Mycoplasma synoviae (MS) is a major pathogen threatening China’s poultry industry, causing severe economic losses, and clarifying its epidemiology is pivotal for disease control and flock purification. In this study, a total of 3215 chicken samples collected from 643 broiler farms across 15 provinces in China in 2024 were analyzed. PCR detected 14% positivity (450 samples), and 18 isolates obtained from these positive samples (4.0% isolation rate). Multilocus sequence typing (MLST, 7 housekeeping genes) and neighbor-joining phylogenetic analysis (integrating 425 reference sequences from public databases) identified 13 distinct sequence types (STs), demonstrating considerable genetic diversity among circulating MS strains. Pathogenicity assessment of the five isolates revealed that the infected chickens exhibited varying degrees of infectious synovitis, while no respiratory signs were observed. In addition, antimicrobial susceptibility testing against 10 commonly used antibiotics was conducted on the 18 strains, providing urgently needed guidance for rational drug use in the clinical treatment of both breeder and broiler flocks. This large-scale epidemiological study yields crucial insights into the current prevalence and genetic diversity of MS in China and lays a scientific foundation for formulating targeted prevention strategies and optimizing management practices.

Infectious bursal disease virus (IBDV) and avian reovirus (ARV) are major immunosuppressive pathogens controlled through the widespread use of live attenuated vaccines. Concerns persist regarding potential immune interference when these vaccines are co-administered, though comprehensive in vivo data are lacking. Here, we reported the immunogenicity and protective efficacy of a live IBDV vaccine (W2512G-61) and a live ARV vaccine (ZJS) administered simultaneously or sequentially at 3-, 5-, and 7-day intervals in specific-pathogen-free (SPF) chickens. The IBDV live vaccine elicits strong, interval-independent humoral immunity and conferred 100% protection, demonstrating no compromise from ARV co-administration. Conversely, ARV-specific immunity was severely impaired by close temporal vaccination. ARV protection rates fell from 86.7% (ARV-only) to 46.7% with simultaneous administration and from 93.3% to 66.7% with a 3-day interval. Extending the interval to five or seven days eliminated this interference, restoring ARV antibody titers and protection to levels equivalent to ARV-only control vaccinated groups. This study provides the first definitive evidence of asymmetric immune interference between live IBDV and ARV vaccines. The results establish a minimum safe interval of five days to prevent interference and ensure robust ARV vaccine efficacy. These findings offer critical, evidence-based guidance for optimizing vaccination schedules to improve disease control in commercial poultry production.