首次从日本血蜱检测出莱姆病螺旋体

１９９３年６－８月间，在河北省涿鹿县捕获部分蜱类，经分离与检测，从全沟硬蜱中肠分离出１株莱母病螺旋体，以ＰＣＲ法从１只全沟硬成蜱及１只日本血蜱若蜱体内检出莱姆病螺旋体ＤＮＡ阳性，从日本血蜱检出莱母病螺旋体ＤＮＡ阳性，从日本血蜱检出莱姆病螺旋体在国内外尚属首次。     

长角血蜱两性生殖株和孤雌生殖株幼蜱发育零点及有效积温的研究

目的研究长角血蜱两性生殖株和孤雌生殖株幼蜱在不同温度下的发育零点和有效积温。方法将长角血蜱两性生殖株和孤雌生殖株饱血幼蜱置于不同温度的环境中,观察发育历期和积温,采用拟合模型方程求出两性株和孤雌株幼蜱发育的零点温度,计算幼蜱发育的有效积温。结果长角血蜱两性株和孤雌株的发育零点分别为14.89℃和14.95℃,平均积温分别为(265.08±99.37)日度和(306.90±176.24)日度,有效积温分别为(101.44±147.66)日度和(116.51±116.79)日度。结论长角血蜱饱血幼蜱的发育历期随温度升高而缩短,两性株比孤雌株发育快,研究结果可以作为长角血蜱生物学的基础理论参数之一。     

长角血蜱饥饿雌蜱cDNA表达文库的构建及免疫学筛选

目的 构建长角血蜱饥饿雌蜱cDNA表达文库,筛选长角血蜱功能性抗原基因。 方法 在无RNA酶污染的条件下提取长角血蜱总RNA,进而纯化mRNA,以寡脱氧胸腺核苷酸［oligo（dT）］为引物合成双链cDNA,并在其两端加EcoRⅠ/HindⅢ定向接头。将cDNA分子定向克隆至具有EcoRⅠ/HindⅢ黏性末端的λSCREEN载体。用噬菌体包装蛋白对以上连接产物进行体外包装以形成完整的噬菌体,转化大肠埃希菌（Escherichia coli）ER1647,从而构建长角血蜱饥饿雌蜱cDNA表达文库。使用兔抗长角血蜱全蜱血清对该文库进行免疫学筛选,经过2次筛选得到的阳性噬菌体转化E. coli BM25.8亚克隆为重组质粒,转化E. coli JM109,提取重组质粒进行PCR和测序分析。 结果 长角血蜱饥饿雌蜱cDNA表达文库的基础库容量为1.8×106 pfu,重组率为100%,扩增后的滴度为2.4×109 pfu/ml。筛选获得42个阳性克隆,序列分析表明有12个新cDNA序列,其编码蛋白与长角血蜱原肌凝蛋白、环状扇头蜱幼蜱未知蛋白、黑腹果蝇染色体2R、褐黄血蜱线粒体DNA、青海血蜱HqL09、Hq05和肌球蛋白轻链mRNA等具有同源性。 结论 构建了长角血蜱饥饿雌蜱cDNA表达文库,获得的阳性克隆为长角血蜱功能性抗原的研究奠定基础。     

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。     

甘肃、湖南和广东三省蜱体内嗜吞噬细胞无形体与伯氏疏螺旋体共感染研究

目的 嗜吞噬细胞无形体和伯氏疏螺旋体是由媒介蜱传播的引起人粒细胞无形体病和莱姆病的病原。目前,对两种病原在蜱体内共感染流行情况的研究相对较少。方法 以甘肃、湖南和广东三个省采集到3个蜱种、共543份样品为研究对象,检测嗜吞噬细胞无形体和伯氏疏螺旋体在蜱体内的感染情况。结果 嗜吞噬细胞无形体和伯氏疏螺旋体在不同地区采集到的蜱体内的感染率不同,分别为3.2%~20.0%和2.3%~19.3%。在检测的样品中,共发现有7份样品中同时感染嗜吞噬细胞无形体和伯氏疏螺旋体。青海血蜱、血红扇头蜱和微小牛蜱中均检测到这两种病原。结论 青海血蜱、血红扇头蜱和微小牛蜱均能够携带嗜吞噬细胞无形体和伯氏疏螺旋体,可能为这两种病原在自然界的持续存在和循环提供了条件。此研究结果丰富了人粒细胞无形体病和莱姆病的流行病学信息,有利于提高这两种病的防控策略。     

