High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines

<p>Abstract</p> <p>Background</p> <p>Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.</p> <p>Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of <it>Aeromonas salmonicida </it>or <it>Moritella viscosa </it>antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response.</p> <p>Results</p> <p>Granulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-β (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing T_H17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of T_H1 T cell lineage (IFN-γ, CD4) or T_H2 (GATA-3) responses were differentially expressed.</p> <p>Conclusions</p> <p>Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-β, IL-17A and its receptor suggests an involvement of T_H17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a T_H2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.</p

Alpha Interferon and Not Gamma Interferon Inhibits Salmonid Alphavirus Subtype 3 Replication In Vitro▿

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-α)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-γ in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-α [rIFN-α] and rIFN-γ) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-α and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-α induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-α is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2α) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-α results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-γ has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-α, while postinfection antiviral responses or postinfection treatment with rIFN-α is not able to limit viral replication

Stress-Induced Reversion to Virulence of Infectious Pancreatic Necrosis Virus in Naïve Fry of Atlantic Salmon (<em>Salmo salar</em> L.)

<div><p>We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (<i>Salmo salar</i> L.) fry. Naïve fry were persistently infected with a virulent strain (T₂₁₇A₂₂₁ of major structural virus protein 2, VP2) or a low virulent (T₂₁₇T₂₂₁) 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 T₂₁₇T₂₂₁ infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T₂₁₇T₂₂₁ 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 <i>in vitro</i> under an antiviral state induced by recombinant IFN-α1. The T₂₁₇A₂₂₁ reverted variant replicated to levels 23-fold higher than the T₂₁₇T₂₂₁ strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an <i>in vivo</i> trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication <i>in vivo</i> and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.</p> </div

Virus detection by cell culture and RT-PCR at persistent period (6 months).

<p>Result of reisolation by cell culture (CC) or detection of virus genome by reverse transcriptase PCR (RT-PCR). Numbers indicate positive/examined for the two methods. When all fish in one group were positive by cell culture they were not examined by RT-PCR. When fish were negative for cell culture, they were subsequently examined by RT-PCR. Strains indicate IPNV strains used for challenge and time points are time post challenge. Nd-not done.</p

Cumulative mortality induced by reverted virus strains.

<p>The cumulative mortality in the fish groups challenged with the TA-reverted strains reached close to 30% while the TT-challenged fish were not different from non-infected controls. Mortality leveled off at day 26 post challenge. Three parallel tanks for each challenge group, plus non-challenged controls (not shown).</p

Virus detection by cell culture and RT-PCR post stress.

<p>Number of fish found positive by cell culture (cc) and reverse transcriptase PCR (RT-PCR) at different time points after the stress period was completed for the TA and TT infected groups. Fish negative by cell culture were subsequently examined by RT-PCR. Results for non-stressed controls and stressed groups are given and show that stress results in higher number of virus positive fish by cell culture at 28 days post stress. N = 6 per group.</p

Expression of IFN-α1 mRNA in TA-, TT-infected and control fish.

<p>IFN-α1 mRNA expression in TA (A) and TT (B) infected, non-stressed and stressed fish is shown. IFNα-1 expression in uninfected fish after stress exposure is also shown (C). Stress down-regulates expression of IFN-α1 in infected fish and also in uninfected controls. P values are indicated, one-sided T-test (+SEM; n = 5 to 6 fish per group).</p

Chromatogram of TA- and TT-infected fish 6 months post challenge.

<p>Detail from chromatogram TA (A) and TT-infected (B) fish (6 months post challenge) from the hypervariable region of VP2. The chromatograms show no mutation for amino acid residues 217, 221 and 247 of VP2. Further there are no double-peaks for these positions. Details of sequencing methods are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054656#s3" target="_blank">Materials and methods</a>.</p

Mortality following primary challenge.

<p>The mortality in groups of naïve fry challenged with a low dose the virulent TA variant of IPNV shows a cumulative mortality of 12% while fish in the TT group has a cumulative mortality of 4%.</p

Antibody measurement in TA and TT-infected fish.

<p>ELISA results (7 months post challenge) in TA- and TT-infected fish tested against TA antigen coat. Pos ctrl are positive controls (TA) and neg ctrl are naïve fish. Blank is background without primary antibodies added. n = 3. As serum is difficult to withdraw from so small fish, we confirmed the protein concentration in the vials used and they were found comparable at 1∶50 dilution protein concentrations between 0.75 to 1.75 mg/ml. The positive control fish sample had a protein concentration around 1.6 mg/ml at the same dilution. Samples were adjusted to equal concentrations.</p