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.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Transcriptional Signatures of Immune, Neural, and Endocrine Functions in the Brain and Kidney of Rainbow Trout (Oncorhynchus mykiss) in Response to Aeromonas salmonicida Infection.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with "sickness behaviors" and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the "sickness behaviors" of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

CXC chemokines and their receptors in black rockfish (Sebastes schlegelii): Characterization, evolution analyses, and expression pattern after Aeromonas salmonicida infection.

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, seventeen CXC chemokine ligands (SsCXCLs) and nine CXC chemokine receptors (SsCXCRs) were systematically identified from Sebastes schlegelii genome. Phylogeny, synteny, and evolutionary analyses were performed to annotate these genes, indicating that the tandem duplications (CXCL8, CXCL11, CXCL32, CXCR2, and CXCR3), the whole genome duplications (CXCL8, CXCL12, CXCL18, and CXCR4), and the teleost-specific members (CXCL18, CXCL19, and CXCL32) led to the expansion of SsCXCLs and SsCXCRs. In addition, SsCXCLs and SsCXCRs were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in head kidney, liver, gill and spleen. Moreover, most SsCXCLs and SsCXCRs were significantly differentially expressed in head kidney, liver, and gill after Aeromonas salmonicida infection, and exhibited tissue-specific and time-dependent manner. Finally, protein-protein interaction network (PPI) analysis indicated that SsCXCLs and SsCXCRs interacted with a few immune-related genes such as interleukins, cathepsins, CD genes, and TLRs, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokines and receptors in teleost.

GAS1: A New ß-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

ß-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two ß-glucan types extracted from wild-type baker's yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-ß-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that ß-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each ß-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard ß-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT ß-glucan, at D36, WT-ß-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-ß-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of ß-glucans (p<0.05). In conclusion, our results suggest that Gas1-ß-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other ß-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

In vitro evaluation of novel (nanoparticle) oral delivery systems allow selection of gut immunomodulatory formulations.

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo, in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further.

Identification and characterization of caspases genes in rainbow trout (Oncorhynchus mykiss) and their expression profiles after Aeromonas salmonicida and Vibrio anguillarum infection.

Caspases are highly conserved cysteine-dependent aspartyl-specific proteases that play an important role in regulating cell death and inflammation. However, the caspase genes have not been systematically studied in rainbow trout (Oncorhynchus mykiss). Rainbow trout experienced 4 rounds (4R) of genome duplication in the evolutionary history. Thereby an increased numbers of paralogs are observed in trout, probably with more complicated gene functions. We identified 18 caspase genes in rainbow trout, including two inflammatory caspases (casp1a, casp1b), six apoptosis executioner caspases (casp3, casp3a1, casp3a2, casp3b, casp6, and casp7), nine apoptosis initiator caspases (casp2a, casp2b, casp8, casp9a, casp9b, casp10a, casp10b, casp20a, and casp20b) and one uncategorized caspase gene (casp17). To investigate the potentially physiological functions of caspase genes, we challenged the rainbow trout with Aeromonas salmonicida (A. salmonicida) and Vibrio anguillarum (V. anguillarum). Results showed that the CASP3-regulated intrinsic apoptosis was activated after A. salmonicida infection, while the CASP8 and CASP6-regulated extrinsic apoptosis exerted the greatest effect on trout challenged with V. anguillarum. In response to V. anguillarum infection, the data of RNA-Seq further showed the casp8 was tightly integrated with the significantly enriched Gene Ontology terms and functional pathways, including apoptosis regulation, pathogen detection and immunomodulation. Our study provides a foundation for the physiological functions and regulatory network of the caspase genes in teleosts.

Early-life infection with a bacterial pathogen increases expression levels of innate immunity related genes during adulthood in zebrafish.

Early-life exposure to different stressors can lead to various consequences on fish health status in later life development. To evaluate the effects of Aeromonas salmonicida achromogenes infection in the early-life on immunity in adulthood, zebrafish were either early-infected at 18 days post-fertilization (dpf), chronically infected from 18 to 35 dpf, or late infected at 35 dpf and then grown up to 61 dpf to be re-infected with the pathogen. The age of first infection was shown to influence both, level and timing of the immune gene expressions, especially for inflammation-related genes. In addition, evidence for an innate immune memory in zebrafish primarily infected with the pathogen at 35 dpf and re-infected at 61dpf provide new insights to consolidate the concept of a "trained" innate immunity in fish.

Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis.

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103  CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A-layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin-killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post-vaccination, respectively, in challenge test.

Response of triploid Atlantic salmon (Salmo salar) to commercial vaccines.

While triploid Atlantic salmon represent a practical and affordable solution to the issues associated with sexual maturation in the salmonid aquaculture industry, empirical evidence suggests triploids are more susceptible to disease and vaccine side-effects than diploids. With vaccination now part of routine husbandry, it is essential their response be studied to confirm their suitability for commercial production. This study tested the response of triploid and diploid Atlantic salmon to vaccination with commercially available vaccines. Triploid and diploid Atlantic salmon siblings were injected with one of three commercial vaccines (or sham-vaccinated) and monitored for performance throughout a commercial production cycle. Sampling at smolt and harvest was undertaken along with individual weight and length assessments through the cycle. Antibody response to Aeromonas salmonicida vaccination was similar in both ploidy, with a positive response in vaccine-injected fish. For both adhesions and melanin, analysis found that higher scores were more likely to occur as the anticipated severity of the vaccine increased. In addition, for adhesion scores at smolt and melanin scores at smolt and harvest, triploids were statistically more likely to exhibit high scores than diploids. Triploids maintained a significantly higher body weight during freshwater and until 11 months post-seawater transfer, with diploids weighing significantly more at harvest. Growth, represented by thermal growth coefficient (TGC), decreased in both ploidy as the severity of adhesions increased, and regression patterns did not differ significantly between ploidy. Vertebral deformity prevalence was consistently higher in triploids (smolt 12.3 ± 4.5%; harvest 34.9 ± 5.9%) than diploids (smolt 0.8 ± 0.5%; harvest 15.9 ± 1.9%), with no significant difference between vaccine groups in each ploidy. This study demonstrates that triploids respond as well to vaccination as diploids and provides further supporting evidence of triploid robustness for commercial aquaculture.

Distinct response of immune gene expression in peripheral blood leucocytes modulated by bacterin vaccine candidates in rainbow trout Oncorhynchus mykiss: A potential in vitro screening and batch testing system for vaccine development in aquaculture.

Fish aquaculture is the world's fastest growing food production industry and infectious diseases are a major limiting factor. Vaccination is the most appropriate method for controlling infectious diseases and a key reason for the success of salmonid cultivation and has reduced the use of antibiotics. The development of fish vaccines requires the use of a great number of experimental animals that are challenged with virulent pathogens. In vitro cell culture systems have the potential to replace in vivo pathogen exposure for initial screening and testing of novel vaccine candidates/preparations, and for batch potency and safety tests. PBL contain major immune cells that enable the detection of both innate and adaptive immune responses in vitro. Fish PBL can be easily prepared using a hypotonic method and is the only way to obtain large numbers of immune cells non-lethally. Distinct gene expression profiles of innate and adaptive immunity have been observed between bacterins prepared from different bacterial species, as well as from different strains or culturing conditions of the same bacterial species. Distinct immune pathways are activated by pathogens or vaccines in vivo that can be detected in PBL in vitro. Immune gene expression in PBL after stimulation with vaccine candidates may shed light on the immune pathways involved that lead to vaccine-mediated protection. This study suggests that PBL are a suitable platform for initial screening of vaccine candidates, for evaluation of vaccine-induced immune responses, and a cheap alternative for potency testing to reduce animal use in aquaculture vaccine development.

Interaction between dietary fatty acids and genotype on immune response in Atlantic salmon (Salmo salar) after vaccination: A transcriptome study.

A pivotal matter to aquaculture is the sourcing of sustainable resources as ingredients to aquafeeds. Levels of plant delivered oils as source of fatty acids (FA) in aquafeeds have reached around 70% resulting in reduced levels of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in salmon fillet composition. EPA and DHA can modulate inflammation and immune response, so it is crucial to understand how fish immune response is affected by low LC n-3 PUFA diet and if this diet can have a detrimental effect on vaccine response. Atlantic salmon (Salmo salar) can produce EPA/DHA from α-linolenic acid (ALA) and this endogenous capacity can be explored to develop families with higher tolerance to low LC n-3 PUFA diets. Here we analyze innate and adaptive immune response in Atlantic salmon to a commercial vaccine after being fed low levels of EPA and DHA, and we also compare three strains of salmon selected by their endogenous capacity of synthesizing LC- n-3 PUFA. A total of 2,890 differentially expressed genes (DEGs) were identified (p-value adjusted < 0.1) when comparing vaccinated fish against control non-vaccinated. Gene ontology (GO) and KEGG analysis with 442 up/downregulated genes revealed that most DEGs were both related to immune response as well as part of important immune related pathways, as "Toll-like receptor" and "Cytokine-Cytokine interaction". Adaptive response was also addressed by measuring antigen specific IgM, and titers were significantly higher than in the pre-immune fish at 62 days post-immunization. However, diet and strain had no/little effect on vaccine-specific IgM or innate immune responses. Atlantic salmon therefore display robustness in its response to vaccination even when feed low levels of LC n-3 PUFA.

Aeromonas salmonicida subsp. salmonicida Early Infection and Immune Response of Atlantic Cod (Gadus morhua L.) Primary Macrophages.

In contrast to other teleosts, Atlantic cod (Gadus morhua) has an expanded repertoire of MHC-I and TLR components, but lacks the MHC-II, the invariant chain/CD74, and CD4+ T cell response, essential for production of antibodies and prevention of bacterial infectious diseases. The mechanisms by which G. morhua fight bacterial infections are not well understood. Aeromonas salmonicida subsp. salmonicida is a recurrent pathogen in cultured and wild fish, and has been reported in Atlantic cod. Macrophages are some of the first responders to bacterial infection and the link between innate and adaptive immune response. Here, we evaluated the viability, reactive oxygen species (ROS) production, cell morphology, and gene expression of cod primary macrophages in response to A. salmonicida infection. We found that A. salmonicida infects cod primary macrophages without killing the cod cells. Likewise, infected Atlantic cod macrophages up-regulated key genes involved in the inflammatory response (e.g., IL-1ß and IL-8) and bacterial recognition (e.g., BPI/LBP). Nevertheless, our results showed a down-regulation of genes related to antimicrobial peptide and ROS production, suggesting that A. salmonicida utilizes its virulence mechanisms to control and prevent macrophage anti-bacterial activity. Our results also indicate that Atlantic cod has a basal ROS production in non-infected cells, and this was not increased after contact with A. salmonicida. Transmission electron microscopy results showed that A. salmonicida was able to infect the macrophages in a high number, and release outer membrane vesicles (OMV) during intracellular infection. These results suggest that Atlantic cod macrophage innate immunity is able to detect A. salmonicida and trigger an anti-inflammatory response, however A. salmonicida controls the cell immune response to prevent bacterial clearance, during early infection.

A recombinant adenovirus targeting typical Aeromonas salmonicida induces an antibody-mediated adaptive immune response after immunization of rainbow trout.

Aeromonas salmonicida, the oldest known fish pathogen and currently endemic throughout most of the world in both fresh and marine waters, causes severe economic losses to the salmon farming industry. Although there have been many studies on the prevention of furunculosis over the past few decades, it is still prevalent in many fisheries. In this study, a recombinant adenovirus vaccine candidate harboring the highly immunogenic Vapa gene (pAd-easy-cmv-Vapa) was successfully constructed and tested. The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout. In addition, relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by quantitative reverse transcription PCR. Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein (encoded by Vapa). Further, survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate (40%) compared to that in the control group (76.6%) and empty vector group (73.6%). This also led to an increase in specific antibodies in peripheral serum. In addition, levels of IgM and IgT in the head kidney and hindgut were increased to varying degrees. In conclusion, our research provides a candidate vaccine for the prevention of Aeromonas salmonicida A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies.

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.

Four selenoprotein P genes exist in salmonids: Analysis of their origin and expression following Se supplementation and bacterial infection.

