Smoltification and seawater transfer of Atlantic salmon (Salmo salar L.) is associated with systemic repression of the immune transcriptome.

Smoltification and seawater adaptation of Atlantic salmon are associated with profound alterations in the endocrine status, osmoregulation and behaviour. Little is known about immunological changes during smoltification, although increased incidences of infectious diseases after seawater transfer (SWT) may indicate weakened protection. We report microarray gene expression analyses in farmed Atlantic salmon during smoltification stimulated with constant light and early seawater adaptation (one and three weeks after SWT). Gene expression changes were large, their magnitude in the head kidney and proximal intestine was greater than in the gill. Among 360 differentially expressed immune genes, 300 genes were down-regulated, and multiple functional groups were affected such as innate antiviral immunity, chemokines, cytokines and receptors, signal transducers, effectors of humoral and cellular innate immunity, antigen presentation and lymphocytes, especially T cells. No recovery was observed after three weeks in seawater. A notable exception was a transient up-regulation of immunoglobulin transcripts in the gill after SWT. Genes involved in stress responses and xenobiotic metabolism were up-regulated in respectively intestine and gill. The duration of this observed immune suppression and the possible consequences for susceptibility to infections and diseases need further exploration.

Transcriptome analyses of Atlantic salmon (Salmo salar L.) erythrocytes infected with piscine orthoreovirus (PRV).

Heart and skeletal muscle inflammation (HSMI) is a widespread disease of farmed Atlantic salmon (Salmo salar L.) and is associated with piscine orthoreovirus (PRV) infection. PRV is detectable in blood long before development of pathology in cardiac- and skeletal muscle appear, and erythrocytes have been identified as important target cells for the virus. The effects of PRV infection on cellular processes of erythrocytes are not known, but haemolytic anemia or systemic lysis of erythrocytes does not seem to occur, even with high virus loads in erythrocytes. In this study, gene expression profiling performed with high-density oligonucleotide microarray showed that PRV infection of erythrocytes induced a large panel of virus responsive genes. These involved interferon-regulated antiviral genes, as well as genes involved in antigen presentation via MHC class I. PRV infection also stimulated negative immune regulators. In contrast, a large number of immune genes expressed prior to infection were down-regulated. Moderate reduction of expression was also found for many genes encoding components of cytoskeleton and myofiber, proteins involved in metabolism, ion exchange, cell-cell interactions as well as growth factors and regulators of differentiation. PRV did not affect expression of genes involved in heme biosynthesis, gas exchange or erythrocyte-specific markers, but some regulators of erythropoiesis showed decreased transcription levels. These results indicate that PRV infection activates innate antiviral immunity in salmon erythrocytes, but suppresses other gene expression programs. Gene expression profiles suggest major phenotypic changes in PRV infected erythrocytes, but the functional consequences remain to be explored.

Comparison of transcriptomic responses to pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) in heart of Atlantic salmon (Salmo salar L).

Pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) are viral diseases associated with SAV (salmonid alphavirus) and PRV (piscine reovirus), which induce systemic infections and pathologies in cardiac and skeletal muscle tissue of farmed Atlantic salmon (Salmo salar L), resulting in severe morbidity and mortality. While general features of the clinical symptoms and pathogenesis of salmonid viral diseases are relatively well studied, much less is known about molecular mechanisms associated with immunity and disease-specific changes. In this study, transcriptomic analyses of heart tissue from PD and HSMI challenged Atlantic salmon were done, focusing on the mature phases of both diseases at respectively 28-35 and 42-77 days post infection. A large number of immune genes was activated in both trials with prevalence of genes associated with early innate antiviral responses, their expression levels being slightly higher in PD challenged fish. Activation of the IFN axis was in parallel with inflammatory changes that involved diverse humoral and cellular factors. Adaptive immune response genes were more pronounced in fish with HSMI, as suggested by increased expression of a large number of genes associated with differentiation and maturation of B lymphocytes and cytotoxic T cells. A similar down-regulation of non-immune genes such as myofiber and mitochondrial proteins between diseases was most likely reflecting myocardial pathology. A suite of genes important for cardiac function including B-type natriuretic peptide and four neuropeptides displayed differential expression between PD and HSMI. Comparison of results revealed common and distinct features and added to the understanding of both diseases at their mature phases with typical clinical pictures. A number of genes that showed disease-specific changes can be of interest for diagnostics.

