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Background   Borrelia burgdorferi  sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus  Ixodes  as vector. For identification of  Borrelia  infections in ticks a TaqMan™ minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an  Ixodes  spp. positive control to verify successful DNA isolation. Besides qPCR, an  osp A-specific conventional PCR for species-specific identification of  B. spielmanii  was established. Afterwards 1000  I. ricinus  flagged in the city of Hanover, Germany, were investigated for  B. burgdorferi  sl infections followed by species identification. Furthermore,  I. hexagonus  ticks were investigated to proof applicability of the PCRs.    Results  Quantitative real time PCR (qPCR) identifying  B. burgdorferi  sl in ticks was able to detect 1-10 copies per reaction.  B. spielmanii osp A-specific conventional PCR was also highly specific and showed no cross reactions with the other tested  Borrelia  species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was  B. garinii , followed by  B. afzelii ,  B. spielmanii ,  B. valaisiana  and  B. burgdorferi  sensu stricto (ss). 70.6% of  I. ricinus  were mono-infected, whereas 28.0% and 1.4% were infected with two and three  Borrelia  species, respectively. From 232  I. hexagonus  collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with  B. burgdorferi  sl, which were identified as  B. afzelii ,  B. garinii  and  B. spielmanii .    Conclusions  The evaluated qPCR to detect  B. burgdorferi  sl in  Ixodes  spp. is highly specific and sensitive. As a duplex qPCR including detection of  Ixodes  spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick. Establishment of a  B. spielmanii  specific conventional PCR filled the gap in PCR identification of principal European  Borrelia  genospecies. Practical application showed that all European pathogenic  Borrelia  spp. were present in  I. ricinus  flagged in recreational areas of the city of Hanover and confirmed  I. hexagonus  as reservoir for pathogenic  Borrelia  spp.

parasites and vectors

Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR