Prevalence and genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ticks in Belgium

Borrelia burgdorferi sensu lato is a genetically diverse group of spirochetes that includes the agent of Lyme borreliosis in which genospecies tend to be associated with specific clinical features. The aim of the study was to determine the prevalence and genetic diversity of Borrelia burgdorferi sensu lato in 524 ticks collected in woods of a western province of Belgium. Presence of spirochetes in ticks was determined by phase contrast microscopy. The mean infection rate of ticks was 12.0%. Variability was observed in the prevalence of infection among the five sites examined, ranging from 2.8 to 21.6%. Identification to genospecies was determined by PCR and sequencing. The most common genomospecies were Borrelia afzelii (55%) and Borrelia garinii (21%). For the first time in Belgium, we detected Borrelia valaisiana and Borrelia spielmanii, representing 14% and 2%, respectively. Borrelia burgdorferisensu stricto counted only for 2%. Co-infections were present in 8% of ticks. We emphasize the need for clinical studies to assess the prevalence of specific genospecies-related clinical manifestations of Lyme borreliosis in Belgium.     

Plasmid requirements for infection of ticks by Borrelia burgdorferi

Borrelia burgdorferi strain B31 MI commonly loses one or more of its complement of 21 extrachromosomal plasmids during normal handling procedures and during genetic manipulations. Certain plasmid losses cause an inability or reduction in the ability of spirochetes to infect mice. In the current study, nine strains of spirochetes with varying plasmid profiles were used to identify plasmids necessary for nymphal tick infection. Nymphal ticks were artificially fed the nine spirochete strains as well as the parental strain containing a full complement of plasmids. The capillary fed nymphs were allowed to feed on mice for at least 63 h and then examined for the presence of spirochetes in their guts and salivary glands. All spirochete strains tested were able to infect ticks guts, but to different degrees. We determined that the plasmids lp5, lp28-1, and cp9 were not required for infecting tick guts, whereas loss of lp25 and lp28-4 was associated with reduced gut infectivity. A reduction in the ability of spirochetes to invade salivary glands was seen in bacteria that did not have lp28-1, whereas cp9 was not required for salivary gland infection. This study has pinpointed specific plasmids whose absence is deleterious to infecting nymphal tick guts and salivary glands.     

Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.     

Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice.

Although recombinant outer surface protein A (OspA) of Borrelia burgdorferi protects mice against injected Lyme disease spirochetes, the mode of protection has not yet been explored. Indeed, the efficacy of vaccine-induced immunity against a realistic vector-mediated challenge remains unexplored. Accordingly, we determined whether this immunogen protects mice against spirochetes delivered by nymphal Ixodes dammini ticks. Following challenge by tick bite, no spirochetes could be cultured from immunized mice, and no characteristic histopathology was found. The spirochete was not detected in ticks that fed on immunized animals and was present in virtually all ticks that fed on nonimmunized mice. We conclude that OspA-immunized mice are protected from spirochetal infection, at least in part, because the spirochete is destroyed in the infecting tick.     

Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.     

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.     

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。     

Neurologic diseases transmitted by ticks

Apart from causing various dermatological disorders, ticks, which also widespread in our part of the world, can transmit diseases that affect the nervous system. The difficulty of carrying out differential diagnosis and the possibility of administering causal treatment in cases of meningopolyneuritis Garin-Bujadoux-Bannwarth and progressive Borrelian encephalomyelitis are giving rise to demands for an improvement in immunodiagnosis.     

Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.     

Characterization of Borrelia burgdorferi isolated from Ixodes persulcatus and Ixodes ovatus ticks in Japan.

The Lyme disease spirochete Borrelia burgdorferi was detected in the adult stage of the ixodid ticks Ixodes persulcatus and I. ovatus collected in various localities in Japan. The total prevalence values were 16.6% of 777 I. persulcatus and 23.6% of 687 I. ovatus. Sixty-two (63.9%) of 97 isolates from I. persulcatus contained the 30-34-kD heterogeneous outer surface protein A (OspA) that reacted with monoclonal antibody H5332. In contrast, all 128 isolates from I. ovatus were homogeneous with regard to the type of 30-kD OspA protein, and were different from the I. persulcatus isolates in their protein composition. These data suggest that the transmission cycle of B. burgdorferi in I. ovatus is not related to that in I. persulcatus. No cases of Lyme disease transmitted by I. ovatus have been confirmed in Japan, in spite of the abundance of human tick bites by this species. Accordingly, the spirochetes of I. ovatus are thought to be a variant of B. burgdorferi with low virulence.     

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.     

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。     

Intra-articular co-infection by Borrelia burgdorferi and Chlamydia trachomatis

OBJECTIVE: Chlamydia trachomatis and Borrelia burgdorferi infections are frequently the cause of unexplained oligoarthritis, as shown by identification of bacteria specific DNA in joint material from patients with reactive arthritis, Lyme arthritis, and undifferentiated oligoarthritis. The aim of this study was to determine whether the two organisms occur simultaneously in joint material from patients with arthritis. METHODS: Seventy six patients with unexplained arthritis were prospectively studied. Synovial fluid was obtained from all patients and examined for DNA from C trachomatis and B burgdorferi using specific polymerase chain reaction (PCR) protocols. Data concerning prior genitourinary infection or a history of tick bite were recorded and serum antibodies to C trachomatis and B burgdorferi were determined. RESULTS: Six patients (8%) had DNA from both C trachomatis and B burgdorferi in the same synovial fluid specimen (mean leucocyte count 11.925/mm(3), 65% granulocytes). These patients (four men, two women; mean age 33.7 years) all had oligoarthritis of the knee, ankle, or both (mean disease duration 11.3 months). From the history and serological examination, four patients had some evidence of actual or previous infection with one or other of the bacteria, while the other two patients had a positive serological test for Chlamydia only. CONCLUSIONS: DNA from two different microorganisms which are known to be triggering agents for arthritis may be present simultaneously in joint material from patients with unexplained oligoarthritis. This finding raises the question as to whether, in such cases, one or both bacteria contribute to the pathogenesis of the disease or whether they are only innocent bystanders.     

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%).     

Clustering of host-seeking nymphal deer ticks (Ixodes dammini) infected by Lyme disease spirochetes (Borrelia burgdorferi).

In areas where the agent of Lyme disease is intensely enzootic, the mouse reservoirs may be universally infected. Because a large proportion of the vector tick population appears to feed upon these hosts, the prevalence of infection in the vectors should approach 100%. However, infection in host-seeking nymphal ticks in nature rarely exceeds 40%. To help reconcile this apparent paradox, we examined whether estimates of prevalence might differ if we did not assume that infected ticks are randomly or uniformly distributed within a site. Nymphal Ixodes dammini were collected by dragging a series of 10-meter replicates within an intensely enzootic site. Estimates of the prevalence of spirochetal infection, based upon the monthly means of individual 10-meter collections, were then compared with estimates derived by pooling all samples. Host-seeking ticks tended to cluster. The Lyme disease spirochete was present in 15.6% of 469 pooled ticks. When the prevalence estimate was based solely on ticks in clusters that contained one or more infected ticks, however, at least 50% of the ticks were infected. We conclude that nymphal deer ticks infected by Lyme disease spirochetes tend to aggregate spatially in nature, and that prevalence estimates based upon a mean value for pools may be misleading.