Effects of functional feeds on the lipid composition, transcriptomic responses and pathology in heart of Atlantic salmon (Salmo salar L.) before and after experimental challenge with Piscine Myocarditis Virus (PMCV).

BACKGROUND: Cardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon (Salmo salar) recently associated with a double-stranded RNA virus, Piscine Myocarditis Virus (PMCV). The disease has been diagnosed in 75-85 farms in Norway each year over the last decade resulting in annual economic losses estimated at up to €9 million. Recently, we demonstrated that functional feeds led to a milder inflammatory response and reduced severity of heart lesions in salmon experimentally infected with Atlantic salmon reovirus, the causal agent of heart and skeletal muscle inflammation (HSMI). In the present study we employed a similar strategy to investigate the effects of functional feeds, with reduced lipid content and increased eicosapentaenoic acid levels, in controlling CMS in salmon after experimental infection with PMCV. RESULTS: Hepatic steatosis associated with CMS was significantly reduced over the time course of the infection in fish fed the functional feeds. Significant differences in immune and inflammatory responses and pathology in heart tissue were found in fish fed the different dietary treatments over the course of the infection. Specifically, fish fed the functional feeds showed a milder and delayed inflammatory response and, consequently, less severity of heart lesions at earlier and later stages after infection with PMCV. Decreasing levels of phosphatidylinositol in cell membranes combined with the increased expression of genes related with T-cell signalling pathways revealed new interactions between dietary lipid composition and the immune response in fish during viral infection. Dietary histidine supplementation did not significantly affect immune responses or levels of heart lesions. CONCLUSIONS: Combined with the previous findings on HSMI, the results of the present study highlight the potential role of clinical nutrition in controlling inflammatory diseases in Atlantic salmon. In particular, dietary lipid content and fatty acid composition may have important immune-modulatory effects in Atlantic salmon that could be potentially beneficial in fish balancing the immune and tissue responses to viral infections.

Cardiac responses to elevated seawater temperature in Atlantic salmon.

BACKGROUND: Atlantic salmon aquaculture operations in the Northern hemisphere experience large seasonal fluctuations in seawater temperature. With summer temperatures often peaking around 18-20°C there is growing concern about the effects on fish health and performance. Since the heart has a major role in the physiological plasticity and acclimation to different thermal conditions in fish, we wanted to investigate how three and eight weeks exposure of adult Atlantic salmon to 19°C, previously shown to significantly reduce growth performance, affected expression of relevant genes and proteins in cardiac tissues under experimental conditions. RESULTS: Transcriptional responses in cardiac tissues after three and eight weeks exposure to 19°C (compared to thermal preference, 14°C) were analyzed with cDNA microarrays and validated by expression analysis of selected genes and proteins using real-time qPCR and immunofluorescence microscopy. Up-regulation of heat shock proteins and cell signaling genes may indicate involvement of the unfolded protein response in long-term acclimation to elevated temperature. Increased immunofluorescence staining of inducible nitric oxide synthase in spongy and compact myocardium as well as increased staining of vascular endothelial growth factor in epicardium could reflect induced vascularization and vasodilation, possibly related to increased oxygen demand. Increased staining of collagen I in the compact myocardium of 19°C fish may be indicative of a remodeling of connective tissue with long-term warm acclimation. Finally, higher abundance of transcripts for genes involved in innate cellular immunity and lower abundance of transcripts for humoral immune components implied altered immune competence in response to elevated temperature. CONCLUSIONS: Long-term exposure of Atlantic salmon to 19°C resulted in cardiac gene and protein expression changes indicating that the unfolded protein response, vascularization, remodeling of connective tissue and altered innate immune responses were part of the cardiac acclimation or response to elevated temperature.

