西北新疆等4省区蜱感染莱姆病螺旋体的分子流行病学调查

目的了解我国西北部分省区蜱中莱姆病螺旋体感染及其基因分型情况。方法应用巢式PCR扩增脾中莱姆病螺旋体5S-23S rRNA间隔区片段,对阳性产物进行限制性片段长度多态性（RFLP）分析。结果共检测11个蜱种2 460只,感染莱姆病螺旋体的有11个种304只,阳性率为12.36%。新疆、宁夏、陕西、甘肃的感染率分别为11.31%、17.96、12.35%及11.64%。不同省区阳性率存在差别;不同蜱种间阳性率亦存在明显差别,其中以青海血蜱阳性率最高,达到59.38%。RFLP分析结果显示西北四省区蜱中莱姆病螺旋体为B.garinii及B.afzelii基因型,其中B.garinii基因型为主要基因型,占80%以上。结论莱姆病螺旋体在我国西北4省区中广泛存在,存在B.garinii及B.afzelii两种基因型。     

吉林林区动物莱姆病螺旋体感染的调查研究

目的了解吉林珲春林区的蜱、野鼠及牛、绵羊感染莱姆病的情况。方法对蜱和野鼠进行莱姆病螺旋体的PCR扩增,阳性标本RFLP分型,应用间接免疫荧光法检测家畜血清中的IgG抗体。结果PCR检测全沟硬蜱和森林革蜱的带菌率为36.0%和30.9%;在五种鼠中检测到莱姆病螺旋体的特异性片段,带菌率分别为14.0%、8.3%、13.0%、25.0%和33.3%。阳性标本RFLP分型结果属于B.garinii和B.afzelii型;牛、羊血清学检测阳性率为27.5%和31.5%。结论野鼠、蜱和牛羊中都检测到伯氏疏螺旋体的感染,证实吉林珲春林区存在莱姆病的自然疫源地。     

我国部分地区蜱中莱姆病螺旋体的检测与基因分型研究

目的 对我国部分地区的多种蜱类进行莱姆病螺旋体的检测和基因分型。方法 选择我国黑龙江、吉林和浙江省部分林区为调查点，采集当地蜱类，用巢式PCR法进行检测，阳性产物进行限制性片段长度多态性（RFLP）分析和单链构象多态性（SSCP）分析，确定莱姆病螺旋体的基因型。结果 共检测蜱512只，阳性126只，阳性率24．61％。其中吉林全沟硬蜱带菌率为37．00％，黑龙江全沟硬蜱带菌率为20．87％，浙江长角血蜱带菌率为28．07％。RFLP分析表明，蜱中莱姆病螺旋体包括B．garinii和B．afzelii两种基因型。SSCP分析显示为7种亚型，其中B．garinii分为5个亚型，B．afzelii分为2个亚型。发现有3只蜱同时感染不同基因（亚）型莱姆病螺旋体。结论 证实B．garinii和B．afzelii基因型为我国莱姆病螺旋体的优势基因型，并在我国蜱中发现莱姆病螺旋体不同基因（亚）型的混合感染。     

我国部分林区鼠中莱姆病螺旋体的分子流行病学调查研究

目的了解我国部分林区鼠中莱姆病螺旋体感染及其基因分型情况。方法应用巢式PCR扩增鼠中莱姆病螺旋体5S-23S rRNA间隔区片段,对阳性产物进行限制性片段长度多态性(RFLP)分析。结果共检测鼠8属19种455只,感染莱姆病螺旋体的有4属8种26只,感染率为5.71%。内蒙古、黑龙江、浙江、贵州林区鼠的感染率分别为3.45%、4.84%、8.00%、7.14%;其中东北林区(包括内蒙古和黑龙江)棕背鼠平的感染率为9.09%;浙江林区社鼠的感染率为9.26%;未发现新疆林区鼠感染莱姆病螺旋体。RFLP分析结果显示内蒙古、黑龙江林区鼠中莱姆病螺旋体均为B.garinii基因型,而浙江、贵州林区鼠感染的莱姆病螺旋体包括B.garinii和B.valaisiana两种基因型。结论东北林区及浙江林区以B.garinii基因型为主,棕背鼠平、社鼠可能分别是两地林区莱姆病螺旋体的主要储存宿主。     