The following research was conducted to elucidate the evolution and expression of salmonid selenoprotein P (SelP), a selenoprotein that is unique in having multiple selenocysteine (Sec) residues, following supranutritional selenium supplementation and infection in rainbow trout. We show that in salmonids SelP is present as four paralogues and that the diversification of SelP genes during vertebrate evolution relates to whole genome duplication events. With 17 and 16 selenocysteine residues for rainbow trout (Oncorhynchus mykiss)/Atlantic salmon (Salmo salar) SelPa1 and SelPa2 proteins respectively and 1 or 2 (trout or salmon) and 4 or 3 (trout or salmon) selenocysteine residues for salmonid SelPb1 and SelPb2 proteins respectively, this is the highest number of (predicted) multiple selenocysteine containing SelP proteins reported for any vertebrate species to date. To investigate the effects of selenium form on SelP expression we added different concentrations (1 nM- 10 µM) of organic or inorganic selenium to a trout cell line (RTG-2 cells) and analysed changes in mRNA abundance. We next studied the impact of supplementation on the potential modulation of these transcripts by PAMPs and proinflammatory cytokines in RTG-2 and RTS-11 cells. These experiments revealed that selenium type influenced the responses, and that SelP gene subfunctionalisation was apparent. To get an insight into the expression patterns in vivo we conducted a feeding trial with 2 diets differing in selenium content and 5 weeks later challenged the trout with a bacterial pathogen (Aeromonas salmonicida). Four tissues were analysed for SelP paralogue expression. The results show a significant induction of SelPa1 in gills and intestine following infection in selenium supplemented fish and for SelPa2 in gills. SelPb1 was significantly reduced in head kidney of both diet groups following infection, whilst SelPb2 was significantly upregulated in skin of both diet groups post infection. Overall these findings reveal differential expression profiles for the SelPa/SelPb paralogues in trout, influenced by selenium supply, cell type/tissue and stimulant. The increase of multiple Sec containing SelP proteins in salmonids could indicate an enhanced requirement for selenium in this lineage.

Induced expression of cathelicidins in trout (Oncorhynchus mykiss) challenged with four different bacterial pathogens.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.

Rainbow trout (Oncorhynchus mykiss) adipose tissue undergoes major changes in immune gene expression following bacterial infection or stimulation with pro-inflammatory molecules.

In mammals, visceral adipose is increasingly seen as playing an important role in immune function with numerous pro-inflammatory, anti-inflammatory and immune-modulating proteins and peptides being identified in adipocytes. Adipose is also now known as a tissue that has an important role in the regulation of peritoneal immune responses. Despite this, only lately has consideration been given to visceral adipose as an important immune tissue in fish, especially in the context of intraperitoneal vaccination. The present study demonstrates that fish visceral adipose is capable of expressing a large range of immune molecules in response to stimulation with a live bacterium (A. salmonicida), a bacterial PAMP (Y. ruckeri flagellin), and the pro-inflammatory cytokines IL-1ß, TNF-α3 and IFN-γ. Following infection and stimulation with flagellin and IL-1ß a large upregulation of pro-inflammatory and antimicrobial molecules was seen, with a high degree of overlap. TNF-α treatment affected relatively few genes and the effects were more modest. IFN-γ had the smallest impact on adipose but IFN-γ inducible genes showed some of the largest effects. Overall, it is clear that adipose tissue should be considered an active immune site in fish, capable of participating in and influencing immune responses.

Vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) at a low temperature leads to a low antibody response against Aeromonas salmonicida.

We present a study on the effect of water temperature on immunization of Atlantic lumpfish. In total, 360 fish were vaccinated with either 50 µl of an oil-based injection vaccine (VAX), with Aeromonas salmonicida and Vibrio salmonicida antigens, or PBS. Fish were vaccinated at three different water temperatures, 5°C, 10°C and 15°C, and sorted into six groups (N = 60). Lumpfish were weighed every 3 weeks after vaccination, sampled at 3, 6, 9 and 18 weeks post-immunization (wpi) and evaluated by modified Speilberg score, ELISA and immunoblotting. Vaccinated fish showed low antibody response against V. salmonicida. Fish vaccinated at 5°C showed significantly lower antibody response against A. salmonicida throughout the study. At higher temperatures, vaccinated fish showed significantly increased antibody responses, at 18 wpi for 10°C and at 6 and 18 wpi for 15°C. Immunoblotting demonstrated specific response against the LPS antigen of A. salmonicida in the 10°C and 15°C VAX groups. Mean body weight increased in all groups throughout the study. Vaccinated fish had low Speilberg scores with no melanization of abdominal tissue. Our results show that vaccinating lumpfish at a lower water temperature may lead to a low antibody response against A. salmonicida.