Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks

Borreliosis is an endemic infection in Denmark. Recent serosurveys have indicated that human anaplasmosis may be equally common. The aim of this study was to look for Anaplasma phagocytophilum and related pathogens in Ixodes ricinus ticks and estimate their prevalence, compared to Borrelia, using PCR. Ticks were collected from three locations in Denmark: Jutland, Funen, and Bornholm. Ticks from Jutland and Funen were analysed individually, ticks from Bornholm were analysed in pools of 20. A. phagocytophilum was found in ticks from all areas. A. phagocytophilum was found in 23.6% of ticks from Jutland and Funen, while 11% were positive for Borrelia burgdorferi. The Borrelia genotype B. afzelii was most prevalent, followed by B. valaisiana, B. burgdorferi s.s. and B. garinii.A. phagocytophilum was found in 14.5% of nymphs and 40.5% of adult ticks, while Borrelia was found in 13% of nymphs and 8% of adult ticks. The difference in prevalence between Anaplasma and Borrelia in adult ticks supports the idea that their maintenance cycles in nature may be different. Ticks were also infected with Rickettsia helvetica. Our study indicates that A. phagocytophilum prevalence in ticks in Denmark is as high as Borrelia prevalence and that human anaplasmosis may be unrecognized.     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n = 15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n = 6), and both OspA and OspC in 39.6% (n = 19) of ticks, while in 16.7% (n = 8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe. Received: 28 July 1998     

Coexistence of ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease recognized in the Western hemisphere. HGE is well known to occur in North America, but records from outside the United States are sparse. The great majority of data from Europe are restricted to seroprevalence studies and molecular biological detection of granulocytic ehrlichiae (GE) in ticks and mammals, but include defined cases from Slovenia. They argue for the existence of this disease in many parts of Europe. In the present study, 510 Ixodes ricinus ticks collected in five different regions of Southern Germany were investigated for the presence of GE and Borrelia burgdorferi sensu lato using polymerase chain reaction. In all, 8 (1.6%) of the 492 ticks that could be evaluated (193 females, 208 males, and 91 nymphs) contained GE and 178 (36.2%) B. burgdorferi s.l.. Four of these ticks were infected with both pathogens. Interestingly, all ehrlichia-infected ticks were adults and all were collected in the English Garden, a recreational park area located in the city of Munich. Sequencing of the 16S rDNA (bp 1–1101) of four of the GE showed 100% sequence identity to each other and greater than 99.9% identity with the published sequence of the HGE agent. The four GE differed in respect to other hitherto described GE by a nucleotide exchange at position 336. These results show that GE that are closely related to the HGE agent are present in Southern Germany, and that coinfection with B. burgdorferi is common in GE-infected ticks. However, in contrast to B. burgdorferi which is endemic everywhere in Southern Germany, the distribution of GE seems to be focal. Received: 30 July 1999     

Demonstration of Borrelia burgdorferi sensu stricto infection in ticks from the northeast of Mexico

Borrelia burgdorferi sensu lato infection has been confirmed in clinical cases in the northeast of Mexico; however, the bacterium has not been identified as infecting the tick vector Ixodes , Amblyomma and Dermacentor ticks were collected from mammals and plants in northeastern Mexico and examined for Borrelia . Eighteen of 214 ticks were PCR-positive for the fla and 16S rRNA genes and 15 for the ospA gene. Southern blotting with a fla probe and sequencing of ospA genes confirmed infection with B. burgdorferi sensu stricto . These findings, together with reports of indigenous cases, fulfil the criteria that allow northeastern Mexico to be considered as a zone endemic for Lyme disease.     

First characterization of Borrelia burgdorferi sensu lato from ticks and skin biopsy in Bulgaria

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragmenth length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe. Received: 21 Juli 1997     

Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks in Austria

Unfed ticks of all instars (Ixodes ricinus, n=853; Haemaphysalis concinna, n=11) collected in all nine federal states of Austria were individually examined for the presence of Borrelia burgdorferi sensu lato (s.l.) using PCR. The mean overall infection rate was 14.4%. Infection rates were 24.5% in adult ticks, 16.1% in nymphs, and 1.6% in larvae. Four genospecies were detected, including B. valaisiana which was detected for the first time in Austria. The most common B. burgdorferi s.l. genospecies was B. garinii (66.9%), followed by B. valaisiana (13.7%), B. afzelii (11.3%), and B. burgdorferi sensu stricto (s.s.) (6.5%). Two specimens (1.6%) could not be identified to the genospecies level. Geographically, the highest infection rates were detected in the federal state of Vorarlberg (33.3%), B. garinii and B. afzelii being the most prevalent genospecies. B. valaisiana occurred most often in the federal state of Lower Austria, and B. burgdorferi s.s. was focally distributed in the Tyrol, in the surroundings of Imst.     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed. Received: 18 August 1997 / Accepted: 12 September 1997     

