Identification Of Major Pathogens In Bovine Mastitis And Prokaryotic Expression Of The Staphylococcal α-Hemolysin

Mastitis was one of the most common disease, usually due to a microbial infection, which caused large economic loses for the dairy industry. So far, we were short of an accurate and quick method to detect the major pathogens; in addition, there were very few data about molecular epidemiology. And there were no very useful vaccine against mastitis.Based on these backgrounds, a series of studies about dairy cows mastitis were carried out. From this study, some results were reported as followings:Part one: Isolate and identify pathogens causing cow mastitis To isolate and identify pathogens causing cow mastitis ,samples of cow mastitis from three regions of Shandong province (Jinan,Taian and Linyi) were investigated. The results of this study indicated that the most common bacterial species causing mastitis were Staphylococci , Streptococci and Escherichia coli. These results were very critical to the prevention and therapy of cow mastitis.Part two: Identification of major pathogens in cow mastitis by PCR Three sets of specific primers were designed for the major pathogens which caused mastitis: Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. The specificity of the assay was 100%.And the sensitivity of the assay was 1.25×103 cfu/ml .In this study, a molecular tool to identify the major pathogens involved in mammary gland infections in cows with rapidity, sensitivity, and specificity was developed.Part three: Prokaryotic expression and bioactivity assay of the Staphylococcalα-Hemolysin.To express the Staphylococcalα-Hemolysin gene in E. coli and study its primary biological characterristics,α-Hemolysin (α-HL) gene without signal peptide sequence was amplified from Staphylococcus aureus by PCR and inserted into pMD18-T vector. The clonedα-HL gene was then inserted into prokaryotic expression vector pET32a+ and transformed into E. coli BL21. The predicted protein was detected by SDS-PAGE after IPTG induction, which had molecular weight approximately 53 kD. Hemolytic experiment demonstrated the expressed protein can lyse mouse red cell and its hemolytic titer attained 2.26×104 HU/mg. Conclusively, the Staphylococcalα-HL gene was successfully cloned and expressed in E. coli. The successful expression ofα-HL in E. coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism and vaccine development.