Novel insights into Radix Pseudostellariae saponins assist Mycoplasma gallisepticum attenuated vaccine to enhance the immunoprotection of chicken.

Mycoplasma gallisepticum (MG) is a major pathogen causing chronic respiratory disease in chickens, and the MG-attenuated vaccine (MGAV) often fails to induce effective mucosal immunity. Radix Pseudostellariae saponins (RPS), important bioactive components of the traditional Chinese herb, have been utilized for immunomodulation, although the underlying mechanisms remain to be elucidated. In this study, chickens were randomly assigned to groups receiving intranasal MGAV alone, MGAV plus RPS at different doses, or appropriate controls. Serum, tracheal mucosa, and respiratory lavage samples were collected for antibody measurement, cytokine profiling, histological assessment, and metagenomic/metabolomic analyses. RPS significantly increased MG-specific antibody levels and enhanced expression of the immunomodulatory cytokines, while suppressing that of pro-inflammatory factors. It also promoted tracheal mucosal integrity by elevating goblet cell numbers and mucosal epithelial height. Through metagenomic and metabolomic analysis, RPS was found to enrich the respiratory bacterium Lactobacillus sp. UMNPBX13, which was positively correlated with the metabolite phosphatidylcholine (PC). Intranasal administration of Lactobacillus increased mucin 2 (MUC2) and PC levels in tracheal mucosa. Similarly, PC combined with MGAV enhanced mucosal immunity by modulating cytokines and increasing MG-specific antibodies. These findings demonstrate that RPS enhances MGAV-induced respiratory mucosal immunity and provides a novel adjuvant strategy for improving poultry respiratory disease vaccines by modulating the respiratory microbiota, specifically through enrichment of Lactobacillus sp. UMNPBX13, which promotes PC production.IMPORTANCEMycoplasma gallisepticum (MG) constitutes a notable challenge to poultry health. Although the MG-attenuated vaccine (MGAV) is widely used for disease control, it often fails to induce robust mucosal immunity. Here, we demonstrate that intranasal co-administration of Radix Pseudostellariae saponins (RPS) with MGAV augments mucosal immunoprotection. Integrated metagenomic and metabolomic analyses revealed that RPS enriches respiratory Lactobacillus, which in turn promotes the production of its key metabolite phosphatidylcholine (PC), thereby supporting mucosal immune enhancement. These findings offer a promising strategy for improving poultry vaccine efficacy and may provide a basis for future exploration of mucosal vaccination approaches in other animal species.