Immune Response In Atlantic Salmon Research Articles

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Salinity change evokes stress and immune responses in Atlantic salmon with microalgae showing limited potential for dietary mitigation.

Smoltification was found to impact both immune and stress responses of farmed Atlantic salmon (Salmo salar), but little is known about how salinity change affects salmon months after completed smoltification. Here, we examined (1) the effect of salinity change from brackish water to seawater on the stress and immune responses in Atlantic salmon and (2) evaluated if functional diets enriched with microalgae can mitigate stress- and immune-related changes. Groups of Atlantic salmon were fed for 8weeks with different microalgae-enriched diets in brackish water and were then transferred into seawater. Samples of the head kidney, gill, liver and plasma were taken before seawater transfer (SWT), 20h after SWT, and 2weeks after SWT for gene-expression analysis, plasma biochemistry and protein quantification. The salmon showed full osmoregulatory ability upon transfer to seawater reflected by high nkaα1b levels in the gill and tight plasma ion regulation. In the gill, one-third of 44 investigated genes were reduced at either 20h or 2weeks in seawater, including genes involved in cytokine signaling (il1b) and antiviral defense (isg15, rsad2, ifit5). In contrast, an acute response after 20h in SW was apparent in the head kidney reflected by increased plasma stress indicators and induced expression of genes involved in acute-phase response (drtp1), antimicrobial defense (camp) and stress response (hspa5). However, after 2weeks in seawater, the expression of antiviral genes (isg15, rsad2, znfx1) was reduced in the head kidney. Few genes (camp, clra, c1ql2) in the gill were downregulated by a diet with 8% inclusion of Athrospira platensis. The results of the present study indicate that salinity change months after smoltification evokes molecular stress- and immune responses in Atlantic salmon. However, microalgae-enriched functional diets seem to have only limited potential to mitigate the related changes.

Immunomodulatory effects of hydrolyzed Debaryomyces hansenii in Atlantic salmon (Salmo salar L): From the in vitro model to a natural pathogen challenge after seawater transfer

Non-Saccharomyces yeast such as Debaryomyces hansenii or its cell wall-components are emerging candidates for novel functional aquafeeds, as they contain several microbe-associated molecular patterns (MAMPs) that can activate and modulate the host's physiological responses, thereby improving fish health and welfare. In this study, we combined in vitro work using SHK-1 cell line and primary cultures of head kidney leukocytes (HKL) from Atlantic salmon (HKL), with an in vivo trial in which two groups of Atlantic salmon were fed 0.1% hydrolyzed D. hansenii (LAN6) in freshwater (7 weeks) and seawater (6 weeks). In vitro results by ELISA showed that after induction with LAN6, SHK-1 cells increased the levels of TNFα and iNOS (at 6 h post-induction), while HKL increased TNFα (6 h post-induction) and IL-10 (24 h post-induction), and reduced TNFα levels at 24 h post-induction. In addition, during an early natural pathogen challenge in the seawater stage (week 5), an increase in specific plasma immunoglobulins against Moritella viscosa, along with an upregulation in pathways associated with humoral immunity and complement activation in the liver was detected in vaccinated fish fed LAN6 (Group A). These data suggest that LAN6 was able to modulate the immune response of Atlantic salmon making it a promising functional feed additive for aquafeeds to achieve a more resilient farmed salmon.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Transcriptomic and targeted immune transcript analyses confirm localized skin immune responses in Atlantic salmon towards the salmon louse.

Atlantic salmon (Salmo salar) are highly susceptible to infestations with the ectoparasite Lepeophtheirus salmonis, the salmon louse. Infestations elicit an immune response in the fish, but the response does not lead to parasite clearance, nor does it protect against subsequent infestations. It is, however, not known why the immune response is not adequate, possibly because the local response directly underneath the louse has been poorly evaluated. The present study describes the transcriptomic response by RNA sequencing of skin at the site of copepodid attachment. Analysing differentially expressed genes, 2864 were higher and 1357 were lower expressed at the louse attachment site compared to uninfested sites in the louse infested fish, while gene expression at uninfested sites were similar to uninfested control fish. The transcriptional patterns of selected immune genes were further detailed in three skin compartments/types: Whole skin, scales only and fin tissue. The elevation of pro-inflammatory cytokines and immune cell marker transcripts observed in whole skin and scale samples were not induced in fin, and a higher cytokine transcript level in scale samples suggest it can be used as a nonlethal sampling method to enhance selective breeding trials. Furthermore, the immune response was followed in both skin and anterior kidney as the infestation developed. Here, newly moulted preadult 1 stage lice induced a higher immune response than chalimi and adult lice. Overall, infestation with salmon louse induce a modest but early immune response with an elevation of mainly innate immune transcripts, with the response primarily localized to the site of attachment.

