福建莱姆病生物媒介的调查

１９９０年３月～１９９３年９月，我们对福建省莱姆病疫区的蜱、虻类进行调查。共捕获蜱类１７种、虻类３种，对部份蜱、虻类肠道进行莱姆病病原体的分离与检测，结果从其中的粒形硬蜱（Ｉ．ｇｒａｎｕｌａｔｕｓ）中肠及其吸血宿主鼠类（社鼠和褐家鼠Ｒ．Ｃｏｎｆｕｃｉａｎｕｓ＆Ｒ．ｎｏｒｖｅｇｉｃｕｓ）肾脏共分离出４株莱姆病病原体－伯氏疏螺旋体，从台湾角血蜱（Ｈ．ｃｏｒｎｉｇｅｒａｔａｉｗａｎａ）中肠观察到同样病原体，表明粒形硬蜱和某些血蜱可能是福建莱姆病疫区传播该病的生物媒介。此外，我们还发现因虻类、蜂类叮咬而发生感染的病例，提示莱姆病的传播媒介可能还不仅局限于蜱类．     

莱姆病概述及吉林省莱姆病调查研究进展

莱姆病（Lyme disease LD）是由伯氏疏螺旋体（Borrelia burgdorferi）经蜱（中国一些地方俗称草爬子）传播的自然疫源性疾病，是一种新发现的人兽共患病，在世界范围内于人和动物中广泛流行，且发病率呈现上升趋势。     

全沟硬蜱在黑龙江小兴安岭莱姆病螺旋体传播中的作用

目的：探讨全沟硬蜱在莱姆病螺旋体传播中的作用。方法：用布旗法在林区草地采蜱，并分类鉴定；用直接荧光抗体法检查蜱类中肠带菌情况，并分离菌株。结果：全沟硬蜱为当地优势蜱种，人群发病与该蜱叮咬率密切相关；该蜱中肠带菌率为４０％，并从１４组（７０只）蜱中分离出２株莱姆病螺旋体；新分离株的单克隆抗体反应与国内分离株Ｍ７相同，而与美国菌株Ｂ３１有区别。结论：当地全沟硬蜱是莱姆病螺旋体的主要生物媒介。     

从粒形硬蜱和社鼠分离出4株莱姆病病原体

从福建省北部莱姆病感染率较高的林区捕获一批野鼠,从鼠体收集到部分粒形硬蜱,取蜱类中肠和社鼠肾脏接种BSKII培养基,结果分离出4株莱姆病病原体—伯氏疏螺旋体(Borrelia burgdorferi)。从粒形硬蜱和社鼠分离出莱姆病病原体,在国内外属首次报告。     

我国莱姆病病原研究和诊断方法的进展

我国莱姆病病原研究和诊断方法的进展中国军事医学科学院（北京１０００７１）张泮河（综述）张启恩（审阅）莱姆病是由伯氏疏螺旋体引起的一种经蜱传播的多系统受累的疾病。我国１９８６年首次在黑龙江省海林地区发现莱姆病［１］，１９８７年分离到病原体，并对其宿主动...     

