Kardiomyopatisyndrom (CMS) hos Atlantisk laks, Salmo salar L. : funksjonelle genomikkstudier av vert-patogenresponser og sykdomsmarkører

Since the first description of CMS in the mid 1980s, little research has been conducted to approach an understanding of host-pathogen responses in affected Atlantic salmon. The research presented in this thesis aimed to address this topic based on the recent success to reproduce CMS in experimental infection trials. Transcriptome analysis based on microarrays represents a powerful method for characterization of host responses to complex and unknown traits like infectious diseases. Paper I describes the development of an oligonucleotide microarray with 20,000 unique probes to genes of Atlantic salmon. Pilot experiments assessed performance of this platform. In parallel, a bioinformatics system was constructed. The raw data from microarray experiments are processed and stored in a custom-build database. This database combines the possibilities of annotating genes automatically and manually as well as comparing different experiments to each other and was used in Paper II-IV. At the beginning of the studies reported in this thesis, the etiology of CMS was still unknown. We conducted a challenge trial by injecting fish with material from cell culture that was expected to contain the causative virus. The challenged fish developed myocardial changes typical for CMS, which were diagnosed by histopathological examination. The biological material of this trial provided the basis of the Papers II and III as well as parts of Paper I and IV. Before Papers II-IV were published, the putative CMS causative virus (piscine myocarditis virus, PMCV) was discovered and sequenced, and viral loads could be measured with real-time RT-PCR. Paper II describes the regulation of immunological pathways and the development of pathology and viral load in CMS challenged fish during 10 weeks of infection. Myocardial pathology associated with CMS was significant from 6 wpi and peaked at 8-9 wpi 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 leveled 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. While Paper II focused on the immune response of pooled groups of fish developing strongest pathology and infection, Paper III describes a study of individual differences between fish with (so called high responder fish, HR) and without (low responder fish, LR) pathological changes in heart tissues. Global gene expression 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 controlling lymphocyte regulation and survival. 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. In contrast, LR fish showed viral clearance and recovery at the late stage, which was associated with activation of genes involved in energy metabolism while adaptive immunity genes were not expressed. These studies (Paper II and III) provide the first characterization of the temporal and spatial regulation of host-virus responses during CMS, as well as correlates and markers of pathology and protection. Paper IV reports the identification and characterization of early virus responsive genes (VRGs) that are commonly activated in different viral diseases. CMS-related gene expression at early time points was represented by data published in Paper II. The expression data from other studies of viral diseases (heart and skeletal muscle inflammation (HSMI), infectious salmon anaemia (ISA) and infectious pancreatic necrosis (IPN)) and poly I:C treatment were from other experiments stored in the internal database. A total of 117 VRGs were identified, characterized by a rapid induced expression, dependence on the virus level and low tissue specificity. Expression of these genes strongly correlated to expression of IFNα. Some of the highest ranked genes of this study have not been described in the context of virus or immune responses in fish before. According to phylogenetic analyses, a large part of the VRGs has undergone rapid evolution and sequence divergence.Norges Forskningsrå

Fast and slow releasing sulphide donors engender distinct transcriptomic alterations in Atlantic salmon hepatocytes

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Recurrent oxidant treatment induces dysregulation in the brain transcriptome of Atlantic salmon (Salmo salar) smolts

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Multiomics Provide Insights into the Key Molecules and Pathways Involved in the Physiological Adaptation of Atlantic Salmon (Salmo salar) to Chemotherapeutic-Induced Oxidative Stress

Although chemotherapeutics are used to treat infections in farmed fish, knowledge on how they alter host physiology is limited. Here, we elucidated the physiological consequences of repeated exposure to the potent oxidative chemotherapeutic peracetic acid (PAA) in Atlantic salmon (Salmo salar) smolts. Fish were exposed to the oxidant for 15 (short exposure) or 30 (long exposure) minutes every 15 days over 45 days. Unexposed fish served as the control. Thereafter, the ability of the remaining fish to handle a secondary stressor was investigated. Periodic chemotherapeutic exposure did not affect production performance, though survival was lower in the PAA-treated groups than in the control. Increased ventilation, erratic swimming, and a loss of balance were common behavioural manifestations during the oxidant exposure. The plasma reactive oxygen species levels increased in the PAA-treated groups, particularly after the third exposure, suggesting an alteration in the systemic oxidative stress status. Plasma indicators for internal organ health were affected to a certain degree, with the changes mainly observed after the second and third exposures. Metabolomics disclosed that the oxidant altered several circulating metabolites. Inosine and guanosine were the two metabolites significantly affected by the oxidative stressor, regardless of exposure time. A microarray analysis revealed that the gills and liver were more responsive to the oxidant than the skin, with the gills being the most sensitive. Moreover, the magnitude of the transcriptomic modifications depended on the exposure duration. A functional analysis showed that genes involved in immunity and ribosomal functions were significantly affected in the gills. In contrast, genes crucial for the oxidation-reduction process were mainly targeted in the liver. Skin mucus proteomics uncovered that the changes in the mucosal proteome were dependent on exposure duration and that the oxidant interfered with ribosome-related processes. Mucosal mapping revealed gill mucous cell hypertrophy after the second and third exposures, although the skin morphological parameters remained unaltered. Lastly, repeated oxidant exposures did not impede the ability of the fish to mount a response to a secondary stressor. This study provides insights into how a chemical oxidative stressor alters salmon physiology at both the systemic and mucosal levels. This knowledge will be pivotal in developing an evidence-driven approach to the use of oxidative therapeutics in fish, with some of the molecules and pathways identified as potential biomarkers and targets for assessing the physiological cost of these treatments.publishedVersio

