Fig 1.
Three groups of Atlantic salmon from Penobscot river strain and St John river strain (six replicate tanks per treatment): sea lice infestation plus ISAv, sea lice infestations no ISAv, and no sea lice infestation no ISAv controls. Fish were bath infested with L. salmonis followed by an ISAv cohabitation infection 18 days post-lice exposure. Twenty-four fish were euthanized per treatment group (n = 12 Penobscot–derived and 12 St John–derived fish, each made up of three distinct families) at days -1, 3, 16, and 37 d.p.e., and the following parameters sampled number of lice per fish, head kidney for immune gene expression and ISAv QRT-PCR kidney, spleen and heart for cell culture. Forty-five Atlantic salmon per treatment group were also monitored until no mortalities had occurred in any group for 7 days. At 51 d.p.e. all survivor fish were sampled as detailed below.
Table 1.
Real-time PCR primers and Taqman MGB probe sequences for ISAv-Segment 8 (Snow et al. 2006).
Table 2.
Mean percentage of Atlantic salmon positive for viable ISAv by cell culture and non-viable ISAv by QRT-PCR detection of ISAv segment 8 RNA pre- and 3, 16, 37 adm 51 days post–ISAv cohab infection.
Fish sampled at 51 d.p.e. to ISAv cohab fish were survivors of the infection.
Table 3.
PCR primer sequences for ISAv-Segment 8 (Mjaaland et al., 1997).
Table 4.
Oligonucleotide primer sets used to amplify Atlantic salmon (Salmo salar) reference genes (EF-1Ab, RPS 20, 18S and elF) and target genes (IL-1β, MMP 9, Mx1, MHC1-b, Galectin 9, TRIM 16 and TRIM 25) for quantitative real-time PCR (qPCR) expression studies.
Fig 2.
Mean number of sessile, mobile or gravid sea lice per Atlantic salmon pre- (day 0) and 3, 16, 37 and 51 days post—ISAv cohab infection which corresponds to 17, 21, 34, 55 and 69 days post-lice infection, respectively.
Fig 3.
Mean percentages of Atlantic salmon positive for ISAv by cell culture pre- and 3, 16, 37 and 51 days post—ISAv cohab infection.
Fish sampled at 51 d.p.e. to ISAv cohab fish were survivors of the ISAv infection. Data is combined from four replicate tanks per treatment with a total n per treatment group of 24 except in the lice plus ISAv treatment group at 37 d.p.e. to ISAv-infected cohab fish where n = 3 due to high mortalities and low survival in this treatment group. At 51 d.p.e. to ISAv-infected cohab fish i.e. survivor fish n = 45, 44 and 14 respectively.
Fig 4.
Percent survival of Atlantic salmon with and without a prior L. salmonis infestation post–exposure to ISAv-infected cohab fish (only mortalities which were positive for viable ISAv were included).
Data combined from duplicate tanks per treatment group: no lice no ISAv, no lice plus ISAv and lice plus ISAv (n = 47, 48 and 33, respectively).
Fig 5.
Percent survival of shedder Atlantic salmon with and without a prior L. salmonis infestation post—ISAv intra-peritoneal injection.
The data is combined from six replicate tanks per treatment (n = 18 per treatment).
Fig 6.
Relative normalized expression of the pro-inflammatory cytokine IL1-β in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 7.
Relative normalized expression of the wound healing gene MMP 9 in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 8.
Relative normalized expression of the pro-inflammatory cytokine IL1-β in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 9.
Mean number of sessile, mobile or gravid sea lice per Atlantic salmon from three combined families of St John or Penobscot derived stocks pre- and 3, 16, 37 and 51 days post—ISAv cohab infection which corresponds to 17, 21, 34, 55 and 69 days post-lice infection, respectively.
No significant differences in sessile, mobile or gravid lice per fish between the two stocks at any of the time points sampled.
Fig 10.
Relative normalized expression of Mx-1 in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 11.
Relative normalized expression of Mx-1 in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 12.
Percent survival of St John- and Penobscot- derived Atlantic salmon with and without a prior L. salmonis infection post–exposure to ISAv-infected cohab fish.
Data combined from duplicate tanks per treatment group: no lice no ISAv, no lice plus ISAv and lice plus ISAv (St John versus Penobscot derived fish, n = 15 and 29, n = 23 and 25, n = 17 and 26, respectively).
Fig 13.
Relative normalized expression of MHC class I β in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).
Fig 14.
Relative normalized expression of ISAv responsive gene galectin 9 in the anterior kidney of Atlantic salmon exposed to both sea lice and ISAv infections.
The three treatment groups (control, ISAv only and ISAv + lice) were sampled at 4 separate time points (17 days post exposure (dpe) to sea lice and minus 1 dpe to ISAv, 21 dpe to lice and 3 dpe to ISAv, 34 dpe to lice and 16 dpe to ISAv and 55 dpe to lice and 37 dpe to ISAv). The asterisk indicates a significant difference between the control and treatment groups for that particular time point (p < 0.05, n = 24).