新疆奶牛乳房炎金黄色葡萄球菌生物学特性及基因分型研究
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摘要
本实验用微生物学和分子生物学方法,对新疆部分地区奶牛场乳房炎病例中金黄色葡萄球菌的生化特性、抗药性、免疫原性、菌体蛋白和基因分型进行了初步研究。
研究结果表明:不同地区牛场检出的304份乳房炎奶样金黄色葡萄球菌的分离率为29.3%。TH-16S生化编码以733720型为主,占所有鉴定菌株的75%。药物敏感性试验表明,所实验菌株对头孢唑林钠、庆大霉素最为敏感,对环丙沙星、头孢拉定、丁胺卡那霉素亦具有良好的抑菌效果,而氨苄西林、青霉素、克林霉素等常用药物的抑菌效果较差。对不同牛场所分离的8个菌株进行免疫原性和抗原相关性测定,不同菌株的免疫原性存在差异,并存在广泛的交叉凝集。用聚丙烯酰氨凝胶电泳对所分离菌株的菌体蛋白进行分析,各菌菌体蛋白图谱相似,有41～52条蛋白条带,其分子量大小在16.7～118.4kDa。根据菌体蛋白图谱之间的差异,69株菌共分为6类。其中,A类和B类共58株,占实验菌株总数的84%,说明新疆地区奶牛乳房炎金黄色葡萄球菌菌体蛋白具有较高的相似性。免疫印迹分析表明,所有实验菌株在35.4kDa、32.0kDa、17.7kDa存在共同性抗原。另外,部分菌株免疫印记图谱在28.7kDa处明显多出一条独特的条带,证明奶牛乳房炎金黄色葡萄球菌菌株间确实存在抗原差异。用三个不同引物以RAPD方法对不同牛场分出的50株金黄色葡萄球菌进行扩增,确定新疆地区奶牛乳房炎金黄色葡萄球菌14个基因型。上述工作为奶牛乳房炎疫苗研制中金黄色葡萄球菌疫苗菌株筛选提供了有益的技术资料。
Preliminary studies on antibiotic susceptibility, immunogenicity, bacterial protein and gene typing of Staphylococcus aureus isolated from cow mastitis were made, with methods of microbiology and molecularbiology.
The study results showed that the isolation rate of Staphylococcus aureus identified from 304 mastitis milk samples collected from different dairy farms in Xinjiang was 29.2%. 75% of all Staphylococcus aureus identified though TH-16S biochemical tests was 733720 type. Drug susceptibility testing showed that strains tested were the most susceptible to cefazolin sodium and Cinoxacin. Ciprofloxacin, cephradine and amikacin had better antibacterial effect. Ampicillin, penicillin and clindamycin etc. common used antibiotics were almost no effective. Immunogenicity and related antigenicity of 8 strains isolated strains from different farms were detected. Diversity of immunogenicity existed and broad across agglutination was found in test strains. Bacterial protein analysis by sodium doedecyl sulfatepolyacrylamide gelelectro -phoresis (SDS-PAGE) shown that all bacterial protein maps were similar, which had 41~52 protein bands with different size and the molecular weight (16.7~118.4kDa). According to differences of bacterial protein maps, 69 strains were divided into six kinds. Kind A and Kind B were 58 strains altogether, accounting for 84% of the total strains. These illustrate bacterial protein of Staphylococcus aureus isolated from cow mastitis in Xinjiang had the high similar. Western blot analysis demonstrated that all test strains had common antigen at 35.4kDa, 32.0kDa and 17.7kDa. But some strains had a unique band at 28.7kDa in western blot mapping, which proved that there were differences among antigen of Staphylococcus aureus from cow mastitis. 50 Staphylococcus aureus isolated from different dairy farms were amplified by three primers with random amplified with polymorphic DNA polymerase chain reaction. There were 14 genotypes for Staphylococcus aureus isolated from cow mastitis in Xinjiang in this study.The above work provided helpful technical data for strain screening of Staphylococcus aureus on cow mastitis vaccine development.
研究结果表明:不同地区牛场检出的304份乳房炎奶样金黄色葡萄球菌的分离率为29.3%。TH-16S生化编码以733720型为主,占所有鉴定菌株的75%。药物敏感性试验表明,所实验菌株对头孢唑林钠、庆大霉素最为敏感,对环丙沙星、头孢拉定、丁胺卡那霉素亦具有良好的抑菌效果,而氨苄西林、青霉素、克林霉素等常用药物的抑菌效果较差。对不同牛场所分离的8个菌株进行免疫原性和抗原相关性测定,不同菌株的免疫原性存在差异,并存在广泛的交叉凝集。用聚丙烯酰氨凝胶电泳对所分离菌株的菌体蛋白进行分析,各菌菌体蛋白图谱相似,有41～52条蛋白条带,其分子量大小在16.7～118.4kDa。根据菌体蛋白图谱之间的差异,69株菌共分为6类。其中,A类和B类共58株,占实验菌株总数的84%,说明新疆地区奶牛乳房炎金黄色葡萄球菌菌体蛋白具有较高的相似性。免疫印迹分析表明,所有实验菌株在35.4kDa、32.0kDa、17.7kDa存在共同性抗原。另外,部分菌株免疫印记图谱在28.7kDa处明显多出一条独特的条带,证明奶牛乳房炎金黄色葡萄球菌菌株间确实存在抗原差异。用三个不同引物以RAPD方法对不同牛场分出的50株金黄色葡萄球菌进行扩增,确定新疆地区奶牛乳房炎金黄色葡萄球菌14个基因型。上述工作为奶牛乳房炎疫苗研制中金黄色葡萄球菌疫苗菌株筛选提供了有益的技术资料。
Preliminary studies on antibiotic susceptibility, immunogenicity, bacterial protein and gene typing of Staphylococcus aureus isolated from cow mastitis were made, with methods of microbiology and molecularbiology.
The study results showed that the isolation rate of Staphylococcus aureus identified from 304 mastitis milk samples collected from different dairy farms in Xinjiang was 29.2%. 75% of all Staphylococcus aureus identified though TH-16S biochemical tests was 733720 type. Drug susceptibility testing showed that strains tested were the most susceptible to cefazolin sodium and Cinoxacin. Ciprofloxacin, cephradine and amikacin had better antibacterial effect. Ampicillin, penicillin and clindamycin etc. common used antibiotics were almost no effective. Immunogenicity and related antigenicity of 8 strains isolated strains from different farms were detected. Diversity of immunogenicity existed and broad across agglutination was found in test strains. Bacterial protein analysis by sodium doedecyl sulfatepolyacrylamide gelelectro -phoresis (SDS-PAGE) shown that all bacterial protein maps were similar, which had 41~52 protein bands with different size and the molecular weight (16.7~118.4kDa). According to differences of bacterial protein maps, 69 strains were divided into six kinds. Kind A and Kind B were 58 strains altogether, accounting for 84% of the total strains. These illustrate bacterial protein of Staphylococcus aureus isolated from cow mastitis in Xinjiang had the high similar. Western blot analysis demonstrated that all test strains had common antigen at 35.4kDa, 32.0kDa and 17.7kDa. But some strains had a unique band at 28.7kDa in western blot mapping, which proved that there were differences among antigen of Staphylococcus aureus from cow mastitis. 50 Staphylococcus aureus isolated from different dairy farms were amplified by three primers with random amplified with polymorphic DNA polymerase chain reaction. There were 14 genotypes for Staphylococcus aureus isolated from cow mastitis in Xinjiang in this study.The above work provided helpful technical data for strain screening of Staphylococcus aureus on cow mastitis vaccine development.
引文
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