Characterization of Borrelia burgdorferi sensu lato strains isolated from Ixodes ricinus in Mecklenburg-Vorpommern,Germany

Epidemiological studies in Mecklenburg-Vorpommern have shown a high prevalence ofBorrelia burgdorferi-infected ticks. A total of 17B. burgdorferi sensu lato strains were isolated from ticks and investigated by Western blots (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). Except for one, all strains could be classified using this system. The majority of strains belonged to theB. garinii-associated OspA serotypes 3, 5 and 6. Three isolates were classified as OspA serotype 2 (B. afzelii).B. burgdorferi sensu stricto strains (Ospa serotype 1) as well asB. garinii-associated OspA serotype 4 were not present.     

Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia

Altogether, 343 adult and 111 nymphal Ixodes ricinus ticks collected from parks in Turku and suburban and rural islands of the Turku archipelago, Finland, and 100 adult I. ricinus ticks collected from Vormsi Island, Estonia, were included in this study. Using the polymerase chain reaction the ticks were examined for 16S rDNA of the Ehrlichia phagocytophila genogroup and for Borrelia burgdorferi sensu lato recA and flagellin genes. None of the Finnish ticks was found to be infected with E. phagocytophila, whereas 3% of the Estonian ticks were positive for this organism. The rate of Finnish ticks infected with B. burgdorferi sensu lato varied from 0% to 11.6% (mean 5%; 9% for adult and 4% for nymphal ticks). The corresponding rate for Estonian ticks was 15%. Borrelia afzelii was the most common genospecies in both Finnish (2.6%) and Estonian (12%) ticks. B. burgdorferi sensu stricto was detected in 2.0% of the Finnish ticks, but in none of the Estonian ticks. These results suggest that the E. phagocytophila genogroup is very rare in Finnish ticks, although the ticks were collected from an area endemic for Lyme borreliosis. In Estonia, E. phagocytophila is found in ticks and may cause disease.     

Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland

Objective: To evaluate by species-specific immunoblots the association of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii with neuroborreliosis in Switzerland.
Methods: Borrelia strains isolated from the cerebrospinal fluid (CSF) of three children with neuroborreliosis were typed by phenotypic and genotypic analysis. The serologic reactions (IgG) of these three patients as well as those of 28 patients, including one of these three children, with confirmed neuroborreliosis were characterized and scored by immunoblots on the three individual Borrelia species antigens. Twenty patients with typical erythema migrans served as a control group.
Results: Phenotypic and genotypic analysis confirmed that all three CSF isolates were B. garinii. In the 28 patients with neuroborreliosis, the comparatively strongest reactions were as follows: 18 to B. garinii , three to B. burgdorferi sensu stricto and two to B. afzelii ; five were inconclusive. In the control group (erythema migrans), the comparatively strongest reactions were as follows: six B. garinii , one to B. burgdorferi sensu stricto and five to B. afzelii ; eight were indeterminate.
Conclusions: Typing of these three CSF isolates and characterization by immunoblots of the antibody reactions of patients with neuroborreliosis give additional evidence of the association of B. garinii and neuroborreliosis. Our serologic results suggest that B. burgdorferi sensu stricto and B. afzelii are also responsible for some neuroborreliosis cases in Switzerland. Our immunoblots and the scoring system proved particularly useful for the serologic typing of patients with late Lyme borreliosis.     

Genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from the Autonomous Province of Trento, Italy

Sequences of the variable intergenic spacer region 5S (rrfA) 23S (rrlB) rRNA were used to identify Borrelia genospecies present in Ixodes ricinus nymphs collected from the Lamar Lakes area of the Province of Trento, Italy (overall prevalence=6.3%). Four genospecies were identified, one for the first time in this Province (B. valaisiana), and three which have been noted previously (B. afzelii, B. garinii, and B. burgdorferi s.s.). In order to compare the genetic variability of these genospecies in Trento with that at a European level, our 21 sequences (15 new haplotypes) and all appropriate European Borrelia sequences registered in GenBank (up to the end of 2004) were subjected to a phylogenetic analysis (for a total of 73 sequences and 43 haplotypes). Clusters of sequences representing the five main European genospecies (afzelii, garinii, burgdorferi s.s., valaisiana, lusitaniae) are well-supported. At least two other groups of haplotypes (genospecies) are suggested by our analysis; moreover, divergent evolution may be occurring in several genospecies. The maximum uncorrected pairwise differences between sequences within genospecies ranges from 1.5% (B. burgdorferi s.s.), to 2.3% (B. garinii and B. valaisiana) to 4.7% (B. afzelii), and are not correlated with geographical distribution. Within the Province of Trento, these values for the same genospecies are 1.5%, 2.3%, 0.9%, 1.9%, respectively. These high mutation rates within genospecies suggest that the sequencing of haplotypes should continue if we are to fully understand and monitor the evolution and epidemiology of Borrelia.     

Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different regions of Poland

Ixodes ricinus ticks, collected in 1996-1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n =48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferi sensu lato. Spirochetes were detected in 130 adult ticks (22.9%) and in a minimum of 32 (5.3%) nymphs. Further identification of 153 B. burgdorferi s.l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferi sensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2%). Within this group B. afzelii was found in 38/153 samples (24.9%), followed by B. burgdorferi sensu stricto (n = 23/153; 15.0%) and B. garinii (n = 22/153; 14.4%). Dual infections with B. burgdorferi s.s. and B. afzelii were detected in 17/121 (14.0%) adults, while both B. burgdorferi s. s./B. garinii and B. afzelii/B. garinii coinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferi s.s., B. afzelii and B. garinii was noted twice (1.6%). In general, B. afzelii was found in 72/153 (47.1%) tick samples and was the predominant species. B. burgdorferi s. s. and B. garinii were detected in a total of 60/153 (39.2%) and 51/153 (33.3%) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferi s.l. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.     

我国东北林区蜱中伯氏疏螺旋体的分离及分型鉴定

目的从我国东北地区全沟硬蜱中分离新的伯氏疏螺旋体菌株,了解其基因型及分布.方法应用BSKH培养基从硬蜱中分离菌株,PCR扩增5S～23S rRNA基因间隔区,进行限制性片段长度多态性分析及序列测定.结果新分离出伯氏疏螺旋体25株,其中11株来自内蒙古自治区,13株来自黑龙江省,1株来自吉林省.RFLP结果显示其中22株属于Borrelia garinii基因型,3株属B.afazenii基因型,1株产生了新的酶切带型,序列测定表明其属于B.garinii.结论东北地区的伯氏疏螺旋体菌株为B.garinii和B.afzelii两种基因型,B.garinii为主要基因型.采用5S～23S rRNA间隔区RFLP分型方法对菌株进行分型研究时应结合序列分析方法,以保证分型结果的可靠性.     

Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland)

To evaluate the importance of vertebrate species as tick hosts and as reservoir hosts in two endemic areas for Lyme borreliosis in Switzerland, we applied molecular methods for the analysis of bloodmeal source and Borrelia infection in questing Ixodes ricinus L. ticks. In total, 1326 questing ticks were simultaneously analyzed for Borrelia and for blood meal remnants by using reverse line blot. An overall infection prevalence of 19.0% was recorded for Borrelia sp., with similar rates in both sites. Using a newly developed method for the analysis ofbloodmeal targeting the 12S rDNA mitochondrial gene, identification of host DNA from field-collected ticks was possible in 43.6% of cases. Success of host identification at the genus and species level reached 72%. In one site, host identification success reached its maximum in spring (93% in May), decreasing in summer (20% in July) and rising in autumn (73% in October). In the other site, identification rate in ticks remained low from April to July and increased in autumn reaching 68% in October and November. The most prevalent identified host DNA was artiodactyls in both sites. Red squirrel DNA was significantly more frequently detected in ticks collected in one site, whereas insectivore DNA was more frequent in ticks in the other site. DNA from more than one vertebrate host was detected in 19.5% of nymphs and 18.9% of adults. Host DNA was identified in 48.4% of the Borrelia infected ticks. Although DNA from all Borrelia species was found in at least some ticks with DNA from mammals and some ticks with DNA from birds, our results confirm a general association of B. afzelii and B. burgdorferi sensu stricto with rodents, and B. valaisiana and B. garinii with birds.     