大兴安岭林区蜱和鼠中莱姆病螺旋体感染及其基因分型研究

目的 了解内蒙古大兴安岭林区蜱和鼠中伯氏疏螺旋体的感染及基因分型情况。方法 应用巢式PCR扩增蜱和鼠中伯氏疏螺旋体5S-23SrRNA间隔区片段，对阳性产物进行限制性片段长度多态性（RFLP）分析和单链构象多态性（SSCP）分析，RFLP分析显示特殊带型的样本测序分析。结果 检测全沟硬蜱1336只，293只阳性，阳性率为21．93％；森林革蜱144只，6只阳性，阳性率为4．17％；嗜群血蜱144只，未发现有伯氏疏螺旋体感染。检测鼠9种145只，感染伯氏疏螺旋体的4种5只，感染率为3．45％；其中检测8只棕背鼠平，2只阳性。RFLP分析及序列分析显示蜱中有B．garinii20047亚型、B．gariniiNT29亚型、B．afzelii基因型以及不同基因型或亚型伯氏疏螺旋体的混合感染；鼠感染的伯氏疏螺旋体包括B．garinii20047亚型和B．gariniiNT29亚型。SSCP分析结果显示带型多于36种。结论 大兴安岭林区蜱及鼠中均存在伯氏疏螺旋体的感染，其中全沟硬蜱的感染率较高；B．garinii型为主要基因型，且该地区伯氏疏螺旋体存在遗传多态性。单只蜱中存在同时感染不同基因型伯氏疏螺旋体的情况，人和宿主动物是否存在不同基因型伯氏疏螺旋体混合感染尚待进一步研究。全沟硬蜱和棕背鼾分别是该林区伯氏疏螺旋体的主要媒介和主要储存宿主。     

莱姆病概述

莱姆病(Iyme disease)是一种由伯氏疏螺旋体(或莱姆病螺旋体)所引起，经硬蜱为主要传播介质的自然疫源性疾病。     

中国20个省、区、市动物莱姆病初步调查研究

目的：广泛调查我国动物莱姆病分布情况。方法：血清流行病学和病原学研究。结果：１９８７～１９９７年间，牛、羊、狗、野兔和８种野鼠有莱姆病的感染存在，其中牛、羊、狗和野鼠的感染率分别为１８．１８％～３２．６１％、１７．１２％～６１．２１％、３８．５％～６０．００％和４１．１８％～８６．０５％；从黑线姬鼠和白腹巨鼠的胎鼠分离到莱姆病螺旋体，证实莱姆病螺旋体可通过胎盘垂直传播。９种蜱的中肠携带莱姆病螺旋体，带菌率以全沟硬蜱、二棘血蜱和粒形硬蜱为高，分别为２０．００％～４５．００％、１６．００％～４０．００％和２４．００％。从华南兔、８种野鼠和８种蜱分离出１２８株莱姆病螺旋体。证实黑线姬鼠和棕背是中国莱姆病螺旋体的重要储存宿主；多种蜱的中肠内携带莱姆病螺旋体，我国北方林区莱姆病螺旋体的主要生物媒介是全沟硬蜱，南方林区二棘血蜱和粒形硬蜱是重要的生物媒介。结论：我国动物莱姆病广泛存在     

全沟硬蜱是我国北方地区莱姆病螺旋体的主要传播媒介

研究我国北方地区莱姆病螺旋体的生物媒介—蜱的种类、带菌状况及在传播莱姆病中的作用。用布旗法和动物诱捕法在山林地区采集蜱，分类鉴定，直接荧光抗体染色法查蜱中肠带菌率，ＢＳＫ培养基分离病原体，单克隆抗体鉴定菌株。调查蜱叮咬率与人群莱姆病感染率、患病率以及蜱的季节消长与莱姆病慢性游走性红斑（ＥＣＭ）病例发生的季节性关系。在北方１０省（市、区）共采集到１００００多只蜱，分属为２科７属１１种，以全沟硬蜱为优势种，占８０％～９５％。４种蜱的中肠携带莱姆病螺旋体，全沟硬蜱的带菌率高达２０．００％～４５．００％。从４种蜱分离到９８株莱姆病螺旋体，其中９０％以上是从全沟硬蜱分离的。全沟硬蜱叮咬率高的人群，莱姆病的感染率和患病率也高。全沟硬蜱的季节消长与ＥＣＭ发生的季节性相一致。证实我国北方地区至少有４种蜱的中肠内携带莱姆病螺旋体，莱姆病螺旋体的主要生物媒介是全沟硬蜱。     

