Distribution of Clinically RelevantBorreliaGenospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR

Abstract

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 ...

Quantitative analysis of Borrelia burgdorferi gene expression in naturally (tick) infected mouse strains

“…Our present experimental protocol, using the most reliable and sensitive primer pairs with specificity for zs7.a68, supports the previous findings that as few as 1-10 B. burgdorferi organisms can be detected by real-time PCR [27,47,48,63,64], though reproducibility is reduced with low numbers of spirochetes [62]. However, as previously shown, artificial spiking of unprocessed mouse tissue with spirochetes led to a dose-dependent inhibitory effect by tissue material on the amplification of B. burgdorferi genes [27,47,58,63].…”

“…Sensitivity and specificity of real-time PCR for detection of B. burgdorferi DNA To define optimal conditions for the quantification of B. burgdorferi DNA by real time PCR, a set of primer pairs specific for lp54-encoded ospA, zs7.a36, zs7.a66, zs7.a68, and cp26-encoded ospC from strain ZS7 were tested on plasmids harboring the respective genes by using various experimental protocols adapted from previous studies [7,39,47,48,62]. Highest sensitivity was obtained for zs7.a68 with a detection limit of about three copies/PCR (Table 1, Fig.…”

“…In light of the notion that needle exposure by no means reflects the situation of tick infection [23,25,54] and the highly improved real-time PCR technique to quantify B. burgdorferi in situ [24,27,43,46,48,62,63,64], we have now performed a detailed quantitative analysis of the spirochete burden and population dynamics in different tissues of mice following natural (tick) infection by B. burgdorferi s.s. (ZS7) for a period of 4 months postinfection (p.i.). For this purpose we have selected one cp26-encoded gene, ospC [40], and four previously described lp54-encoded genes, i.e.…”

Survival of Ixodes ricinus (Acari: Ixodidae) Under Challenging Conditions of Temperature and Humidity Is Influenced by Borrelia burgdorferi sensu lato Infection

“…They reported a median of 4,000 spirochetes per tick, which is higher than the one observed in the current study (2,760 per nymph; 1,496 per male; 2,078 per female). However, Rauter et al (2002) did not discriminate between life stages or between Borrelia genospecies (both factors have been shown to signiÞcantly inßu-ence infection load).…”

“…Infection load in questing I. ricinus in Europe has been investigated by Rauter et al (2002), who conducted a real-time PCR targeting the ospA gene (compared with a fragment of the ßagellin gene here). They reported a median of 4,000 spirochetes per tick, which is higher than the one observed in the current study (2,760 per nymph; 1,496 per male; 2,078 per female).…”