健康与腹泻仔猪源A型产气荚膜梭菌的多位点序列分型  

Multilocus sequence typing analysis of Clostridium perfringens type A isolates from diarrheic and healthy piglets

作　　者：王婧祺 戴益民[1] 王娇 韩春生 曾秀 吴允正 王林康 张锦华[1] WANG Jingqi;DAI Yimin;WANG JiaoA;HAN Chunsheng;ZENG Xiu;WU Yunzheng;WANG Linkang;ZHANG Jinhua(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学,动物科学技术学院,江西南昌330045

出　　处：《中国兽医学报》2021年第1期68-73,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31860694,31660720)。

摘　　要：A型产气荚膜梭菌(CpA)存在于健康仔猪肠道,同时也是仔猪肠毒血症的关键病原。为研究两者的差异,本试验在江西省4个地区采集健康和腹泻仔猪粪便样本分离得到CpA,通过16S rRNA鉴定所有CpA分离株,并通过多重PCR确定分离株的毒素分型。采用MLST多位点序列分型技术对选取的24株CpA(腹泻或健康来源CpA各12株)进行聚类分析,根据菌株的ST型用UPGMA和NJ两种方法构建系统进化树。结果显示,8个管家基因中glpk基因相对保守,recA和plc基因变异程度较大,等位基因中存在可变位点的比例为1.8%(tpi)至6.1%(plc),腹泻源与健康源CpA遗传差异不明显;无论是否增添plc基因,对整体聚类分析结果无明显影响,但腹泻源CpA(75%)的plc等位基因比例明显高于健康源(33.3%)。根据24株CpA的ST型的聚类分析发现,75%的健康源CpA聚集在同一亚组CC1中,而CC2中聚类了2个地区的腹泻源CpA,有较高的遗传多态性。在12株腹泻源CpA的ST型聚类分析中,同一地区的CpA之间存在更近的亲缘关系,这表明其基因突变有一定的规律和方向性。因此推测,CpA的产毒致病性可能与地域环境密切相关。Clostridium perfringens type A(CpA)widely exists in the intestines of healthy piglets and it is the key pathogen of piglet enterotoxemia.The aim of this study was to analyze the genetic relatedness of CpA from the intestinal tract of healthy and diarrheic piglets which were collected from four regions of Jiangxi Province.All CpA isolates were identified by 16 S rRNA and typed by multiplex PCR.24 CpA strains(12 healthy isolates and 12 diarrhea isolates)were selected to cluster analysis by the method of MLST.Minimum phylogenetic tree was constructed by UPGM and NJ method according to the STs of 24 CpA strains.Analysis results showed that the glpk gene was relatively conservative among the 8 housekeeping genes,and the recA and plc genes were highly variable.The rate of variable sites in the allele is 1.8%(tpi)to 6.1%(plc).The genetic difference of CpA between diarrheic piglets and healthy piglets is not obvious,the overall cluster analysis result of the 24 CpA did not change whetherα-toxin gene(plc)was present or not.The plc allele ratio(75%)of CpA from the diarrhea group was significantly higher than that of the CpA from healthy group(33.3%).24 STs were identified from 24 CpA by MLST,they gathered in two subgroups with high genetic polymorphism.According to the results of cluster analysis of the STs of 24 CpA strains,it was found that 75%of the healthy source CpA were gathered in the subgroup CC1,while the CpA of diarrhea source from 2 regions were gathered in subgroup CC2,showing high genetic polymorphism.The gene mutations of 12 CpA from diarrhea samples have a certain regularity and directivity,indicating that ST of CpA from same region clustered together.Therefore,it is speculated that the pathogenicity of CpA may still be closely related to the environment.

关 键 词：A型产气荚膜梭菌 仔猪 多位点序列分型 ST型 系统发育分析 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]