Portal Of Entry For Two Pathogens And A Live Attenuated Vaccine Into Fish

Vibrio anguillarum is a causative agent of vibriosis in a variety of commercial farmed fish species in China, taking the mucosal tissues as the portal of entry to get into the fish. Presently, mucosal injuries resulted from rude handling, ectoparasite infestations and enteritis are common in aquaculture due to the increasing intensive cultivation. However, the effect of mucosal injuries on the virulence of V. anguillarum has not been reported detailedly. In the current study, the wild-type pathogenic V. anguillarum MVM425load in the mucosal tissues of healthy zebrafish was investigated after bath challenge using qPCR assays. As a result, MVM425was found to persist in the intestine and skin of zebrafish for a few hours and proliferate rapidly in the skin. Moreover, two injured models of skin and intestine were established to study V. anguillarum infection when zebrafish was under the stress of mucosal injury. As a result, the LD50values of healthy, skin-injured and intestine-injured zebrafish caused by V. anguillarum MVM425were106,103, and106CFU/ml, respectively. Furthermore, V. anguillarum was proved to break through the physical barrier via skin injury, proliferate rapidly, and cause a systemic vibriosis symptom finally. Therefore, it is supposed that strengthening the daily aquaculture management and avoiding mucosal tissue injuries, especially skin mucus injury, will be effective to defense against V. anguillarum infection.Vaccination is one of the effective means of preventing vibriosis. A live attenuated V. anguillarum vaccine MVAV6203has been successfully developed in our laboratory, protecting Scophthalmus maximus and Paralichthys olivaceus efficiently against V. anguillarum infection. However, the portal of entry for the vaccine as well as the mucosal immune responses induced by the vaccine has not been clear yet. In the study, the vaccine load in the mucosal tissues after bath vaccination was investigated by qPCR assays and TEM. Intestine and skin of zebrafish were the preferable tissues for MVAV6203proliferation and bacteria persisted in the intestine for a longer time than those in the skin. As the results shown in TEM images, only a few structures of vaccine bacteria were observed in the gills at0h post vaccination (p.v.) and in the skin at3h p.v. while they were detected in the intestine until72h p.v. Moreover, the relative expressions of TLR5in intestine, skin and gills at12h p.v. were4.7-,7.4-, and3.8-folds, respectively. Besides, the relative expressions of TLR2and TLR4in intestine were up-regulated significantly and a MyD88-dependent signaling pathway was triggered to produce cytokines such as IL-1β, IL-6, IL-8and TNF-a. Furthermore, slight inflammatory responses occurred in the intestine by histopathologic analysis with an obvious increase of intraepithelial lymphocytes from6h p.v. Therefore, the intestine of zebrafish was the mainly portal of entry for MVAV6203and the origin site for mucosal immune responses.It was confirmed that MVAV6203took the intestine as the main access to zebrafish and triggered the mucosal immune responses. However, the process of local and systemic immune responses induced by the vaccine and the relationship between them has not been clear yet. In the study, the process and effect of immune responses induced by MVAV6203at local tissues and systemic organs were investigated by RT-qPCR. In the early stage of vaccination, mononuclear phagocytes in the kidney proliferated rapidly and expressed TLR5. The expressions of b7r and TLR5were5.7-and4.5-folds at3d p.v. Then the cells transported to mucosal tissues via blood circulation for phagocytosis and antigen delivery. At21d p.v.,3.3-and1.9-fold elevation of b7r and TLR5were observed in the skin. Moreover,1.7-and5.5-fold increased transcriptions of MHC Ⅰ and MHC Ⅱ were detected in the spleen and kidney after bath-vaccination for3days while a delayed elevation of these two genes was detected in the intestine at7d p.v. At21d p.v., both MHC Ⅰ and MHC Ⅱ in the skin (>1.7-fold) showed obvious elevations and the expressions of CD4and CD8(>3.4-fold) increased as well. It is speculated that immune cells migrated from systemic organs to local tissues. Furthermore, T cell differentiation related genes were firstly increased in systemic organs and then upregulated in local tissues. Significant up-regulations were observed in the kidney at3d p.v. and in the skin and intestine from14to21d p.v. Therefore, both systemic and local immune responses were induced by MVAV6203and immune components were transported between the two parts.An efficient immune protection could be obtained by MVAV6203vaccination via both immersion and injection. However, the immune process induced by the different vaccination modes has not been clear yet. In the study, this issue was clarified using RT-qPCR. The immune cells were activated for antigen recognization and processing with the proliferation and differentiation of T cells after MVAV6203vaccination to enhance the nonspecific and specific immune defenses. However, the immune responses induced by injection vaccination happened earlier than that induced by immersion vaccination. Moreover, antibody-mediated immunity was induced by the two inoculation modes finally. Therefore, it was confirmed that both injection and immersion vaccinations obtained a similar protection.As well, Edwardsiella tarda is a causative agent of edwardsiellosis in China, resulting in large-scale death and great losses in aquaculture. It was demonstrated that zebrafish was not susceptible to E. tarda infection even with a high infective dose in our previous work. However, the effect of mucosal injuries on the virulence of E. tarda has not been reported detailedly as well. Thus, this issue was determined in the current study. The LD50values of skin-and intestine-injured zebrafish caused by E. tarda EIB202were107and105CFU/ml, respectively, suggesting that intestine injury would be the inducement of E. tarda infection. Moreover, EIB202induced inflammatory responses in several tissues of intestine-injured zebrafish. Antimicrobial responses such as neutrophil recruitment and antibacterial peptides release during E. tarda infection might inhibit the growth of intestinal microbiota that E. tarda could resist. Besides, bacteria that were not eliminated by immune system colud colonize in intestine for a long time. Therefore, it might be helpful to prevent enteritis for the control of edwardsiellosis in aquaculture.In conclusion, injured skin and intestine of zebrafish were the inducement for V. anguillarum and E. tarda infection. Intestine was the main portal of entry for MVAV6203and the origin of mucosal immune responses. Moreover, both systemic and local immune responses were induced by MVAV6203, and immune components were transported between two responses. Furthermore, both injection and immersion vaccinations obtained a similar protective effect.