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。     

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。     

甘肃麦积山区媒介及宿主动物中莱姆病螺旋体检测及基因型别研究

目的 了解甘肃麦积山景区蜱及野鼠体内莱姆病螺旋体感染及其基因分型情况。方法 采用布旗法采集蜱标本,采用夹夜法捕捉野鼠。经种类鉴定后,提取病原体DNA,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4个蜱种1 273只蜱标本,其中若蜱1 126只,成蜱147只,若蜱分组进行处理和检测,共分为67组,其中13组检测到莱姆病螺旋体DNA片段;成蜱15只检测阳性。检测4个鼠种42只野鼠,其中10只检测阳性,不同鼠种阳性率存在差别(χ²=16.93, P=0.00),4个鼠种中以大林姬鼠的阳性率为最高,达到33.33%。基因分型分析结果显示蜱及野鼠标本中莱姆病螺旋体为B. garinii 及B. afzelii基因型,其中B. garinii基因型占78.95%。结论 麦积山景区存在莱姆病螺旋体,并至少有B. garinii 及B. afzelii两种基因型。     

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。     

海南省关节炎和神经系统疾病患者莱姆病调查

目的 了解我国海南省关节炎或神经性疾病患者的莱姆病感染状况,并确认海南省是否存在莱姆病人。方法 海南省三亚市人民医院提供血清542份,均为关节炎或神经性疾病患者。采用间接免疫荧光法(Indirect Fluorescent-Antibody Test, IFA)和免疫印迹法(Western Blot,WB)对血清标本进行莱姆病抗体检测;并采用巢式PCR法对WB抗体阳性的血清标本进行病原学检测。结果 IFA方法检测血清样本542份,54份阳性,阳性率为9.96%。应用WB法对上述54份IFA阳性血清样本进行检测,12份阳性。采用巢式PCR方法检测12份WB阳性样本,2份阳性,测序结果表明两份均为B.garinii基因型。结论 本研究证实了海南省人群中存在莱姆病。建议当地医生在诊治关节炎和神经性疾病患者时,可以考虑其是否罹患莱姆病。     

基于PCR蜱体内巴贝虫检测方法的评价

目的分别对4种牛体表蜱体内巴贝虫和两种犬体表蜱体内巴贝虫基于PCR的检测方法进行评价。方法在广西壮族自治区6个市的62个村,现场采集犬和牛体表蜱。巢式PCR特异性扩增巴贝虫18s rDNA,纯化阳性样本的PCR产物、测序、比对,以确定感染的病原体。应用双重PCR、双重巢式PCR、双重环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测牛体表蜱内病原体双芽巴贝虫(Babesia bigemina)和牛巴贝虫(B.bovis)的感染情况。应用多重PCR检测犬体表蜱的犬巴贝虫(B.canis canis)、佛氏巴贝虫(B.canis vogeli)和罗氏巴贝虫(B.canis rossi)的感染情况。将各方法的检测结果进行比较,对检测方法进行评价。结果采集牛体表蜱102只。巢式PCR、双重PCR、双重巢式PCR检测牛体表蜱内双芽巴贝虫和牛巴贝虫均为阴性,双重LAMP的浊度仪检测结果为5例阳性,颜色观测结果阳性样本为29例,该29例包含浊度仪检测的5例阳性。采集犬体表蜱184只。巢式PCR检测出犬体表蜱佛氏巴贝虫阳性样本9例。其中4例被多重PCR检测出佛氏巴贝虫阳性。同时多重PCR检测检出罗氏巴贝虫阳性样本4例,经测序均为罗氏巴贝虫阴性。结论双重PCR、双重巢式PCR检测牛体表蜱体内巴贝虫未出现假阳性情况,巢式PCR检测微小巴贝虫,双重PCR、双重巢式PCR未发生交叉反应,说明其具有一定特异性,但因无阳性样本,敏感度需要进一步评价。双重LAMP颜色观测结果阳性率15.76%,浊度仪测出阳性率2.72%,其中双重LAMP颜色观测法2例阳性样本为巢式PCR微小泰勒虫阳性(Theieria microti)。双重LAMP法直观快捷,但对其检测的阳性样本需进行进一步确认,同时由于该方法的高度敏感性,需注重预防实验污染。犬体表蜱多重PCR可一次鉴别3种犬巴贝虫,鉴于在本研究中的假阳性和假阴性情况,多重PCR不适用,建议将病原体独立检测。     

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。     

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。     

天津地区莱姆病疫源地的发现与研究

目的通过对天津地区自然人群、动物宿主和媒介生物进行研究,以确定该地区是否为莱姆病疫源地。方法采用分层整群抽样方法选取3个代表性地区10个调查点的居民进行人群的莱姆病血清流行病学调查,同时用间接免疫荧光试验、病原分离培养和PCR方法对动物宿主和媒介生物进行血清学和病原学研究。并通过测序确定基因型。结果905份人群血清样本检测结果显示该地区人群的莱姆病感染率为5.97%;100份野鼠血清样本的感染率是32.00%,并从大林姬鼠、社鼠、小林姬鼠的脾脏和/或肾脏中检查到莱姆病螺旋体的特异片段。经聚类分析定为Borrelia garinii基因型,因此可以推断鼠类可能为此地区莱姆病螺旋体的重要储存宿主。共收集到1226只蜱,以长角血蜱为优势种。选取300只长角血蜱经PCR检测,其带菌率为4.67%(14/300)。结论首次发现天津蓟县山林地区可能为我国莱姆病的疫源地之一。     

Acarologic risk of exposure to emerging tick-borne bacterial pathogens in a semirural community in northern California

