| Bovine mastitis is a common frequently-occurring disease causing great damage and economic losses on modern cow cultivation.To investigate the flora distribution of common pathogens isolated from bovine mastitis in China, 201 milk samples of mastitis were collected from some large dairy farms all over the country. Streak cultivation and biochemistry methods were applied to isolate and identify the main pathogenic bacteria of mastitis. The results showed that 9 kinds of bacteria and 239 strains were isolated and identified from 201 samples with mastitis. 23 samples were bio-clean. Plating efficiency is 88.6%. The pathogens of mastitis in 239 strains were 89 strains of Streptococcus agalactiae(37.23%), 53 strains of Streptococcus dysgalactiae(22.18%), 47 strains of Staphylococcus flavus(19.67%) and 31 strains of Escherichia coli(12.97%). The test ascertained the flora distribution of common pathogens islated from bovine mastitis in China furtherly . It is important for .prevention of bovine mastitis.To investigate the antimicrobial resistance of common pathogens isolated from bovine mastitis in China, antimicrobial susceptibility tests of S. agalactiae, S. dysgalactiae, S. aureus and E. coli, which were isolated from milk samples of dairy cow with clinical mastitis , were performed using slip method. The results showed that the four pathogens were resistant to antibilties in varying degrees with resistance rate ranging from 10% to 100%. No antibiotic was found with high susceptibility to the four pathogens. The four pathogens were all susceptive to florfcnicol, levofloxacin, lomcfloxacin, norfloxacin and cephalosporins and the sensitivity ranged from 60% to 100%. The four pathogens had high resistance to Penicillin G, streptomycin and SMZ-TMP with resistance rate ranging from 30% to 100%. The test ascertained the antimicrobial resistance of common pathogens islated from bovine mastitis in China furtherly . It is important for clinical use of antibiotics.The separation and purification for capsular polysaccharide of Streptococcus dysgalactiae of bovine mastitis was studied for the preparation of the effective capsular polysaccharides-protein conjugate vaccine of Streptococcus of bovine mastitis. The effect of incubation time on the bacterial concentration and the yield rate of capsular polysaccharides, the effect of pH value of fermentation on the yield rate of capsular polysaccharides, the ethanol concentration of removing the nucleic acids, the concentration, temperature and time of ethanol required to precipitate polysaccharides, the frequency of extraction of cold phenol to remove the complex protein had finished. Finally, the following purification process for capsular polysaccharide were established. To harvested broth after 8h fermentation from Streptococcus dysgalactiae, to adjust the pH value of the broth to 7.0, and then the centrifugation supernatant was collected, sterilized with 0.22um filter membrane, 10 times was ultrafilter concentration with 10KD membrane bag, to precipitate and remove the nucleic acids with 25% ethanol, the supernatant was collected after centrifugation, and the polysaccharides were got after precipitate with 80% ethanol, extracted 3 times with cold phenol to remove the unwanted proteins, collected sugar level, dialysis to remove the residual phenol, collected the internal dialysis fluid, and then precipitated with 80% ethanol to obtain purified capsular polysaccharide antigens of streptococcal. The study showed that the technology to extract and purify capsular polysaccharides had lower cost and easy to operate, and had higher yield rate of capsular polysaccharide, it could also ensured that all indicators meet the requirement of'Bio-logical Products'published in 2000, and the technology had highly feasibility and effectiveness. The research provided a good foundation for the preparation of capsular polysaccharides-protein conjugate vaccine for bovine mastitis in the future. |
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