Immunostimulants and vaccines are important for controlling infectious diseases in fish aquaculture. In this study we assess the potential of flagellin to be used for such purposes in rainbow trout (Oncorhynchus mykiss). A recombinant flagellin from the salmonid pathogen Yersinia ruckeri (YRF) has been produced previously by us and shown to be a potent activator of inflammatory cytokines, acute phase proteins and antimicrobial peptides in vitro. Here we show that YRF is the most potent inflammatory activator of three bacterial PAMPs (LPS, peptidoglycan and flagellin) tested. The host response to flagellin was next studied in vivo. The YRF modulated gene expression was examined in two systemic (spleen and liver) and two mucosa-associated (gills and skin) tissues. YRF injection initiated a transient systemic inflammatory response with key pro-inflammatory cytokines (IL-1β, TNFα, IL-6, and IL-11 etc.) and chemokines (CXCL_F4 and CXCL-8) induced rapidly (by 6 h) but subsiding quickly (by 24 h) in multiple tissues. Consequently, a variety of anti-microbial pathways were activated systemically with heightened expression of acute phase proteins, antimicrobial peptides and complement genes in multiple tissues, which was sustained to 24 h in the liver and mucosal tissues. The Th17 cytokine IL-17A/F1 was also induced in the spleen and liver, and Th2 cytokine IL-4/13 was induced in the liver. However, the anti-inflammatory IL-10 and the Th1 cytokine IFNγ were refractory. A secreted form of TLR5 (TLR5s) was induced by flagellin in all tissues examined whilst the membrane form was refractory, suggesting that TLR5s may function as a negative feedback regulator. Trout liver appeared to be an important organ responding to flagellin stimulation, with marked induction of IL-11, IL-23P19, IL-17C1, SAA, and cathelicidin-2. YRF induced a strong antibody response. These antibodies reacted against the middle domain of YRF and were able to decrease YRF bioactivity. Intact YRF was necessary for its bioactivity, as deletion of the N-terminal, C terminal or middle domain of YRF led to functional loss. This study suggests that flagellin could be a potent immunostimulant and vaccine adjuvant for fish aquaculture.

The SOCS family are key negative regulators of cytokine and growth factor signaling. Typically, 8–17 SOCS genes are present in vertebrate species with eight known in mammals, classified as type I (SOCS4–7) and type II (CISH and SOCS1–3) SOCS. It was believed that the type II SOCS were expanded through the two rounds of whole genome duplication (1R and 2R WGDs) from a single CISH/SOCS1–3 precursor. Previously, 12 genes were identified in rainbow trout but here we report 15 additional loci are present, and confirm 26 of the genes are expressed, giving rainbow trout the largest SOCS gene repertoire identified to date. The discovery of the additional SOCS genes in trout has led to a novel model of SOCS family evolution, whereby the vertebrate SOCS gene family was derived from CISH/SOCS2, SOCS1/SOCS3, SOCS4/5, SOCS6, and SOCS7 ancestors likely present before the two WGD events. It is also apparent that teleost SOCS2b, SOCS4, and SOCS5b molecules are not true orthologues of mammalian SOCS2, SOCS4, and SOCS5, respectively. The rate of SOCS gene structural changes increased from 2R vertebrates, to 4R rainbow trout, and the genes with structural changes show large differences and low correlation coefficient of expression levels relative to their paralogues, suggesting a role of structural changes in expression and functional diversification. This study has important impacts in the functional prediction and understanding of the SOCS gene family in different vertebrates, and provides a framework for determining how many SOCS genes could be expected in a particular vertebrate species/lineage.

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.