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Here we have studied the impact of lice (Lepeophtheirus salmonis) infestation of donor fish on the ability of isolated peripheral blood monocytes (PBMCs) to control the replication of salmonid alphavirus (SAV) ex vivo. PBMCs were collected by Percoll gradients at eight and nine weeks post copepodid infestation of Atlantic salmon post smolt. Uninfested fish were controls. PBMCs were then infected ex vivo with SAV (subtype 3), and samples were collected for analysis at two, four, and six days post virus infection. Virus titer in the supernatant was assayed in CHH-1 cells, and in addition, the relative expression of the virus structural protein E2 and selected host antiviral genes, IRF9, ISG15, Mx, and IFIT5, were assayed using real-time PCR. Significantly higher virus replication was detected in cells collected from lice-infested fish compared to controls. Higher virus titer coincided with an inability to upregulate the expression of different immune genes, IFIT5, IRF9, and Mx. These findings point towards compromised ability of PBMCs from lice-infested fish to control virus replication, and, to our knowledge, is the first report showing the direct effect of lice infestation on the interplay between viruses and immune cells. There is a possible impact on the dynamic spread of viral diseases in the aquatic environment.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.

Intensive salmon smolt production normally includes reduced water flow and hyperoxygenation (added oxygen) of remaining water. There is little information on how different water quality parameters influence the fish health and the susceptibility to infectious diseases. The current experiment was carried out to evaluate if the combination of hyperoxygenation and reduced water flow (hyperoxic) can act as a chronic stressor to salmon in freshwater (FW) in such a way that it increases the susceptibility to IPN virus (IPNV) following seawater transfer. In FW, after 22 days of hyperoxic exposure plasma ion, TBARS and cortisol were measured. The cortisol levels were significantly (p = 0.011) higher in the hyperoxic group compared to controls maintained under normal oxygen saturation and water flow (normoxic), indicating chronic stress. Hyperoxygenation in FW caused decreased plasma [Cl−] compared to the normoxic group (p = 0.037), while [K+] tended to be higher in the hyperoxic group (p = 0.088). No significant differences were observed in plasma [Na+], total osmolality, TBARS or hematocrit, but there was a tendency towards a lower hct in the hyperoxic compared to the normoxic group. In SW the mortality was higher in the hyperoxic group challenged with IPNV (34%) compared to the normoxic group challenged with IPNV (20%) (p = 0.02), and no mortality was observed in the PBS injected fish. The challenged fish showed an overall increase in plasma cortisol day 8, 10, 12 and 14 post-challenge (p = 0.015, p = 0.000, p = 0.046 and p = 0.022 respectively). After SW transfer and challenge, plasma [K+] was elevated in both challenged groups, but no consistent trends were found for plasma [Cl−], [Na+] or total osmolality during the SW phase. There were no significant differences in the gene expression level of IFN 1α, Mx and IL 1β prior to challenge, suggesting that the basic expression level of these genes were not affected by hyperoxygenation. IPNV was detected in kidney and pylorus, by immunohistochemistry, cell culture, and RT-PCR in head kidney. This experiment indicates that chronic stress induced by a combination of low water flow and hyperoxygenation increases the susceptibility to IPNV challenge.

Atlantic salmon post-smolts were exposed to either chronic hypoxic (Hy) or normal oxygen (No) conditions in seawater tanks for 58 days, mimicking conditions typical of sea cages for farmed salmon at some periods of the year. By day 29 head kidney macrophages were isolated and subjected to in vitro poly I:C stimulation to simulate viral infection, and samples were collected over 48 h. By day 58 fish were subjected to in vivo stimulation using poly I:C or a Vibrio water-based vaccine to simulate viral or bacterial infection, respectively. The fish were monitored for stress responses and expression of several pro-inflammatory genes in head kidney and intestinal tissue up to five days post-injection. Stress load was monitored by plasma cortisol estimation at days 29 and 58, and on days 1, 2, 3 and 5 post-injection in the in vivo trial. Hy exposure resulted in elevated plasma cortisol levels on day 29 compared to No, while on day 58 cortisol levels were higher in the control group. Additionally, both poly I:C and the Vibrio vaccine gave significantly increased cortisol levels one day post-injection compared to PBS treated controls, irrespective of previous oxygen exposure. In vitro stimulation of macrophages with poly I:C revealed higher IFNα mRNA levels at 6, 12 and 24 h and for Mx at 12 and 24 h post-stimulation, for both No and Hy individuals. Moreover, IFNα levels were higher in No than in Hy individuals at all time points, and a similar difference was seen in Mx at 48 h. In vivo stimulation with poly I:C elicited strong elevation of the IL-1β, IFNγ, Mx and IP10 mRNA transcripts in head kidney, while TNFα1 and IFNα were found unaffected. The Vibrio vaccine elicited a strong up regulation of IL-1β, IFNγ and IP10 mRNA, whereas Mx, TNFα1 and IFNα appeared unchanged. Significant differences in expression between different oxygen exposure groups were found for all genes and both stimuli. The overall trend suggests that long-term hypoxia either reduces or delays the expression of these genes in head kidney. Expression of IFNγ and Mx in intestinal tissues also showed a strong up regulation of the genes following poly I:C stimulation, and also here the overall trend suggests that chronic hypoxia results in a lower or delayed expression of the measured genes. In summary, our results indicate that chronic hypoxia modulates the expression of important immune related genes putatively altering the immune response. As the effect is present in isolated macrophages as well as head kidney and intestinal tissue the modulation appears to be affecting local as well as systemic responses.

