| Vibrio anguillarum is the causative agent of vibriosis in maricultured fish. A live attenuated vaccine Vibrio anguillarum MVAV6203was successfully constructed in our laboratory, which showed a remarkable immunogenicity against V. anguillarum in bath-vaccinated and injection-vaccinated Scophthalmus maximus. However, the mechanism of its immunoprotection induced by bath-vaccination has not yet been fully understood. To elucidate the molecular mechanisms underlying the vaccine protection, we used microarray technology to compare the spleen transcriptomes of bath-vaccinated and unvaccinated zebrafish at28days post vaccination. The results showed that a total of2164genes and transcripts were differentially expressed. In addition to iron metabolism related to the innate immune and signaling pathways, these differentially expressed genes also involved in the adaptive immunity, mainly including the genes associated with B and Th17cells differentiation. RT-qPCR analysis revealed the transcription profiles of Th17-related transcription factors, cytokines, and cytokine receptors during35days post-vaccination, suggesting that Thl7-related pathways were activated in bath-vaccinated zebrafish with the live attenuated V. anguillarum.Furthermore, our study investigated the mucosal immunity and immunoprotection elicited by live attenuated V. anguillarum in fish. Zebrafish were immunized by bath or injection vaccination, and underwent bath challenge at28days post vaccination with wild type strain. The results showed that bath vaccination induced the slightly higher immunoprotection against bath challenge than injection vaccination in zebrafish. The expressions of Th17-related genes in the mucosal tissues of vaccinated and challenged zebrafish were analyzed. Up-regulated expressions of IL-17AF, IL-21, IL-22, and defensin suggested that Thl7-like immune responses were induced in mucosal tissues by vaccination via bath and injection routes during28days after vaccination and72hours after challenge. Thl-like mucosal immune response was elicited in gills of injection-vaccinated zebrafish and Th2-like mucosal immune response elicited in gut and gills of bath-vaccinated zebrafish. However, the expressions of Thl-and Th2-relgated genes were not significantly up-regulated when compared to their control groups during3days after bath challenge, which implied they played a minor role in protective mucosal immunity. Th17-like mucosal immune occurred mainly in gut and gills during bath-vaccination and bath-challenge, compared to gills and skill during injection-vaccination and bath-challenge. Bath-vaccination elicited the intense Th17-like immune responses in the gut tissue of zebrafish, suggesting that immunity elicited in gut tissue might play a critical role in resistance to pathogen infection. Gills are a tissue in which Thl7-like mucosal immune response is elicited during both bath-vaccination and injection-vaccination in zebrafish. Additionally, Th17-like mucosal immunity elicited by injection-vaccination occurred later than that by bath-vaccination. These results also suggested that mucosal immuninty was independent of system immunity during immune response to bath-vaccination. In bath-vaccinated turbot, effective immune protection against virulent V. anguillarum MVM425was obtained and the Th17-like immune responses were found in mucosal and systemic tissues. The antibody levels in turbot were increased during vaccination and challenge, suggesting that humoral immunity also contributed to the RPS.The gene of leukotriene B4receptor(BLT1) was cloned and characterized using SMART RACE PCR methods. The results showed that BLT1cDNA was1577bp in length while the ORF,5’UTR, and3’UTR were1119bp,250bp, and208bp, respectively. The gene is homologous to the BLT1from fish species. A web-based online software predicted this gene encode a seven-transmembrane G protein-coupled receptor. Phylogenetic tree analysis showed that its distance is close to Takifugu rubripes and Oryzias latipes. The abundant transcriptions of BLT1were highly expressed in spleen and kidney. In terms of the mucosal tissues BLT1expression was detected in skin and gills higher than in gut. Furthermore, the expression changes of BLT1were analyzed in turbot bath-vaccinated with the live attenuated vaccine V. anguillarum MVAV6203and challenged with wide-type V. anguillarum MVM425. BLT1expression was up-regulated after21dpv in spleen and7dpv in gill and gut, which suggested its involvement in the innate and adaptive immune of turbot. Recombinant BLT1protein fused with EGFP was transiently transfected into and expressed in CHO cells. These results imply that turbot BLTl is a chemoattractant receptor and plays a role in immune response to the vaccine.In conclusion, Th17-like adaptive immune and mucosal immunity play an important role against vibriosis in zebrafish and turbot bath-vaccinated with the live attenuated V. anguillarum. In addition, the cloned and identified BLTl gene in turbot will be benefit to understanding the immune response against pathogen infection. |
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