浙江省衢州地区鼠类携带莱姆病螺旋体研究

目的 了解衢州市鼠类携带莱姆病螺旋体状况以及莱姆病螺旋体基因型。方法 采用针对莱姆病螺旋体5S~23S基因间隔区的巢式PCR方法检测鼠脾标本,对阳性样本进行基因测序及分析,构建系统发育树。结果 在378份鼠标本中检出莱姆病螺旋体核酸阳性57份,阳性率15.08%。有Borrelia garinii(B.g)基因型、Borrelia valaisiana(B.v)基因型Borrelia spielmanii(B.sp)基因型。结论 衢州境内鼠类存在至少3个型别莱姆病螺旋体感染,应加强该病原体的检测和莱姆病的防制。     

内蒙古中西部草原蜱媒病原体多样性及基因型分析

目的了解内蒙古中西部草原蜱类的群落结构、携带病原体多样性及基因型。方法于2016-2019年春夏季,在内蒙古中西部草原,采用动物体表搜集法采集蜱标本,进行蜱种鉴定。解剖摘取蜱的唾液腺并提取基因组DNA,以斑点热立克次体柠檬酸合成酶A (gltA)、疏螺旋体以鞭毛蛋白B (flaB)、埃立克体属以外膜蛋白质1 (omp1)、无形体属主要表面蛋白2 (msp2)为靶基因进行PCR扩增初筛。立克次体gltA初筛阳性样品经限制性片段长度多态性(RFLP)分类,再根据蜱种和地区每类选20~30个代表性样品进行gltA、立克次体外膜蛋白A (rOmpA)基因测序。扩增序列测序后用BLAST、 Clustal W和MEGA 7.0软件进行同源性分析,以邻接法构建系统进化树。结果共采集成蜱3 822只,经形态学特征和特异性18S r RNA基因分型法鉴定,隶属于2属3种,分别为草原革蜱、亚东璃眼蜱和边缘璃眼蜱,其中草原革蜱占55.7%(2 129/3 822)、亚东璃眼蜱占30.0%(1 147/3 822),为该地区的优势蜱种。PCR检测结果显示,立克次体gltA基因阳性蜱1 899只,阳性率为49.7%(1 899/3 822), gltA基因阳性样品根据RFLP结果分为两类,两类样品的gltA基因序列均为581 bp,与R. raoultii (DQ365804)或R. aeschlimanni (KT873466)的同源性为100%;两类样品的rOmpA基因均长367 bp,与R. raoultii (AH015610)或R. aeschlimanni (U83466)的同源性为100%,与gltA基因的结果相符。3 822只蜱中,R. raoultii和R. aeschlimanni的阳性率分别为37.2%(1 422/3 822)和12.5%(477/3 822),其中草原革蜱中分别为58.5%(1 245/2 129)和11.1%(477/2 129);亚东璃眼蜱中分别为15.4%(177/1 147)和0;边缘璃眼蜱中分别为0和44.0%(240/546)。疏螺旋体flaB基因阳性蜱28只,阳性率为0.7%(28/3 822),其中草原革蜱中为0.8%(16/2 129),亚东璃眼蜱为1.0%(12/1 147)。共获得疏螺旋体flaB基因序列10条,与莱姆病主要病原体B. garinii (AB035602)和B. afzelii PKo (NC008277)的同源性分别为90.6%~100%和95.6%~100%。亚东璃眼蜱中B. garinii和B. afzelii的阳性率分别为0.9%(10/1 147)和0.2%(2/1 147),草原革蜱中均为0.4%(8/2 129)。3 822只蜱中omp1基因阳性1只,TA克隆后获得8个氨基酸序列相同的克隆和3个氨基酸序列存在差异的克隆,11个克隆的氨基酸序列与E. muris的同源性最高,但仅为65%~69%。3 822只蜱中均未检出无形体属菌群。系统进化树分析结果显示,3种蜱感染的立克次体均与R. raoultii和R. aeschlimanni聚在一簇。在获得的10条疏螺旋体菌群flaB基因序列中,源于草原革蜱和亚东璃眼蜱的1条序列与B. garinii聚在一簇,草原革蜱的另1条序列与B. afzelii聚在一簇,其余8条序列与B. garinii和B. afzelii的flaB基因序列处在不同的分支。草原革蜱感染的埃立克体属菌与目前已知的埃立克体属菌群关系较远,形成独自的聚类。结论内蒙古中西部草原存在草原革蜱、亚东璃眼蜱和边缘璃眼蜱,蜱类中广泛存在斑点热立克次体和莱姆病螺旋体的感染,是R. raoultii、R. aeschlimanni、 B. garinii和B. afzelii潜在的自然疫源地。有必要加强该地区蜱媒传染病的预防控制工作。     