Diversity in prevalence and genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and rodents in Lithuania and Norway

A total of 1679 questing Ixodes ricinus ticks collected in Lithuania and 535 I. ricinus ticks collected in Norway from locations with different habitats were investigated for the presence of Borrelia burgdorferi sensu lato. In Lithuania, 223 ticks (13.3%) were infected with B. burgdorferi s.l., and in Norway, 28 ticks (5.2%). The highest prevalence of B. burgdorferi s.l was found in deciduous and mixed forests (19.4% in Lithuania, 8.6% in Norway). A lower prevalence was determined in pine forests (8.6% in Lithuania) and costal zones (4.3% in Norway), and the least prevalence was found in grasslands (2.5% in Lithuania, 1% in Norway). A total of 398 rodents belonging to 9 species were live-captured in Lithuania and Norway. Prevalence of B. burgdorferi s.l. in rodents varied between species and sampling sites in both countries. In Lithuania, the prevalence of infection was higher in Microtus arvalis (range 25–57% in different sampling sites) and in Myodes glareolus (range 14–71%) than in Apodemus flavicollis (range 0–37%) and in A. agrarius (range 11–33%). In Norway, the prevalence of B. burgdorferi s.l. in rodents was lower (range from 5% in A. sylvaticus to 6% in A. flavicollis). B. afzelii was the predominant genospecies in ticks and rodents in Lithuania and Norway. In Lithuania, B. afzelii was found in 76%, B. garinii in 10%, B. burgdorferi sensu stricto in 7%, and Borrelia spp. in 6% of infected ticks. Double infections were observed in 1% of the infected ticks. In Norway, B. afzelii was found in 68%, B. garinii in 21%, and B. burgdorferi s.s. in 11% of infected ticks. All infected rodents from both countries hosted B. afzelii genospecies. Only the red squirrel (Sciurus vulgaris) harbored both B. afzelii and B. burgdorferi s.s.     

Characterization of Borrelia burgdorferi sensu lato isolated in Moscow province--a sympatric region for Ixodes ricinus and Ixodes persulcatus

The relationships between Borrelia species and the vector ticks, Ixodes persulcatus and Ixodes ricinus were examined in a molecular epidemiological study. We conducted a survey in the Moscow region which is a sympatric region for both species of tick. We examined 630 unfed I. ricinus and I. persulcatus, ticks collected from four different regions around Moscow within an area of 250 km2. Eighty-four ticks were culture positive (13.3%) and the prevalence rate varied in each region from 5.7% to 42.3%. No difference was found between the total prevalence rate for both species. Eight Borrelia afzelii-like variant isolates from I. ricinus and Clethrionomys glareolus were identified as B. afzelii by flagellin gene and 16S rDNA sequence analyses. Most isolates from I. ricinus were identified as Borrelia garinii type 20047 and B. afzelii. Two isolates were identified as Borrelia burgdorferi sensu stricto (s.s.) and Borrelia valaisiana, respectively, but no B. garinii type NT29 was found. In contrast, isolates from I. persulcatus were identified as both types 20047 and NT29 of B. garinii, and B. afzelii. No B. burgdorferi s.s. isolate was found among isolates from I. persulcatus.     

Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa

Free-living adult Ixodes ricinus L, were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.     

Ixodes ricinus density, and distribution and prevalence of Borrelia burgdorferi sensu lato infection along an altitudinal gradient

In this study, we measured the phenology of Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) simultaneously along an altitudinal gradient to assess the impact of climate on the phenology of ticks and on their infection with B. burgdorferi sl. From 1999 to 2001, free-living I. ricinus ticks were collected monthly by flagging vegetation at three different altitudes (620, 740, and 900 m above sea level) on the slope of a mountain in Chaumont (Neuchatel, Switzerland). I. ricinus ticks were examined for the presence of B. burgdorferi sl by using direct fluorescent antibody assay and isolation of spirochetes. Borrelia species were characterized by polymerase chain reaction followed by restriction fragment-length polymorphism. Tick density and tick phenology varied with altitude. Although the peak tick density decreased and the onset of ticks was delayed with altitude, the phenology was much more stable among years at the highest altitudes than at the lowest. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection differed significantly among years, and it was significantly higher in adults (30%) than in nymphs (21%). B. burgdorferi infection in adults was positively related with adult density, but this was not observed for nymphs. Five B. burgdorferi sl genospecies were successfully isolated: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, and B. lusitaniae. Mixed infections were obtained from five of 140 infected ticks. The greatest diversity in Borrelia species was observed at the lowest altitude where all five Borrelia species were present, whereas at the two highest altitudes, B. lusitaniae was not observed.     

Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay

Objective   To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens.
Methods   A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii , Borrelia garinii and Borrelia valaisiana . A recombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard.
Results   The specificity of the assay was documented with 48 different clinically relevant Borrelia burgdorferi strains. No cross-reaction occurred with unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed. The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence. Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated. None of these samples showed measurable amounts of Borrelia burgdorferi DNA.
Conclusion   By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.