Project description: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 underlying profile of the 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. Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscose 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. Granulomatous lesions were observed in all vaccinated fish. Presence of severe granulomas were 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, IL-17A (Th17) was significantly up-regulated (p=0.009) in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of Th1 T cell lineage (IFN-γ, CD4) or Th2 (GATA-3) differentiation were differentially expressed. Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon has the profile of strong expression of genes related to innate immune responses. The expression of IL-17A suggests an involvement of Th17 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 a Th2-bias. To what extent the IL-17A profile reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon vaccinated with oil-based vaccines.

Project description: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 underlying profile of the 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. Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscose 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. Granulomatous lesions were observed in all vaccinated fish. Presence of severe granulomas were 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, IL-17A (Th17) was significantly up-regulated (p=0.009) in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of Th1 T cell lineage (IFN-γ, CD4) or Th2 (GATA-3) differentiation were differentially expressed. Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon has the profile of strong expression of genes related to innate immune responses. The expression of IL-17A suggests an involvement of Th17 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 a Th2-bias. To what extent the IL-17A profile reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon vaccinated with oil-based vaccines. RNA from 12 head kidney samples (12 fish) per group were pooled into groups of 4. The RNA was amplified prior to hybridization to arrays. Three biological and 2 technical replicates (dye-swapped) were utilized in different combinations to hybridize to 18 arrays using RNA from fish with the least injection-site inflammatory reactions (FO-1) as the reference group. The assignment of microarrays to treatment groups for hybridization was randomised by using a random number generator. To minimize technical variability, target synthesis was done in batches of 6 where all treatment groups were equally represented.

Project description:BackgroundTwo 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.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa 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.ResultsGranulomatous 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-beta (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of T(H)1 T cell lineage (IFN-gamma, CD4) or T(H)2 (GATA-3) responses were differentially expressed.ConclusionsGranulomatous 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-beta, IL-17A and its receptor suggests an involvement of T(H)17 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(H)2-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.

Project description:Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.

Dataset Information

Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-?, IL-4/13A, and IL-17A Expression in Atlantic Salmon.

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Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-γ, IL-4/13A, and IL-17A Expression in Atlantic Salmon.

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