The Research Of The Of The Virulence Factors Of Staphylococcus Aureus In Dairy Cattle Mastitis

The bacteria from the milk were separated, cultivated, counted and identified in this experiment. The bacteria separated from the milk in this test were staphylococcus, streptococcus, coliform, mycetozoan and microzyme.Then Sthapylococcus aureus was identified by specific primer.Hemolysin is a kind of ectotoxin, which is the most important virulence factor of Staphylococcus aureus virulence factor, includingα,β,γandδ-toxin. Hemolysin has the hemolytic reaction effect on many kinds of mammal blood cell, and cause the toxic mastitis of mammary gland. To detect staphylococcus aureusαandβ-toxin involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the gene ofα-toxin andβ-toxin. Two pairs of oligonucleotide primers special to the sequence ofαandβ-toxin gene were designed according to the sequences which encode theα-toxin andβ-toxin. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. Result showed that gene ofα-toxin were found in more than 80% of the Staphylococcus aureus with the 704bp specific fragment on gel, while theβ-toxin gene was found only in 59% isolates with the 496bp specific fragment.To detect staphylococcus aureus adhesins gene Fnbp A and Fnbp B involved in dairy cow mastitis, multiplex polymerase-chain reaction was used to amplify the gene of Fnbp A and Fnbp B. Two pairs of oligonucleotide primers special to the sequence of Fnbp A and Fnbp B gene were designed according to the sequences which encode the Fnbp A and Fnbp B. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of 524bp and 642bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus Fnbp A and Fnbp B gene tested. The detection of Staphylococcus aureus which were isolated from dairy cow with mastitis showed that adhesin gene of Fnbp A were found in more than 95% of the Staphylococcus aureus, while the FnbpB gene was found only in 10% isolates.Coagulase is one of the most important indexes in detection of the pathogenic Staphylococcus aureus, which can enable the blood plasma of the rabbit or human to clotting. It conduce the pathogenic bacterium to resist the effect of the phagocytic cell and the germicide inside of the host. A pair of oligonucleotide primers special to the sequence of coagulase gene was designed according to the sequence of the gene which encodes the coagulase of staphylococcus aureus. DNA of the bacteria isolated from the milk of dairy cow with mastitis was used for template to identify the Staphylococcus aureus. The PCR products were detected by agarose gel electrophoresis analysis. The gene amplified was cloned into the pMD18-T plasmid, and the positive stain detected was sequenced by Songon. The Staphylococcus aureus revealed positive results of the presence of a 761bp specific fragment on gel, while other bacteria gave negative results demonstrating that the primers should be specific for Staphylococcus aureus tested.As a kind of bacteriotropin, the antibody to the Fnbp can not only prevent the adhesion of the staphylococcus aureus to the cell, but also promot the removing of the body. Hemolysin is a kind of ectotoxin of Staphylococcus aureus, the anatoxine of which can partly protect goat from mastitis induced by Staphylococcus aureus. In this experiement, the engineered protein of rFnbp and rα-HL were mixed with adjuvant.The mice were vaccined with the protein vaccine and the eukaryotic expression plasmid pcDNA3.1+-Clfa, and the level of the antibody in the serum was tested by ELISA. The result showed that the specific antibody was found in all the serum of the mice vaccined with the protein. Through the initial test of aggression with the Staphylococcus aureus, the engineered protein vaccine could lower the infection rate and the death rate of the mice, which offered a new way to prevent the infection of Staphylococcus aureus.