青藏高原喜马拉雅旱獭分离布鲁氏菌的MLVA分型与溯源分析

Genotyping and traceability analysis of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau by MLVA

目的观察青海省青藏高原喜马拉雅旱獭分离的布鲁氏菌的多位点可变数目串联重复序列分析(multiple locus variable-number tandem repeat analysis,MLVA)分型,探讨其与既往青海省布鲁氏菌分离菌株间的亲缘关系。方法2019年3月至2020年10月,采集青海省青藏高原喜马拉雅旱獭全血样本,对虎红平板凝集试验(rose bengal test,RBT)布鲁氏菌抗体阳性样本进行病原体分离培养,采用传统生物学方法和分子生物学方法(BCSP31-PCR和AMOS-PCR方法)进行菌株鉴定。同时,应用MLVA方法对分离菌株进行基因分型,并结合既往青海省不同宿主分离的70株布鲁氏菌的基因型,应用聚类分析方法探讨菌株间的亲缘关系。结果共采集青藏高原喜马拉雅旱獭全血样本1466份,从其中64份RBT阳性样本中分离培养出2株布鲁氏菌,分别命名为QH2013054、QH2013062,经传统生物学和分子生物学方法鉴定为羊种布鲁氏菌生物Ⅲ型。MLVA分型结果显示,QH2013054与QH2013062菌株在Bru16位点有差异,为不同的MLVA基因型。聚类分析结果显示,QH2013054菌株与7株菌株具有相同的MLVA基因型,其中6株来自共和县同一个家庭的3名农民和3只羊,1株来自门源回族自治县农民;QH2013062菌株与4株菌株具有相同的MLVA基因型,其中3株来自门源回族自治县的3名农民,1株来自互助土族自治县农民。结论从青海省青藏高原喜马拉雅旱獭中分离的布鲁氏菌与在本省人、羊中分离的部分布鲁氏菌具有相同的MLVA基因型,推测宿主人、羊、青藏高原喜马拉雅旱獭可能具有共同的传染源。

Objective To observe multiple locus variable-number tandem repeat analysis(MLVA)typing of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province,and to explore the relationship between the strains and strains previous isolated from Qinghai Province.Methods Blood samples of Himalayan marmot were collected in Qinghai-Tibet Plateau of Qinghai Province from March 2019 to October 2020.Pathogens were isolated and cultured from Brucella antibody positive samples identified by using the rose bengal test(RBT).Conventional biological methods and molecular biological methods(BCSP31-PCR and AMOS-PCR)were used for strain identification.At the same time,MLVA method was used to genotype the isolated strains,and cluster analysis was used to analyze the genetic relationships between the strains based on the genotype of 70 Brucella isolated from different hosts in Qinghai Province.Results A total of 1466 blood samples of Himalayan marmot were collected from Qinghai-Tibet Plateau.Two strains of Brucella were isolated and cultured from 64 RBT-positive samples,named QH2013054 and QH2013062,respectively.They were identified as Brucella ovis biotypeⅢby conventional and molecular biological methods.The MLVA genotyping results showed that QH2013054 and QH2013062 were different at the Bru16 locus,indicating different MLVA genotypes.Cluster analysis showed that strain QH2013054 had the same MLVA genotype as 7 strains,among which 6 strains were from 3 farmers and 3 sheep from the same family in Gonghe County,and 1 strain was from a farmer in Menyuan Hui Autonomous County.The strain QH2013062 had the same MLVA genotype as 4 strains,including 3 strains from 3 farmers in Menyuan Hui Autonomous County and 1 strain from a farmer in Tu Autonomous County of Huzhu.Conclusions The strains of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province have the same MLVA genotype as some strains of Brucella isolated from humans and sheep in Qinghai Province.It is speculated that the host humans,sheep and Himalayan marmot in Qinghai-Tibet Plateau may have a common source of infection.