MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro.

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.

Macrophage Heterogeneity in the Intestinal Cells of Salmon: Hints From Transcriptomic and Imaging Data.

The intestine has many types of cells that are present mostly in the epithelium and lamina propria. The importance of the intestinal cells for the mammalian mucosal immune system is well-established. However, there is no in-depth information about many of the intestinal cells in teleosts. In our previous study, we reported that adherent intestinal cells (AIC) predominantly express macrophage-related genes. To gather further evidence that AIC include macrophage-like cells, we compared their phagocytic activity and morphology with those of adherent head kidney cells (AKC), previously characterized as macrophage-like cells. We also compared equally abundant as well as differentially expressed mRNAs and miRNAs between AIC and AKC. AIC had lower phagocytic activity and were larger and more circular than macrophage-like AKC. RNA-Seq data revealed that there were 18309 mRNAs, with 59 miRNAs that were equally abundant between AIC and AKC. Integrative analysis of the mRNA and miRNA transcriptomes revealed macrophage heterogeneity in both AIC and AKC. In addition, analysis of AIC and AKC transcriptomes revealed functional characteristics of mucosal and systemic macrophages. Five pairs with significant negative correlations between miRNA and mRNAs were linked to macrophages and epithelial cells and their interaction could be pointing to macrophage activation and differentiation. The potential macrophage markers suggested in this study should be investigated under different immune conditions to understand the exact macrophage phenotypes.

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

Sockeye salmon demonstrate robust yet distinct transcriptomic kidney responses to rhabdovirus (IHNV) exposure and infection.

Aquatic rhabdoviruses are globally significant pathogens associated with disease in both wild and cultured fish. Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes the internationally regulated disease infectious hematopoietic necrosis (IHN) in most species of salmon. Yet not all naïve salmon exposed to IHNV become diseased, and the mechanisms by which some individuals evade or rapidly clear infection following exposure are poorly understood. Here we used RNA-sequencing to evaluate transcriptomic changes in sockeye salmon, a keystone species in the North Pacific and natural host for IHNV, to evaluate the consequences of IHNV exposure and/or infection on host cell transcriptional pathways. Immersion challenge of sockeye salmon smolts with IHNV resulted in approximately 33% infection prevalence, where both prevalence and viral kidney load peaked at 7 days post challenge (dpc). De novo assembly of kidney transcriptomes at 7 dpc revealed that both infected and exposed but noninfected individuals experienced substantial transcriptomic modification; however, stark variation in gene expression patterns were observed between exposed but noninfected, infected, and unexposed populations. GO and KEGG pathway enrichment in concert with differential expression analysis identified that kidney responses in exposed but noninfected fish emphasised a global pattern of transcriptional down-regulation, particularly for pathways involved in DNA transcription, protein biosynthesis and macromolecule metabolism. In contrast, transcriptomes of infected fish demonstrated a global emphasis of transcriptional up-regulation highlighting pathways involved in antiviral response, inflammation, apoptosis, and RNA processing. Quantitative PCR was subsequently used to highlight differential and time-specific regulation of acute phase, antiviral, inflammatory, cell boundary, and metabolic responsive transcripts in both infected and exposed but noninfected groups. This data demonstrates that waterborne exposure with IHNV has a dramatic effect on the sockeye salmon kidney transcriptome that is discrete between resistant and acutely susceptible individuals. We identify that metabolic, acute phase and cell boundary pathways are transcriptionally affected by IHNV and kidney responses to local infection are highly divergent from those generated as part of a disseminated response. These data suggest that primary resistance of naïve fish to IHNV may involve global responses that encourage reduced cellular signaling rather than promoting classical innate antiviral responses.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in recruiting the ESCRT pathway through three ways in the host cells of fish during IHNV budding.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.

Synergistic osmoregulatory dysfunction during salmon lice (Lepeophtheirus salmonis) and infectious hematopoietic necrosis virus co-infection in sockeye salmon (Oncorhynchus nerka) smolts.

