Identification of housekeeping genes of Candidatus Branchiomonas cysticola associated with epitheliocystis in Atlantic salmon (Salmo salar L.).

Candidatus Branchiomonas cysticola is an intracellular, gram-negative Betaproteobacteria causing epitheliocystis in Atlantic Salmon (Salmo salar L.). The bacterium has not been genetically characterized at the intraspecific level despite its high prevalence among salmon suffering from gill disease in Norwegian aquaculture. DNA from gill samples of Atlantic salmon PCR positive for Cand. B. cysticola and displaying pathological signs of gill disease, was, therefore, extracted and subject to next-generation sequencing (mNGS). Partial sequences of four housekeeping (HK) genes (aceE, lepA, rplB, rpoC) were ultimately identified from the sequenced material. Assays for real-time RT-PCR and fluorescence in-situ hybridization, targeting the newly acquired genes, were simultaneously applied with existing assays targeting the previously characterized 16S rRNA gene. Agreement in both expression and specificity between these putative HK genes and the 16S gene was observed in all instances, indicating that the partial sequences of these HK genes originate from Cand. B. cysticola. The knowledge generated from the present study constitutes a major prerequisite for the future design of novel genotyping schemes for this bacterium.

Correction: Triploid atlantic salmon (Salmo salar L.) post-smolts accumulate prevalence more slowly than diploid salmon following bath challenge with salmonid alphavirus subtype 3.

[This corrects the article DOI: 10.1371/journal.pone.0175468.].

Triploid atlantic salmon (Salmo salar L.) post-smolts accumulate prevalence more slowly than diploid salmon following bath challenge with salmonid alphavirus subtype 3.

Triploid Atlantic salmon (Salmo salar L.) may play an important role in the sustainable expansion of the Norwegian aquaculture industry. Therefore, the susceptibility of triploid salmon to common infections such as salmonid alphavirus (SAV), the causative agent of pancreas disease (PD), requires investigation. In this study, shortly after seawater transfer, diploid and triploid post-smolts were exposed to SAV type 3 (SAV3) using a bath challenge model where the infectious dose was 48 TCID50 ml-1 of tank water. Copy number analysis of SAV3 RNA in heart tissue showed that there was no difference in viral loads between the diploids and triploids. Prevalence reached 100% by the end of the 35-day experimental period in both infected groups. However, prevalence accumulated more slowly in the triploid group reaching 19% and 56% at 14 and 21 days post exposure (dpe) respectively. Whereas prevalence in the diploid group was 82% and 100% at the same time points indicating some differences between diploid and triploid fish. Both heart and pancreas from infected groups at 14 dpe showed typical histopathological changes associated with pancreas disease. Observation of this slower accumulation of prevalence following a natural infection route was possible due to the early sampling points and the exposure to a relatively low dose of virus. The triploid salmon in this study were not more susceptible to SAV3 than diploid salmon indicating that they could be used commercially to reduce the environmental impact of escaped farmed fish interbreeding with wild salmon. This is important information regarding the future use of triploid fish in large scale aquaculture where SAV3 is a financial threat to increased production.

Salmonid alphavirus infection causes skin dysbiosis in Atlantic salmon (Salmo salar L.) post-smolts.

Interactions among host, microbiota and viral pathogens are complex and poorly understood. The goal of the present study is to assess the changes in the skin microbial community of Atlantic salmon (Salmo salar L.) in response to experimental infection with salmonid alphavirus (SAV). The salmon skin microbial community was determined using 16S rDNA pyrosequencing in five different experimental groups: control, 7 days after infection with low-dose SAV, 14 days after infection with low-dose SAV, 7 days after infection with high-dose SAV, and 14 days after infection with high-dose SAV. Both infection treatment and time after infection were strong predictors of the skin microbial community composition. Skin samples from SAV3 infected fish showed an unbalanced microbiota characterized by a decreased abundance of Proteobacteria such as Oleispira sp. and increased abundances of opportunistic taxa including Flavobacteriaceae, Streptococcaceae and Tenacibaculum sp. These results demonstrate that viral infections can result in skin dysbiosis likely rendering the host more susceptible to secondary bacterial infections.

Relationship between viral dose and outcome of infection in Atlantic salmon, Salmo salar L., post-smolts bath-challenged with salmonid alphavirus subtype 3.

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.