内蒙古大兴安岭林区牙克石段的蜱种及蜱携病原体感染调查

目的 调查内蒙古东部大兴安岭林区牙克石段的蜱种分布及病原体感染状况。方法 2016-2019年,在东部林区蜱活跃高峰期(5-6月)采用布旗法采集游离蜱标本,使用体视显微镜对蜱初步分类;提取蜱全基因组DNA,采用斑点热立克次体、无形体属、埃立克体属、莱姆病螺旋体和新型回归热螺旋体病原体的特异性基因进行PCR检测。结果 本次共采集蜱虫2 786只,通过体视显微镜和基因检测分析,隶属于1科3属4种,分别为全沟硬蜱(Ixodes persulcatus)、嗜群血蜱(Haemaphysalinae concinna)、日本血蜱(Haemaphysalinae japonica)、森林革蜱(Dermacentor silvarum)。其中,全沟硬蜱(78.6%,2 191/2 786)、嗜群血蜱(15.9%,442/2 786)为本地区的优势蜱种。在大兴安岭林区牙克石段的7个采样地区的蜱中,乌奴耳镇斑点热立克次体检出率最高,为74.2%(222/299);博克图镇无形体属检出率较高,为18.9%(39/206);库都尔镇埃立克体属、莱姆病螺旋体、新型回归热螺旋体的检出率均较高,分别为26.1%(12/46)、76.1%(35/46)、13.0%(6/46)。除新型回归热螺旋体外,其他4种病原体的检出率雄蜱均高于雌蜱(P<0.05);成蜱的无形体、埃立克体、莱姆病螺旋体检出率均高于幼蜱(P<0.05)。大兴安岭林区牙克石段蜱的复合感染率为19.5%(544/2 786)。其中,免渡河镇蜱携2种病原体复合感染率为40.6%(186/458),乌奴耳镇蜱携3种及以上病原体复合感染率分别是47.4%(36/76)、90%(9/10)。结论 大兴安岭林区牙克石段共存在4种蜱,蜱携莱姆病螺旋体最普遍,其中免渡河镇和乌奴耳镇的蜱普遍携带多种病原体,需重点加强该地区蜱媒传染病的监测和预防。     

湖北省长角血蜱携带嗜吞噬细胞无形体的调查

目的调查湖北省长角血蜱是否携带嗜吞噬细胞无形体。方法运用聚合酶链反应方法对湖北随州地区采集的蜱标本进行嗜吞噬细胞无形体16S rRNA基因片段进行扩增和序列分析,将扩增序列与GenBank注册的基因序列进行比较。结果共检测游离蜱72只,蜱种鉴定均为长角血蜱,嗜吞噬细胞无形体最小感染率为4.22%;所检出的嗜吞噬细胞无形体16S rRNA基因与我国已在GenBank注册的24个相对应序列同源性100%。结论湖北省长角血蜱携带嗜吞噬细胞无形体。     

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。     

甘肃省迭部和铧尖地区4种蜱自然感染莱姆病伯氏疏螺旋体的调查

目的 调查甘肃省迭部和铧尖地区4种蜱自然感染莱姆病伯氏疏螺旋体(Bb)情况,为莱姆病的防治提供科学依据。方法 2010年3月至6月,在甘肃省迭部和铧尖地区岷山北麓迭部林区（秦岭山脉）和肃南祁连山北麓铧尖林区,对4种优势蜱（森林革蜱、草原革蜱、日本血蜱和青海血蜱）自然感染莱姆病Bb进行流行病学检测。采用夹夜法,每隔10 m布夹,晚放晨收,将捕获的啮齿类动物逆毛检虫法采集寄生蜱,同时采用拖旗法采集游离蜱。对采集的4种活的成蜱,清洗消毒后解剖取其中肠内容物分别涂片,置暗视野镜下观察莱姆病Bb;对所分离到的螺旋体再用Bb单克隆和多克隆抗体鉴别试验加以证实。结果 共采集到蜱类2科8属36种,即硬蜱科6属33种,软蜱科2属3种。解剖森林革蜱、草原革蜱、日本血蜱和草原硬蜱4种201只蜱的肠道,暗视野观察出携带莱姆病Bb的阳性蜱25只,阳性率为12.44％(25/201);接种培养森林革蜱、草原革蜱和日本血蜱3种65只蜱,从12只蜱体内培养分离出莱姆病Bb,阳性率为18.46％(12/65)。结论 森林革蜱、草原革蜱和日本血蜱均有程度不同地莱姆病Bb的自然感染。     