内蒙古奇乾地区媒介蜱携带伯氏疏螺旋体的调查研究

目的了解内蒙古奇乾地区媒介蜱携带伯氏疏螺旋体(Borrelia burgdorferii,B.b)的感染情况。方法用布旗法采集游离蜱,利用PCR和基因测序法对媒介蜱标本中B.b的感染进行检测和基因分型。结果采集媒介蜱共320只,其中全沟硬蜱293只(91.56%),森林革蜱22只(6.88%),嗜群血蜱5只(1.56%)。媒介蜱B.b阳性携带率为26.88%,可携带B.garinii、B.afzelii和B.miyamotoi 3种基因型。其中全沟硬蜱、森林革蜱和嗜群血蜱B.b阳性携带率分别为29.01%(85/293)、4.55%(1/22)、0(0/5)。结论内蒙古奇乾地区全沟硬蜱为优势蜱种,携带B.b基因型存在多样性,应引起公共卫生部门的高度重视。     

中国莱姆病螺旋体菌株bmpA基因中人B细胞表位多态性分析

目的 研究我国伯氏疏螺旋体(Borrelia burgdorferi sensu lato),即莱姆病螺旋体菌株中bmpA基因及其人B细胞表位区的序列多态性。方法 选取61株莱姆病螺旋体分离株,PCR扩增其bmpA基因并测序后,结合4种基因型参考菌株序列,与从免疫表位数据库(Immune Epitope Database,IEDB)中查询到的B细胞表位序列进行比对分析。结果 中国B.garinii基因型菌株的cluster2、cluster3以及B.afzelii和B.valaisiana基因型所有菌株与参考序列比对后发现分别有3、9、5和18个异义突变。B细胞表位改变主要发生在B.afzelii基因型全部分离株和B.garinii基因型的cluster3及5株非成簇菌株。B.afzelii基因型菌株在B细胞表位区的异义突变A125S影响了2个B细胞表位;B.garinii基因型的cluster3和5株非成簇菌株B细胞表位区的3个异义突变导致5个B细胞表位均发生改变。结论 中国莱姆病螺旋体bmpA基因在4种基因型间和B.garinii基因型内存在较大多态性。     

中国6省莱姆病螺旋体主要宿主动物鼠的初步调查

目的了解中国吉林、山西、甘肃、青海、贵州、湖南6省林区莱姆病螺旋体宿主动物鼠的感染情况。方法在每个省各选取两个采样点进行捕鼠,采用病原分离培养和巢式PCR方法对野鼠的脾脏、肾脏和膀胱进行了病原学检测。并通过基因测序方法确定基因型。结果从贵州黑线姬鼠中分离到了2株莱姆病螺旋体;从5省(贵州未检测到)野鼠的脾脏和/或肾脏中检查到了莱姆病螺旋体的特异片段,其中青海黄南(28.85%)和湖南石门(19.6%)两地标本的PCR阳性率较高,各地区阳性率的差异有统计学意义。通过序列同源性分析确定吉林、青海、甘肃和山西的基因型均为Borrelia garinii。湖南的基因型为Borreliavalaisiana。结论本次调查表明各地宿主动物鼠的感染状况不同,各地应根据具体情况进一步扩大调查以明确当地的主要宿主动物种类及携带病原体情况。     

