Mycoplasma bovis is an important cause of bovine mastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M. bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced
bovine mastitis. Cytotoxicity (as indicated by release of
lactate dehydrogenase, LDH) from bMEC depended on multiplicity of
infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (
hematoxylin and
eosin) of cytoplasm were apparent 24 h after
infection with ST172 or ST173 M. bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after
infection with MOI of 1:1,000. Both production of
reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after
infection. Increased Bax/Bcl-2 ratios and activation of
caspase-3 implied involvement of mitochondria-dependent pathways of apoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved
caspase-3 were mitigated by
N-acetyl-l-cysteine, a ROS scavenger. Both
interleukin (IL)-1β and
IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of
tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway of apoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced
bovine mastitis.