Maximum heart rate measurements.

<p>Maximum heart rate (<i>f</i>_Hmax) measurements at 10 WPI in non-infected controls (Ctrl, grey line), PRV-infected (PRV, black line) and PRV-infected exposed to periodic hypoxic stress (PRV-H, red line). A: Average <i>f</i>_Hmax (±SE) during temperature increase for each group. Dashed lines between dots indicate that half or more individuals of the initial population were missing or had cardiac arrhythmia and therefore were removed from the measurement. Grey areas and asterisks indicate significant differences between groups (ANOVA, *: <i>p</i> < 0.05). Point "acc" shows <i>f</i>_Hmax after acclimation at 12°C, just before atropine injection. B: Average <i>f</i>_Hmax (±SE) of the three groups between 13 and 19°C. C: T_opt for aerobic scope calculated from Arrhenius breakpoint temperature of <i>f</i>_Hmax for each group.</p

PRV RNA levels in blood and heart.

<p>PRV RNA levels (Ct values) in blood (A) and heart (B) from naïve fish sampled at Day 0 (white dots), non-infected controls (Ctrl, grey dots), PRV-infected (PRV, black dots) and PRV-infected fish exposed to periodic hypoxic stress (PRV-H, red dots), at each time-point during the infection trial. Weeks post-infection (WPI) are indicated on the x-axis. Ct value ≥ 37.0 indicates no virus RNA detected. A non-parametric Mann-Whitney unpaired rank test was performed between the groups at all time-points detecting no significant differences between the groups (<i>p</i> > 0.05).</p

Kaplan-Meier plots of tolerance time during hypoxia challenge test.

<p>Percent cumulative incipient lethal oxygen saturation (ILOS) levels over time during acute hypoxia challenge of the Ctrl (grey line), PRV (black line) and PRV-H (red line) groups at 4 (A), 7 (B) and 10 (C) weeks post-infection (WPI). Secondary y-axis and dotted line (blue) shows water oxygen levels (% air saturation). Statistical significance levels are indicated in the bottom left of each plot after performing a Peto & Peto modification of the Gehan-Wilcoxon test between the curves for each group; Ctrl vs PRV (4, 7 and 10 WPI), Ctrl vs PRV-H (7 and 10 WPI) and PRV-H vs PRV (10 WPI). NS indicates not significant.</p

Measures from the hypoxia challenge test.

<p>Measures from the hypoxia challenge test.</p

Blood oxygen binding affinity.

<p>Blood oxygen binding affinity in PRV-infected fish exposed to periodic hypoxic stress (PRV-H, red curve/line) and non-infected controls (Ctrl, black curve/line) at 10 weeks post-infection (WPI). A: Hb-O₂ dissociation curves (ODC) relating partial pressure of oxygen (P_O2; x-axis) with Hb-oxygen saturation (y-axis). ATP concentrations are shown in inset with significance level (<i>p</i> = 0.07, indicated by +) according to non-parametric Mann-Whitney unpaired rank test. B: Linear regression of ODC from log-transformed data showing Hill coefficients and K_d (zero intercept) values for the groups.</p

PRV RNA levels–HCT.

<p>PRV RNA levels (Ct values) in blood (A) and heart (B) from fish included in the HCT. Groups are non-infected controls (Ctrl, grey dots), infected (PRV, black dots) and infected exposed to periodic hypoxic stress (PRV-H, red dots). A non-parametric Mann-Whitney unpaired rank test was performed between the groups at all time-points detecting no significant differences between the groups (<i>p</i> > 0.05). Weeks post-infection (WPI) are indicated on the x-axis.</p

Histological scoring of inflammatory changes in the heart–HCT.

