The Phenotypic Divergence and Potential Microevolution of a Dominant Mycoplasmopsis bovis ST-52 Clone Within a Closed Dairy Herd in China

<i>Mycoplasmopsis bovis</i> is a significant pathogen causing substantial economic losses in cattle, yet its within-herd microevolution remains poorly understood. This study aimed to characterize phenotypic and genomic variations within a dominant ST-52 clone circulating in a closed dairy herd. We isolated <i>M. bovis</i> from respiratory (<i>n</i> = 11) and milk (<i>n</i> = 5) samples. Phenotypic characterization included biofilm formation, antimicrobial susceptibility testing, and cellular invasion assays. Whole-genome sequencing was performed on four representative isolates to identify genetic variations. All isolates were genetically identical according to MLST (ST-52). However, significant phenotypic diversity was observed. Biofilm formation capacity varied significantly (OD595 from 0.25 to 1.10), and resistance to doxycycline was higher in nose swabs (100%) than milk isolates (20%). Cellular invasion assays demonstrated that all isolates could invade bovine-derived cells (MDBK, MAC-T, EBL, and PBMC), but the invasion efficiency differed by strain and cell type. These findings confirm the circulation of a single genetic lineage within a closed herd while highlighting significant phenotypic diversification in biofilm formation, antibiotic resistance, and cellular invasiveness. The results provide evidence consistent with microevolution and underscore the adaptive potential of <i>M. bovis</i>. This study underscores the adaptive potential of <i>M. bovis</i> during within-host colonization and cross-tissue transmission, providing critical insights for optimizing herd management and treatment strategies.