宁夏地区牛源无乳链球菌主要毒力基因和耐药基因的检测与序列分析

发布时间：2018-08-09 11:39

【摘要】：奶牛乳房炎是危害奶牛养殖业健康发展的常见疾病之一。该病增加了奶牛淘汰率与保健治疗费用,给奶业造成严重的经济损失。据有关部门统计,在中国仅因奶牛乳房炎淘汰的奶牛占总淘汰牛的9%-11%,每年造成的经济损失超过135亿元。引起奶牛乳房炎的病原微生物极为复杂,而链球菌和葡萄球菌是奶牛乳房炎的主要致病菌,链球菌中又以无乳链球菌占主要比例。无乳链球菌按兰氏划分法属于B群链球菌,该菌不但可引起奶牛、山羊、绵羊等动物的乳房炎,且可导致人类新生儿败血症、脑膜炎和肺炎等,严重危害动物及人类的健康。因此快速准确地进行诊断以及研究出有效的防治制剂和方法对控制奶牛乳房炎具有重大而深远的意义。1.本实验从银川市及周边地区牛场采集大量乳房炎乳样,通过细菌培养、形态学观察和生化试验等一系列检测方法,分离出266株链球菌。之后结合分子生物学鉴定法,共鉴定出无乳链球菌22株,停乳链球菌19株,乳房链球菌15株。试验结果表明宁夏地区奶牛乳房炎链球菌感染中,无乳链球菌的分离率最高。因此,无乳链球菌是宁夏地区奶牛乳房炎链球菌感染的主要流行菌株。2.采用CLSI推荐的纸片扩散法,对22株临床型奶牛乳房炎无乳链球菌分离株进行了19种常用药物的敏感试验。结果表明,分离菌株对19种抗菌药物具有不同程度的耐药性,其中对磺胺异恶唑的耐药率达到95.5%；对复方新诺明的耐药率达到77.3%；对萘啶酸和氨苄青霉素的耐药率达到68.2%；对四环素和林可霉素的耐药率达到59.1‰对头孢他啶的耐药率达到54.5%；对克林霉素、羧苄西林、诺氟沙星的耐药率均达到50%；对阿莫西林的耐药率也达到45%以上。另外22株无乳链球菌共出现18种耐药谱,最高的达到13耐。其中耐药类型最少的是2耐,仅1株(4.5%),最多的是9耐,达到8株(36.4%)。3.根据GenBank公开的相关序列,设计磺胺类sull、sul2、sul3和四环素类tetE的PCR反应特异性引物,建立了相应的PCR检测方法。结果表明,该方法灵敏可靠,可以用作临床检测方法的使用。对22株奶牛乳房炎无乳链球菌临床分离株进行检测,4种耐药基因的检出率分别为：sull(18.2%)、sul2 (22.7%)、sul3 (27.3%)、tetE (18.2%)。本研究对宁夏地区奶牛乳房炎无乳链球菌分离株耐磺胺类药物性和耐四环素类药物性进行检测,为预防奶牛乳房炎无乳链球菌耐药性提供科学的理论基础。4.对22株无乳链球菌,分别扩增其Sip、cpsE、CAMP三种主要毒力基因并进行测序,之后对其序列进行对比分析。结果表明,三种基因与GenBank公开的序列相似性非常高,其中,Sip基因的相似性均高于94%；cpsE基因的相似性为99%；而CAMP基因的相似性均为98%以上。
[Abstract]:Cow mastitis is one of the common diseases that endanger the healthy development of dairy cattle breeding industry. The disease increases the rate of dairy cow elimination and the cost of health care treatment, and causes serious economic losses to the dairy industry. According to the statistics of relevant departments, only the cows eliminated because of mastitis accounted for 9- 11% of the total cattle eliminated in China, resulting in economic losses of more than 13.5 billion yuan each year. The pathogenic microorganisms causing cow mastitis are very complicated, and Streptococcus and Staphylococcus are the main pathogens of dairy mastitis. Streptococcus actinomycetes belongs to group B streptococcus according to the method of Lance's division. This bacterium can not only cause mastitis of cows, goats, sheep and other animals, but also lead to neonatal septicemia, meningitis and pneumonia, and seriously harm the health of animals and human beings. Therefore, rapid and accurate diagnosis and the development of effective preparation and methods for the control of cow mastitis has great and far-reaching significance. In this experiment, a large number of mastitis milk samples were collected from cattle farms in Yinchuan city and surrounding areas. 266 strains of streptococcus were isolated by a series of methods such as bacterial culture, morphological observation and biochemical test. Combined with molecular biological identification, a total of 22 strains of streptococcus lactobacillus, 19 strains of Streptococcus brevis and 15 strains of Streptococcus brevis were identified. The results showed that the isolation rate of streptococcus mastitis was the highest in Ningxia. Therefore, Streptococcus acuminata is the main epidemic strain of Streptococcus mastitis in Ningxia. Using the disk diffusion method recommended by CLSI, the sensitivity tests of 19 commonly used drugs were carried out on 22 clinical isolates of Streptococcus acuminatum from dairy cow mastitis. The results showed that the isolates were resistant to 19 antimicrobial agents to varying degrees, among which the resistance rate to sulfamethoxazole was 95.5.The resistant rate to compound sulfamethoxazole was 77.3%, the resistance rate to nalididinic acid and ampicillin was 68.2%, the drug resistance rate to sulfamethoxazole reached 95.5%, and the resistance rate to compound sulfamethoxazole reached 77.3%, and the resistance rate to ampicillin was 68.2%. The resistance rate to tetracycline and lincomycin was 59.1 鈥，

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