巢式PCR与LAMP方法在蜱的莱姆病螺旋体检测中的应用

目的 应用巢式PCR方法与环介导等温扩增(LAMP)方法检测蜱中莱姆病螺旋体带菌率。方法 从青海省循化县与辽宁省新宾县共采集蜱112只,采用巢式PCR与LAMP两种方法检测蜱的莱姆病螺旋体带菌率。结果 采用两种方法检测青海循化蜱标本51份,12份阳性,阳性率为23.53%;辽宁省新宾县蜱标本61份,18份阳性,阳性率为29.51%。采用巢式PCR方法检测112份蜱标本,20份阳性,阳性率为17.86%;采用LAMP方法检测112份蜱标本,15份阳性,阳性率为13.39%,两种方法差异无统计学意义(χ²=0.85,P＞0.05)。结论 巢式PCR与LAMP方法均可用于蜱标本莱姆病螺旋体的检测,两种方法共同使用可以提高莱姆病螺旋体的检出率。同时,本研究首次报道了青海循化县和辽宁新宾县蜱的莱姆病螺旋体带菌率,完善了我国高林区覆盖率地区的蜱媒带菌率数据。     

Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi sensu lato in Switzerland: spatio-temporal pattern at a regional scale

Lyme borreliosis, the most important vector-borne disease in the Northern hemisphere, causes health problem for populations in endemic areas. In the present study, the density of questing Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) was examined in 11 areas located on the Swiss Plateau and in an alpine valley. From 1999 to 2001, free-living I. ricinus ticks were collected on a monthly basis by flagging vegetation in these areas. Each tick was examined for the presence of B. burgdorferi sl using direct fluorescent antibody assay, and for isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. Density of questing ticks varied greatly between studied areas. Borreliae were observed in ticks collected in all investigated sites. However, the prevalence of infection differed significantly among areas. Infection prevalence varied from 9% to 40% in nymphs and from 22% to 47% in adults. Adult ticks were significantly more infected (129/366, 35%) than nymphs (109/552, 20%). There was no correlation between nymphal density and infection prevalence as well as between adult density and infection prevalence, but there was a correlation between density of ticks and density of infected ticks. During the spring peak of questing tick density, a range of 2-30.3 infected ticks per 100 m(2) was observed. B. burgdorferi sl isolates (n = 129) were obtained from ticks collected in 10/11 areas. Five Borrelia species were identified: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, B. lusitaniae, and six mixed infections were also obtained. Borrelia species were heterogeneously distributed in the different areas.     

Co-infection of Borrelia garinii and B. afzelii in a population of wild rodents from woodland

The maintenance of Borrelia burgdorferi s.l. in the environment is dependent on the zoonotic cycle involving tick vectors and certain reservoir hosts. It is well known, that the same species of wild rodents, as well as the vector Ixodes ricinus, are often co-infected with at least two genomospecies of B. burgdorferi s.l.: B. afzelii and B. garinii. The ticks collected from two rodent species: Clethrionomys glareolus and Apodemus flavicollis were examined for the presence of B. burgdorferi s.l., as well as for B. garinii and B. afzelii. In this study, an immunofluorescent antibody assay (IFA) and polymerase chain reaction (PCR) protocols were used. The high level of infestation in rodents (90% for C. glareolus and nearly 100% for A. flavicollis) shows that wild rodents are important hosts of the immature stages of I. ricinus. A high percent of Borrelia positive ticks collected from bank voles and yellow necked mice; above 7% determined by IFA and 2% determined by PCR, clearly revealed that these species of animals are competent zoonotic reservoirs of B. burgdorferi s.l.     

Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in The Netherlands

In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.     

