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Staphylococcus aureus  intramammary infection is one of the most common causes of chronic mastitis in dairy cows, whose development may be associated with epigenetic changes in the expression of important host defense genes. This study aimed to construct a genome-wide DNA methylation profile of the mammary gland of Chinese Holstein cows ( n  = 3) following experimentally induced  S. aureus  mastitis, and to explore the potential gene regulatory mechanisms affected by DNA methylation during  S. aureus  mastitis. DNA was extracted from  S. aureus -positive ( n  = 3) and  S. aureu s-negative ( n  = 3) mammary gland quarters and subjected to methylation-dependent restriction-site associated DNA sequencing (Methyl-RAD Seq). Results showed that C m CGG/C m CWGG DNA methylation sites were unevenly distributed and concentrated on chromosomes 5, 11, and 19, and within intergenic regions and intron regions of genes. Compared with healthy control quarters, 9,181 significantly differentially methylated (DM) C m CGG sites and 1,790 DM C m CWGG sites were found in the  S. aureus -positive quarters ( P  < 0.05, |log2FC| > 1). Furthermore, 363 C m CGG differently methylated genes (DMGs) and 301 C m CWGG DMGs (adjusted  P  < 0.05, |log2FC| > 1) were identified. Gene ontology and KEGG enrichment analysis indicated that C m CGG DMGs are involved in immune response pathways, while the C m CWGG DMGs were mainly enriched in gene ontology terms related to metabolism. The mRNAs of 526 differentially methylated C m CGG genes and 124 differentially methylated C m CWGG genes were also significantly differentially expressed (RNA-Seq data) in the same samples, herein denoted differentially methylated and expressed genes (DMEGs) ( P  < 0.05). Functional enrichment analysis of DMEGs revealed roles related to biological processes, especially the regulation of immune response to diseases. C m CGG DMEGs like  IL6R ,  TNF ,  BTK ,  IL1R 2, and  TNFSF8  enriched in several immune-related GO terms and pathways indicated their important roles in host immune response and their potential as candidate genes for  S. aureus  mastitis. These results suggest potential regulatory roles for DNA methylation in bovine mammary gland processes during  S. aureus  mastitis and serves as a reference for future epigenetic regulation and mechanistic studies.

Genome-Wide DNA Methylation Analysis of Mammary Gland Tissues From Chinese Holstein Cows With Staphylococcus aureus Induced Mastitis