规模化养殖肉鸡泄殖腔拭子弯曲菌分离及PFGE分型分析  

Detection and subtyping of Campylobacterfrom broilers reared in intensive farms

作　　者：邓义贞[1,2,3] 顾一心[3] 何利华[3] 张艾煜 梁昊[3] 张建中[3] 张茂俊[3] 邓俊良[1,2] 

机构地区：[1]四川农业大学动物医学学院环境公害与动物疾病四川省高校重点实验室,四川雅安625014 [2]四川农业大学动物疫病与人类健康四川省高校重点实验室,四川雅安625014 [3]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《中国兽医学报》2016年第5期778-783,789,共7页Chinese Journal of Veterinary Science

基　　金：国家"973"发展计划资助项目(2013CB127204);国家自然科学基金资助项目(81271789)

摘　　要：为了了解规模化养殖肉鸡弯曲菌的带菌现状,比较2种基础培养基对于粪便标本弯曲菌检出差异,获得菌株菌型特征。本研究选择规模化肉鸡养殖场共300份肉鸡泄殖腔拭子,分别应用Columbia、Karmali 2种基础培养基,采用滤膜过滤法进行弯曲菌的分离培养。结合革兰染色、生化鉴定以及PCR方法对疑似菌落进行弯曲菌菌种鉴定;选择60株空肠弯曲菌进行脉冲场凝胶电泳分析并用BioNumerics软件对脉冲场凝胶电泳结果进行聚类分析。结果显示,300份泄殖腔拭子样品中,弯曲菌的检出率为51.33%(154/300)。空肠弯曲菌、结肠弯曲菌检出率分别为40%(120/300)和14.67%(44/300),空、结肠弯曲菌混合感染的感染率为3.33%(10/300)。检测标本中空肠弯曲菌的感染率显著大于结肠弯曲菌(P<0.01)。应用Columbia、Karmali培养基对弯曲菌的检出率分别为41.67%(125/300)、42.67%(128/300),2种基础培养基检出率无显著性差异(P>0.05),2种培养基的补充使用样本的检出率增加了15%(45/300)。本次滤膜过滤法同时获得相同培养条件下标本中其他细菌3种,分别为:Cellulosimicrobiumsp.、Microbacteriumsp.、Helicobacter brante,共45株。60株空肠弯曲菌脉冲场凝胶电泳共分为39个带型。结果表明,本研究发现规模化养殖肉鸡中弯曲菌的携带情况较为严重,相同鸡场及不同鸡场分离菌株脉冲场凝胶电泳带型呈多样性特征。滤膜过滤法能够有效检测家禽肛拭子标本中弯曲菌,缩减了因使用选择性抗生素添加剂的成本。本研究没有检出除空肠弯曲菌和结肠弯曲菌外的其他弯曲菌菌种。In order to obtain the prevalence situation and molecular sub-typing characteristics of Campylobacter spp.in broilers from intensive farms,Campylobacterisolation,species differentiation and pulse field gel electrophoresis were investigated in this study.300 cloacal swabs were collected from 5intensive reared broilers farms.The filtration method was used to isolate the bacteria.The Karmali agar base and Columbia agar base were used for the culture for each sample.The suspected colonies from each plate were picked and conformed by Gram stain,biochemical tests,and multiply PCR analysis.Pulse field gel electrophoresis was performed on 60 Campylobacter jejuni isolates,clustering analysis for the pulse field gel electrophoresis patterns were analyzed with BioNumerics Verision 4.0software.Totally,164（164/300,54.67%）isolates were obtained from300 samples.Among the 164 isolates,120（120/300,40%）were Campylobacter jejuni,and 44（44/300,14.67%）were Campylobacter coli.The mix infection ratio for both C.jejuni and C.coli is 3.33%（10/300）.The isolation ratio of C.jejuni was significantly higher than C.coli（P〈0.01）.The isolate ratios from Columbia agar and Karmali agar were 41.67%（125/300）and42.67%（128/300）,respectively.There is no significant difference（P〉0.05）between the isolation ratio of Columbia agar and Karmali agar（P〉0.05）.However,the complemental using of these two culture mediums made the isolation increased by 15%（45/300）.39 PFGE patterns were observed in 60 C.jejuni isolates.The C.jejuni presented frequently in the broilers of the intensive farms,and a large diversity among isolates from one farm or different farms were detected by PFGE.The isolation method using filtration with membrane is one effective way for Campylobacterisolation,which saves the cost of the selective supplement in the agar base with conventional methods.

关 键 词：弯曲菌 分离率 COLUMBIA Karmali PFGE1 

分 类 号：S858.31[农业科学—临床兽医学]