山羊化脓隐秘杆菌LM01株分离鉴定及其毒力基因PLO遗传进化分析

Isolation and identification of Trueperella pyogenes LM01 strain from goats and phylogenetic analysis of PLO virulence gene

为确诊福州某羊场患病山羊皮下脓肿的病因,采集脓液进行细菌分离、形态染色检镜、培养特性观察、生化试验及16S rRNA测序分析,结果分离鉴定出1株革兰阳性、形态不规则、具有β溶血现象的化脓隐秘杆菌,命名为LM01株。用分离菌皮下接种小鼠,能复制出与山羊自然感染相似的皮下脓肿临床病例,半数感染量(ID_(50))为1.65×10^(7)CFU/m L。病理组织学观察可见皮肤损伤处形成一个大的囊腔,腔内充满大量脓细胞,囊腔与肌层之间有大量成纤维细胞增生及中性粒细胞浸润。毒力基因检测显示分离株至少存在PLO、NanH、NanP、FimA、FimC、FimG等6个毒力基因,其中溶血素PLO是化脓隐秘杆菌最主要的毒力因子,又是宿主保护性抗原。PCR分段扩增PLO基因,得到全长1605 bp,编码534个氨基酸,与GenBank中收录的其他参考株相比,核苷酸和氨基酸同源性分别为95.0%~99.8%和97.0%~99.8%。进化树分析显示,化脓隐秘杆菌分成Group 1(牛型)、Group 2(猪型)和Group 3(羊型)3个独立分支,本分离株归属在Group 3(羊型)分支,与重庆2012CQ-ZSH株亲缘关系最近。变异性分析发现,PLO基因仅发生点突变,没有插入或缺失现象;羊型与猪型、牛型相比,存在25处核苷酸特异性位点及5个特异性氨基酸位点:62(N/D)、340(T/A)、355(V/A)、384(F/Y)和470(A/L)。药敏试验表明,分离菌对恩诺沙星、强力霉素、阿米卡星等22种药物敏感,对阿奇霉素、复方新诺明、红霉素等6种药物耐药。结果表明,本次山羊患病的病原菌是化脓隐秘杆菌,本试验为病原学研究提供了参考。

In order to identify the etiology of subcutaneous abscess of a goat farm in Fuzhou,the pus was collected for bacterial isolation,morphological staining microscopy,culture characteristics observation,biochemical test and 16 S rRNA sequencing analysis,respectively.A Gram-positive bacterium with irregular form andβhemolytic characteristic was isolated and identified as Trueperella pyogenes,named LM01 strain.Using the isolated bacteria solution to subcutaneously inoculate to mice,clinical symptoms of subcutaneous abscess similar to natural infection of goats could be replicated,and median infective dose(ID_(50))was 1.65×10^(7) CFU/m L.Histopathological observation showed that a large cystic cavity was formed at the skin lesion,which was filled with a large number of pus cells,and there were a large number of fibroblast proliferation and neutrophils infiltration between the cystic cavity and the muscular layer.Virulent gene detections showed that the isolate carries at least 6 virulence genes,such as PLO,Nan H,Nan P,Fim A,Fim C and Fim G.Among them,hemolysin PLO is the most important virulence factor of Clostridium pyogenes,and it is also a host protective antigen.The full length of PLO gene was 1605 bp by PCR,encoding 534 amino acids.Compared with other reference strains in Gen Bank,nucleotide and amino acid homologies were 95.0%—99.8%and 97.0%—99.8%,respectively.The phylogenetic tree showed that Trueperella pyogenes was divided into three independent clades:Group 1(Bos type),Group 2(Sus type)and Group 3(Ovis type),and this isolate belonged to Group 3(Ovis type)clades,with the closest genetic relationship with Chongqing 2012 CQ-ZSH strain.Variability analysis showed that there were only point mutations,without insertion or deletion in PLO gene.There were 25 nucleotide specific sites and 5 amino acid specific sites:62(N/D),340(T/A),355(V/A),384(F/Y)and 470(A/L)in Ovis type compared with Sus type and Bos type.Drug sensitivity test showed that the isolate was sensitive to enrofloxacin,doxycycline and amicacin,and resistant to azithromycin,cotrimoxazole and erythromycin.The results showed that Trueperella pyogenes was the pathogen that caused the disease of goats in the case,which provides theoretical reference for etiology research and the development of host-specific vaccine.