莱姆病的部分传播媒介

莱姆（Lyme）病是由伯氏疏螺旋体（Borreliaburgdorferi）引起的蜱传自然疫源性人畜共患病。全世界五大洲30多个国家有病例报告，据我国初步调查，20个省、直辖市、自治区都存在人群莱姆病。该病累及人体多器官、多系统，危害严重，有人称为“当今除爱滋病外的第一号新病”[1]。莱姆病螺旋体贮存宿主多，传播途径多，能携带该螺旋体的节肢动物多。动物宿主主要为啮齿类，还有野生动物、家畜、鸟类和爬行类。传染源是感染B．burgdorferi的人、动物和硬蜱。硬蜱既是莱姆病的传播媒介，又是传染源和保菌宿主。全世界已从2科9属30余种蜱（附表…     

Vector interactions and molecular adaptations of lyme disease and relapsing fever spirochetes associated with transmission by ticks

Pathogenic spirochetes in the genus Borrelia are transmitted primarily by two families of ticks. The Lyme disease spirochete, Borrelia burgdorferi, is transmitted by the slow-feeding ixodid tick Ixodes scapularis, whereas the relapsing fever spirochete, B. hermsii, is transmitted by Ornithodoros hermsi, a fast-feeding argasid tick. Lyme disease spirochetes are generally restricted to the midgut in unfed I. scapularis. When nymphal ticks feed, the bacteria pass through the hemocoel to the salivary glands and are transmitted to a new host in the saliva after 2 days. Relapsing fever spirochetes infect the midgut in unfed O. hermsi but persist in other sites including the salivary glands. Thus, relapsing fever spirochetes are efficiently transmitted in saliva by these fast-feeding ticks within minutes of their attachment to a mammalian host. We describe how B. burgdorferi and B. hermsii change their outer surface during their alternating infections in ticks and mammals, which in turn suggests biological functions for a few surface-exposed lipoproteins.     

Protective efficacy of an oral vaccine to reduce carriage of Borrelia burgdorferi (strain N40) in mouse and tick reservoirs

Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted through the bite of infected Ixodes ticks. Vaccination of mice with outer surface protein A (OspA) of B. burgdorferi has been shown to both protect mice against B. burgdorferi infection and reduce carriage of the organism in feeding ticks. Here we report the development of a murine-targeted OspA vaccine utilizing Vaccinia virus to interrupt transmission of disease in the reservoir hosts, thus reducing incidence of human disease. Oral vaccination of mice with a single dose of Vaccinia expressing OspA resulted in high antibody titers to OspA, 100% protection of vaccinated mice from infection with B. burgdorferi, and significant clearance of B. burgdorferi from infected ticks fed on vaccinated animals. The results indicate the vaccine is effective and may provide a manner to reduce incidence of Lyme disease.     

Oral vaccine that breaks the transmission cycle of the Lyme disease spirochete can be delivered via bait

Borrelia burgdorferi causes Lyme disease, a potentially debilitating human disease for which no vaccine is currently available. We developed an oral bait delivery system for an anti-B. burgdorferi vaccine based in OspA. Mice were immunized orally via gavage and bait feeding. Challenge was performed via Ixodes scapularis field nymphs carrying multiple B. burgdorferi strains. Vaccination protected 89% of the mice and the systemic immune response was skewed toward IgG2a/2b production. Moreover, this oral vaccine reduced the pathogen in the tick vector by eight-fold. We conclude that this oral vaccine induces a protective systemic immune response against a variety of infectious B. burgdorferi strains found in nature and therefore it can eliminate this zoonotic pathogen from its major host reservoirs. Because we observed elimination of the spirochete from the tick vector, a broad delivery of this oral vaccine to wildlife reservoirs is likely to disrupt the transmission cycle of this pathogen.     

