The Detection Of Three Cytokines In Milk Of Dairy Cattle Mastitis And Effect Of Tumor Necrosis Factor-α On Proliferation And Apoptosis In Primary Cultured Mammary Cell

The dairy cattle mastitis is a kind of important disease. It brought about large economy losses to bovine breed aquatics. The pathogenic bacteria infection is a significant cause of mastitis incidence,cytokine was released to participate in immune response and regulate local inflammatory response after the pathogenic bacteria infections. So cytokine is of paramount importance to diagnose and treat dairy cattle mastitis.In this study, the bacteria was isolated and identified in natural case (clinical mastitis and subclinical mastitis) milk. The three main type of cytokine (IL-1,IL-2,TNF-α)was detected using radioimmunoassay, and investigated the relationship among them .The Somatic Cell Count(SCC) in the milk from patients of mastitis was tested by Automated Somatic Cell Counter Observation , and cell morphology was carried out using Wright's staining assay in the milk, We used the primary cultured mammary glandular cell (mammary epithelial cell and fibroblast) as a model in vitro, The mammary epithelial cell were treated with various concentration of tumor necrosis factor-α(1ng/ml, 5ng/ml, 8ng/ml, 10ng/ml, 15ng/ml) for five time point (3h, 6h, 24h, 48h, 72h), the apoptotic cells were determined using a ?ow cytometer. For evaluation fibroblast proliferation, the fibroblast were treated with various concentration of tumor necrosis factor-α(0.1ng/ml, 1ng/ml, 5ng/ml, 10ng/mL, 50ng/mL) for five time points (3h, 6h, 24h, 48h, 72h), Cell viability was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.The result indicated that the several main type of cytokine concentration in the milk from the patient of mastitis infected different pathogenic bacteria were enhanced to different degrees, TNF-αconcentration was significant difference (p<0.05) in the milk serum between mastitis case and normal dairy cattle, TNF-αconcentration in the milk serum of subclinical mastitis is higher than clinical mastitis.The bacteria isolated and identified result showed that the pathogenic bacteria was Gram-Negative bacillus and Streptococcus by primarily in clinical mastitis case, the great majority were E.coli; while the pathogenic bacteria was Staphylococcus by primarily in subclinical mastitis case. TNF-αconcentration in the milk serum of subclinical mastitis case by Staphylococcus infected were obviously higher, followed by Gram-Negative bacillus.The SCC in the milk of mastitis case increased remarkably (p<0.01) than normal dairy cattle. In addition, the SCC was positively correlated with TNF-αconcentration in the milk from patients of mastiti(sr=0.59612), the milk Wright's staining analysis demonstrated that the number of neutrophile granulocyte, mononuclear-macrophage and lymphocyte increased significantly.With an increase of TNF-αconcentration, TNF-αcould evidently promote fibroblast proliferation in the mammary gland. and tended dose effect relationship, all concentration gradient of TNF-αcould facilitate fibroblast proliferation at different time point (24h, 48h, 72h, 96h), various concentration of TNF-αhad started an effect for mammary gland fibroblast from 24h. The proliferation effect of TNF-αaffected mammary gland fibroblast is the most significant at 72h.TNF-αcould induce mammary epithelial cell apoptosis. With an increase of TNF-αconcentration cellular apoptosis rate showed typical dose effect relationship. The apoptosis was closely related to the action of TNF-αat different time point. Earlier cells apoptosis was induced by TNF-αat 24h primarily.