Gene Expression Profiles And Methylation Analysis On The Mammary Tissue Challenged With Streptococcus In Chinese Holstein

Streptococcus is one of the most important bacteria which causing dairy chronic infectious mastitis. Dairy mastitis seriously affected the quality of the raw milk, the economical utilization age limit of the cows, the economic benefits of the cow industry, the quality of its products and the consumer’s health. The disease has been studied by generations of scholars over a hundred years, and remarkable achievements have been achieved from the perspective of antibiotics, vaccines and resistance-associated genetic markers, the problem, however, still exists.1. In this study, Streptococcus was administered in healthy dairy cows to establish the disease model of the clinical mastitis. Using the "Agilent4×44K bovine microarray" to screen the differentially expressed genes closely related to streptococcal mastitis in dairy cows from the whole genome-wide respect, the regulatory mechanism of the immune system would be explored and elucidated preliminarily during streptococcal dairy mastitis. The results showed that:the disease model of Streptococcus-induced mastitis was successfully established. Through the observation and detection of the clinical symptoms of cows infected with Streptococcus, the model was confirmed to reflect the disease process of streptococcal dairy mastitis. The microarray analysis exposed136genes significantly differentially expressed (Fold change≥2.0, P<0.05), of which58genes were up-regulated in the Streptococcus group while78were down-regnlated. The GO analysis of differentially expressed genes showed that there are73 immune-related GO Terms. Pathway analysis with KEGG database of differentially expressed genes screened out13immune-related Pathyway, namely:Complement and coagulation cascades, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy, Leukocyte transendothelial migration, Chemokine signaling pathway, Viral myocarditis, Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, Basal transcription factors, MAPK signaling pathway, Cell adhesion molecules (CAMs), TGF-beta signaling pathway, Homologous recombination. Considering function and Pathyway of the differentially expressed genes, we ultimately selected19key genes as candidate genetic markers: C4BPB, CCL17, EPO, FST, IL12A, METTL13, TAF4B, ACTN2, CACNA1A, CD34, CD55, CDH13, CDH5, CXCR1, ELMO1, ITGA1, ITGB4, PROS1, VWF. The regulation mechanisms in the pathogenesis of dairy mastitis and the relationship with resistance to mastitis of these genes can be further studied.2. Using bisulfite sequencing technology to compare methylation degree of five candidate gene (CDH13, EPO, METT13, CXCR1, IL12A) promoters between streptococcal infected and normal mammary tissue and analyze the correlation between gene expression level and the degree of methylation. We want to elucidate regulation of DNA methylation during streptococcal dairy mastitis preliminarily. The results showed that:The degree of methylation of the promoter region of the EPO, METTL13gene in Streptococcus group was significantly greater than which in the PBS group(P<0.01), while the expression quantity of these two genes was significantly lower(P<0.05). This result can verified the principle that the negative correlation between gene expression level and the degree of methylation in the promoter region of DNA. We concluded preliminarily that these two genes involved in the regulation of DNA methylation during streptococcal dairy mastitis.