乳房炎奶牛金黄色葡萄球菌毒素基因的检测及PFGE分型研究  

Virulence Genes and PFGE Profiles of Staphylococcus aureus Isolated from Cows with Subclinical and Clinical Mastitis

作　　者：王新[1] 韦艺媛[2] 张静[3] 周婷[3] 梁珍娟[1] 杨保伟[1] 席美丽[1] 夏效东[1] 孟江洪[1] 俞英[3] 

机构地区：[1]西北农林科技大学食品学院,杨凌7121002 [2]中国农业大学动物科技学院,北京100193 [3]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2011年第7期974-980,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：长江学者讲座教授奖励计划项目(Z111020001);博士科研启动费(01140407);教育部基础科研业务项目(2011JS006);转基因生物新品种培育科技重大专项(2009ZX08009-146B);北京市农业局试验示范项目(20100222);"948"项目(2010-C14)

摘　　要：作者针对临床及亚临床乳房炎奶牛乳汁中金黄色葡萄球菌分离株的毒素基因进行检测和脉冲场凝胶电泳(PFGE)基因分型,比较2种类型乳房炎金黄色葡萄球菌分离株的差异。无菌法采集奶样,采用国际标准方法从中分离金黄色葡萄球菌,用多重PCR方法扩增nuc基因和mecA基因以确证金黄色葡萄球菌(SA)和耐甲氧西林金黄色葡萄球菌(MRSA)。进一步用PCR方法检测SA的各种毒素基因(SEs、ETs、TSST-1和PVL基因等)。利用限制性内切酶SmaⅠ对SA基因组DNA进行酶切和PFGE分析,最后利用BioNumerics软件进行聚类分析。结果:19.3%(23/119)的临床乳房炎奶样和14.8%(26/176)的亚临床乳房炎奶样确定为金黄色葡萄球菌阳性样品,分别从中分离鉴定出43株和26株金黄色葡萄球菌,其中临床乳房炎分离株中有5株为mecA基因阳性。临床乳房炎奶牛奶样中检测到SA的SEA、SEB、SED、SEJ和PVL毒素基因,检出率分别为3.8%(1株)、11.5%(3株)、19.2%(5株)、7.7%(2株)和31.2%(10株);亚临床乳房炎奶牛乳样中仅检测到SA的SEA和PVL毒力基因,检出率分别为7.0%(3株)和84.1%(37株)。表明临床与亚临床乳房炎奶牛乳汁中SA菌株携带的毒素基因不一样,SEs可能是临床乳房炎菌株的重要致病基因,PVL可能是亚临床乳房炎菌株的重要致病基因。69株SA使用SmaⅠ酶切分型后,可分为7个大簇、50个基因型,来源相同的SA分型后大部分位于同一簇内。临床乳房炎奶牛乳汁中检测到MRSA菌株,PVL基因在亚临床乳房炎中的检出率为临床乳房炎的2.7倍。PFGE方法能较好的区分临床乳房炎和亚临床乳房炎的SA分离菌株。The aim of the present study was to investigate toxin genes properties and PFGE profiles of S.aureus strains（SA） isolated from cows with subclinical mastitis and clinical mastitis.For this purpose,milk samples were sterilely collected from Chinese Holstein cows with subclinical and clinical mastitis.SA strains or methicillin-resistant S.aureus（MRSA） strains were confirmed by multiplex PCR based on nuc and mecA genes.SA isolates were recovered and characterized by kinds of toxin genes of SA（SEs,ETs,TSST-1 and PVL genes） with PCR and PFGE assays.Results were as follows： 69 S.aureus isolates were recovered from 19.3% subclinical mastitis samples（43 isolates） and 14.8% clinical mastitis samples（26 isolates）,in which 5 isolates from clinical mastitis were mecA-positive S.aureus.From clinical mastitis isolates,SEA,SEB,SED,SEJ and PVL genes were detected with the rate of 3.8%（1 isolate）,11.5%（3 isolates）,19.2%（5 isolates）,7.7%（2 isolates） and 31.2%（10 isolates）,respectively,whereas SEA and PVL were detected from subclinical mastitis isolates with the rates of 7.0%（3 isolates） and 84.1%（37 isolates）,respectively.The results implied that SEs may be important pathogenic genes of S.aureus strains in the cows with clinical mastitis,while PVL may be key pathogenic gene for the cows with subclinical mastitis.Fifty pulsotypes and seven PFGE lineage groups from A to G were obtained by PFGE,and S.aureus strains isolated from the same source were classified into the same cluster.MRSA strains were only isolated from cows with clinical mastitis.The detection rates of PVL genes in cows with subclinical mastitis were 2.7 times higher than that with clinical mastitis.The distribution of virulence genes in S.aureus isolates was distinctly different between the cows with subclinical mastitis and clinical mastitis via PFGE assays.

关 键 词：奶牛 金黄色葡萄球菌 MECA基因 毒素基因 PFGE分型 

分 类 号：R378.22[医药卫生—病原生物学]