The Separation, Identification And Polyclonal Antibody Preparation Of Streptococcus From Mastitis In Dairy Cow

Bovine mastitis is one of the most serious diseases causing loss of cow industry; Streptococcus is a genus of common pathogens of bovine mastitis, including S.agalactiae, S.dysgalactiae, uberis, S.pyogenes and beast Streptococcus etc. It infects glands by causing bovine mammary tissue damage the long-term reduction of milk. It also causes mammary gland losing function of producing milk, makes lots of cow culled, and leads to economic losses in the cow industry. Therefore, in this study the prevalence of bovine mastitis causing by Streptococcus was investigated in Guangxi, drug sensitive test for Streptococcus was conducted, and the anti-streptococcal polyclonal antibody was prepared so as to continue to do some rapid diagnosis study.In total,75samples of mastitis milk were collected from six large-scale cow farms in Guangxi, Streptococcus from these samples were isolated and identified by traditional pathogenic microbiology, Gram stain and microscopy, PCR and biochemical experiments. The incidence of mastitis infection of Streptococcus was also preliminarily analyzed.14Kunming mice were used to virulence test of the14Streptococcus strain selected randomly from this study, the mice divided into three groups with different concentrations of Streptococcus. We elected another six mice injected with saline as control group and observed their symptom for3days. At the same time, we analyzed the incidence of streptococcus mastitis. Rhubarb, berberine, Scutellaria, Schisandra, honeysuckle and other11kinds of traditional Chinese medicine were used as the single herb sensitive experiments, while drug sensitive test was done in16kinds of drug often used in dairy cow farm. Two strains of virulent Streptococcus were selected as a particulate antigen. Rabbits, rats were immunized for several times. The antiserums with saturated ammonium acetate antibody purification were obtained, and titer and specificity of antiserum against Streptococcus were detected through an indirect ELISA and SDS-PAGE electrophoresis. Results:1、In total160strains of Streptococcus were isolated and identified from75samples of mastitis milk.(1)160strains of Streptococcus were identified by Gram staining from328strains of microbe, it was of48.78%of the total. The incidence of streptococcus mastitis was the highest in F farm among6dairy farms, the infection rate was up to90.48%. The infection rates were51.24%and51.85%, in A and C dairy farm, respectively. The infection rates in remaining dairy farms also were between40%and50%.(2)152were identified as positive from160strains of germs by PCR from cultivation broth, it was of46.34%of the total.2、By Streptococcus virulent experiments on mice, the median lethal dose of Streptococcus to mice was2.04×108cfu/mL. Results of drug sensitive test: some drugs often usd in dairy farm had certain inhibition against Streptococcus; Among chinese medicine, rhubarb, baicalin, berberine have very good effects against Streptococcus, but licorice, honeysuckle, Houttuynia root almost had no effect.3、By determination of the polyclonal antibody titer by indirect ELISA,the highest antibody titers were1:819200and1:512000in rabbits and rats respectively. Antisera were purified twice by a nucleic acid and their results were14.29μg/mLand11.44μg/mL. The antisera detected by SDS-PAGE vertical electrophoresis visible specificity and higher purity of the antibody strip having a molecular weight of about55KD,25KD match with the theoretical molecular size. Candida detected by indirect ELISA, there was no cross-reactivity to other strains such as Staphylococcus aureus, E. coli and others.Conclusion:The average infection rate of streptococcal mastitis was48.78%in six dairy farms in Guangxi, and the detection range was40.5%to90.48%. Streptococcus virulent experiments showed that the half lethal dose of bacterium in mice was2.04×108cfu/ml, the bacterium have strong pathogenicity, pathogenic rate was100%, and lesions occur on multiple organs in mice, parts of the mice acutely dead within12h. Extremely sensitive drugs to Streptococcus were Mo Jian, ampicillin sodium in western medicine, and berberine, rhubarb, Scutellaria in traditional Chinese medicine. These results provide measures for clinical therapy of streptococcal mastitis. Through the white rabbit, rat immunity for many times and the establishment of the antiserum indirect ELISA, high titer anti-streptococcal polyclonal antibody was obtained, it is useful for rapid diagnostic kits and test strips development.