Oxidative-Stress-Mediated AMPK/mTOR Signaling in Bovine Mastitis: An Integrative Analysis Combining 16S rDNA Sequencing and Molecular Pathology

The bovine mammary gland, the exclusive site of milk synthesis, is a structurally specialized tissue that houses distinct cellular subsets, yet it remains highly susceptible to major mastitis pathogens, including <i>Staphylococcus aureus</i>, <i>Streptococcus agalactiae</i>, and <i>Escherichia coli</i>. Infection disrupts redox homeostasis, leading to excessive accumulation of reactive oxygen species (ROS) and rapid activation of antioxidant pathways. In this study, we integrated 16S DNA sequencing, histopathology (hematoxylin and eosin), and immunohistochemistry to map the mastitis-associated microbiota and visualize oxidative-damage foci in mammary tissues challenged by <i>Staphylococcus aureus</i>, <i>Streptococcus agalactiae</i>, or <i>Escherichia coli</i>. Quantitative reverse transcription polymerase chain reaction and Western blot analyses were subsequently performed on the same samples to measure the kinetic response of six oxidative-stress-related signalling nodes: adenosine 5'-monophosphate-activated protein kinase, cytochrome P450 1A1, heme oxygenase 1, nitric oxide synthase, mammalian target of rapamycin, and superoxide dismutase. By correlating the temporal expression patterns of these genes/proteins with ROS accumulation and histological severity, this study delineates the molecular cascade linking oxidative imbalance to mastitis pathology, providing data-driven targets for future preventive and therapeutic strategies.