| Staphylococcus aureus is one of the most important pathogenic bacteria which causing dairy mastitis, and the infection rate is very high. In this study, S. aureus was used to infect dairy mammary gland in Chinese Holstein cows. After infection, the clinical diagnosis of mastitis, bacterial detection, and pathological examination of breast tissue were observed. By using Affymetrix Bovine cDNA microarray, the differences in genes expression profiles in the whole genome-wide were analyzed. Q-PCR, ELISA and Western Blot methods were used to verify the microarray results, then the differentially expressed genes features were detected by using GO and Pathway analysis. Bovine CACNA2D1 gene was considered to be a candidate gene for mastitis resistance traits, and the association between gene and Somatic Cell Score (SCS) was analyzed. The main results are as follows:1. The disease model of the clinical mastitis which caused by artificially infected with S. aureus was successfully established, after the cows infected by the S.aureus bacteria, cows showed the obvious signs of clinical mastitis. The model reflected the same process of mammary inflammation of the natural disease.2. Microarray analysis of differentially expressed genes were screened out of 305 genes, of which 161 genes were upregulated and 144 downregulated genes (FC>2, P<0.05). Using Q-PCR mathod on the part of significant differentially expressed genes were verified, including the IL8, CSNISI, CD14, TLR4, STAT5A, S100A12, LBP,IL-1β, MFGE8, SELENBP1, the results proved that Q-PCR analysis and statistical analysis of microarray data were consistent, which indicating that this microarray data of differentially expressed genes obtained with reliability.3. The differentially expressed genes were analyzed by GO analysis, we found significant differences in the expression of genes involved in 138 GO Terms (P<0.05). Pathway analysis screened out a total of 9 significant Pathway, namely:TOLL-like receptor signaling pathway, NOD-like receptor signaling pathway, Cytosolic DNA-sensing pathway, Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, RIG-I-like receptor signaling pathway, chemokine signaling pathway, MAPK signaling pathway, B cell receptor signaling pathway. Further detected in the hub of the Pathway of differentially expressed genes, and ultimately got 27 key genes:IL8, TLR4, TLR2, CD36, CD14, CD40, LBP, CXCL10, IL6, CXCL5, IL1A, IL1B, IL6R, BIRC3, CACNA2D1, CCL2, DAPP1, DUSP10, IKBKE, LGP2, MKNK1, VAV1, PRKCA, PTPN5, SOS2, TNFSF13B, NFKB1A. These candidate genes maybe used for resistance to mastitis.4. ELISA method was used to analyzed clinical mastitis which caused by the artificial infection of S.aureus. In the inflammatory process caused by breast tissue, the content of inflammatory cytokines IL8 was highly expressed in the infected mammary tissue compared with the normal breast tissue. However, as one of the most abundant and highly expressed protein in milk, CSN1S1 was significantly lower expression of the normal group, this result was matched the actual physiological process of inflammation in the dairy mastitis. 5. Western Blot results shown that IL8 and STAT5A protein were expressed in the mammary gland both experimental group and normal group, but its protein expression were significant differences. In the infected mammary tissue, IL8 protein expression was significantly higher than the normal group, while STAT5A gene expression showed a significantly lower expression of the normal group, the results also proved that the microarray results was reliably in the protein expression level.6. The association analysis showed that the SCS values of GG genotype of G519663A and A526745G were significantly lower than the AG and AA genotypes. The results of this research will be useful in further studies to determine the role of the CACNA2D1 gene in mastitis resistance and further work will be necessary to investigate whether the CACNA2D1 gene play a role in defending the host from mastitis.
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