CpG- and LPS-activated MAPK signaling in in vitro cultured salmon (Salmo salar) mononuclear phagocytes.

The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes.

Induced erythropoiesis during acute anemia in Atlantic salmon: a transcriptomic survey.

Anemia is a common pathophysiological response to stressors, malnutrition and infections in salmonid fish. In order to improve our understanding of the molecular mechanisms and markers associated with induced erythropoiesis (EP) during acute anemia in Atlantic salmon (Salmo salar L.), we performed transcriptome analysis of fish injected with the hemolytic compound phenylhydrazine (PHZ). Treatment with a low dose of PHZ resulted in moderate but significant reduction of hematocrit (Hct) and increased transcription of cardiac erythropoietin (epo) at 2 days post challenge (dpc), and epo receptor (epor) in spleen from 2 to 4 dpc. Oligonucleotide microarrays were used to characterize the events of EP in the spleen, an important organ for expansive EP during acute erythropoietic stress in rodents, and these were compared to gene expression profiles of untreated mature red blood cells (RBC) in order to search for erythroid-specific genes. Splenic responses suggested a prevalence of protective mechanisms at the first stage, characterized by induced xenobiotic metabolism and responses to oxidative and protein stress. Erythroid-specific regulation was evident at 2 dpc and enhanced by 4 dpc, and gene expression profiles witnessed a rapid establishment of RBC phenotype although Hct levels remained low. A large group of genes showed a strong correlation to globins by expression profiles. In addition to epor this included genes of heme and iron metabolism, scavengers of free radicals and chaperones, channels and transporters, markers of erythrocytes, regulators of proliferation and cell cycle arrest and many genes with unidentified roles in RBC differentiation. Induced EP in spleen was characterized by specific features, such as upregulation of innate antiviral immune genes and sustained high expression of proapoptotic genes including caspases. Transcriptome changes suggested an association between EP and suppression of several developmental programs including adaptive immune responses. In conclusion, acute hemolysis and resulting anemia rapidly induced EP in the spleen of Atlantic salmon, which showed both common characteristics for all vertebrates as well as fish-specific properties.

Comparison of Atlantic salmon individuals with different outcomes of cardiomyopathy syndrome (CMS).

BACKGROUND: Cardiomyopathy syndrome (CMS) is a severe disease of Atlantic salmon (Salmo salar L.) associated with significant economic losses in the aquaculture industry. CMS is diagnosed with a severe inflammation and degradation of myocardial tissue caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV), with structural similarities to the Totiviridae family. In the present study we characterized individual host responses and genomic determinants of different disease outcomes. RESULTS: From time course studies of experimentally infected Atlantic salmon post-smolts, fish exhibited different outcomes of infection and disease. High responder (HR) fish were characterized with sustained and increased viral load and pathology in heart tissue. Low responder (LR) fish showed declining viral load from 6-10 weeks post infection (wpi) and absence of pathology. Global gene expression (SIQ2.0 oligonucleotide microarray) in HR and LR hearts during infection was compared, in order to characterize differences in the host response and to identify genes with expression patterns that could explain or predict the different outcomes of disease. Virus-responsive genes involved in early antiviral and innate immune responses were upregulated equally in LR and HR at the first stage (2-4 wpi), reflecting the initial increase in virus replication. Repression of heart muscle development was identified by gene ontology enrichment analyses, indicating the early onset of pathology. By six weeks both responder groups had comparable viral load, while increased pathology was observed in HR fish. This was reflected by induced expression of genes implicated in apoptosis and cell death mechanisms, presumably related to lymphocyte regulation and survival. In contrast, LR fish showed earlier activation of NK cell-mediated cytotoxicity and NOD-like receptor signaling pathways. At the late stage of infection, increased pathology and viral load in HR was accompanied by a broad activation of genes involved in adaptive immunity and particularly T cell responses, probably reflecting the increased infiltration and homing of virus-specific T cells to the infected heart. This was in sharp contrast to LR fish, where recovery and reduced viral load was associated with a significantly reduced transcription of adaptive immunity genes and activation of genes involved in energy metabolism. CONCLUSIONS: In contrast to LR, a stronger and sustained expression of genes involved in adaptive immune responses in heart tissue of HR at the late stage of disease probably reflected the increased lymphocyte infiltration and pathological outcome. In addition to controlled adaptive immunity and activation of genes involved in cardiac energy metabolism in LR at the late stage, recovery of this group could also be related to an earlier activation of NOD-like receptor signaling and NK cell-mediated cytotoxicity pathways.

Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon.

BACKGROUND: Cardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis primarily in adult farmed Atlantic salmon (Salmo salar L.), caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV) with structural similarities to the Totiviridae family. Here we present the first characterisation of host immune responses to CMS assessed by microarray transcriptome profiling. RESULTS: Unvaccinated farmed Atlantic salmon post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays (SIQ2.0 chip), six gene sets representing a broad range of immune responses were identified, showing significant temporal and spatial regulation. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 4 wpi suggesting a broad tissue tropism. High correlation between viral load and cardiac histopathology score suggested that cytopathic effect of infection was a major determinant of the myocardial changes. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that levelled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi, suggesting a complement-dependent activation of humoral Ab-responses. Peak of cardiac pathology and viral load coincided with cardiac-specific upregulation of T cell response genes and splenic induction of complement genes. Preceding the reduction in viral load and pathology, these responses were probably important for viral clearance and recovery. CONCLUSIONS: By comparative analysis of gene expression, histology and viral load, the temporal and spatial regulation of immune responses were characterised and novel immune genes identified, ultimately leading to a more complete understanding of host-virus responses and pathology and protection in Atlantic salmon during CMS.

IPNV with high and low virulence: host immune responses and viral mutations during infection.

BACKGROUND: Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described. METHODS: In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney. RESULTS: Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates). CONCLUSIONS: Mortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties.

Aerobic training stimulates growth and promotes disease resistance in Atlantic salmon (Salmo salar).

Improving fish robustness is of utmost relevance to reducing fish losses in farming. Although not previously examined, we hypothesized that aerobic training, as shown for human studies, could strengthen disease resistance in Atlantic salmon (Salmo salar). Thus, we exercised salmon pre-smolts for 6 weeks at two different aerobic training regimes; a continuous intensity training (CT; 0.8bls(-1)) and an interval training (IT; 0.8bl s(-1) 16h and 1.0bl s(-1) 8h) and compared them with untrained controls (C; 0.05bl s(-1)). The effects of endurance training on disease resistance were evaluated using an IPN virus challenge test, while the cardiac immune modulatory effects were characterized by qPCR and microarray gene expression analyses. In addition, swimming performance and growth parameters were investigated. Survival after the IPN challenge was higher for IT (74%) fish than for either CT (64%) or C (61%) fish. While both CT and IT groups showed lower cardiac transcription levels of TNF-α, IL-1ß and IL-6 prior to the IPN challenge test, IT fish showed the strongest regulation of genes involved in immune responses and other processes known to affect disease resistance. Both CT and IT regimes resulted in better growth compared with control fish, with CT fish developing a better swimming efficiency during training. Overall, interval aerobic training improved growth and increased robustness of Atlantic salmon, manifested by better disease resistance, which we found was associated with a modulation of relevant gene classes on the cardiac transcriptome.

In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon.