While co-infections are common in both wild and cultured fish, knowledge of the interactive effects of multiple pathogens on host physiology, gene expression and immune response is limited. To evaluate the impact of co-infection on host survival, physiology and gene expression, sockeye salmon Oncorhynchus nerka smolts were infected with the salmon louse Lepeophtheirus salmonis (V-/SL+), infectious hematopoietic necrosis virus (IHNV; V+/SL-), both (V+/SL+), or neither (V-/SL-). Survival in the V+/SL+ group was significantly lower than the V-/SL- and V-/SL+ groups (p = 0.024). Co-infected salmon had elevated osmoregulatory indicators and lowered haematocrit values as compared to the uninfected control. Expression of 12 genes associated with the host immune response was analysed in anterior kidney and skin. The only evidence of L. salmonis-induced modulation of the host antiviral response was down-regulation of mhc I although the possibility of modulation cannot be ruled out for mx-1 and rsad2. Co-infection did not influence the expression of genes associated with the host response to L. salmonis. Therefore, we conclude that the reduced survival in co-infected sockeye salmon resulted from the osmoregulatory consequences of the sea lice infections which were amplified due to infection with IHNV.

Dietary exposure to a binary mixture of polybrominated diphenyl ethers alters innate immunity and disease susceptibility in juvenile Chinook salmon (Oncorhynchus tshawytscha).

Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in consumer products and are now found in the aquatic environment. The presence of PBDEs puts the health and survival of aquatic species at risk due to the various toxic effects associated with exposure to these compounds. The effects of a binary dietary mixture of PBDEs on innate immunity and disease susceptibility of juvenile Chinook salmon (Oncorhynchus tshawytscha) were examined in the present study. Salmon were fed roughly 1:1 mixtures of two environmentally predominant PBDE congeners, BDE-47 and BDE-99. The six resulting whole body total PBDE concentrations ranged from less than the limit of quantification to 184 ng/g, wet weight (ww). The innate immune system was assessed by using two in vitro macrophage function assays. Specifically, assays that examined the ability of head kidney macrophages to: (1) engulf sheep red blood cells (SRBCs); and (2) produce a respiratory burst, as determined by the production of a reactive oxygen species, superoxide anion. Macrophages from salmon fed the BDE-47/99 mixture diets engulfed more SRBCs and produced greater superoxide anion than salmon fed the control diet. An increase in macrophage function was observed in fish with whole body total PBDE concentrations ranging from 2.81 ng/g, ww to 184 ng/g, ww. The mechanism for this increase in macrophage function due to PBDE exposure is currently unknown, but may be due to the ability of PBDEs to act as an endocrine receptor agonist and/or antagonist. Salmon exposed to the BDE-47/99 mixture diets were also challenged with the pathogenic bacteria, Vibrio (Listonella) anguillarum to determine disease susceptibility. Kaplan-Meier survival curves of fish exposed to the BDE-47/99 mixture and control diets were significantly different. The Cox proportional hazard risk ratios of disease-induced mortality in juvenile Chinook salmon with whole body concentrations of total PBDEs of 10.9, 36.8, and 184 ng/g, ww were significantly greater than the fish fed the control diet by 1.56, 1.83 and 1.50 times, respectively. Not all concentrations of the binary mixture diets had significant hazard ratios relative to the control diet, due to a non-monotonic concentration response curve. The mixture of PBDE congeners resulted in interactive effects that were generally non-additive and dependent upon the congener concentrations and metric examined. Consequently, predicting the interactive effects in juvenile Chinook salmon exposed to mixtures of PBDE congeners on innate immunity and disease susceptibility cannot be readily determined from the adverse effects of individual PBDE congeners.

Acquired Protective Immunity in Atlantic Salmon Salmo salar against the Myxozoan Kudoa thyrsites Involves Induction of MHIIß+ CD83+ Antigen-Presenting Cells.

