2021年贵州省环境致病性钩端螺旋体分离培养及基因种分析

Culture and genospecies analysis of pathogenic Leptospira in leptospirosis epidemic areas in Guizhou Province in 2021

目的对贵州省2021年钩端螺旋体(钩体)病疫区环境标本进行致病性钩体分离培养,了解其基因种特征,为钩体病防控提供科学依据。方法采集钩体病疫情发生地环境水和土壤样品,采用EMJH培养基进行钩体分离培养。提取疑似钩体生长培养物基因组DNA,采用致病性钩体特异性PCR进行检测,同时采用16S rRNA序列分析法对其进行基因种鉴定,并应用MEGA 7.0软件构建系统发育树,分析菌株间进化关系。结果从112份环境样品中分离出50株疑似钩体菌株,致病性钩体特异性PCR检测显示其中3株为致病性钩体,包含环境水样分离出的2株和土壤样品中分离出的1株。16S rRNA序列比对分析显示,经PCR检测为致病性钩体的3株菌株属于钩体致病性基因种,其中2株为Leptospira noguchii基因种,1株为Leptospira borgpetersenii基因种;另外47株菌株中5株菌株属于钩体中间种,42株菌株属于钩体非致病性基因种。系统发育树显示,致病性钩体菌株与其基因种代表性菌株进化关系较近。结论贵州省钩体病疫区环境钩体基因种复杂,同时存在致病性基因种和非致病性基因种,本次检出的2种致病性钩体基因种为贵州省尚未报道的基因种,可为贵州省钩体病疫情防控提供科学依据。

Objective In order to provide scientific basis for the control and prevention of leptospirosis,the Leptospira in the environment in leptospirosis epidemic area of Guizhou province in 2021 was investigated.Methods Water and soil samples that may be contaminated with Leptospira were collected from leptospirosis epidemic areas and culture-biotechnical assays were performed by applying EMJH medium containing 5-fluoruracil.Genomic DNA of the suspected Leptospira growth cultures was extracted and detected by pathogenic leptospirosis specific PCR,and then nearly full length of 16S rRNA gene of the cultures were amplified with PCR and subsequently sequenced.Phylogenetic tree were constructed by MEGA 7.0 software to demonstrate the evolutionary relationship between the isolates and the representative strains of Leptospira genospecies.Results A total of 50 suspected strains of Leptospira isolated from 112 samples,of which 3 strains were identified as pathogenic Leptospira,and the rate of isolating pathogenic Leptospira from environmental samples was 2.68%.The 3 pathogenic Leptospira strains isolated include 2 strains isolated from the environmental water samples and 1 strain isolated from the soil samples.Three strains of pathogenic Leptospira were identified as pathogenic genospecies Leptospira,with 2 strains being Leptospira noguchii genospecies and 1 strain being Leptospira borgpetersenii genospecies by the 16S rRNA gene assay.Among the other 47 strains,5 strains belonged to the intermediate genospecies of Leptospira,and 42 strains belonged to the non-pathogenic genospecies of Leptospira.The clustering relationship between most of isolates and their genospecies representative strains was close.Conclusion The genospecies of Leptospira in environmental in leptospirosis epidemic areas in Guizhou Province are complex,with both pathogenic genospecies and non-pathogenic genospecies.The 2 pathogenic Leptospira genospecies detected are unreported gene species in Guizhou Province,which can provide scientific basis for the prevention and control of leptospirosis epidemic in Guizhou Province.