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。     

Ixodes sinensis: competence as a vector to transmit the Lyme disease spirochete Borrelia garinii

In order to determine the principal vectors of Lyme disease in South China, the capability of Ixodes sinensis to transmit Lyme disease spirochetes transstadially was estimated in the laboratory. Results suggest that I. sinensis can acquire active Lyme disease spirochetes by feeding on infected mice of Kunming strains (KM), and the positive rates of larvae and nymph I. sinensis infected were 94.0% and 92.0%, respectively. I. sinensis maintained active spirochetes during blood digestion and molting periods; the subsequent questing tick stages were also found to be infected and infectious to naive KM mice, which resulted in more than 66.7% spirochete-positive KM mice. I. sinensis can transmit the Lyme disease spirochete Borrelia garinii from infected KM mice to naive KM mice during larvae-nymph and nymph-adult periods. Therefore, I. sinensis might be considered as a competent vector of Lyme disease in South China.     

Growth kinetics of the Lyme disease spirochete (Borrelia burgdorferi) in vector ticks (Ixodes dammini)

Lyme disease spirochetes (Borrelia burgdorferi) multiplied rapidly in larval Ixodes dammini, reaching a mean density of 2,735 spirochetes/tick on day 15 post-repletion. A 5-fold drop in spirochete levels occurred during the subsequent premolting period. Recently molted nymphs contained a mean of less than 300 spirochetes/tick. Following nymphal repletion, spirochete multiplication renewed, reaching a mean abundance of 61,275 spirochetes/nymph on day 75 post-repletion. A 10-fold drop in spirochete abundance occurred again when ticks molted to the adult stage. Tick-derived spirochetes proved to be infectious when greater than 10(4) spirochetes were inoculated ip into hamsters (4 of 4 animals infected). Inocula of 10(3-4) spirochetes were not always infectious (8 of 23 animals infected), and inocula of less than 10(3) spirochetes were insufficient for establishing infection (0 of 8 animals infected).     

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.     

Detection of Borrelia burgdorferi Sensu Lato and Anaplasma phagocytophilum in small mammals and ectoparasites in Hungary

The aim of our study was to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in small mammals and ticks using polymerase chain reaction and to gain information about the prevalence and possible coexistence of these pathogens at a selected site in Hungary. Two hundred seventy-seven small mammals were trapped in South-Eastern Hungary during 2009. Tissue samples and a total of 831 ectoparasites (Ixodes ricinus, Ixodes acuminatus, Haemaphysalis concinna, Ctenophtalmus assimilis, and Nosopsyllus fasciatus) were collected from small mammals. One thousand one hundred and six I. ricinus and 476 H. concinna were collected from the vegetation during the investigation. Neither A. phagocytophilum nor B. burgdorferi s.l. was detected in any of the mammal tissue samples. A. phagocytophilum was not found in ticks collected from small mammals. Very low minimum prevalence was found for all pathogens (0.62% for Borrelia afzelii in ticks collected from small mammals, and 0.57%, 0.06%, and 0.19% for A. phagoctyophilum, B. afzelii, and Borrelia garinii, respectively, in questing ticks). The present study is the first report of borreliae from I. acuminatus and H. concinna from Hungary.     

Occurrence of Borrelia afzelii and Borrelia garinii in Ixodes ricinus ticks from southern Moravia, Czech Republic

During the years 1996-2000, a total of 2398 Ixodes ricinus ticks were collected in three areas of southern Moravia and eastern Bohemia, Czech Republic, and examined by dark-field microscopy for the presence of spirochetes. The prevalence of B. burgdorferi sensu lato in Ixodes ricinus ticks varied and depended upon the year. In 1996, the prevalence 6.8% was observed, during 1997 and 1998, it increased to 8.4% and 12.3%, respectively. The lowest prevalence was observed in 1999 (3.6%), and in 2000 it increased to 4.0%. The mean rate of infection was 6.5%, and the proportions of infected ticks were 12.2% in 263 male ticks, 8.3% in 289 female ticks, 6.0% in 1621 nymphs, and 1.3% in 225 larvae. From the total of 156 highly infected ticks (>100 spirochetes per sample) transferred into BSK-H medium for isolation attempts, 13 isolates were obtained. PCR-RFLP and electrophoresis (SDS-PAGE) were used for the identification and characterization of Borrelia strains. Ten tick isolates were identified as Borrelia afzelii, and the other three isolates were found to be Borrelia garinii. The results indicate the epidemiological importance of B. afzelii and B. garinii in central Europe, and emphasize the role of I. ricinus in the ecology of B. burgdorferi and in the epidemiology and epizootiology of Lyme disease.     