Salmonid alphavirus type-3 (SAV-3) is the causative agent of pancreas disease (PD) in Atlantic salmon. It is associated with significant economic losses to the aquaculture industry. Vaccines can overcome losses through vaccination. In this study, anti-SAV-3 antibodies in plasma samples from Atlantic salmon vaccinated with DNA, inactivated whole virus vaccines (IWV) and combined delivery of were quantified through indirect enzyme-linked immunosorbent assay (ELISA). Plasma samples at 1:50 dilution was found to have neutralizing antibodies using 100 TCID50 SAV-3, but additional testing showed no neutralization at 1:20, 1:40, 1:80, and 1:160 dilution of plasma samples tested on both chinook salmon embryo (CHSE-214) and chum salmon heart (CHH-1) cell lines. At 1:20 dilution, cytotoxicity was observed in CHSE-214 and CHH-1 cell lines. Furthermore, there was no significant effect of complement on neutralization, although a tendency towards delayed CPE was observed. In summary, immunization of Atlantic salmon with plasmid-encoded (DNA), inactivated whole virus vaccines or combined delivery of intraperitoneal and intranasal induced low levels of circulating antibodies tested by ELISA. Classical virus neutralization assays gave very low to no neutralizing titer. However, employing an indirect fluorescent antibody test (IFAT), titers were found to be 40-80 with some variation between vaccines while for some fish in the DNA vaccinated group, above 80 (no endpoint dilution obtained). It is concluded that an ELISA and IFAT-based neutralization assay can be combined to estimate and document the presence of anti-SAV-3 antibodies post vaccination but correlation with protection against disease is not known. Keywords: Atlantic salmon, SAV-3, CHSE-214, CHH-1, Indirect ELISA, Neutralization, Antibody, Complement, Dilutions, TCID50, Vaccines ABSTRACT Salmonid alphavirus type-3 (SAV-3) is the causative agent of pancreas disease (PD) in Atlantic salmon. It is associated with significant economic losses to the aquaculture industry. Vaccines can overcome losses through vaccination. In this study, anti-SAV-3 antibodies in plasma samples from Atlantic salmon vaccinated with DNA, inactivated whole virus vaccines (IWV) and combined delivery of were quantified through indirect enzyme-linked immunosorbent assay (ELISA). Plasma samples at 1:50 dilution was found to have neutralizing antibodies using 100 TCID50 SAV-3, but additional testing showed no neutralization at 1:20, 1:40, 1:80, and 1:160 dilution of plasma samples tested on both chinook salmon embryo (CHSE-214) and chum salmon heart (CHH-1) cell lines. At 1:20 dilution, cytotoxicity was observed in CHSE-214 and CHH-1 cell lines. Furthermore, there was no significant effect of complement on neutralization, although a tendency towards delayed CPE was observed. In summary, immunization of Atlantic salmon with plasmid-encoded (DNA), inactivated whole virus vaccines or combined delivery of intraperitoneal and intranasal induced low levels of circulating antibodies tested by ELISA. Classical virus neutralization assays gave very low to no neutralizing titer. However, employing an indirect fluorescent antibody test (IFAT), titers were found to be 40-80 with some variation between vaccines while for some fish in the DNA vaccinated group, above 80 (no endpoint dilution obtained). It is concluded that an ELISA and IFAT-based neutralization assay can be combined to estimate and document the presence of anti-SAV-3 antibodies post vaccination but correlation with protection against disease is not known. Keywords: Atlantic salmon, SAV-3, CHSE-214, CHH-1, Indirect ELISA, Neutralization, Antibody, Complement, Dilutions, TCID50, Vaccines