The Changes Of Relative Inflammatory Cytokines In The Acute Phase Of Mastitis Induced By Staphylococcus Aureus In Rats

Bovine mastitis is an important infectious disease for dairy cattle, which can affect the production of milk. One of the most important causes of bovine mastitis is the intramammary infection caused by Staphylococcus aureus. Up to date, few methods have been found to prevent or treat mastitis by Staphylococcus aureus infection. The aim of this experiment was to observe clinical symptoms, the hematological changes, the change in immune organs and histopathological changes, and to detect the level of the inflammatory cytokines in mRNA and protein by establishing mastitis model by injecting Staphylococcus aureus via teat duct in rats.Eighty-four healthy and pregnant wistar rats were selected, with a body weight of (275±25) g and of the same age. The rats were randomly divided into experimental groups and control groups(n=42).0.1ml Staphylococcus aureus liquid (2×107cfu/mL) was inoculated into one side of the fourth(abdominal) mammary gland via the teat duct96hours post parturition.The rats of the control groups were injected with0.1ml physiological saline solution.At intervals of Oh,6h,12h,24h,48h,72h and96h (n=6) after infusion, six rats from the control and experimental groups respectively,were euthanized.Blood samples were collected for blood routine examination, serum samples were used to check relative cytokines by ELISA,the weisht and the index of thymus and spleen were measured, mammary tissue was used to observe histomorphological changes,the mammary gland was collected to count Staphylococcus aureus, the gene expression of TLR2,NOD2,MyD88,RIP2,NF-κB,IL-1β,IL-6,CXCL1,IL-10, TNF-a,TGF-β1,IFN-y in mammary tissue were detected by qRT-PCR. The results are as follows:1. After injected Staphylococcus aureus, mammary gland became swollen and fever. Apart from the48h, the index of spleen in experimental groups became significantly larger after injection (P<0.05or P<0.01). The index of thymus in experimental groups also became significantly larger after injection (P<0.05or P<0.01).The amount of Staphylococcus aureus in mammary glands decreased significantly in after injected (P<0.01). Compared to Oh, the number of WBC and Lymphocytes increased significantly at96h after injected in experimental groups (P<0.01). Compared to Oh, the number of PMN significantly increased at6h and12h after injected in experimental groups (P<0.01). There was no change in mammary gland of control groups post injection. Bacteria were not isolated from the control group. The index of spleen and thymus, the number of various types of inflammatory cells in the blood showed no significant changes after injected in control groups (P>0.05).2. At6h after injected, a few inflammatory cells were located in mammary gland. A large number of inflammatory cells appeared in mammary gland at12h and24h after injected. Mammary gland alveoli began to rupture and shrink, while the number of inflammatory cells located in the mammary gland began to decrease at48h,72h and96h after injected. The mammary gland in control groups showed no histopathologic changes after injected.3. After injecting Staphylococcus aureus, the mRNA expression of TLR2,NOD2,MyD88, RIP2,NF-KB,TNF-a,IL-1β,IL-6and CXCL1showed a significant up-regulation in mammary gland (P<0.05or P<0.01). The mRNA expression of TGF-β1showed a significant down-regulation (P<0.05) then up-regulation gradually. The mRNA expression of IFN-y showed a significant up-regulation at48h after injected (P<0.01). The mRNA expression of IL-10showed a significant up-regulation at48h after injected (P<0.01). After injected, the protein expression of TNF-a showed a significant down-regulation (P<0.05or P<0.01) in serum. The protein expression of IFN-y showed a downward trend in serum and a significant down-regulation in serum at48h after injected (P<0.05). The protein expression of IL-1β, IL-6, IL-8and IL-10in serum displayed no significant changes. The expression of these genes in mammary gland and the content of these protein in the serum had no significant changes in control groups (.P>0.05).In conclusions:when staphylococcus aureus was injected into the mammary gland of rats, then the mRNA expression of TLR2and NOD2increased. Innate immune response was activated. Finally a large number of inflammatory cells recruited to sites of inflammation in chemokines (such as TNF-a and CXCL1) effect and then more cytokines (such as IL-1β,IL-6, CXCL1and IFN-y) were released. In the cooperation of cytokines, immune cells eliminated invasive bacteria and caused damage to the mammary tissues. As the infection was controlled, Suppression of inflammatory cytokines(such as IL-10and TGF-β1) began to play a leading role. So after staphylococcus aureus infected into the mammary gland, the innate immunity of TLR and NOD was activated, which participated in the immune regulation of the local mammary gland.