It is assumed that the mobilisation of a strong cellular immune response is important for the survival of Atlantic salmon infected with infectious salmon anaemia virus (ISAV). In this study, the characterisation of immune cell populations in tissues of non-ISAV infected Atlantic salmon and during the early viraemia of ISAV was undertaken. Immunohistochemical investigations of spleen, head kidney and gills using monoclonal antibodies against recombinant proteins from MHC I, II and CD8 were performed on tissues from Atlantic salmon collected day 17 post-challenge in a cohabitant infection model. The localisations of MHC I and II in control salmon were consistent with previous reports but this study presents novel observations on the distribution of CD8 labelled cell populations in Atlantic salmon including the description of significant mucosal populations in the gills. The distribution of MHC I, MHC II and CD8 positive cell populations differed between control salmon and cohabitant salmon in the early stages of ISAV infection. The changes in MHC I labelled cells differed between organs in ISAV cohabitants but all investigated organs showed a decreased presence of MHC II labelled cells. Together with a clustering of CD8 labelled cells in the head kidney and a reduced presence of CD8 labelled cells in the gills, these observations support the early mobilisation of cellular immunity in the response of Atlantic salmon to ISAV infection. However, differences between the present study and the findings from studies investigating immune gene mRNA expression during ISAV infection suggest that viral strategies to interfere with protein expression and circumvent the host immune response could be operative in the early response to ISAV infection.

Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis.

BACKGROUND: Furunculosis, a disease caused with gram negative bacteria Aeromonas salmonicida produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon. RESULTS: Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance) and survivors (HR - high resistance) were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip) and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB) or greater (Jun) in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and exogenous toxic compounds. A number of genes with marked expression difference between HR and LR can be considered as positive and negative correlates of vaccine protection against furunculosis. CONCLUSION: Efficiency of vaccination against furunculosis depends largely on the ability of host to neutralize the negative impacts of immune responses combined with efficient clearance and prevention of tissue damages.

Modulation of splenic immune responses to bacterial lipopolysaccharide in rainbow trout (Oncorhynchus mykiss) fed lentinan, a beta-glucan from mushroom Lentinula edodes.

Immunostimulants (IS) are considered a promising approach for improving resistance to pathogens in fish aquaculture. At present, development of IS are complicated due to limited knowledge on the mechanisms of their action. To assess the use of global gene expression analysis for screening of candidate IS we applied lentinan, a beta-glucan from the mushroom Lentinula edodes, as a model. After feeding rainbow trout (Oncorhynchus mykiss) with lentinan-supplemented (L) and control (C) diets for 37 days, fish were injected with bacterial lipopolysaccharide (LPS), a classical inducer of inflammation. Gene expression was analyzed in LPS-challenged compared to saline-injected fish using a salmonid 1.8k cDNA microarray (SFA2.0 immunochip) and real-time qPCR. Spleen was selected for data analyses due to highest magnitude of responses and its key role in the fish immune system. A group of genes implicated in acute inflammatory responses was higher induced in C versus L, including IFN-related and TNF-dependent genes (galectins and receptors, signal transducers and transcription factors), genes involved in MHC class I antigen presentation and leukocyte recruitment. A similar trend was observed in metabolism of iron and xenobiotics, markers of oxidative and cellular stress. Interestingly, differences between C and L were similar to those observed between salmon with low and high resistance to infectious salmon anemia virus. Genes with equal responses to LPS in L and C were related to cell communication (cytokines, chemokines and receptors), signal transduction, activation of immune cells, apoptosis, cellular maintenance and energy metabolism. In conclusion, lentinan decreased the expression of genes involved in acute inflammatory reactions to the inflammatory agent while major parts of the immune response remained unchanged. Such effects are expected for IS, which should modify immunity by enhancing beneficial and reducing detrimental responses.

Gene expression analyses in Atlantic salmon challenged with infectious salmon anemia virus reveal differences between individuals with early, intermediate and late mortality.