我国莱姆病螺旋体生物媒介的初步调查

自1987年以来,我们在全国开展了莱姆病调查,从黑龙江、吉林、辽宁、内蒙、河北、新疆、湖北、四川、福建和北京等省、市采集的全沟硬蜱、粒形硬蜱、壤塘硬蜱、嗜群血蜱、二棘血蜱和长角血蜱分离出71株莱姆病螺旋体,其中55株从全沟硬蜱分离。全沟硬蜱是东北和西北针阔叶林区的优势蜱种,其带菌率为20~45%,表明全沟硬蜱是我国北方莱姆病螺旋体的主要生物媒介。虽然从我国南方的某些硬蜱属和血蜱属种类分离到莱姆病螺旋体,但是南方的主要生物媒介还不清楚。从我国蜱体内分离的螺旋体在超微结构、单克隆抗体反应和外表蛋白等方面与美国菌株有某些区别。     

Transgenic functional complementation with a transmission -associated protein restores spirochete infectivity by tick bite

The relapsing fever spirochete Borrelia hermsii and the Lyme disease spirochete Borrelia burgdorferi sensu stricto each produces an abundant, orthologous, outer membrane protein, Vtp and OspC, respectively, when transmitted by tick bite. Gene inactivation studies have shown that both proteins are essential for spirochete infectivity when transmitted by their respective tick vectors. Therefore, we transformed a vtp-minus mutant of B. hermsii with ospC from B. burgdorferi and examined the behavior of this transgenic spirochete in its soft tick vector Ornithodoros hermsi. IFA staining indicated up to 97.8 % of the transgenic B. hermsii upregulated OspC in the ticks’ salivary glands compared to no more than 12.8 % in the midgut, similar to our previous findings with wild-type B. hermsii producing Vtp. Transformation with ospC also restored B. hermsii infectivity to mice when fed upon by infected ticks. Previous sequence analysis of Vtp for 79 isolates and DNA samples of B. hermsii in our laboratory showed this protein is highly polymorphic with 9 divergent amino acid types, yet strikingly the signal peptide is identical among all samples and the same for all OspC signal peptides for B. burgdorferi and related species examined to date. Searches in multiple genome sequences for other species of relapsing fever spirochetes failed to find the same signal peptide sequence to help identify potential transmission-associated proteins. However, some candidate signal peptides with highly similar sequences were found and worthy of future efforts with other species. While OspC of B. burgdorferi restored infectivity to a Vtp-minus mutant of B. hermsii, the functions of these proteins are not known. Our results should stimulate investigators to search for orthologous transmission-associated proteins in other tick-borne spirochetes to better understand how this group of pathogens has coevolved with diverse tick vectors.     

Co-feeding transmission and its contribution to the perpetuation of the lyme disease spirochete Borrelia afzelii

To determine whether direct passage of spirochetes between co-feeding vector ticks contributes to the likelihood that the Lyme disease spirochete Borrelia afzelii will perpetuate in nature, we compared the effects of time and space on transmission efficiency between simultaneously feeding ticks. The likelihood of co-feeding transmission increases with duration of attachment of the infecting tick. Co-feeding transmission becomes less efficient as distance from the infecting tick increases. Approximately 6 times as many ticks acquire infection when feeding on infected mice than when co-feeding with infected ticks. Both subadult stages of the wood tick Ixodes ricinus infrequently co-infest mice and voles in nature; on approximately 1 in 20 small rodents, larvae co-feed with spirochete-infected nymphs. Because only 1 in 100 larvae in nature appear to acquire spirochetal infection when co-feeding with infected nymphs, perpetuation of B. afzelii depends largely on horizontal transmission of such pathogens from previously infected mice to noninfected larvae.     

Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses.

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.     

Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease

Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.     

Oral vaccination with vaccinia virus expressing the tick antigen subolesin inhibits tick feeding and transmission of Borrelia burgdorferi

Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin, a conserved tick protein that can act as a regulator of gene expression, expressed from vaccinia virus for use as an orally delivered reservoir - targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing vaccinia virus inhibited tick infestation by 52% compared to control vaccination with vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts.     

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

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(polymerase chain reactio,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，选择阳性标准本进行克隆和序列测定，与已知序进行同源性比较，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，57只寄全沟硬蜱若蜱中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。50份野鼠血清有5份莱姆病螺旋体IgG抗体阳性，采集采的160只长角血蜱（20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体，野鼠可能是贮存宿主。