| Calf diarrhea is one of the major diseases that cause the death of calves in the large-scale dairy farm. Intestinal pathogenic E. coli(EPEC) and enterotoxigenic Escherichia coli(ETEC) are the main pathogen that cause diarrhea and septicemia of newborn calves, among which K99, F41 fimbriae pathogenic strains have highest morbidity. Immunization is the most effective way to control the disease, but there is no commercially available vaccine used in cattle at home and abroad.This study used O8(K99),O78(F41)strain isolated and identified from material of calf diarrhea and pathogenic get from local area,made pili antigens and whole cell antigen as oil adjuvant inactivated vaccine,and through the challenge protection test in immunized mice,testing the serum antibodies produced by immune cow colostrum and its calf with indirect ELISA method.Calf diarrhea cases were recorded to determine the incidence of natural immune effects,and the results are as follows:1.O8,O78 strains were inoculated LB broth and improvement Minca broth.Inactivated after cultured them.Extracted pili at 60℃by water bath shaking method.And extracted protein from saturated ammonium sulfate salting.Then added ISA206 biphasic oil adjuvant and prepared calf diarrhea E.coli K99-F41 pili oil adjuvant vaccine and whole-cell vaccines.2.Vaccine Test:two of the vaccine antigen content was measured physical properties inspection,sterility testing,safety testing,experiments in mice immunized with poison attack,the immune cows antibody test,the results showed that the protein content of 0.5 pili seedlings~1.0 mg/m L,the number of whole-cell bacterial vaccine 40 to 150 billion/m L.Immunized mice poison attack results in protection rate pili vaccines and whole-cell vaccine were 100%and 93.3%,indicating that the two vaccines sterility,safety,character and stability,immunogenicity good indicator.3.Use O8,O78 strain extracted K99-F41 pili antigen as coating antigen,by checkerboard titration established indirect ELISA method,K99-F41 pili vaccine antibody negative serum were measured to calculate the value of OD450 negative threshold,with results showed that multiple antigen diluted 1:80,was seized and whey serum dilution of 1:100 for the best conditions.4.The two vaccines were given at different doses 2-4 weeks pregnant cows prenatal immunization immunohistochemical.The detection of postpartum cows by indirect ELISA method has been established,3h,6 h,12 h,24 h,36 h,72 h of colostrum and colostrum fed calves produced 1d,3d,5d,7d corresponding serum antibody titers of E.coli to compare two different E.coli oil adjuvant vaccine dose of difference pregnancy cows produced calves of antibody titers.The results showed that a 5 m L dose of whole cell vaccine mother colostrum has group’s highest antibody titers;pili vaccine 5 m L group calf serum antibody titers was in the highest;colostrum two vaccine dose 5 m L group E.coli and calf serum antibody titers corresponding were significantly higher than 3 m L group;unvaccinated female calf colostrum and produced by E.coli antibodies was significantly lower than immunohistochemistry,and newborn calf diarrhea incidence was significantly higher than natural Immunohistochemistry. |
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