Next-generation live vector vaccine targeting Mycoplasma synoviae and Mycoplasma gallisepticum via recombinant Salmonella.

Avian mycoplasmosis has a considerable financial impact on the global chicken industry with Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) being the dominant causes. Currently, inactivated vaccines provide minimal cross-protection and can't induce high levels of mucosal immunity, indicating the need to develop more effective immunisation platforms. This study aimed at designing and testing rationally engineered live recombinant vaccines using Salmonella enterica serovar Typhimurium expressing MS- and MG-specific antigens, GrpE and CrmA, to produce a robust immune response. Three recombinant Salmonella &#x394;phoPQ strains, including SG1 (GrpE), SG2 (CrmA), and SFGs (GrpE + CrmA), were designed and verified by western blotting. The humoral (IgG), mucosal (IgA), cytokine (IFN- &#x3b1;, IL-1&#x3b2;, TNF- &#x3b1;), and innate immune (TLR15, iNOS) responses were determined using ELISA and qRT-PCR. The effectiveness of protection was determined by the use of clinical indicators, bacterial load measurements, histological examination, and survival parameters following the homologous challenge of MS or MG. The results showed that SG1 and SG2 significantly increased mucosal IgA titers (OD: 0.82 ± 0.02 and 0.77 ± 0.01, respectively) and antigen-specific IgG (mean OD: 1.66 ± 0.03 and 3.17 ± 0.06, respectively) relative to inactivated vaccine and control groups (p < 0.0001). The IgG (1.24 ± 0.04) and IgA (0.73 ± 0.01) of SFGs were moderate, indicating a dual expression of antigens in them. These reactions were accompanied by the upregulation of IFN-alpha, IL-1beta, TNF-alpha, TLR15, and iNOS gene expression, indicating the activation of Th1-biased and innate immune responses. Recombinant strains showed strong protective performance, with a survival rate of 90 to 95% after challenge and also reduced shedding of bacteria as well as histological changes. As a result, strains of Recombinant Salmonella expressing GrpE and CrmA resulted in robust humoral, innate, and mucosal immune responses, which provide significant protection against MS and MG.