Bovine Mastitis-Derived Bacillus cereus in Inner Mongolia: Strain Characterization, Virulence Factor Identification, and Pathogenicity Validation

This study aimed to investigate the epidemiological characteristics and antimicrobial resistance patterns of <i>Bacillus cereus</i> (<i>B. cereus</i>) in bovine mastitis within Inner Mongolian dairy herds, with a focus on virulence gene distribution and their clinical implications. A cross-sectional epidemiological investigation was conducted across three large-scale dairy farms. A total of 340 bacterial strains were isolated from milk samples collected from 108 cows with clinical mastitis, all of which underwent comprehensive PCR testing. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method against eight antibiotics. Virulence gene profiling was conducted for all <i>B. cereus</i> isolates, and murine challenge experiments were performed to assess virulence-factor-dependent pathogenicity. Bacteriological analysis identified <i>B. cereus</i> as the predominant pathogen (104 strains, 30.58%), followed by <i>Staphylococcus</i> spp. (74 strains, 21.76%). Antimicrobial susceptibility testing revealed high resistance to tetracycline (38.46%), cotrimoxazole (15.38%), and ciprofloxacin (7.69%), while complete sensitivity (100%) was observed for gentamicin, amikacin, and roxithromycin. Virulence gene profiling demonstrated universal presence of <i>nheA</i>, <i>nheB</i>, and <i>entFM</i> genes in all isolates, with <i>bh1D</i> detected in only 21.15% (22/104) of strains. Murine challenge experiments confirmed virulence-factor-dependent pathogenicity, with strains harboring nine virulence factors inducing significant upregulation of hepatic inflammatory markers (<i>p</i> < 0.05) and histopathological alterations in hepatic and renal tissues compared to strains with three virulence factors. Our findings highlight <i>B. cereus</i> as an emerging virulent pathogen in Inner Mongolian dairy herds, necessitating enhanced surveillance of virulence factors and antimicrobial stewardship in mastitis management. This study provides critical epidemiological data to inform clinical veterinary practices and targeted intervention strategies.