BACKGROUND: Infectious salmon anemia virus (ISAV) causes a multisystemic disease responsible for severe losses in salmon aquaculture. Better understanding of factors that explain variations in resistance between individuals and families is essential for development of strategies for disease control. To approach this, we compared global gene expression using microarrays in fish dying early and late in the time course following infection from a highly pathogenic ISAV. RESULTS: Tissues (gill, heart, liver and spleen) from infected Atlantic salmon (cohabitation, ISAV Glesvaer 2/90 isolate) were collected from three stages over the time course of the experiment; early (EM, 0-10% cumulative mortality (CM), 21-25 days post-infection (DPI)), intermediate (IM, 35-55% CM, 28-31 DPI) and late (LM, 75-85% CM, 37-48 DPI) mortality. Viral loads were equal in EM and IM but dropped markedly in LM fish. Gene expression analyses using a 1.8 K salmonid fish cDNA microarray (SFA2.0) and real-time qPCR revealed a large group of genes highly up-regulated across tissues in EM, which were mainly implicated in innate antiviral responses and cellular stress. Despite equal levels of MHC class I in EM and LM, increase of splenic and cardiac expression of immunoglobulin-like genes was found only in LM while a suite of adaptive immunity markers were activated already in IM. The hepatic responses to ISAV were characterized by difference between EM and LM in expression of chaperones and genes involved in eicosanoid metabolism. To develop classification of high and low resistance phenotypes based on a small number of genes, we processed results from qPCR analyses of liver using a linear discriminant analysis. Four genes (5-lipoxygenase activating protein, cytochrome P450 2K4-1, galectin-9 and annexin A1) were sufficient for correct assignment of individuals to EM, LM and uninfected groups, while IM was inseparable from EM. Three of four prognostic markers are involved in metabolism of inflammatory regulators. CONCLUSION: This study adds to the understanding of molecular determinants for resistance to acute ISAV infection. The most susceptible individuals were characterized by high viral replication and dramatic activation of innate immune responses, which did not provide protection. The ability to endure high levels of infection for sustained periods could be associated with lower inflammatory responses while subsequent protection and viral clearance was most likely conferred by activation of adaptive immunity.

Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells.

The aquatic orthomyxovirus infectious salmon anemia virus (ISAV) is an important pathogen for salmonid aquaculture, however little is known about protective and pathological host responses to infection. We have investigated intracellular responses during cytopathic ISAV infection in the macrophage-like Atlantic salmon kidney (ASK) cell line by microarray analysis (1.8k SFA2.0 immunochip) and a functional assay for glutathione. Gene transcription changed rapidly and consistently with time and with minor differences between two virus isolates. While several pro-inflammatory and antiviral immune genes were induced, genes involved in cell signaling and integrity were down-regulated, suggesting isolation of infected cells from cell-to-cell interaction and responses to external signals. Differential expression of genes regulating cell cycle and apoptosis implied opposite cues from host cell and virus. This was in pace with massive down-regulation of genes involved in biosynthesis and processing of nucleotides and nucleic acids. Significant down-regulation of several genes involved in metabolism of reactive oxygen species suggested increased oxidative stress, which was confirmed by a functional assay showing reduced levels of glutathione during infection. Testing of expression data against a microarray database containing diverse experiments revealed candidate marker genes for ISAV infection. Our findings provide novel insight into cellular host responses and determinants for acute cytopathic ISAV infection.

Atlantic salmon skin barrier functions gradually enhance after seawater transfer.

Atlantic salmon farming operates with high production intensities where skin integrity is recognized as a central factor and indicator for animal health and welfare. In the described trial, the skin development and its immune status in healthy Atlantic salmon reared in two different systems, a traditional open net-pen system and a semi-closed containment system, were investigated. Freshwater smolts were compared to post-smolts after 1 and 4 months in seawater. Growth performance, when adjusted for temperature, was equal between the systems. Skin analyses, including epidermis and dermis, showed that thickness and mucus cell numbers increased in pace with the growth and time post seawater transfer (PST). Gene expression changes suggested similar processes with development of connective tissue, formation of extracellular matrix and augmented cutaneous secretion, changes in mucus protein composition and overall increased immune activity related to gradually enforced protection against pathogens. Results suggest a gradual morphological development in skin with a delayed recovery of immune functions PST. It is possible that Atlantic salmon could experience increased susceptibility to infectious agents and risk of diseases during the first post-smolt period.