An acarologic study was conducted in a semirural community in northern California to determine the relative abundance of, and the prevalence of infection with, three emerging bacterial pathogens in the western black-legged tick (Ixodes pacificus). These included the agents causing Lyme disease (Borrelia burgdorferi), human granulocytic ehrlichiosis [Ehrlichia phagocytophila (formerly Ehrlichia equi)], and human monocytic ehrlichiosis (Ehrlichia chaffeensis). The study area in Sonoma County consisted of two properties each with four residents and an uninhabited adjacent comparison area. Six of the eight residents had been either physician-diagnosed or serodiagnosed previously with Lyme disease, and, of these, one also had been serodiagnosed with human monocytic ehrlichiosis. Direct immunofluorescent/culture assays and bacterial species-specific polymerase chain reaction assays were used to test whole ticks individually for presence of B. burgdorferi and Ehrlichia spp., respectively. Overall, 6.5% of the nymphal (n = 589) and 1.6% of the adult ticks (n = 318) from the same generational cohort were found to contain B. burgdorferi. In contrast, none of 465 nymphs and 9.9% of 202 adults were infected with E. phagocytophila. Excised tissues from another 95 adult ticks yielded a comparable E. phagocytophila infection prevalence of 13.7%. E. chaffeensis was not detected in either nymphal or adult ticks. Using a combination of culture and polymerase chain reaction assays, coinfection of I. pacificus adults with B. burgdorferi and E. phagocytophila was demonstrated for the first time. The marked disparity in the infection prevalence of these pathogens in nymphal and adult ticks suggests that their maintenance cycles are inherently different.     

Genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in north-west Poland

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).     

Detection of Borrelia burgdorferi DNA in tick feces provides evidence for organism shedding during vector feeding

Borrelia burgdorferi, the bacterium that causes Lyme disease, is transmitted to a susceptible host by Ixodes spp. tick bites. However, there is uncertainty whether B. burgdorferi are shed from ticks by the fecal route. In this study, B. burgdorferi-infected ticks were fed on mice while confined to a certain area of the skin by a capsule. During and after feeding, tick feces were collected and placed in Barbour-Stoenner-Kelley (BSK)-II media for cultivation and in sterile water for polymerase chain reaction (PCR) analysis. Although none of the tested samples were culture positive for B. burgdorferi, all but one of the fecal DNA samples from infected ticks were PCR positive. These results indicated that B. burgdorferi were shed from feeding ticks during defecation and suggest that the spirochetes did not remain viable once exposed to the outside environment. This finding has important ramifications for investigators interpreting B. burgdorferi-specific PCR results when conducting tick transmission experiments.     

First isolation of Borrelia burgdorferi sensu lato from Ixodes ricinus ticks in Morocco

To determine the infection rate of Ixodes ricinus (I. ricinus) ticks with Borrelia burgdorferi sensu lato (B. burgdorferi sl) and to assess the frequency of the individual Borrelia species in this tick species, a total of 295 I. ricinus were collected in Taza region (Northeast of Morocco), from January to June 2002. The presence of B. burgdorferi sl was determined by direct fluorescence antibody assay (DFA) and by PCR after culture. B. burgdorferi sl isolates were identified at the species level by restriction fragment length polymorphism analysis of amplified products. The mean rate of I. ricinus infection with B. burgdorferi sl was 47.8%. Isolation attempts in BSK II medium resulted in 26 pure isolates. However, PCR performed on culture medium allowed to identify 82 Borrelia DNAs. B. lusitaniae has been identified from 76 out of 82 infected I. ricinus ticks (92.7%). Three ticks were infected by B. burgdorferi ss, and three other ticks were infected by B. garinii. This is the first report of the presence of B. burgdorferi sl in Morocco and more specifically of B. burgdorferi ss in North Africa.     

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany)

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.     

Longitudinal study of infection with Borrelia burgdorferi in a population of Peromyscus leucopus at a Lyme disease-enzootic site in Maryland.

The maintenance of Borrelia burgdorferi in a population of Peromyscus leucopus was investigated from 202 mark and recapture mice and 61 mice that were removed from a site in Baltimore County, Maryland. Borrelia burgdorferi infection was detected by culture and polymerase chain reaction (PCR) of ear tissue, and exposure to the spirochete was quantified by serology. Overall prevalence of B. burgdorferi, as determined by culture and PCR of ear tissue at first capture, was 25% in the longitudinal sample and 42% in the cross-sectional sample. Significantly more juvenile mice were captured in the longitudinal sample (18%) than in the cross-sectional sample (0%). Among 36 captured juvenile mice, only one was infected with B. burgdorferi; this contributed to a significant trend for infection with B. burgdorferi with age. Recovery from infection with B. burgdorferi was not detected among 77 mice followed for an average of 160 days. The incidence rate of infection with B. burgdorferi was 10 times greater in mice captured during two periods of high risk of exposure to nymphal Ixodes scapularis ticks compared with a period of low risk. Maintenance of B. burgdorferi in this population was dependent on indirect transmission of the organism from infected ticks to susceptible mice and development of chronic infection with the spirochete, which had no measurable effect on the survival of infected mice.     

Co-detection of Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum in Ixodes pacificus ticks from California, USA

Presence of Bartonella DNA was explored in 168 questing adult Ixodes pacificus ticks from Santa Cruz County, California. Bartonella henselae type I DNA was amplified from 11 ticks (6.55%); previously, two (1.19%) were found to be infected with Borrelia burgdorferi and five (2.98%) with Anaplasma phagocytophilum. Detection of B. henselae was not dependent on co-infection. The present study offers additional evidence that Ixodes spp. ticks may act as hosts and possibly vectors for B. henselae.