| Vibrio anguillarum is a main aetiological agent of vibriosis, a common disease along coast of China. A live attenuated vaccine V. anguillarum strain MVAV6203was established in our previous work. This strain was proven to be protective of fish out of vibriosis in some farms. But the mechanism of the immuneprotection is still unclear. In this study, the zebrafish, a powerful model animal in science, was utilized to reveal the mechanism.Firstly, the zebrafish were injected with V. anguillarum MVAV6203(1×105CFU/fish). After4weeks, the zebrafish were challenged with wild-type V. anguillarum MVM425. The vaccinated groups significantly survived from the infection of wild-type V. anguillarum MVM425with about90%RPS, which was similar to the results on industrial fish such as flounder. It is indicated that zebrafish could be used as a model animal to investigate the mechanism of protection. Moreover, the serum specific antibody level was up-regulated following14days post vaccination, which suggested the responses of humoral immunity was triggered. In addition, the vaccination led to cross-protection to Vibrio harveyi and Vibrio vulnificus with about40%RPS, while no protection to Vibrio parahaemolyticus.Secondly, zebrafish were protected against the wild pathogenic strain with remarkable RPS of90%when bath-vaccinated with the live attenuated vaccine (1×108CFU/ml). The specific antibody response of vaccinated zebrafish against V. anguillarum was found to gradually increase during28days post-vaccination. Moreover, the expressions of several immune-related genes including IL-1β、IL-8、MHC and MHC Ⅱ were enhanced in the spleens and livers of zebrafish, which was a symbol of the immune-response from vaccination to emerge the specific protection. In addition, the entrance of the live attenuated V. anguillarum was detected with PCR using toxR gene as a molecule marker after bath-vaccination for the entrance is critical to the administration of the live attenuated vaccine. As shown in the results, the toxR gene was detected in the gills and intestines samples on0.5h post bath-vaccination, which stated the gill and intestine could be the tissues for entrance of V. anguillarum MVAV6203. The gene was also found in the skin samples on0.5-3h post vaccination, which was consistent to the reported founding that skin is vital for the wild-type V. anguillarum to infect fish. It was indicated the intaking with water or the colonization of shin could be the route for the live attenuated V. anguillarum to induce immune responses. Furthermore, the immune responses including specific antibody and immune-related genes expression were investigated to clarify the mechanism responsible for protection in bath-vaccinated and non-vaccinated zebrafish after challenge. As the results, specific antibody response of fish was stronger in non-vaccinated zebrafish than that in vaccinated group in3days post infection. On the aspect of gene transcription, genes encoding pro-inflammatory cytokine and chemokine were much more up-regulated in non-vaccinated group than in vaccinated group after infection. On the contrast, the expression levels of adaptive immune-related genes were enhanced in vaccinated group after challenge. These results suggested that zebrafish vaccinated with the live attenuated vaccine was triggered the protection to avoid hurt by repressing the inflammation and strengthen the adaptive immunity at the early stage of infection.Meanwhile, the effects of booster vaccination with live attenuated V. anguillarum were investigated using bath-vaccination in a zebrafish model. Zebrafish that received booster doses at2weeks or both2and4weeks after primary vaccination were better protected compared to those in the non-booster group in the8weeks post vaccination. In addition, the booster did not induce a stronger specific antibody response. No correlation between a weak specific antibody response and strong protection was observed, indicating the complicated role of IgM in fish immunity.The maternal transfer is also an important aspect of fish immunity. The level of specific IgM against V. anguillarum did not rise in the embryo cytosol at14days post vaccination from the female broodstock injection-vaccinated with V. anguillarum MVAV6203. Meanwhile the bacteriolytic activity to V. anguillarum of the embryo cytosol did not change, but was up-regulated of that to E. coli. With Western blotting, the bands of lysozyme were detected with anti-zebrafish lysozyme monoclonal antibody in both embryo cytosol samples from vaccinated and non-vaccinated broodstock, but there was no difference between the two groups. The transcriptions of IL-8and lysozyme were initiated early in the embryo development. To the contrast, the mRNA of IgM was relied on maternal transfer. The vaccination on broodstock did not impact on the mRNA level of immune-related genes in the embryo. The larvaes at6days post fertilization from vaccinated and non-vaccinated zebrafish broodstock were bath-challenged with V. anguillarum. The V. anguillarum in larvaes from vaccinated group was eliminated more quickly than in non-vaccinated group, which suggests stronger clearness in larvaes from vaccinated broodstock. Meanwhile, the transcriptional levels of genes of innate immune were lower in larvaes from vaccinated zebrafish than those from non-vaccinated zebrafish after challenge. The results on the maternal transfer and the immunity of larvaes indicated that injection-vaccination with V. anguillarum MVAV6203has impact on the zebrafish immunity maternal transfer at14days post vaccination. In conclusion, the mechanism of immuneprotection of live attenuated V. anguillarum was comprehensively investigated using the zebrafish as a model animal. The results will facilitate the further study on the relationship between vaccine and fish immunity. |
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