猪肺炎支原体Mhp-1株的分离鉴定及其全基因组测序分析

Isolation and identification of Mycoplasma hyopneumoniae Mhp-1 strain and its whole genome sequencing analysis

为了分离与鉴定引起的猪群疑似支原体性肺炎的病原体,本实验收集了20份病死猪肺脏病变组织,采用猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)特异性引物进行PCR鉴定、分析培养特性、颜色变化单位(CCU)测定、传代培养后扩增16S rRNA基因并测序分析等,并进一步采用BLAST分析分离菌16S rRNA基因序列的同源性,采用邻接法构建16S rRNA基因的系统进化树,对该分离株MLST分型、全基因组测序和毒力基因预测。结果显示,20份临床样品仅3份样品为Mhp单阳性,接种KM2液体培养基后仅1份生长良好,颜色规律性变黄;接种KM2固体培养基培养7 d后呈现特异性“煎蛋样”的菌落形态;瑞氏姬姆萨染色结果可见环状、点状、丝状、两级状等形状且大小不等的菌体形态,传12代后经特异性PCR检测结果为Mhp单阳性,表明该分离株可以稳定传代,且无Mhr污染;该分离株在KM2培养基上为1×10^(7)CCU/mL;16S rRNA基因序列比对结果显示该分离株与GenBank中登录的Mhp菌株ES-2(CP038641.1)同源性达100%。以上结果确定本研究分离出一株Mhp,并将其命名为Mhp-1。16S rRNA基因系统发育树分析结果显示,分离株Mhp-1与国外猪源Mhp分离株F7.2(KY264125.1)处于同一分支,亲缘关系最近;MLST分型结果显示Mhp-1分离株属于ST184型;全基因组测序获得序列大小为0.91 Mb,与已报道的Mhp基因组大小基本一致;毒力基因预测结果显示其存在EF-Tu、Lttp、P216、P102、P46、P97、PDH-B、Capsule、HlyA等毒力因子编码基因。本研究进一步丰富了Mhp的研究数据,为规模化猪场猪支原体性肺炎的防控提供参考数据。

In order to isolate and identify the pathogen causing suspected Mycoplasma pneumonia in the pig population,20 pig lung lesion tissues were collected in this experiment.The species of Mycoplasma hyopneumoniae(Mhp)and Mycoplasma hyorhinis(Mhr)were identified by using PCR identification,culture characteristics,16S rRNA gene sequencing and color changing units(CCU)determination.The characteristics of the strain were analyzed through phylogenetic tree analysis,multilocus sequence typing(MLST),whole genome sequencing,and virulence genes prediction.The results of PCR identification showed that 3 samples detected positive for Mhp.When inoculated with KM2 liquid culture medium,only one sample grew well and the medium color turned yellow regularly.In addition,it showed specific"fried egg-like"colony morphology on solid culture medium after 7 days of culture.The Rui's Giemsa staining results showed the bacterial punctate,filamentous,circular,and of varying sizes of the visible morphology.The PCR detection of the isolate after 12 passages was only positive for Mhp,indicating that the isolate can be stably passaged without Mhr contamination.The CCU of the isolate on KM2 medium is 1×10^(7)CCU/mL.The BLAST results showed that the isolate displayed 100%homology with the Mhp strain ES-2(Accession:CP038641.1)registered in the GenBank database.The above results confirmed that a strain of Mhp was successfully isolated,and the isolate named as Mhp-1 in this study.The 16S rRNA gene phylogenetic tree analysis showed that it was in the same branch as the foreign swine-derived strain F7.2(KY264125.1)and had the closest genetic relationship.The results of MLST revealed that the Mhp-1 isolate belonged to the ST184.The whole genome size was 0.91 Mb,which is similar to the size of reported Mhp genome size.In addition,the whole genome sequencing showed that multiple virulence factor encoding genes were found in the isolate,including EF-Tu,Lttp,P216,P102,P46,P97,PDH-B,Capsule,and HlyA.This study further enriches the research data on Mhp,providing important theoretical data for the prevention and control of Mhp in large-scale pig farms.