The histozoic myxozoan parasite Kudoa thyrsites causes postmortem myoliquefaction and is responsible for economic losses to salmon aquaculture in the Pacific Northwest. Despite its importance, little is known about the host-parasite relationship, including the host response to infection. The present work sought to characterize the immune response in Atlantic salmon during infection, recovery, and reexposure to K. thyrsites After exposure to infective seawater, infected and uninfected smolts were sampled three times over 4,275 degree-days. Histological analysis revealed infection severity decreased over time in exposed fish, while in controls there was no evidence of infection. Following a secondary exposure of all fish, severity of infection in the controls was similar to that measured in exposed fish at the first sampling time but was significantly reduced in reexposed fish, suggesting the acquisition of protective immunity. Using immunohistochemistry, we detected a population of MHIIß+ cells in infected muscle that followed a pattern of abundance concordant with parasite prevalence. Infiltration of these cells into infected myocytes preceded destruction of the plasmodium and dissemination of myxospores. Dual labeling indicated a majority of these cells were CD83+/MHIIß+ Using reverse transcription-quantitative PCR, we detected significant induction of cellular effectors, including macrophage/dendritic cells (mhii/cd83/mcsf), B cells (igm/igt), and cytotoxic T cells (cd8/nkl), in the musculature of infected fish. These data support a role for cellular effectors such as antigen-presenting cells (monocyte/macrophage and dendritic cells) along with B and T cells in the acquired protective immune response of Atlantic salmon against K. thyrsites.

Sockeye salmon immunoglobulin VH usage and pathogen loads differ between spawning sites.

The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.

Infectious hematopoietic necrosis virus (IHNV) persistence in Sockeye Salmon: influence on brain transcriptome and subsequent response to the viral mimic poly(I:C).

BACKGROUND: Sockeye Salmon are an iconic species widely distributed throughout the North Pacific. A devastating pathogen of Sockeye Salmon is infectious hematopoietic necrosis virus (IHNV, genus Novirhabdovirus, family Rhabdoviridae). It has been postulated that IHNV is maintained in salmon populations by persisting over the life of its host and/or by residing in natural reservoirs other than its susceptible hosts. Herein we demonstrate the presence of IHNV in the brain of Sockeye Salmon that survived an experimentally-induced outbreak, suggesting the presence of viral persistence in this susceptible species. To understand the viral persistent state in Sockeye Salmon we profiled the transcriptome to evaluate the host response in asymptomatic carriers and to determine what effects (if any) IHNV exposure may have on subsequent virus challenges. RESULTS: A laboratory disease model to simulate a natural IHNV outbreak in Sockeye Salmon resulted in over a third of the population incurring acute IHN disease and mortality during the first four months after initial exposure. Nine months post IHNV exposure, despite the absence of disease and mortality, a small percentage (<4 %) of the surviving population contained IHNV in brain. Transcriptome analysis in brain of asymptomatic virus carriers and survivors without virus exhibited distinct transcriptional profiles in comparison to naïve fish. Characteristic for carriers was the up-regulation of genes involved in antibody production and antigen presentation. In both carriers and survivors a down-regulation of genes related to cholesterol biosynthesis, resembling an antiviral mechanism observed in higher vertebrates was revealed along with differences in nervous system development. Moreover, following challenge with poly(I:C), survivors and carriers displayed an elevated antiviral immune response in comparison to naïve fish. CONCLUSIONS: IHN virus persistence was identified in Sockeye Salmon where it elicited a unique brain transcriptome profile suggesting an ongoing adaptive immune response. IHNV carriers remained uncompromised in mounting efficient innate antiviral responses when exposed to a viral mimic. The capacity of IHNV to reside in asymptomatic hosts supports a virus carrier hypothesis and if proven infectious, could have significant epidemiological consequences towards maintaining and spreading IHNV among susceptible host populations.

Dietary Exposure to Individual Polybrominated Diphenyl Ether Congeners BDE-47 and BDE-99 Alters Innate Immunity and Disease Susceptibility in Juvenile Chinook Salmon.

