河南省某养殖场猪源弯曲杆菌的分离鉴定及系统发育分析

Identification and phylogenetic analysis of Campylobacter of a pig farm in Henan province

目的探究某生猪养殖场中弯曲杆菌的污染状况,分析猪源弯曲杆菌分离株的种类,表型和基因型。方法采集生猪养殖场鼻拭子、粪便、污水、土壤、环境(地面和墙壁)涂抹物和饲料等83份样品,选择性增菌后采用滤膜法培养,根据菌落形态、生化表型和多重PCR检测进行弯曲杆菌的分离鉴定,扩增16srRNA后克隆测序,获得序列与Genbank数据库弯曲菌全基因组序列进行Blast分析,构建系统发育树,并确定弯曲菌种属。结果从猪粪便中分离9株弯曲杆菌,分离率10.84%,包括4株结肠弯曲菌(C.coli)、3株拉尼尔弯曲菌(C.lanienae)、2株豚肠弯曲菌亚种(C.hyointestinalis)。结论该生猪养殖场中的猪肠道内弯曲菌污染以结肠弯曲菌为主,种属和基因型具有多样性,分子系统发育树结果表明与国外猪源弯曲菌属具有较高同源性。

Objective To investigate the contamination status of Campylobacterin a pig farm;to analyze the subspecies,phenotypes and genetypes of Campylobacter isolates.Methods Eighty-three samples of nasal swabs,faeces,sewage,soil,environmental smears（ground smears and wall smears）and pig feeds were collected in a pig farm.After selective enrichment,the samples were simultaneously cultured for Campylobacter by filtration method.Isolation and identification of Campylobacter strains were performed by colony morphology,biochemical tests and multiplex PCR.After amplification,16 srRNA was cloned and sequenced,and the sequences were subjected to Blast analysis in Genbank database.The phylogenetic tree was constructed and the Campylobacter genus was identified.Results A total of 9 strains of Campylobacter were isolated from pig faeces and the detection rate was 10.84%.Among them,4 strains were identified as Campylobacter coli,3 strains were identified as Campylobacter lanienae and 2 strains were identified as Campylobacterhyointestinalis.Conclusion The mainly contamination of Campylobacterin the pig farm was caused by Campylobacter coli,with diverse biochemical phenotypes and genotypes.The molecular phylogenetic tree showed isolates had high homology with Campylobacter isolates from pig abroad.