This study aimed to characterize Mycoplasmosis bovis strain 16M—a highly virulent isolate from one Chinese outbreak—as a candidate for challenge models and inactivated vaccine development. We assessed strain 16M through morphological observation, PCR identification, drug susceptibility testing, growth titer and biofilm quantification, immunological profiling, and calf challenge experiments. We used genomic resequencing to evaluate the genetic stability across 150 passages. Classified as the prevalent ST52 lineage in China, strain 16M showed phylogenetic proximity to strain 08M and exhibited multidrug resistance (notably to macrolides). It achieved higher titers and stronger biofilm formation than other isolates and the reference strain PG45. In calves, intratracheal inoculation with 16M induced universal infection, severe pulmonary consolidation, and peribronchial cuffing, with significantly higher disease scores (p < 0.01). The inactivated 16M vaccine elicited elevated antigen-specific IgG titers, PBMC proliferation, and IFN-γ production versus PG45. Post challenge, immunized calves showed reduced pathological lesions, shorter bacterial shedding, and lower disease scores than the infected controls (p < 0.05). Genetic stability was confirmed for virulence-associated genes (e.g., adhesion proteins), with stable titers and biofilm production within 50 generations. Strain 16M combines high virulence for challenge modeling and industrial-scale vaccine suitability, owing to its robust growth, stable immunogenicity, and genetic consistency.

Mycoplasmopsis bovis 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 M. bovis from respiratory (n = 11) and milk (n = 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 M. bovis. This study underscores the adaptive potential of M. bovis during within-host colonization and cross-tissue transmission, providing critical insights for optimizing herd management and treatment strategies.

Mycoplasmopsis bovis is a major causative agent of bovine mastitis and causes substantial economic losses in the dairy industry because of the chronic and refractory nature of the infection. This study aimed to determine how M. bovis PG45 infection dynamically alters host metabolism and gene expression over time and whether these changes are accompanied by progressive redox imbalance and enrichment of multiple programmed cell death pathways. Transcriptomic and untargeted metabolomic profiles were analyzed at 8, 24, 48, and 72 h post-infection using three biological replicates for RNA sequencing and six biological replicates for metabolomic analysis. RNA-seq identified 779, 1777, 4631, and 4855 differentially expressed genes (DEGs) at 8, 24, 48, and 72 h, respectively, revealing a progressive expansion of transcriptional responses. KEGG enrichment analysis identified significant temporal shifts from early TLR, NF-κB, and TNF signaling toward cell-cycle regulation and DNA replication at later stages (FDR < 0.05). Metabolomic analysis identified 721, 1505, 1270, and 849 differentially abundant metabolites (DAMs), respectively, with prominent alterations in lipid, amino acid, and redox-related metabolism. Integrated transcriptomic–metabolomic analysis identified significant stage-specific gene–metabolite associations (|r| > 0.8, p < 0.05), including strong associations of (S)-Leucic acid with SAA3 and SGSH and of N-acetyl-L-aspartic acid with TANC1. A dynamic pattern of glutathione depletion, together with disruption of sphingolipid metabolism and enrichment of ferroptosis-related pathways, provided convergent correlative evidence of increasing redox and lipid metabolic stress during infection. Programmed cell death-related pathway analysis showed increasing prominence of apoptosis and cellular senescence at later stages. The RNA-seq findings were further supported by qRT-PCR validation, with strong concordance at all four time points (R = 0.99, p ≤ 1.62 × 10⁻⁴). Collectively, these findings support a time-dependent transition from inflammatory activation to metabolic reprogramming, redox imbalance, and programmed cell death, and provide correlative evidence that ferroptosis may contribute to injury of bovine mammary epithelial cells during M. bovis infection.