Lyme disease (LD) is caused by Borrelia burgdorferi and displays different stages, including localized, early disseminated, and persistent infection, all of which are associated with profound inflammatory reactions in the host. Induction of proinflammatory cytokines by B. burgdorferi is mainly mediated by outer surface proteins interacting with TLR-2/TLR-1 heterodimers. In this study, we show that TNF-α induction by Borrelia lysate was impaired in heterozygous TLR-2 knockout mice, while reactivity to lipoteichoic acid, another TLR-2 ligand signaling via TLR-2/TLR-6 heterodimers, was unaffected. Blood from individuals heterozygous for the TLR-2 polymorphism Arg753Gln was tested for cytokine release upon stimulation with Borrelia lysate, and induction of TNF-α and IFN-γ was significantly lower as compared with individuals not exhibiting this variation. Overexpression of TLR-2 carrying the Arg753Gln polymorphism in HEK 293 cells led to a significantly stronger impairment of activation by TLR-2/TLR-1 ligands as compared with TLR-2/TLR-6 ligands. To study whether heterozygosity for the Arg753Gln variant of TLR-2 influenced susceptibility for LD, we analyzed 155 patients for this polymorphism. The Arg753Gln variant occurs at a significantly lower frequency in LD patients as compared with matched controls (5.8 vs 13.5%, odds ratio 0.393, 95% confidence interval 0.17–0.89, p = 0.033), with an even more pronounced difference when late stage disease was observed (2.3 vs 12.5%, odds ratio 0.163, 95% confidence interval 0.04–0.76, p = 0.018). These data suggest that Arg753Gln may protect from the development of late stage LD due to a reduced signaling via TLR-2/TLR-1.

A LightCycler-based PCR protocol was developed which targets the ospA gene for the identification and quantification of the different Borrelia burgdorferi sensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three different B. burgdorferi sensu lato genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of different Borrelia species in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence of B. burgdorferi sensu lato infection in Ixodes ricinus ticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution of B. burgdorferi species by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas was B. afzelii (53%), followed by B. garinii (18%) and B. burgdorferi sensu stricto (11%); 0.8% of the infecting Borrelia could not be identified. (24) and the United States (25)-is a complex multisystem disorder caused by Borrelia burgdorferi sensu lato, a group of genetically diverse spirochetes. The principal vectors of these spirochetes are ticks belonging to the genus Ixodes (2). Lyme borreliosis (LB)-the most common arthropod-borne infection in EuropeThe development of an erythema migrans rash at the site of the tick bite often characterizes the onset of LB. If left untreated, the infection can persist for years and may result in a range of clinical symptoms, which vary depending on the duration of the infection and the organs affected.Isolates of B. burgdorferi sensu lato can be classified into different genomic species (1, 11). Only one of them, B. burgdorferi sensu stricto, has been implicated as the cause of disease in North America, but in Europe three genospecies, Borrelia afzelii, Borrelia garinii, and B. burgdorferi sensu stricto, are known to be pathogenic, and still others, such as Borrelia valaisiana and Borrelia lusitaniae, have been identified but are of unknown pathogenicity (7). Coinfections by two or more genomic groups of B. burgdorferi sensu lato have been found in ticks (13,14) and patients with LB (5).There is strong evidence that different species are involved in distinct clinical manifestations of the disease (28). Different studies have presented indirect evidence for the association of B. garinii with predominantly neurological symptoms (5), while infections by B. burgdorferi sensu ...

If left untreated, infection withCross-tolerance towards all tested stimuli was induced. Furthermore, using primary bone marrow cells from TLR2-deficient mice and from mice with a nonfunctional TLR4 (strain C3H/HeJ), we demonstrated that the TLR2 was required for tolerance induction by Borrelia, and using neutralizing antibodies, we identified interleukin-10 as the key mediator involved. Although peripheral blood mononuclear cells tolerized by Borrelia exhibited reduced TLR2 and TLR4 mRNA levels, the expression of the respective proteins on monocytes was not decreased, ruling out the possibility that tolerance to Borrelia is attributed to a reduced TLR2 expression. In summary, we characterized tolerance induced by B. burgdorferi, describing a model of desensitization which might mirror the immunosuppression recently attributed to the persistence of Borrelia in immunocompetent hosts.

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Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at ؊20°C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 ؋ g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.Diagnosis of Lyme borreliosis (LB) is currently based on clinical criteria confirmed by serological tests. However, serology has a number of limitations, such as serological crossreactivity, the delayed production of antibodies in early stages of LB, and an inability to distinguish between ongoing and previous infections, due to the persistence of antibodies. In patients with ongoing or recurring symptoms after antibiotic therapy, it is often impossible to definitely attribute clinical symptoms to a persistent infection with Borrelia burgdorferi sensu lato, the causative agent of LB. A way out of this dilemma might be the direct detection of this pathogen in clinical samples, such as the detection of Borrelia DNA by PCR. The use of urine as a sample material is an attractive approach, since urine is obtained without invasive procedures.Since 1991, when the detection of Borrelia DNA in urine was shown for the first time by Goodman et al. (14), many studies have been carried out (for reviews, see references 11, 19, and 29). A comparison between the different reports is nearly impossible because of significant differences in patient selection, study design, and DNA extraction and PCR methods. Nevertheless, a meta-analysis of urine PCR assays for diagnosis of LB showed an overall sensitivity of 68% (range, 13 to 100%) and a specificity of 99% (range, 95 to 100%) (11).Even when comparing only reports on urine of patients with erythema migrans (EM) who clearly have an active infection, detection of Borrelia DNA in urine ranged between 13% and Ͼ90% (1,5,20,25,28,30). A caution was issued by Brettschneider et al., who were not able to confirm the presence of Borrelia DNA in the 27% PCR-positive urine speci-