Basal and immune-responsive transcript expression of two Atlantic salmon interferon regulatory factor 2 (irf2) paralogues.

Atlantic salmon (Salmo salar) is one of the most economically important aquaculture species globally. However, disease has become a prevalent threat to this industry. A thorough understanding of the genes and molecular pathways involved in the immune responses of Atlantic salmon is imperative for selective breeding of disease-resistant broodstock, as well as developing new diets and vaccines to mitigate the impact of disease. Members of the interferon regulatory factor (IRF) family of transcription factors play roles in the induction of interferons and other cytokines involved in host immune responses to intracellular and parasitic pathogens. IRF family members also play diverse roles in other biological processes, such as stress response, reproduction and development. The current study focused on one member of the IRF family: interferon regulatory factor 2 (irf2). As previously shown, due to the genome duplication that occurred ∼80 million years ago in the salmonid lineage, there are two irf2 paralogues in the Atlantic salmon genome. In silico analyses at the cDNA and deduced amino acid levels were conducted followed by phylogenetic tree construction with IRF2 amino acid sequences from various ray-finned fishes, cartilaginous fish and tetrapods. qPCR was then used to analyze paralogue-specific irf2 constitutive expression across 17 adult tissues, as well as responses to the viral mimic pIC (i.e., synthetic double-stranded RNA analog) in cultured macrophage-like cells (in vitro) and to infection with the Gram-negative bacterium Moritella viscosa in skin samples (in vivo). The qPCR studies showed sex- and paralogue-specific differences in expression across tissues. For example, expression of both paralogues was higher in ovary than in testes; expression (considering both sexes together) was highest for irf2-1 in gonad and for irf2-2 in hindgut. Both irf2 paralogues were responsive to pIC stimulation, but varied in their induction level, with irf2-1 having an overall stronger response than irf2-2. Only one paralogue, irf2-2, was significantly responsive to M. viscosa infection. Differences in irf2-1 and irf2-2 transcript expression levels constitutively across tissues, and in response to pIC and M. viscosa, may suggest neo- or subfunctionalization of the duplicated genes. This novel information expands current knowledge and provides insight into how genome duplication events may impact host regulation of important immune markers.

Microencapsulation of Piscirickettsia salmonis Antigens for Fish Oral Immunization: Optimization and Stability Studies.

The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 μm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering protein release. 62.42% of the protein content was delivered at the intestinal level after in vitro digestion. Finally, micro-CT-scanning images confirmed microparticle incorporation in fish feed pellets. In conclusion, the AAJ system is effective at encapsulating P. salmonis antigens in alginate with a high EE% and a size small enough to be incorporated in fish feed and produce an oral vaccine.

Steroidogenic and innate immune responses in Atlantic salmon are influenced by dietary total lipid, long chain polyunsaturated fatty acids and dissolved oxygen