Absence of Lyme disease spirochetes in larval Ixodes ricinus ticks

To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.     

A relapsing fever group spirochete transmitted by Ixodes scapularis ticks

A species of Borrelia spirochetes previously unknown from North America has been found to be transmitted by Ixodes scapularis ticks. Infected ticks are positive for Borrelia spp. by DFA test but negative for Borrelia burgdorferi by polymerase chain reaction (PCR) using species-specific primers for 16S rDNA, outer surface protein A, outer surface protein C, and flagellin genes. A 1,347-bp portion of 16S rDNA was amplified from a pool of infected nymphs, sequenced, and compared with the homologous fragment from 26 other species of Borrelia. The analysis showed 4.6% pairwise difference from B. burgdorferi, with the closest relative being Borrelia miyamotoi (99.3% similarity) reported from Ixodes persulcatus in Japan. Phylogenetic analysis showed the unknown Borrelia to cluster with relapsing fever group spirochetes rather than with Lyme disease spirochetes. A 764-bp fragment of the flagellin gene was also compared with the homologous fragment from 24 other Borrelia species. The flagellin sequence of B. burgdorferi was 19.5% different from the unknown Borrelia and showed 98.6% similarity with B. miyamotoi. A pair of PCR primers specifically designed to amplify a 219-bp fragment of the flagellin gene from this spirochete was used to survey field-collected I. scapularis nymphs from five northeastern states (Connecticut, Rhode Island, New York, New Jersey, and Maryland). Positive results were obtained in 1.9-2.5% of 712 nymphs sampled from four states but in none of 162 ticks collected from Maryland. Transovarial transmission was demonstrated by PCR of larval progeny from infected females with filial infection rates ranging from 6% to 73%. Transstadial passage occurred from larvae through adults. Vertebrate infection was demonstrated by feeding infected nymphs on Peromyscus leucopus mice and recovering the organism from uninfected xenodiagnostic larvae fed 7-21 days later. Considering the frequency of contact between I. scapularis and humans, further work is needed to determine the potential public health significance of yet another zoonotic agent transmitted by this tick species.     

The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi

To determine the significance of the western black-legged tick, Ixodes pacificus, as a vector of the Lyme disease spirochete, Borrelia burgdorferi, a tick/spirochete survey was conducted in northern California and southwestern Oregon from 1982 to 1984. Of 1,687 adult ticks collected off vegetation, 25 (1.48%) contained spirochetes. Of 715 ticks from Oregon, 14 (1.96%) were infected whereas 11 (1.13%) of 972 ticks from California harbored spirochetes. An isolate of 1 of the spirochetes reacted specifically when treated with monoclonal antibodies to B. burgdorferi. Polyacrylamide gel electrophoresis of a lysate of the isolate showed it to be nearly identical with 2 isolates of B. burgdorferi. Of the 25 infected I. pacificus, 17 had spirochetes in their midgut only; the remaining 8 ticks showed a generalized infection of all the tissues, with midgut, central ganglion and ovary or testes showing heavy spirochetal infections. Decreased immunofluorescent staining reactivity of spirochetes in tissues other than midgut in 6 of 8 I. pacificus with generalized infection may reflect adverse physiologic conditions for the development of spirochetes in the hemocele.     

Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle.

A human-derived strain of the agent of human granulocytic ehrlichiosis, a recently described emerging rickettsial disease, has been established by serial blood passage in mouse hosts. Larval deer ticks acquired infection by feeding upon such mice and efficiently transmitted the ehrlichiae after molting to nymphs, thereby demonstrating vector competence. The agent was detected by demonstrating Feulgen-positive inclusions in the salivary glands of the experimentally infected ticks and from field-derived adult deer ticks. White-footed mice from a field site infected laboratory-reared ticks with the agent of human granulocytic ehrlichiosis, suggesting that these rodents serve as reservoirs for ehrlichiae as well as for Lyme disease spirochetes and the piroplasm that causes human babesiosis. About 10% of host-seeking deer ticks were infected with ehrlichiae, and of these, 20% also contained spirochetes. Cotransmission of diverse pathogens by the aggressively human-biting deer tick may have a unique impact on public health in certain endemic sites.