Transcriptome Responses of Atlantic Salmon (Salmo salar L.) to Viral and Bacterial Pathogens, Inflammation, and Stress

Transcriptomics provides valuable data for functional annotations of genes, the discovery of biomarkers, and quantitative assessment of responses to challenges. Meta-analysis of Nofima’s Atlantic salmon microarray database was performed for the selection of genes that have shown strong and reproducible expression changes. Using data from 127 experiments including 6440 microarrays, four transcription modules (TM) were identified with a total of 902 annotated genes: 161 virus responsive genes – VRG (activated with five viruses and poly I:C), genes that responded to three pathogenic bacteria (523 up and 33 down-regulated genes), inflammation not caused by infections – wounds, melanized foci in skeletal muscle and exposure to PAMP (180 up and 72 down-regulated genes), and stress by exercise, crowding and cortisol implants (33 genes). To assist the selection of gene markers, genes in each TM were ranked according to the scale of expression changes. In terms of functional annotations, association with diseases and stress was unknown or not reflected in public databases for a large part of genes, including several genes with the highest ranks. A set of multifunctional genes was discovered. Cholesterol 25-hydroxylase was present in all TM and 22 genes, including most differentially expressed matrix metalloproteinases 9 and 13 were assigned to three TMs. The meta-analysis has improved understanding of the defense strategies in Atlantic salmon. VRG have demonstrated equal or similar responses to RNA (SAV, IPNV, PRV, and ISAV), and DNA (gill pox) viruses, injection of bacterial DNA (plasmid) and exposure of cells to PAMP (CpG and gardiquimod) and relatively low sensitivity to inflammation and bacteria. Genes of the highest rank show preferential expression in erythrocytes. This group includes multigene families (gig and several trim families) and many paralogs. Of pathogen recognition receptors, only RNA helicases have shown strong expression changes. Most VRG (82%) are effectors with a preponderance of ubiquitin-related genes, GTPases, and genes of nucleotide metabolism. Many VRG have unknown roles. The identification of TMs makes possible quantification of responses and assessment of their interactions. Based on this, we are able to separate pathogen-specific responses from general inflammation and stress.publishedVersio

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

<p>Abstract</p> <p>Background</p> <p>Infectious pancreatic necrosis virus (IPNV) is an aquatic member of the <it>Birnaviridae </it>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans

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Association between swimming performance, cardiorespiratory morphometry, and thermal tolerance in Atlantic salmon (Salmo salar L.)

This experiment tested the hypothesis that swimming performance in Atlantic salmon (Salmo salar) parr is connected to cardiorespiratory performance and morphology, as well as maximum heart rate (fHmax) related measures of thermal tolerance. Moreover, it was hypothesized that the cardiorespiratory differences between poor and strong swimmers will be retained in a later life stage, i.e., 15 weeks post-smoltification and seawater transfer. This experiment screened a population of 3200 parr (11.2 ± 0.25 g) for their swimming performance, classifying them as poor and good swimmers based on their critical swimming speeds (4.4 ± 0.1 body length s−1 and > 6.8 ± 0.1 body length s−1, respectively). Compared with poor performing parr, good swimmers had a significantly thicker compact myocardium (by 23.7%) and taller gill secondary lamellae (by 16.2%). In contrast, there was no significant difference in maximum oxygen consumption between the two groups as assessed using a “chase” protocol, and the relationship between heart rate specific measures of thermal tolerance and swim performance was variable. For example, three measures did not differ between the two groups, whereas the Arrhenius breakpoint temperature for fHmax and the highest fHmax value were lower and higher, respectively, in the poor swimmers. Importantly, the identified morphological and difference in the highest fHmax value at the parr stage persisted after 15 weeks of common garden rearing in seawater, and they were associated with an increase in relative ventricular mass and a small, but significant, improvement in growth rate. Therefore, it seems that an early assessment of swimming performance can effectively screen for morphological capacities related to oxygen supply and growth rate, but less so for heart rate related measures of thermal tolerance.</p

Cardiac responses to elevated seawater temperature in Atlantic salmon

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Mucosal and systemic physiological changes underscore the welfare risks of environmental hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar)

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