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Flagellin is the principal component of bacterial flagellum and a major target of the host immune system. To provide new insights into the role of flagellin in fish immune responses to flagellated microorganisms, a recombinant flagellin from Yersinia ruckeri (rYRF) was produced and its bioactivity investigated in the trout macrophage cell line RTS-11 and head kidney cells. rYRF is a potent activator of pro-inflammatory cytokines, acute phase proteins, antimicrobial peptides and subunits of the IL-12 cytokine family. This and the synergy seen with IFN-γ to enhance further expression of specific IL-12 and TNF-α isoforms may suggest that flagellin could be a useful immune stimulant or adjuvant for use in aquaculture. Gene paralogues were often differentially modulated, highlighting the need to study all of the paralogues of immune genes in fish to gain a full understanding of the effects of PAMPs or other stimulants, and the potential immune responses elicited. [ABSTRACT FROM AUTHOR]

Highlights: [•] We investigated flagellin in vaccines for enteric redmouth disease of salmonids. [•] Strains of Yersinia ruckeri differ in pathogenicity and flagellin expression. [•] Flagellin is not an important factor in the effectiveness of whole cell vaccines. [•] Pure flagellin is a potent vaccine against Yersinia ruckeri and other pathogens. [•] Flagellin acts as a vaccine even if the pathogen does not express flagellin. [ABSTRACT FROM AUTHOR]

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3–5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish. • 4 monoclonal antibodies (mAbs) specifically react to rainbow trout interferon (IFN)-γ. • MAb VAB3 detected IFN-γ+ cells by intracellular staining and immunohistochemistry. • IFN-γ+ cells increased significantly when blood leucocytes were stimulated in vitro. • IFN-γ+ cells increased in vivo after injection of live bacteria, poly I:C or vaccine. • Several types of cells produce IFN-γ in trout that contribute to immune protection. [ABSTRACT FROM AUTHOR]

IL-22 is a critical cytokine which is involved in modulating tissue responses during inflammation, and is produced mainly by T cells and innate leucocytes. In mammals, IL-22 is a key component in mucosal defences, tissue repair, epithelial cell survival and proliferation. In teleosts, IL-22 has been cloned and studied in several species, and the transcript is highly expressed in mucosal tissues and induced by pathogen associated molecular patterns (PAMPs), suggesting IL-22 also functions as an important component of the innate immune response in fish. To investigate these immune responses further, we have validated and characterised two monoclonal antibodies (mAbs) which were raised against two different peptide immunogens of salmonid IL-22. Our results show that both mAbs specifically react to their own peptide immunogens and recombinant IL-22, and are able to detect the induction of native protein expression after stimulation. In flow cytometry, an increase in IL-22 positive cells was detected after stimulation in vitro with cytokines and PAMPs and in vivo after bacterial challenge. The immunohistochemistry results showed that IL-22 is highly upregulated in the gills after challenge, both in cells within the gill filaments and in the interbranchial lymphoid tissue. Such results suggest IL-22 may have a role in triggering local antimicrobial defences in fish that may facilitate efficient microbial clearance. Hence monitoring IL-22 producing cells/protein secretion may provide an alternative mean to assess the effectiveness of mucosal vaccines. • Two monoclonal antibodies (L7 & L8) were developed and characterised against rainbow trout IL-22. • Both mAbs specifically react to their peptide immunogens, recombinant IL-22, and induced native IL-22 protein. • IL-22 producing cells were detected by intracellular staining in flow cytometry following in vitro and in vivo stimulation. • IL-22 transcript and protein expression is up-regulated in gills and PBLs in response to bacterial infection. • IL-22+ cells were found in the gill epithelium of lamellae and interbranchial lymphoid tissue after bacterial infection. [ABSTRACT FROM AUTHOR]