云南省边境地区卵形硬蜱中中华基因型伯氏疏螺旋体的检出和鉴定

Detection and identification of Borrelia sinica in Ixodes ovatus from the border region of Yunnan province,China

目的对云南省耿马傣族佤族自治县(耿马县)采集的蜱进行伯氏疏螺旋体分子生物学检测,为当地莱姆病的调查提供参考。方法对2016年4月在云南省耿马县采集的游离蜱单只进行DNA提取后,采用巢式PCR扩增伯氏疏螺旋体5S^23S rRNA间隔区作为初筛实验,阳性者进一步检测16S rRNA和FLA鞭毛蛋白基因确证。结果 3种蜱共94只,检出阳性14只,阳性率为14.89%。阳性蜱均为卵形硬蜱,不同性别间差异无统计学意义(χ~2=0.746,P=0.388)。锐跗硬蜱和金泽革蜱均未检出阳性。检出的伯氏疏螺旋体3个目的基因片段序列均与中华基因型伯氏疏螺旋体同源性达98%~99%,构建的系统发育树显示,与我国四川、安徽省检出的中华基因型伯氏疏螺旋体聚在一个分支上;与日本疏螺旋体接近,而阿弗西尼疏螺旋体、狭义伯氏疏螺旋体、法雷斯疏螺旋体等基因型聚在外围。结论耿马县采集的卵形硬蜱携带中华基因型伯氏疏螺旋体,其宿主和媒介的种类、分布及其对人的致病性等,值得进一步调查研究。

Objective To identify Borrelia species in ticks collected in Gengma Dai and Va Autonomous county of Yunnan province using molecular biology detection methods,and to provide a basis for the investigation of local Lyme disease.Methods DNA was extracted from individual free-living ticks collected in Gengma county of Yunnan province in April2016,and Borrelia DNA was identified by nested-PCR amplification of the 5 S-23 S rRANA intergenic spacer region.Positive samples were further tested by 16 S rRANA amplification and verified with the flagellin(FLA)gene.Results A total of 94 ticks belonging to 3 species were captured,and 14 ticks(14.89%)were positive for Borrelia.All Borrelia-positive ticks were Ixodes ovatus,and there was no significant difference in the rate of Borrelia positivity between sexes(χ~2=0.746,P=0.388).Ixodes acutitarsus and Dermacentor auratus tested negative for Borrelia.The sequences of the three target gene fragments identified from Borrelia were 98%-99%homologous to those in B.sinica.Phylogenetic analysis showed that the bacterial species identified in this study clustered with B.sinica detected in Sichuan and Anhui provinces,China,and was close to B.japonica but distinct from B.afzelii,B.burgdorferi sensu stricto,and B.valaisiana.Conclusion Ixodes ovatus collected from Gengma county carries B.sinica.The types and distribution of hosts and vectors of B.sinica and its pathogenicity in humans warrant further investigation.