Recent studies have found that feeding high levels of long chain polyunsaturated fatty acids (LC-PUFA) influenced the steroid biosynthesis pathway of Atlantic salmon on a transcriptomic level, although research observed on a metabolic level is lacking. Dietary lipid and chronic stress have also been suggested to play a role in steroidogenesis. A study was performed on Atlantic salmon post-smolts (184 g) fed with diets of high and low levels of lipid with and without high levels of LC-PUFA under hypoxic (stressed) or normoxic (unstressed) conditions. Liver, head kidney and blood samples were collected after 35 and 116 days to determine short- and long-term effects. On day 35, dissolved oxygen affected the levels of the steroid hormones 11-deoxycortisol, cortisone and testosterone as well as the relative expression of cyp11a involved in steroidogenesis and ud2a2 involved in sex steroid metabolism. On day 116, an interaction between lipid x LC-PUFA x oxygen was found for cyp11a, hsd3b (steroidogenesis) and ud2a2 genes, whereas LC-PUFA and/or oxygen affected the steroid levels of 11-dehydrocorticosterone, corticosterone, cortisol, dehydroepiandrosterone, pregnenolone and testosterone. The observation that both LC-PUFA and oxygen affected cyp11a expression and the levels of pregnenolone, provides evidence for the influence of both parameters on the rate-limiting point of steroid production, which can take >35 days to establish. The 3-way interaction between lipid, LC-PUFA and dissolved oxygen on the expression of steroid genes suggests that the chronic stressor of hypoxia may be over-riding dietary influences of the lipid and LC-PUFA, though the relative proportion of LC-PUFA within the lipid may play a role as well. In terms of fatty acid composition, low dietary lipid and high LC-PUFA increased the levels of total n-6 and n-3 PUFA in the liver, particularly EPA and DHA, and the head kidney to a lesser extent. In terms of immune pathways, hypoxia influenced cytokine gene expression (tgfb, il10), cellular repair gene expression (gr, hsp70 and hsp90) and eicosanoid levels (PGE2 and LTB5) in the head kidney, while dietary lipid and LC-PUFA influenced cytokines (ifnγ and il1β) and eicosanoids (PGE2 and LTB5). These findings demonstrate that feeding different levels of LC-PUFA significantly alters steroidogenesis and innate immune response in Atlantic salmon post-smolts with additional interactions from dietary lipid and hypoxia stress. These results will significantly impact the aquaculture industry since it demonstrates salmon fed high levels of LC-PUFA are likely more resilient to cope with chronic stressors (e.g. prolonged hypoxia) due to lower corticosteroid levels and upregulated immune response.

Dietary Inclusion of Hydrolyzed Debaryomyces hansenii Yeasts Modulates Physiological Responses in Plasma and Immune Organs of Atlantic Salmon (Salmo salar) Parr Exposed to Acute Hypoxia Stress.

Stress related to salmon aquaculture practices (handling, sub-optimal nutrition, diseases, and environmental problems) may compromise fish welfare. This study describes the effects of two hydrolyzed Debaryomyces hansenii yeast-based products (LAN4 and LAN6) on physiological and immune responses of Atlantic salmon (Salmo salar) parr exposed to short hypoxia stress. A commercial-like diet (control diet: CD) and two experimental diets (CD supplemented with 0.1% of either component LAN4 or LAN6) were fed to fish for 8 weeks. At the end of the feeding experiment, fish were exposed to 1-min hypoxia and samples were collected at 0, 1, 3, 6, 12, and 24 h post-stress. Results showed that plasma cortisol reached a peak at 1 h post-stress in CD and LAN6 groups, whereas no significant increase in cortisol levels was detected in the LAN4 group. Moreover, the LAN6 group enhanced IL-10 responses to hypoxia, when compared to the control and LAN4 group. This suggests a regulation of immunosuppressive profiles in fish fed LAN4. Hypoxia stress increased TNFα in all groups, which indicates that fish may compensate for the short-term stress response, by modulating innate immune molecules. The apparent suppression of hypoxia responses in the LAN4 group coincided with the detection of differences in goblet cells size and Muc-like proteins production in DI; and upregulation (1 h post-stress) of pathways related to oxygen transport, hemoglobin complex, and glutathione transferase activity and the downregulation of fatty acid metabolism (6 h post-stress) in gills. To conclude, a 1-min hypoxia stress exposure affects the response to stress and immunity; and D. hansenii-based yeast products are promising components in functional aquafeeds for salmon due to their ability to counteract possible consequences of hypoxic stress.

Genome-wide integrated analysis reveals functions of lncRNA-miRNA-mRNA interactions in Atlantic salmon challenged by Aeromonas salmonicida

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 108 CFU/mL), low-dose A. salmonicida infection (3.06 × 105 CFU/mL), and a PBS (100 μL) control. We identified 65 differentially expressed lncRNAs, 41 miRNAs, and 512 mRNAs between the control group and infection groups. Functional analysis showed that these genes were significantly enriched in the p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, and Toll-like receptor signaling pathway. In addition, we predicted key genes in immune-related pathways and constructed a lncRNA-miRNA-mRNA network based on whole transcriptomic analysis. We further predicted three lncRNA-miRNA-mRNA axes as potential novel biomarkers in regulating the immune response of Atlantic salmon against A. salmonicida infection.

