Enhanced mucosal immunity and protection against Aeromonas veronii infection in crucian carp via synergistic immunization with Bacillus coagulans and recombinant Lactobacillus casei expressing mshB gene.

Aeromonas veronii (A. veronii) is a pathogen that causes zoonotic diseases in humans, animals, and aquatic organisms, leading to symptoms such as scale loss, red and swollen anus, and hemorrhaging of tissues and organs in aquatic life. To better combat infections caused by A. veronii, this study employed a previously constructed and stored recombinant Lactobacillus casei (L. casei) strain that expresses the mshB gene from A. veronii TH0426, coding for type IV pili (MSHA). This strain was used in conjunction with Bacillus coagulans (B. coagulans) for oral immunization, and its immunological efficacy in carp was evaluated. The results indicated that combining immunization with the recombinant L. casei expressing mshB and B. coagulans significantly enhances the mucosal immune response compared to using the recombinant L. casei alone. In vitro coculture experiments showed that B. coagulans BC1 promotes the growth of recombinant Lc-pPG612-mshB. Antibacterial assays demonstrated that the recombinant Lc-pPG612-mshB exhibits antibacterial activity against seven common pathogenic bacteria. The group that received combined oral immunization with recombinant Lc-pPG612-mshB and BC1 showed significantly higher levels of specific IgM antibodies and activities of ACP, AKP, SOD, LZM, C3, and C4 enzymes in their serum compared to the group that received only the recombinant Lc-pPG612-mshB. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of IL-10, IL-1β, TNF-α, and TGF-β cytokines in various tissues were significantly elevated in the group receiving combined immunization with Lc-pPG612-mshB and BC1 compared to those receiving only Lc-pPG612-mshB. In challenge tests, the recombinant lactic acid bacteria could effectively resist infection by A. veronii TH0426, with relative protection rates of 48.0 % and 56.0 % for the groups using it alone and in combination, respectively. Bacterial load testing showed that the group receiving combined immunization with Lc-pPG612-mshB and BC1 had a significantly lower bacterial load of A. veronii TH0426 three days post-immunization compared to the group receiving only Lc-pPG612-mshB, indicating that the combined immunization group better inhibits the proliferation of A. veronii TH0426 in carp, thereby increasing the protection rate. In summary, the results of this study indicate that B. coagulans BC1 can promote the growth of recombinant L. casei Lc-pPG612-mshB and enhance its antibacterial effects. Additionally, it can induce the production of more specific IgM antibodies in carp, enhance the activity of immune-related enzymes in the serum, promote the transcription level of cytokines in tissues, and improve resistance to A. veronii TH0426. The findings of this research provide a theoretical basis for the application of orally administered recombinant lactic acid bacteria as vaccine carriers in aquaculture.