Polybrominated diphenyl ethers (PBDEs), used as commercial flame-retardants, are bioaccumulating in threatened Pacific salmon. However, little is known of PBDE effects on critical physiological functions required for optimal health and survival. BDE-47 and BDE-99 are the predominant PBDE congeners found in Chinook salmon collected from the Pacific Northwest. In the present study, both innate immunity (phagocytosis and production of superoxide anion) and pathogen challenge were used to evaluate health and survival in groups of juvenile Chinook salmon exposed orally to either BDE-47 or BDE-99 at environmentally relevant concentrations. Head kidney macrophages from Chinook salmon exposed to BDE-99, but not those exposed to BDE-47, were found to have a reduced ability in vitro to engulf foreign particles. However, both congeners increased the in vitro production of superoxide anion in head kidney macrophages. Salmon exposed to either congener had reduced survival during challenge with the pathogenic marine bacteria Listonella anguillarum. The concentration response curves generated for these end points were nonmonotonic and demonstrated a requirement for using multiple environmentally relevant PBDE concentrations for effect studies. Consequently, predicting risk from toxicity reference values traditionally generated with monotonic concentration responses may underestimate PBDE effect on critical physiological functions required for optimal health and survival in salmon.

Immune response genes and pathogen presence predict migration survival in wild salmon smolts.

We present the first data to link physiological responses and pathogen presence with subsequent fate during migration of wild salmonid smolts. We tagged and non-lethally sampled gill tissue from sockeye salmon (Oncorhynchus nerka) smolts as they left their nursery lake (Chilko Lake, BC, Canada) to compare gene expression profiles and freshwater pathogen loads with migration success over the first ~1150 km of their migration to the North Pacific Ocean using acoustic telemetry. Fifteen per cent of smolts were never detected again after release, and these fish had gene expression profiles consistent with an immune response to one or more viral pathogens compared with fish that survived their freshwater migration. Among the significantly upregulated genes of the fish that were never detected postrelease were MX (interferon-induced GTP-binding protein Mx) and STAT1 (signal transducer and activator of transcription 1-alpha/beta), which are characteristic of a type I interferon response to viral pathogens. The most commonly detected pathogen in the smolts leaving the nursery lake was infectious haematopoietic necrosis virus (IHNV). Collectively, these data show that some of the fish assumed to have died after leaving the nursery lake appeared to be responding to one or more viral pathogens and had elevated stress levels that could have contributed to some of the mortality shortly after release. We present the first evidence that changes in gene expression may be predictive of some of the freshwater migration mortality in wild salmonid smolts.

Acquired immunity and vaccination against infectious pancreatic necrosis virus of salmon.

Acquired immunity plays an important role in the protection of salmonids vaccinated against infectious pancreatic necrosis virus (IPNV) infections. In recent years, vaccine research has taken a functional approach to find the correlates of protective immunity against IPNV infections. Accumulating evidence suggests that the humoral response, specifically IgM is a correlate of vaccine protection against IPNV infections. The role of IgT on the other hand, especially at the sites of virus entry into the host is yet to be established. The kinetics of CD4+ and CD8+ T-cell gene expression have also been shown to correlate with protection in salmonids, suggesting that other arms of the adaptive immune response e.g. cytotoxic T cell responses and Th1 may also be important. Overall, the mechanisms of vaccine protection observed in salmonids are comparable to those seen in other vertebrates suggesting that the immunological basis of vaccine protection has been conserved across vertebrate taxa.

Cloning of the IL-1ß3 gene and IL-1ß4 pseudogene in salmonids uncovers a second type of IL-1ß gene in teleost fish.

