Sea Lice (Lepeophtheirus salmonis) Infestation Reduces the Ability of Peripheral Blood Monocytic Cells (PBMCs) to Respond to and Control Replication of Salmonid Alphavirus in Atlantic Salmon (Salmo salar L.).

Here we have studied the impact of lice (Lepeophtheirus salmonis) infestation of donor fish on the ability of isolated peripheral blood monocytes (PMBCs) to control the replication of salmonid alphavirus (SAV) ex vivo. PMBC 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 PMBCs 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.

Stress-induced reversion to virulence of infectious pancreatic necrosis virus in naïve fry of Atlantic salmon (Salmo salar L.).

We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (Salmo salar L.) fry. Naïve fry were persistently infected with a virulent strain (T(217)A(221) of major structural virus protein 2, VP2) or a low virulent (T(217)T(221)) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T(217)T(221) infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T(217)T(221) infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied in vitro under an antiviral state induced by recombinant IFN-α1. The T(217)A(221) reverted variant replicated to levels 23-fold higher than the T(217)T(221) strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an in vivo trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication in vivo and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.

Modulation of innate immune responses in Atlantic salmon by chronic hypoxia-induced stress.

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-1ß, 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-1ß, 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.