Mycoplasma synoviae (M. synoviae) is a serious avian pathogen that causes significant economic losses to chicken and turkey producers worldwide. The currently available live attenuated and inactivated vaccines provide limited protection. The objective of this study was to identify potential subunit vaccine candidates using immunoproteomics and reverse vaccinology analyses and to evaluate their preliminary protection. Twenty-four candidate antigens were identified, and five of them, namely RS01790 (a putative sugar ABC transporter lipoprotein), BMP (a substrate-binding protein of the BMP family ABC transporter), GrpE (a nucleotide exchange factor), RS00900 (a putative nuclease), and RS00275 (an uncharacterized protein), were selected to evaluate their immunogenicity and preliminary protection. The results showed that all five antigens had good immunogenicity, and they were localized on the M. synoviae cell membrane. The antigens induced specific humoral and cellular immune responses, and the vaccinated chickens exhibited significantly greater body weight gain and lower air sac lesion scores and tracheal mucosal thicknesses. Additionally, the vaccinated chickens had lower M. synoviae loads in throat swabs than non-vaccinated chickens. The protective effect of the RS01790, BMP, GrpE, and RS00900 vaccines was better than that of the RS00275 vaccine. In conclusion, our study demonstrates the potential of subunit vaccines as a new approach to developing M. synoviae vaccines, providing new ideas for controlling the spread of M. synoviae worldwide.

Mycoplasma synoviae (M. synoviae) is a serious avian pathogen causing severe economic losses to chicken and turkey producers worldwide. The currently available live attenuated vaccine and inactivated vaccine on the market elicit only limited protection. The aim of this study was to identify antigenic proteins of M. synoviae as potential subunit vaccine candidates. In immunoproteomics and reverse vaccinology analyses, a total 24 candidate antigens were picked out. Five candidate antigens were selected to evaluate immunogenicity and preliminary protection in a chicken model, including RS01790 (lipoprotein, putative sugar ABC transporter), BMP (BMP family ABC transporter substrate-binding protein), GrpE (nucleotide exchange factor), RS00900 (putative nuclease) and RS00275 (Uncharacterized protein). The results showed that the five antigens had good immunogenicity. They could be localized on the M. synoviae cell membrane and adhere to DF-1 cells using indirect immunofluorescence assay. They induced specific humoral and cellular immune responses. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes. As a result, the vaccinated chickens revealed significantly higher body weight gain, with lower air sac lesion scores and tracheal mucosal thicknesses. The vaccinated chickens also showed lower M. synoviae loads in throat swabs than nonvaccinated chickens. The protective effect of BMP, GrpE and RS00900 vaccines is better than that of RS01790 and RS00275. In conclusion, our study demonstrates the potential of using subunit vaccine, providing new ideas for the development of M. synoviae vaccines. The vaccine candidates may contribute to the future development of therapeutic strategies to control the spread of M. synoviae worldwide.