Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.     

Comparison of the spirochete Borrelia burgdorferi S. L. isolated from the tick Ixodes scapularis in southeastern and northeastern United States

Thirty-five strains of the Lyme disease spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s. l.) were isolated from the blacklegged tick vector Ixodes scapularis in South Carolina, Georgia, Florida, and Rhode Island. They were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. PCR-RFLP analysis indicated that the strains represented at least 3 genospecies (including a possible novel genospecies) and 4 different restriction patterns. Thirty strains belonged to the genospecies B. burgdorferi sensu stricto (B. burgdorferi s. s.), 4 southern strains were identified as B. bissettii, and strain SCCH-5 from South Carolina exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Complete sequences of rrf-rrl intergenic spacers from 14 southeastern and northeastern strains were determined and the phylogenetic relationships of these strains were compared. The 14 strains clustered into 3 separate lineages on the basis of sequence analysis. These results were confirmed by phylogenetic analysis based on 16S rDNA sequence analysis.     

Examination of the Borrelia burgdorferi transcriptome in Ixodes scapularis during feeding

Borrelia burgdorferi gene expression within the guts of engorging Ixodes scapularis ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library. Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks. These data demonstrate a new approach to the global analysis of B. burgdorferi genes that are preferentially expressed within the vector during feeding.     

Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the Northeastern United States

Over 80% of reported cases of Lyme disease in the United States occur in coastal regions of northeastern and mid-Atlantic states. The genetic structure of the Lyme disease spirochete (Borrelia burgdorferi) and its main tick vector (Ixodes scapularis) was studied concurrently and comparatively by sampling natural populations of I. scapularis ticks along the East Coast from 1996 to 1998. Borrelia is genetically highly diverse at the outer surface protein ospC. Since Borrelia is highly clonal, the ospC alleles can be used to define clones. A newly designed reverse line blotting (RLB) assay shows that up to 10 Borrelia clones can infect a single tick. The clone frequencies in Borrelia populations are the same across the Northeast. On the other hand, I. scapularis populations show strong regional divergence (among northeastern, mid-Atlantic, and southern states) as well as local differentiation. The high genetic diversity within Borrelia populations and the disparity in the genetic structure between Borrelia and its tick vector are likely consequences of strong balancing selection on local Borrelia clones. Demographically, both Borrelia and I. scapularis populations in the Northeast show the characteristics of a species that has recently expanded from a population bottleneck. Major geological and ecological events, such as the last glacial maximum (18,000 years ago) and the modern-day expansion of tick habitats, are likely causes of the observed "founder effects" for the two organisms in the Northeast. We therefore conclude that the genetic structure of B. burgdorferi has been intimately shaped by the natural history of its main vector, the northern lineage of I. scapularis ticks.     

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Norway: evaluation of a PCR test targeting the chromosomal flaB gene

A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169–4176, 2010) on material consisting of 242 Ixodes ricinus ticks collected from dogs and cats in Northern Norway (n?=?139) and Telemark County in Southern Norway (n?=?103). Ticks positive in either test were further tested by nested PCR amplification of the 5S-23S rRNA intergenic-spacer region followed by sequencing for species identification. A tick was defined as Borrelia positive if two of three tests were positive. Thirty-four of the 242 (14?%) ticks satisfied this definition of positivity. Of these ticks 32 were positive both in the rRNA and flaB test, while two were positive only in the rRNA test. One tick was positive only in the rRNA test and was considered false positive since PCR for sequencing failed. The sensitivity of the flaB test was 94?% and the specificity 100?%. It was possible to determine the species present using Tm analysis. Among ticks from Northern Norway the prevalence of Borrelia was 13?%, whereas the prevalence in Telemark was 16?%. Among identified species (n?=?33) B. afzelii was found in 16 (47?%), B. garinii in 15 (44?%) and B. valaisiana in 2 (6?%) ticks, respectively. The flaB test is a rapid, sensitive and specific test for detection and quantification of Borrelia burgdorferi s.l. in I. ricinus ticks. This is the first report on Borrelia prevalence in I. ricinus in Northern Norway.     

Genotypic diversity of Borrelia burgdorferi strains detected in Ixodes scapularis larvae collected from North American songbirds

We genotyped Borrelia burgdorferi strains detected in larvae of Ixodes scapularis removed from songbirds and compared them with those found in host-seeking I. scapularis nymphs sampled throughout the eastern United States. Birds are capable of transmitting most known genotypes, albeit at different frequencies than expected based on genotypes found among host-seeking nymphs.     

Dynamics of Borrelia burgdorferi infection in nymphal Ixodes ricinus ticks during feeding

We report the sequential developmental events of Borrelia burgdorferi in histological sections of Ixodes ricinus nymphs before, during and after feeding. During the blood meal a decrease of approximately 50% in the number of infected ticks was recorded (eight out of 76, 11%) in comparison with the infection rate of unfed ticks (12 out of 56, 21%). Spirochetes were detected in tick salivary glands only after 2 days of attachment. From day 3 until drop-off, the number of infected ticks increased to 31% (15 out of 49). A quadratic logistic regression analysis showed that the variation in the number of infected ticks was significant, but only during the blood meal. The drop in the percentage of infected ticks during the first hours following attachment to the host is explained by our observation of spirochetes in the faeces of the ticks. The increase in the infection rate of replete ticks may be due to an uptake of spirochetes from the host skin at the feeding site.     