Chitosan nanoparticle formulation attenuates poly (I:C) induced innate immune responses against inactivated virus vaccine in Atlantic salmon (Salmo salar)

Many vaccine formulations, in particular vaccines based on inactivated virus, needs adjuvants to boost immunogenicity. In aquaculture, mineral and plant oil are used as adjuvant in commercial vaccines, and the advent of oil-adjuvanted vaccines was crucial to aquaculture development. Nevertheless, some of these approved vaccines display suboptimal performance in the field compared to experimental conditions. Therefore, there is a need to improve adjuvants and delivery methods for fish vaccines against viruses. We used RNA sequencing of Atlantic salmon head kidney to analyse the difference in gene expression 24 h after injection of different experimental vaccine formulations. We compared five different formulations in addition to a PBS control: inactivated virus alone (group V), soluble poly (I:C) (group P), nanoparticles containing poly (I:C) (group N), soluble poly (I:C) + inactivated virus (group PV) and finally nanoparticles containing poly (I:C) + inactivated virus (group NV). Our results showed poly (I:C)’s ability as adjuvant and its capacity influence innate immune genes expression in Atlantic salmon. Soluble poly (I:C) upregulated multiple immune related genes and was more effective compared to poly (I:C) formulated into chitosan nanoparticles (more than 10 fold increase in differentially expressed genes, DEGs). However, inclusion of inactivated ISA virus in the nanoparticle vaccine, increased the number of DEGs fivefold suggesting a synergistic effect of adjuvant and antigen. Our results indicate that the way poly (I:C) is formulated and the presence of antigen is important for the magnitude of the innate immune response in Atlantic salmon.

Dietary Inclusion of Black Soldier Fly (Hermetia Illucens) Larvae Meal and Paste Improved Gut Health but Had Minor Effects on Skin Mucus Proteome and Immune Response in Atlantic Salmon (Salmo Salar).

The present study investigated effects of dietary inclusion of black soldier fly larvae (BSFL) (Hermetia illucens) meal and paste on gut health, plasma biochemical parameters, immune response and skin mucus proteome in pre-smolt Atlantic salmon (Salmo salar). The seven-week experiment consisted of seven experimental diets: a control diet based on fishmeal and plant protein (Control-1); three BSFL meal diets, substituting 6.25% (6.25IM), 12.5% (12.5IM) and 25% (25IM) of protein; two BSFL paste diets, substituting 3.7% (3.7IP) and 6.7% (6.7IP) of protein and an extra control diet with 0.88% of formic acid (Control-2). The 6.25IM diet reduced enterocyte steatosis in pyloric caeca, improved distal intestine histology, and reduced IgM in distal intestine. The fish fed 12.5IM diet reduced enterocyte steatosis in pyloric caeca, improved distal intestine histology, had a higher plasma lysozyme content compared to 6.25IM, and tend to increase phagocytic activity in head-kidney macrophages-like cells. On the other hand, 25IM diet improved distal intestine histology, but showed mild-moderate enterocyte steatosis in pyloric caeca, increased IFNγ and reduced IgM in distal intestine. In the case of BSFL paste diets, 3.7IP diet caused mild inflammatory changes in distal intestine, although it reduced enterocyte steatosis in pyloric caeca. The 6.7IP diet reduced enterocyte steatosis in pyloric caeca and improved distal intestine histology. Increasing level of BSFL meal in the diet linearly decreased plasma C-reactive protein, whereas increasing level of BSFL paste linearly increased plasma antioxidant capacity. Dietary inclusion of BSFL meal and paste had minor effects on the expression profile of proteins in skin mucus and no effects on immune markers in splenocytes. BSFL meal showed no negative effect on liver and muscle health as indicated by plasma alanine aminotranseferase, asparate aminotransferase and creatine kinase. The present study showed that replacing conventional protein sources with low to moderate levels of BSFL meal (6.25% and 12.5%) or paste (3.7% and 6.7%) reduced enterocyte steatosis in pyloric caeca, while replacing up to 25% with BSFL meal or 6.7% with paste improved distal intestine histology. Further, dietary inclusion of BSFL meal and paste had minor effects on skin mucus proteome and immune response in Atlantic salmon.

Transcriptomic Profiling of the Adaptive and Innate Immune Responses of Atlantic Salmon to Renibacterium salmoninarum Infection.

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Impact of a candidate vaccine on the dynamics of salmon lice (Lepeophtheirus salmonis) infestation and immune response in Atlantic salmon (Salmo salar L.).

Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. To address this, vaccination is a cost-effective and environmentally friendly control approach. In this study, efficacy of a vaccine candidate, containing a peptide derived from ribosomal protein P0, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the vaccine candidate when administered intraperitoneally in the host, in reducing the ectoparasite load, through reduction of adult female lice counts and fecundity and with greater presumptive effect in F1 lice generation. The sampling results correlated well with the differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level at different lice stages. Overall, the results supports approximately 56% efficacy when administered by intraperitoneal injection. However, additional validation is necessary under large-scale laboratory trial for further application under field conditions.

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.

Assessing the Immune Response of Atlantic Salmon (Salmo salar) after the Oral Intake of Alginate-Encapsulated Piscirickettsia salmonis Antigens.

Salmon rickettsial septicaemia (SRS) is the infectious disease that produces the highest losses in the Chilean salmon industry. As a new strategy for the control of SRS outbreaks, in this study we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in the feed as an oral vaccine to induce the immune response in Atlantic salmon (Salmo salar). Fish were distributed into three vaccination groups (injectable, oral high dose, oral low dose). Feed intake and fish growth were recorded during the trial. The P. salmonis-specific IgM levels in blood plasma were measured by ELISA. Alginate microparticles containing the antigen were effectively incorporated in fish feed to produce the oral vaccine. Incorporation of AEPSA did not affect the palatability of the feed or the fish appetite. Furthermore, the oral vaccine did not have a negative effect on fish growth. Finally, the oral vaccine (high and low dose) produced an acquired immune response (IgM) similar to the injectable vaccine, generating a statistically significant increase in the IgM levels at 840-degree days for both experimental groups. These findings suggest that AEPSA incorporated in the feed can be an effective alternative to boost the immune response in Atlantic salmon (S. salar).

Down-stream processing of baker's yeast (Saccharomyces cerevisiae) – Effect on nutrient digestibility and immune response in Atlantic salmon (Salmo salar)

The increased demand for high-quality feed ingredients for the aquaculture industry has led to an increased focus on microbial ingredients as nutrient sources. Limited information exists, however, on the effect of down-stream processing of yeast on nutrient digestibility and health in Atlantic salmon (Salmo salar). In the present study, three laboratory-scale down-stream processing methods were used: direct inactivation by spray-drying, autolysis, or cell crushing. All yeast was inactivated with sray-drying post treatment. The yeast products were evaluated for their nutritional value and health effects in diets for Atlantic salmon. The experimental diets consisted of a high-quality fishmeal-based reference diet (FM) and six experimental diets were 300 g kg−1 of each of the processed yeast candidates were added to the FM control mixture. Fish were fed the diets for 21 days, and at the end of the experiment, feces were collected, and spleen, head kidney, distal intestine (DI) and plasma were sampled for assessment of immune response.Processing of yeast resulted in increased level of soluble protein in the yeast cream, with the highest level for yeast disrupted by cell crushing (20 K PSI). This was confirmed with scanning electron microscopy, where disrupted and cracked cells could be observed. Significant lower protein digestibility of the directly inactivated yeast coincided with the increased detection of whole yeast cells by flow cytometry in the DI content of fish fed the directly inactivated yeast. Autolysis for 16 h resulted in the highest protein digestibility, which was similar to the FM control.Interestingly, 16 h autolyzed yeast induced the secretion of IL-8, while cell crushed yeast induced the secretion of TNFα in the DI as analyzed by ELISA. To conclude, different down-stream processing of S. cerevisiae led to increased protein and β-glucan solubility that further increased protein digestibility in Atlantic salmon. Furthermore, different processing of the yeast triggered different immune stimulatory effects in Atlantic salmon.

Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon (Salmo salar).

