Lindsey Moore

Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue

In modern bony fishes, or teleost fish, the general lack of leucocyte markers has greatly hampered investigations of the anatomy of the immune system and its reactions involved in inflammatory responses. We have previously reported the cloning and sequencing of the salmon CD3 complex, molecules that are specifically expressed in T cells. Here, we generate and validate sera recognizing a peptide sequence of the CD3ε chain. Flow cytometry analysis revealed high numbers of CD3ε+ or T cells in the thymus, gill and intestine, whereas lower numbers were detected in the head kidney, spleen and peripheral blood leucocytes. Subsequent morphological analysis showed accumulations of T cells in the thymus and spleen and in the newly discovered gill‐located interbranchial lymphoid tissue. In the latter, the T cells are embedded in a meshwork of epithelial cells and in the spleen, they cluster in the white pulp surrounding ellipsoids. The anatomical organization of the salmonid thymic cortex and medulla seems to be composed of three layers consisting of a sub‐epithelial medulla‐like zone, an intermediate cortex‐like zone and finally another cortex‐like basal zone. Our study in the salmonid thymus reports a previously non‐described tissue organization. In the intestinal tract, abundant T cells were found embedded in the epithelium. In non‐lymphoid organs, the presence of T cells was limited. The results show that the interbranchial lymphoid tissue is quantitatively a very important site of T cell aggregation, strategically located to facilitate antigen encounter. The interbranchial lymphoid tissue has no resemblance to previously described lymphoid tissues.

CD4 homologues in Atlantic salmon.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.

Atlantic salmon (Salmo salar L.) post-smolts challenged two or nine weeks after seawater-transfer show differences in their susceptibility to salmonid alphavirus subtype 3 (SAV3)

BackgroundPancreas 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.MethodsPost-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.ResultsVirus 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 < 0.05) and a similar trend was also seen in the BI groups. Necrosis of exocrine pancreatic cells was observed in all infected groups. Extensive histopathological changes were found in Phase-A fish whereas milder PD-related histopathological lesions were seen in Phase-B fish. The presence of SAV3 in pancreas tissue from all infected groups was also confirmed by immuno-histochemical staining.ConclusionOur results suggest that post-smolts are more susceptible to SAV3 infection two weeks after seawater-transfer than nine weeks after transfer. In addition, the BI challenge model described here offers an alternative SAV3 infection model when better control of the time-of-infection is essential for studying basic immunological mechanisms and disease progression.

Triploid atlantic salmon (Salmo salar L.) postsmolts 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.

Immune gene profiles in Atlantic salmon (salmo salar L.) post-smolts infected with SAV3 by bath-challenge show a delayed response and lower levels of gene transcription compared to injected fish

ABSTRACT Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post‐smolts were infected by either i.m.‐injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.‐injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT‐qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN‐stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.‐injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time‐points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m‐injected fish at later time‐points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups. HighlightsThe route of SAV3 infection affects the innate response in Atlantic salmon post‐smolts recently transferred to seawater.SAV3 bath immersion challenge induces lower and more sustained innate immune response compared to injection challenge.Recently smoltified Atlantic salmon have a poor interferon response to salmonid alphavirus.

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.

Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge

ABSTRACT Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra‐muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM‐B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI‐B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM‐B and BI‐B groups. Transcription of the analysed genes peaked at 7 dpi in the IM‐B group and at 14 dpi in the BI‐B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI‐B group showed higher transcription of IL‐1&bgr;, IFN&ggr; and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM‐B group. Histopathological changes in the heart were restricted to the IM‐B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non‐infected control fish in Phase B (CT‐B) was also significantly different when compared to Phase A control fish (CT‐A). HIGHLIGHTSSalmon adapted to seawater for a longer time have a higher and longer interferon response to SAV.Immune response after bath challenge follows that for a i.m. challenge in fish transferred to seawater 9 weeks earlier.Non‐infected control fish have higher basal levels of several immune genes when adapted for a longer time in seawater.After 9 weeks in seawater salmon can maintain a good immune response for a longer period.

Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus

Abstract 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. HighlightsNeutralizing abs to SAV were detected only in salmon adapted for longer to seawater.Salmon adapted for longer to SW develop stronger cell‐mediated immunity to SAV.Upon shorter adaptation to SW, salmon show IL‐10 upregulation at early time points.SOCS1 gene was upregulated in all infected groups in both phases.Upon shorter adaptation to SW, SOCS1 upregulation was maintained for longer time.