一种莱姆病螺旋体real-time PCR方法的建立及其在鼠标本检测中的应用评价

目的 基于莱姆病螺旋体recA 基因,建立一种检测鼠中莱姆病螺旋体的real-time PCR方法。方法 通过GenBank分析比较莱姆病螺旋体recA 基因,选择其保守序列设计MGB探针及引物并进行方法学评估。并应用建立的real-time PCR方法和nested PCR方法对收集的123份鼠标本进行检测分析。结果 本研究建立的real-time PCR方法仅对莱姆病螺旋体检测阳性,其最小检出浓度为10¹ copies/μL。标准曲线各浓度点Ct值批内、批间平均变异系数(CV)分别为1.56%和2.30%。123份鼠标本中,real-time PCR检测59例阳性,nested PCR检测43例阳性。结论 新建立的real-time PCR方法具有快速、敏感和特异的优点,可用于鼠标本中莱姆病螺旋体的检测。     

新疆克拉玛依地区蜱媒病原体的调查

目的 调查新疆克拉玛依地区蜱种类及其携带病原体。方法 于克拉玛依市不同区采集游离蜱和寄生蜱,鉴定蜱种类,利用荧光定量PCR或套氏PCR/半套氏PCR检测发热伴血小板减少综合征布尼亚病毒、斑点热群立克次体、克里米亚-刚果出血热病毒、蜱传脑炎病毒、巴贝斯虫/泰勒虫、贝氏立克次体、查菲埃立克次体、恙虫病东方体和伯氏疏螺旋体等9种病原体。结果 共采集蜱683只,分3属3种,其中亚东璃眼蜱数量(n=362)最多,其次为银盾革蜱(n=311)、图兰扇头蜱(n=10)。银盾革蜱中斑点热群立克次体的阳性率为3.2%(10/311),巴贝斯虫的阳性率为1.0%(3/311);亚东璃眼蜱中巴贝斯虫的阳性率为0.8%(3/362),泰勒虫阳性率为0.3%(1/362);其他病原体检测结果均呈阴性。PCR扩增阳性基因序列分析显示银盾革蜱携斑点热群立克次体阳性与劳氏立克次体最相关,银盾革蜱和亚东璃眼蜱携带驽巴贝斯虫。结论 新疆克拉玛依地区优势蜱为亚东璃眼蜱和银盾革蜱,银盾革蜱携带劳氏立克次体,银盾革蜱和亚东璃眼蜱携带驽巴贝斯虫,提示该地区存在斑点热、巴贝斯虫病的自然疫源地,因此有必要加强对这些蜱携疾病的防控。     

Significance of red deer (Cervus elaphus) in the ecology of Borrelia burgdorferi sensu lato

BACKGROUND: Red deer (Cervus elaphus) is one of the most important host of the adult tick (Ixodes ricinus) which is the basic vector of the Lyme disease causative agent--Borrelia burgdorferi sensu lato in Europe. The aim of the present study was to establish the role of red deer in the transmission of B. burgdorferi s.1. Material and methods. Tissues from 74 red deers were evaluated and the presence of B. burgdorferi s.1 DNA was identified using nested PCR technique based on fla gene. The identification of species belonging to B. burgdorferi s.1 complex was performed after restriction digestion of nested PCR product with Ddel enzyme and sequencing of nested PCR product. The study included also 55 isolates of I. ricinus females removed from red deer and 466 ticks (73 adult and 393 nymphs) collected from the vegetation in the area where the red deer lives. RESULTS: There were no DNA of B. burgdorferi s.1 complex in the red deer tissues and in ticks removed from deer, however in one tick removed from deer the DNA of other Borrelia species--B. miyamotoi was identified. In ticks collected from vegetation 3 species belonging to B. burgdorferi s.1. complex were identified: B. garinii (3.2% ticks studied), B. afzelii (6.9%) and B. valaisiana (3.6%), however DNA of B. miyamotoi was absent. These results confirm inability of survival of B. burgdorferi s.1. species in tick I. ricinus feeding on red deer blood. However there is a possibility of survival of B. miyamotoi in presence of deer blood at least in ticks feeding on red deer. The main role of red deer in keeping the constant infection level of B. burgdorferi s.1. in the whole population of I. ricinus ticks does not concern B. miyamotoi.