| Bovine mastitis is a common and frequently occurring disease of dairy cows, and its complicated etiology often result in recurrent attacks, which causes the most, serious economic loss in dairy farming. Streptococcus agalactiae (S. agalactiae) is one of the most important pathogens causing mastitis. This research utilized the traditional methods of bacteriological identification and molecular biology technology to isolate and identify the S. agalactiae from the recessive mastitis milk samples collected from the region of Northern China from 2012 to 2016. Then the disc diffusion method and PCR method were used respectively to detect the drug sensitivity, molecular typing, surface protein gene and virulence factor of the isolated strains. Based on the understanding of the S. agalactiae biological characteristics, polysaccharide-protein conjugate subunit vaccines were prepared and the preliminary study on relevant immune effects were conducted.1. In the survey and analyze of 592 milk samples,63 strains of S. agalactiae were isolated; the isolation rate was 10.64%. All serum type of S. agalactiae isolates is I a, its surface proteins are unidentified. The detection for the 7 kinds of virulence factors of S. agalactiae showed that there was no obvious difference in the distribution of 7 kinds of virulence genes for the 63 strains of S. agalactiae. The S. agalactiae is sensitive to most of antibiotics. 90%-100% of isolates were sensitive to penicillin G, cephalosporin cefotaxime, ampicillin, erythromycin, ofloxacin, clindamycin and vancomycin. 82.14% of the S. agalactiae had a strong resistance to tetracycline. In the detection of resistance genes, the gene tetK was first detected in bovine S. agalactiae.2. The research considered serum type I a that is prevalent in Northern China as the object and made the preliminary research on the influence of nutritional and cultural conditions on the formation of capsular. Meanwhile, it researched the influencing factors of liquid culture for S. agalactiae from multiple aspects. The optimized culture medium composition and condition for the S. agalactiae were preliminarily obtained, which laid the foundation for large-scale industrial fermentation.3. Surface immunogenic protein Sip and Surface protein fibrinogens binding protein (FbsA) and Sip-FbsA series were used as a carrier protein to conjugated capsular polysaccharide to construct subunit vaccine. Western-blot analysis showed that the subunit vaccine had the immune response, based on which we took capsular polysaccharides (CP), SIP protein, FbsA protein, Sip-FbsA protein and protein polysaccharide conjugates CP+Sip-FbsA as vaccines to conduct immunologic tests on Westar rat to evaluate the immunological characteristics by detecting the specific antibody titer, conducting T lymphocytes proliferation test, blood phagocytic conditioning test, flow cytometry, and challenging with virus protection test. The results showed that conjugates CP+Sip-FbsA could induce well humoral immune responses and cellular immune responses by first intramuscular immunization together with strengthen immunity via the breast basal injection and had the best immune effects among all the immune groups. Westar rats immunized with conjugates CP+Sip-FbsA could not only generate good system immune effects but also enhance the local immune response in breasts.The results above suggested that the bovine mastitis associated S. agalactiae subunit vaccine can induce pretty well humoral responses and cellular immunity responses and provide theoretical and experimental basis for further researching for the prevention and treatment of bovine mastitis associated S. agalactiae. |
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