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. 2014 Jun 11;15(1):462.
doi: 10.1186/1471-2164-15-462.

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)

Laura Martinez-Rubio  1 , Øystein Evensen, Aleksei Krasnov, Sven Martin Jørgensen, Simon Wadsworth, Kari Ruohonen, Jose L G Vecino, Douglas R Tocher
Affiliations

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)

Laura Martinez-Rubio et al. BMC Genomics. .

Abstract

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.

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Figures

Figure 1
Figure 1
Proportions of lipid classes in total lipid of heart tissue from fish fed the reference (REF) and functional (CMS1 and CMS2) feeds at different times before (PreCh) and after (6, 8, 10, 12 and 14 weeks) infection with PMCV. Data were analysed by 2-way ANOVA with “time-course” and “diet” as the two factors. Different letters above time-points indicate significant differences between time-points over the time-course of the infection. The factor diet showed significant differences in PC, PE, total phospholipids and TAG (not indicated). PC, phosphatidycholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerol.
Figure 2
Figure 2
Histopathology results in heart tissue. Figures on the left are representing incidence (percentage of fish sampled) and severity of histopathology (based on the criteria described in methods) in both parts of the heart, atrium and ventriculum at 6-, 8-, 10-, 12-, 14-weeks post-challenge in fish fed the Reference diet (R), and the two functional feeds CMS1 (1) and CMS2 (2). Figures on the right are representing the statistical analysis of the atrium and ventriculum histoscores. Estimated effects of CMS1 and CMS2 diets in comparison to the REF diet by sampling weeks. Negative estimates mean there are lower scores and positive that there are higher scores than for the REF dietary group. Error bars denote approximate 95% confidence limits.
Figure 3
Figure 3
Histopathology results in liver tissue. Figure on the left is representing incidence (percentage of fish sampled) and severity of histopathology (based on the criteria described in methods) at 6-, 8-, 10-, 12-, 14-weeks post-challenge in fish fed the Reference diet (R), and the two functional feeds CMS1 (1) and CMS2 (2). Figure on the right is representing the statistical analysis of liver histoscores. Estimated effects of CMS1 and CMS2 diets in comparison to the REF diet by sampling weeks. Negative estimates mean there are lower scores and positive that there are higher scores than for the REF dietary group. Error bars denote approximate 95% confidence limits.
Figure 4
Figure 4
Viral load in heart tissue of the different dietary groups at 6- and 8-weeks post-challenge. Viral load was determined by quantitative real-time PCR analysis of Piscine Myocarditis Virus. Results are presented as CT values (normalized) as a basis for showing the relative level of virus expression (n = 6) Levels of significant differences (t-test) were calculated using the relative expression software tool (REST 2009).
Figure 5
Figure 5
Time-course of four immune pathways to PMCV infection examined with microarray in salmon fed with REF diet. Boxes show median log2 gene expression ratio (total number of features for each pathway indicated in panel header) between pre-challenge and time points post infection, with the 25th (dark grey) and 75th (light gray) percentile, and whiskers indicate minimum and maximum values. Letters indicate significant differences between time points (p < 0.05, Student t-test).
Figure 6
Figure 6
Effect of functional feeds on gene expression in heart tissue of Atlantic salmon examined with microarray. A: Number of differential expressed genes (y-axis) per time point post infection (x-axis). B: Hierarchical clustering by 2419 features (Pearson r, Ward’s method)
Figure 7
Figure 7
Effect of the two functional feeds on immune gene expression in heart tissue of Atlantic salmon pre (week 0) and post (weeks 6, 8, 14) PMCV infection, as examined with microarray. Data are log2 expression ratios between functional feeds (CMS1 and 2, noted respectively as -1 and -2 after each time points) and reference feed (REF). Boxes show median values (total number of features for each pathway indicated in panel header) with the 25th (dark grey) and 75th (light gray) percentile, and whiskers indicating minimum and maximum values. Letters indicate significant differences between time points (p < 0.05, Student t-test).
Figure 8
Figure 8
Expression between functional feeds (CMS1 and CMS2) and reference diet (REF) of genes related with phosphatidyl inositol signalling pathway. At cut off log2-ER = 0.8 (1.75-fold). Red/orange colour intensity indicates higher expression and green/blue colour intensity indicates lower expression.
Figure 9
Figure 9
Expression of fatty acyl desaturase (Fadsd6) gene. A) Normalized expression of different probes of the oligoarray from delta-6 fatty acyl desaturase (Fadsd6) gene over the time course of the PMCV infection on fish fed with the REF diet and the functional feeds (CMS1 and CMS2). B) Expression ratios (fold changes) of different probes of the oligoarray from delta-6 fatty acyl desaturase (Fadsd6) gene. Data are fish fed the functional feeds (CMS1 and CMS2) relative to the REF diet during the course of the infection. Red/orange colour intensity indicates higher expression and green/blue colour intensity indicates lower expression.
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References

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