compared with Phase-A fish was also observed. Viral RNA in hearts from IM Phase-A fish was higher than in IM Phase-B fish at all sampling points (p