Profiling of milk miRNAs associated with the innate immune system and pathway analysis in dairy cows with <i>S. aureus</i>-infected subclinical mastitis

We address the hypothesis that bioinformatics analysis can effectively identify miRNAs and target genes associated with the innate immune response to Staphylococcus aureus-induced mastitis in cows. Gram-positive bacteria, such as S. aureus, are some of the mastitis pathogens that cause subclinical infection and pose one of the most severe threats to dairy cattle due to their contagiousness and incidence. Therefore, newer molecular markers must be identified to predict bovine mastitis non-invasively with low error and high specificity. To address this, we conducted in-silico analysis to identify hub genes related to the innate immune system specific to the S. aureus pathogen in subclinical mastitis infections in cows, and experimental validation of the miRNAs of these determined hub genes was completed. As a result of bioinformatic analysis, we identified five hub genes (TLR2, MYD88, CASP4, NOD2 and TBK1) related to the innate immune system that are specific to the S. aureus pathogen. We investigated the expression levels of miRNAs associated with these genes (bta-miR-15a, bta-miR-16b, bta-miR-23a, bta-miR-27a-3p, bta-miR-103, bta-miR-146b, and bta-mir-374b) using real-time quantitative PCR. Except for bta-miR-15a and bta-miR-23a, all miRNAs studied varied in expression between healthy cows and cows with subclinical mastitis infected with S. aureus. This is the first study to bioinformatically determine hub genes specific to the innate immune system and the S. aureus pathogen in subclinical mastitis infections and then validate the determined miRNAs in milk between healthy and subclinical mastitis infected cows. The findings of our study expand our understanding of the roles of these miRNAs in cows with S. aureus-infected subclinical mastitis.