<p>Scoring of the inflammatory changes for the fish included in the HCT are displayed by groups. Ctrl: non-infected control group, PRV: PRV-infected group, PRV-H: PRV-infected exposed to periodic hypoxic stress. Inflammatory changes in epicardium, compactum and spongiosum were scored from sections of the heart ventricle using a continuous visual analogue scale ranging from 0–3. The total HSMI score was calculated from the mean of scores from the separate heart compartments. The lower and upper border of boxes indicates the 25^th and 75^th percentiles, respectively and the centerline indicates the 50^th percentile. The upper and lower whiskers correspond to the highest and lowest value of the 1.5*IQR (inter-quartile range). A non-parametric Mann-Whitney unpaired rank test was performed between the groups at all time-points detecting no significant differences between the groups (<i>p</i> > 0.05). Weeks post-infection (WPI) are indicated on the x-axis.</p

Histological scoring of inflammatory changes in the heart.

<p>A. Section of heart tissue (hematoxylin and eosin staining) showing epicarditis and myocarditis from a heart having an average cardiac inflammation score of 1.86. The PRV RNA Ct value in heart and blood of the fish in A was 17.2 and 20.5, respectively. B. Picture of a non-infected heart. The hearts in A and B were sampled 7 weeks post infection. *, ** and *** indicate epicardium, compactum and spongiosum, respectively. Arrowhead indicate myocardial necrosis. Bars indicate the approximate thickness of the respective layers. 20x magnification in A and B. C—E. Development of inflammatory changes is displayed for each group throughout the challenge trial. Ctrl: non-infected controls, PRV: PRV-infected fish, PRV-H: PRV-infected fish exposed to periodic hypoxic stress. Inflammatory changes in epicardium, compactum and spongiosum were scored from sections of the heart ventricle using a continuous visual analogue scale ranging from 0 (no inflammatory changes) to 3 (severe panmyocarditis). The total HSMI score was calculated from the mean of scores from the separate heart compartments. The lower and upper border of boxes indicates the 25^th and 75^th percentiles, respectively and the centerline indicates the 50^th percentile. The upper and lower whiskers correspond to the highest and lowest value of the 1.5*IQR (inter-quartile range). A non-parametric Mann-Whitney unpaired rank test was performed between the groups at all time-points. Weeks post-infection (WPI) are indicated on the x-axis.</p

Hypoxia tolerance and responses to hypoxic stress during heart and skeletal muscle inflammation in Atlantic salmon (<i>Salmo salar</i>)

<div><p>Heart and skeletal muscle inflammation (HSMI) is associated with <i>Piscine orthoreovirus</i> (PRV) infection and is an important disease in Atlantic salmon (<i>Salmo salar)</i> aquaculture. Since PRV infects erythrocytes and farmed salmon frequently experience environmental hypoxia, the current study examined mutual effects of PRV infection and hypoxia on pathogenesis and fish performance. Furthermore, effects of HSMI on hypoxia tolerance, cardiorespiratory performance and blood oxygen transport were studied. A cohabitation trial including PRV-infected post-smolts exposed to periodic hypoxic stress (4 h of 40% O₂; PRV-H) at 4, 7 and 10 weeks post-infection (WPI) and infected fish reared under normoxic conditions (PRV) was conducted. Periodic hypoxic stress did not influence infection levels or histopathological changes in the heart. Individual incipient lethal oxygen saturation (ILOS) was examined using a standardized hypoxia challenge test (HCT). At 7 WPI, i.e. peak level of infection, both PRV and PRV-H groups exhibited reduced hypoxia tolerance compared to non-infected fish. Three weeks later (10 WPI), during peak levels of pathological changes, reduced hypoxia tolerance was still observed for the PRV group while PRV-H performed equal to non-infected fish, implying a positive effect of the repeated exposure to hypoxic stress. This was in line with maximum heart rate (<i>f</i>_Hmax) measurements, showing equal performance of PRV-H and non-infected groups, but lower <i>f</i>_Hmax above 19°C as well as lower temperature optimum (<i>T</i>_opt) for aerobic scope for PRV, suggesting reduced cardiac performance and thermal tolerance. In contrast, the PRV-H group had reduced hemoglobin-oxygen affinity compared to non-infected fish. In conclusion, Atlantic salmon suffering from HSMI have reduced hypoxia tolerance and cardiac performance, which can be improved by preconditioning fish to transient hypoxic stress episodes.</p></div