To date two closely related interleukin-1ß genes (IL-1ß1 and IL-ß2) have been found in salmonids. The cloning of trout and salmon IL-1ß3, and a salmon IL-1ß4 pseudogene reveals that two types of IL-1ß genes exist in teleost species. Type I teleost IL-1ß genes, including salmonid IL-1ß3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1ß genes, e.g. salmonid IL-1ß1-2, lack one or two coding exons at their 5'-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1ß genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1ß3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1ß3 also has a role in inflammation and host defence.

Additive, non-additive and maternal effects of cytokine transcription in response to immunostimulation with Vibrio vaccine in Chinook salmon (Oncorhynchus tshawytscha).

Estimation of quantitative genetic parameters is important for improving salmonid broodstock management in commercial and government hatcheries. Using a replicated 2 × 2 factorial breeding design (48 families and 192 individuals), we partitioned early immune response transcription variation into additive genetic, non-additive genetic, and maternal components in juvenile Chinook salmon (Oncorhynchus tshawytscha). Transcription of four cytokine genes (IL1, TNF-α, IL-8, IL8-R) and two control genes (IgM and RPS-11) was measured relative to an endogenous control (EF1a) before and 24 h after immune stimulation with Vibrio vaccine. Additive genetic variation was not significant for cytokine transcription and heritability ranged from 0.44 (in pre-challenge IL1) to 0.04 (in post-challenge TNF-α). Non-additive genetic variance was significant in post-challenge IL1 (18 %) and TNF-α (12 %) while maternal effects contributed to pre-challenge cytokine transcription. Cytokine transcription co-expressed within but not between pre- and post-challenge states. The lack of additive genetic effects indicates that cytokine transcription is not a likely candidate for selection programs to improve immune function in Chinook salmon. Our results add to the growing evidence that non-additivity in salmon is common and contributes to our understanding of the genetic architecture of transcription. This indicates that transcription variation may act to maintain genetic variation and facilitate rapid adaptive response in salmonids.

Salmon allergen exposure, occupational asthma, and respiratory symptoms among salmon processing workers.

BACKGROUND: This investigation was triggered by three cases of asthma-about 10% of the workforce-occurring in a salmon processing plant over a short period of time. The aim of the investigation was to characterize the work exposure of inhalable organic particles with personal measurements. Respiratory symptoms at work among workers were also assessed. METHODS: Exposures to airborne salmon allergen, airborne mold spores, and endotoxin in water and air were measured during work. To assess the Atlantic salmon (Salmo salar) Sal s 1 allergen exposure a polyclonal sandwich enzyme-linked immunosorbent assay (ELISA) was developed. Current workers (n = 26) answered questionnaires and underwent allergy and lung function tests. RESULTS: Using the sensitive ELISA method (0.05 ng/ml), we found that workers were exposed to high levels of salmon major allergen at the filleting machine and at the filleting table. Airborne endotoxin levels were low, and mold levels were elevated. Only the three initial asthma cases had IgE to salmon. Of the other workers, 65% reported respiratory symptoms at work. These had lower pulmonary function than workers without such symptoms. CONCLUSIONS: We developed a sensitive method to measure salmon antigen in air and found that filleting workers were most exposed. It is important to reduce aerosols by improving the ventilation system, machines and organization of work since respiratory symptoms at work among workers were common.

Immunoprotective activity of a Salmonid Alphavirus Vaccine: comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I:C alone or combined with an oil adjuvant.

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response - a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.

Comparative defense-associated responses in salmon skin elicited by the ectoparasite Lepeophtheirus salmonis.

