Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

IgM+ and IgT+ B Cell Traffic to the Heart during SAV Infection in Atlantic Salmon.

B cells of teleost fish differentiate in the head kidney, and spleen, and either remain in the lymphatic organs or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites of vaccination and infection and their functional roles at these sites. In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV). In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ (heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the heart in vaccinated fish compared to those that were unvaccinated. Transcriptome analyses performed at 21 days post-challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated salmon appeared to alleviate the disease. Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an important part of the adaptive immune response of Atlantic salmon to SAV.

A time-course study of gene expression and antibody repertoire at early time post vaccination of Atlantic salmon.

The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0-35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.