Borrelia burgdorferi Promotes the Establishment of Babesia microti in the Northeastern United States

Babesia microti and Borrelia burgdorferi, the respective causative agents of human babesiosis and Lyme disease, are maintained in their enzootic cycles by the blacklegged tick (Ixodes scapularis) and use the white-footed mouse (Peromyscus leucopus) as primary reservoir host. The geographic range of both pathogens has expanded in the United States, but the spread of babesiosis has lagged behind that of Lyme disease. Several studies have estimated the basic reproduction number (R ₀) for B. microti to be below the threshold for persistence (<1), a finding that is inconsistent with the persistence and geographic expansion of this pathogen. We tested the hypothesis that host coinfection with B. burgdorferi increases the likelihood of B. microti transmission and establishment in new areas. We fed I. scapularis larva on P. leucopus mice that had been infected in the laboratory with B. microti and/or B. burgdorferi. We observed that coinfection in mice increases the frequency of B. microti infected ticks. To identify the ecological variables that would increase the probability of B. microti establishment in the field, we integrated our laboratory data with field data on tick burden and feeding activity in an R ₀ model. Our model predicts that high prevalence of B. burgdorferi infected mice lowers the ecological threshold for B. microti establishment, especially at sites where larval burden on P. leucopus is lower and where larvae feed simultaneously or soon after nymphs infect mice, when most of the transmission enhancement due to coinfection occurs. Our studies suggest that B. burgdorferi contributes to the emergence and expansion of B. microti and provides a model to predict the ecological factors that are sufficient for emergence of B. microti in the wild.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

全沟硬蜱体内伯氏疏螺旋体的分离及特征

本文报道从黑龙江省全沟硬蜱分离的伯氏疏螺旋体H7株的特征。H7株细胞长9．8—26．5μm,宽0．1 3—0．35μm,有l—11个波,波长1．2—3．Oμm,波幅0．59一1．13μm,两端尖乩每端有7根鞭毛,菌体左旋。体外培养最适温度为31℃,具有2lk、32k、34k主要结构和抗原蛋白,能与新疆及黑龙江莱姆病人血清反应。这些结果表明,该菌株具备伯氏疏螺旋体的特征,它是与其它地区或媒介分离株不同的“亚型”。     

Molecular characterization of Borrelia isolates from ticks and mammals from the southern United States

Fifty Borrelia isolates from ticks and rodents from several geographic regions of the southern United States were analyzed by genomic macrorestriction analysis. Significant genetic diversity was observed among them. These isolates segregated into 4 major clusters and 10 subclusters, which are correlated with the genospecies distribution. Nineteen pulsed-field gel electrophoresis (PFGE) types were recognized among the isolates. The genospecies Borrelia andersonii and Borrelia bissettii consisted of 5 and 2 subclusters, respectively. Two subclusters comprised the Borrelia burgdorferi sensu stricto (s. s.) strains. These results indicated that PFGE is a suitable molecular typing method for B. burgdorferi at both the genospecies and strain levels. Seventeen representative isolates from different PFGE groups were analyzed by restriction fragment length polymorphism (RFLP) and sequence analysis of flaB. Twenty-three AluI, 3 CelII, and 11 DdeI RFLP patterns were found among strains from the B. burgdorferi sensu lato (s. l.) complex and the relapsing fever borreliae complex. Three genospecies in the B. burgdorferi s. l. complex and 1 species in the relapsing fever borreliae complex were recognized. Phylogenetic analysis based on nucleotide sequences of flaB indicated that all the Borrelia strains analyzed here could be divided into 2 parts, i.e., B. burgdorferi s. l. complex and the relapsing fever borreliae complex. The flaB appears to be a useful target gene to screen and identify strains from both B. burgdorferi s. l. and relapsing fever borreliae complexes.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Local variations in the distribution and prevalence of Borrelia burgdorferi sensu lato genomospecies in Ixodes ricinus ticks.

Unfed nymphal and adult Ixodes ricinus ticks were collected from five locations within the 10,000-ha Killarney National Park, Ireland. The distribution and prevalence of the genomospecies of Borrelia burgdorferi sensu lato in the ticks were investigated by PCR amplification of the intergenic spacer region between the 5S and 23S rRNA genes and by reverse line blotting with genomospecies-specific oligonucleotide probes. The prevalence of ticks infected with B. burgdorferi sensu lato was significantly variable between the five locations, ranging from 11.5 to 28.9%. Four genomospecies were identified as B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and VS116. Additionally, untypeable B. burgdorferi sensu lato genomospecies were identified in two nymphs. VS116 was the most prevalent of the genomospecies and was identified in 50% of the infected ticks. Prevalences of B. garinii and B. burgdorferi sensu stricto were similar (17 and 18%, respectively); however, significant differences were observed in the prevalence of these genomospecies in mixed infections (58.8 and 23.5%, respectively). Notably, the prevalence of B. afzelii was low, comprising 9.6 and 7.4%, respectively, of single and mixed infections. Significant variability was observed in the distribution and prevalence of B. burgdorferi sensu lato genomospecies between locations in the park, and the diversity and prevalence of B. burgdorferi sensu lato genomospecies was typically associated with woodland. The distributions of B. burgdorferi sensu lato genomospecies were similar in wooded areas and in areas bordering woodland, although the prevalence of B. burgdorferi sensu lato infection was typically reduced. Spatial distributions vegetation composition, and host cenosis of the habitats were identified as factors which may affect the distribution and prevalence of B. burgdorferi sensu lato genomospecies within the park.