Susceptibility among salmonids to the ectoparasite Lepeophtheirus salmonis is related to inflammatory reactions at the site of parasite attachment. Salmon from two susceptible (Salmo salar, Oncorhynchus keta) and one resistant (Oncorhynchus gorbuscha) species were exposed to adult L. salmonis. After 24 and 48h, skin samples directly below the attachment site and at non-attachment sites were assessed for transcriptomic profiles of select innate defense genes. Abrasion of the skin permitted comparisons between abrasion-associated injury and louse-associated injury. Infection responses were consistently higher than those caused by abrasion. Temporal patterns of expression were evident in all species for the transcription factor CCAAT/enhancer-binding protein ß (C/EBP-ß), the cytokine interleukin-6 (IL-6) and the enzyme prostaglandin D synthase (PGDS) at attachment sites. O. gorbuscha was the highest responder in a number of genes while there was an absence of C-reactive protein (CRP) gene expression in S. salar and O. keta, indicating an altered acute-phase response. Moreover, O. keta displayed distinct interleukin-8 (IL-8) and serum amyloid P (SAP) responses. Impaired genetic expression or over-expression in these pathways may be evidence for species-specific pathways of susceptibility to the parasite. At L. salmonis attachment sites, reduced expression compared to non-attachment sites was observed for C/EBP-ß (S. salar), CRP (S. salar), SAP (S. salar, O. gorbuscha, O. keta), PGDS (S. salar, O. gorbuscha, O. keta), and major histocompatibility class II (MH class II, S. salar), suggesting local immunodepression.

Transcriptional differences between triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) during live Vibrio anguillarum challenge.

Understanding how organisms function at the level of gene expression is becoming increasingly important for both ecological and evolutionary studies. It is evident that the diversity and complexity of organisms are not dependent solely on their number of genes, but also the variability in gene expression and gene interactions. Furthermore, slight differences in transcription control can fundamentally affect the fitness of the organism in a variable environment or during development. In this study, triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) were used to examine the effects of polyploidy on specific and genome-wide gene expression response using quantitative real-time PCR (qRT-PCR) and microarray technology after an immune challenge with the pathogen Vibrio anguillarum. Although triploid and diploid fish had significant differences in mortality, qRT-PCR revealed no differences in cytokine gene expression response (interleukin-8, interleukin-1, interleukin-8 receptor and tumor necrosis factor), whereas differences were observed in constitutively expressed genes, (immunoglobulin (Ig) M, major histocompatibility complex (MHC) -II and beta-actin) upon live Vibrio anguillarum exposure. Genome-wide microarray analysis revealed that, overall, triploid gene expression is similar to diploids, consistent with their similar phenotypes. This pattern, however, can subtly be altered under stress (for example, handling, V. anguillarum challenge) as we have observed at some housekeeping genes. Our results are the first report of dosage effect on gene transcription in a vertebrate, and they support the observation that diploid and triploid salmon are generally phenotypically indistinguishable, except under stress, when triploids show reduced performance.

Influence of reduced feed ration on Lepeophtheirus salmonis infestation and inflammatory gene expression in juvenile pink salmon.

The effect of reduced feed ration on infestation levels with the sea louse Lepeophtheirus salmonis and gene expression in juvenile pink salmon Oncorhynchus gorbuscha was tested in three laboratory trials. Body weight was significantly lower among fish on the reduced ration for 27, 34, or 65 d than fish on the full ration. Neither the prevalence nor the abundance of L. salmonis differed between fish on full and reduced rations at any time in any trial. In trial 2, sea louse rejection was delayed among fish on reduced rations; however, the parasite was ultimately rejected from all fish in this trial regardless of ration. Proinflammatory gene expression in salmon exposed to L. salmonis was modulated by reduced rations. There was a reduction in the expression of interleukin-8 in pink salmon on reduced rations 7 d after exposure but not 14 d after exposure. In contrast, the 7-d expression of interleukin-1 beta (IL-1beta) was reduced in exposed pink salmon regardless of ration. By day 14, however, expression of IL-1beta was increased in association with reduced rations among exposed salmon. Similarly, the expression of tumor necrosis factor alpha (TNFalpha) was increased 14 d after exposure among salmon on a reduced ration. There was no evidence that short-duration exposure of otherwise healthy juvenile pink salmon to a reduced ration affected susceptibility to L. salmonis. The expression data do not suggest an obvious mechanism of louse rejection; rather, they indicate that a more comprehensive suite of inflammatory pathways should be surveyed to better understand the early pink salmon response to L. salmonis.