Cloning and expression analysis of an Atlantic salmon (Salmo salar L.) tapasin gene.

Loading of the major histocompatibility complex (MHC) class I molecule with peptide is mediated by the multimeric peptide-loading complex in the ER where the glycoprotein tapasin (TAPBP) is required for stabilization of the complex and for control of peptide loading onto MHC class I. To expand our knowledge on antigen presentation genes in Atlantic salmon, we isolated a full-length salmon tapasin cDNA sequence (Sasa-TAPBP). It encoded a 443 bp amino acid sequence with two N-glycosylation sites, two conserved mammalian tapasin signature motifs, two Ig superfamily (IgSf) domains, a transmembrane (TM) domain and an ER-retention KK motif at the C-terminal end, indicative of a similar function as mammalian tapasins. We analysed the regulation of Sasa-TAPBP under immunostimulatory conditions and found an mRNA-upregulation during early infectious salmon anemia virus (ISAV) infection and poly I:C stimulation in vivo and in vitro, in line with our previous findings for other MHC class I pathway genes.

Ig-seq: Deep sequencing of the variable region of Atlantic salmon IgM heavy chain transcripts.

Immunoglobulin M plays a key role in systemic protection of Atlantic salmon against pathogens. Until recent, studies have focused on antigen-specific antibodies and little is known about the IgM repertoire: its size, developmental changes and responses to antigens. We report the development of deep sequencing protocol to characterize the repertoire of IgM heavy chain variable region. Its structure and changes were examined at the early stages of life and after infection with virus of cardiac myopathy. Clonotypes are identified by the V and J gene segments and amino acid sequences of CDR3, which determine the contribution of the heavy chain to the antigen binding properties. A major fraction of transcripts are functional while the rest are either sterile (transcribed from noncoding parts of Ig loci) or include stop codons. Despite marked difference in frequencies of combinations of V and J genes, the size of repertoire is large. The IgM diversity steadily increases after hatch followed with temporal reduction during smoltification and recovery after seawater transfer. Most clonotypes are present only in one fish. However multiple transcripts in uninfected fish are produced exclusively from a small fraction of shared clonotypes. While only 4.7% of clonotypes are detected in three and more fish, they comprise 35% of transcripts. Increased frequencies of most abundant clonotypes were detected in the head kidney and blood at ten weeks after viral infection and all were shared. Occurrence of the same clonotypes in multiple individuals can be explained with either their simple structure or exposure to common antigens. Complexity of CDR3 assessed by contents of non complementary nucleotides is slightly lower in shared clonotypes but difference is small. High nucleotide diversity of CDR3 with identical amino acid sequences suggests selection.

Short-term starvation at low temperature prior to harvest does not impact the health and acute stress response of adult Atlantic salmon.

A period of starvation is regarded as a sound practice in aquaculture prior to handling, transportation and harvest, to minimise impacts on welfare and ensure proper hygiene after harvest. However, documentation of welfare issues such as stress following starvation and handling in adult Atlantic salmon are lacking. This study aimed to examine gut emptying and potential stress during a two week starvation period, and whether this starvation period changed the tolerance for physical stress. The study confirmed slower emptying of the gut segments at low temperature. Plasma and bile cortisol, and selected clinical analyses were used to characterize potential stress, as well as the response to acute physical crowding stress during the starvation period. Neither the general stress level nor the ability to cope with handling stress was affected by a 14 day starvation period. Down-regulation of selected nutritional related gene markers in liver indicated classical starvation responses, with reduced metabolism and oxidative pressure, and sparing of nutrients. The response to acute handling stress was not affected by two weeks of starvation. There were minor effects of starvation on stress and health markers, as evaluated by plasma lysozyme activity and gene expression of selected inflammation marker proteins in heart and skin tissues.