用聚合酶链方法对北京林区莱姆病疫源地及莱姆病病原体基因型的探索性研究

Exploratory study on natural focus and its causative agent of genotype of Lyme disease by polymerase chain reaction in the forest areas of Beijing

目的 进一步明确北京林区是否存在莱姆病的自然疫源地及其分布。方法 基于莱姆病螺旋体外膜蛋白A基因建立半巢式聚合酶链式反应 (polymerasechainreaction ,PCR)方法 ,对从北京 6个林区采集的蜱和鼠进行检测和基因分型 ,选择阳性标本进行克隆和序列测定 ,与已知序列进行同源性比较。间接免疫荧光法检测抗莱姆病螺旋体IgG抗体 ,从长角血蜱中分离莱姆病螺旋体。结果 从门头沟区东灵山采集的标本中检测到莱姆病螺旋体DNA片段 ,3只游离全沟硬蜱 1只检测阳性 ,5 7只寄生全沟硬蜱若蜱中 1只检测阳性 ;119只野鼠中 9只检测阳性 ,其中 8只B .garinii阳性 ,1只B .afzelii阳性。 5 0份野鼠血清有 5份莱姆病螺旋体IgG抗体阳性 ,采集的 16 0只长角血蜱(2 0只 /组 )未分离到莱姆病螺旋体菌株。结论 北京门头沟区东灵山可能存在莱姆病的自然疫源地 ,包括两个基因型 ,全沟硬蜱可能是莱姆病的传播媒介 ,野鼠可能是贮存宿主。

Objective To learn the existance of natural focus of Lyme disease and its distribution. Methods A semi nested polymerase chain reaction (PCR) method was developed for detection and genotyping of Borrelia burgdorferi on basis of outer surface protein A (OspA) gene. Ticks and mice collected from 6 forest areas in Beijing were detected with above methods. The positive PCR products were cloned and sequenced. The sequences were compared with published sequences for homology. IFA as used to detect IgG antibody on Borrelia burgdorferi . Lyme disease spirochete were isolated from H.longicornis were also attempted. Results B.Burgdorferi sensu lato were detected from 939 ticks and 250 mice specimens collected from above 6 study sites using primer pairs OA 1/OA 4 and SL/OA 4. Only the specimens collected from Dongling mountain showed positive amplification. One in three adult Ixodes persulcatus with one of 57 nymph Ixodes persulcatus showed positive while 9 of 119 ( 7.56 %) mice specimens showed positive, of which 8 were B.grinii and one B.afzelii . In this study, we attempted to isolate B.burgdorferi sensu lato strains from 160 H.longicornis ticks (20/group) but failed. Serological survey showed a 9.1 %(5/55) infection rate with B.burgdorferi sensu lato in the mice of Dongling mountain forest areas. Conclusions The natural focus of Lyme disease including B.garinii and B.afzelii might have existed in Dongling mountain of Mentougou district, Beijing. Ixodes persulcatu and mice may serve as vectors and reservoirs, respectively.