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes (cc motif chemokine 19 precursor [ccl19], integrin β2 (itβ2, also named cd18), glutathione transferase omega-1 [gsto-1], heat shock 70 KDa protein [hsp70], major histocompatibility complex class I [mhc-I]) were analyzed to identify SNPs by data mining. These genes were chosen based on their previously reported expression on infectious pancreatic necrosis virus (IPNV)-infected Atlantic salmon phenotype. The available EST sequences for these genes were obtained from the Unigene database. Twenty-eight SNPs were found in the genes evaluated and identified most of them as transition base changes. The effect of the SNPs located on the 5’-untranslated region (UTR) or 3’-UTR upon transcription factor binding sites and alternative splicing regulatory motifs was assessed and ranked with a low-medium predicted FASTSNP score risk. Synonymous SNPs were found on itβ2 (c.2275G > A), gsto-1 (c.558G > A), and hsp70 (c.1950C > T) with low FASTSNP predicted score risk. The difference in the relative synonymous codon usage (RSCU) value between the variant codons and the wild-type codon (ΔRSCU) showed one negative (hsp70 c.1950C > T) and two positive ΔRSCU values (itβ2 c.2275G > A; gsto-1 c.558G > A), suggesting that these synonymous SNPs (sSNPs) may be associated to differences in the local rate of elongation. Nonsynonymous SNPs (nsSNPs) in the gsto-1 translatable gene region were ranked, using SIFT and POLYPHEN web-tools, with the second highest (c.205A > G; c484T > C) and the highest (c.499T > C; c.769A > C) predicted score risk possible. Using homology modeling to predict the effect of these nonsynonymous SNPs, the most relevant nucleotide changes for gsto-1 were observed for the nsSNPs c.205A > G, c484T > C, and c.769A > C. Molecular dynamics was assessed to analyze if these GSTO-1 variants have significant differences in their conformational dynamics, suggesting these SNPs could have allosteric effects modulating its catalysis. Altogether, these results suggest that candidate SNPs identified may play a crucial potential role in the immune response of Atlantic salmon.

Impact of co-infection with Lepeophtheirus salmonis and Moritella viscosa on inflammatory and immune responses of Atlantic salmon (Salmo salar).

This study was conducted to determine the effects of a co-infection with Moritella viscosa at different exposure levels of sea lice Lepeophtheirus salmonis in Atlantic salmon (Salmo salar). M. viscosa (1.14×106 cfu/ml) was introduced to all experimental tanks at 10days post-lice infection (dpLs). Mean lice counts decreased over time in both the medium lice co-infection (31.5±19.0 at 7dpLs; 16.9±9.3 at 46dpLs) and high lice co-infection (62.0±10.8 at 7dpLs; 37.6±11.3 at 46dpLs). There were significantly higher mortalities and more severe skin lesions in the high lice co-infected group compared to medium lice co-infected group or M. viscosa-only infection. Quantitative gene expression analysis detected a significant upregulation of genes in skin from the high lice co-infection group consistent with severe inflammation (il-8, mmp-9, hep, saa). Skin lesions retrieved throughout the study were positive for M. viscosa growth, but these were rarely located in regions associated with lice. These results suggest that while M. viscosa infection itself may induce skin lesion development in salmon, co-infection with high numbers of lice can enhance this impact and significantly reduce the ability of these lesions to resolve, resulting in increased mortality.

Induced‑iron overdose modulate the immune response in Atlantic salmon increasing the susceptibility to Piscirickettsia salmonis infection

Although iron has been proved as a vital element for the development of living organisms, high levels of this element can impact the normal function of different biological processes, including immune response. Therein, this study aimed to characterize the transcriptional changes in Atlantic salmon during the exposure to iron overload, evaluating the susceptibility to bacterial infection. Atlantic salmon were injected with 1 or 5 mg of iron dextran and an iron chelator (DFOM), and subsequently challenged with the intracellular bacterium Piscirickettsia salmonis. Iron levels in plasma and the transcriptional responses of the head kidney, liver, and spleen during the infection processes were evaluated. Here, iron transport, response to oxidative stress, and immune related genes were strongly modulated in individuals exposed to the metal. Fish overloaded with iron showed significant downregulation of CD83, IL-17, IL-1, Toll-like receptors, immunoglobulin, and T-cell receptors genes, suggesting an overall deleterious effect on the immune system. Furthermore, histopathological analysis evidenced that iron overdoses can strongly increase the clinical signs during the P. salmonis infection. Notably, fish exposed to 1 mg of iron and infected with P. salmonis evidenced high mortality and bacterial burden. Also, this group showed significant downregulation of immune-related genes comparing with fish with 5 mg of iron. In addition, Atlantic salmon injected with deferoxamine mesylate salt (DFOM) showed high tolerance to P. salmonis infection and an increase of IL-1b expression, suggesting a cytokines response activation. These results suggest that iron overload disrupts fish immune response although not in a doses dependent manner, and when a chelator is injected, fish present higher resistance to P. salmonis. This study reveals novel insights about teleost's immune system modulation by iron availability.