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

Abstract

BackgroundBorrelia 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 ospA-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.ResultsQuantitative real time PCR (qPCR) identifying B. burgdorferi sl in ticks was able to detect 1-10 copies per reaction. B. spielmanii ospA-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.ConclusionsThe 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.

Rickettsiaspp. and Coinfections With Other Pathogenic Microorganisms in Hard Ticks From Northern Germany

“…From this cohort, a total of 1,089 I. ricinus ticks, consisting of 669 adults (341 males/328 females), 332 nymphs, and 88 larvae, were examined for Rickettsia DNA using duplex real time polymerase chain reaction (PCR). Isolation of genomic DNA was performed as described by Strube et al (2010).…”

“…Plasmid preparation was carried out using the NucleoSpin Plasmid kit (Marcherey-Nagel, Dü ren, Germany) and the inserted sequence was veriÞed by sequencing. The Ixodes plasmid was prepared as described by Strube et al (2010).…”

Natural Focal Transmissible Infections with Neurological Manifestations in Ukraine

“…The first description of LB was given in 1977 in the US, and in 1982 they deciphered its etiology. The use of modern molecular-biological techniques enabled detecting of more than 10 (up to 15) genotypes of LB causative agent [Strube et al, 2010], which were consolidated in Borrelia burgdorferi sensu lato complex. Apart from known 'classical' pathogenic representatives -Borrelia (B.)…”

The ecology of Lyme borreliosis risk : interactions between lxodes ricinus, rodents and Borrelia burgdorferi sensu lato

“…Infected rodents transmit the spirochaetes to feeding larvae, which in turn moult into infected nymphs, closing the B. afzelii lifecycle (Radolf et al 2012). There are, however, occasional records of B. burgdorferi s.l.-infected larvae (Fingerle et al 2008, Kjelland et al 2010, Strube et al 2010, Tappe et al 2014), but it is unknown whether these larvae are able to transmit the spirochaetes to a host. It was, therefore, investigated whether larvae can infect rodents with B. afzelii.…”

“…However, B. burgdorferi s.l. is also, but rarely, found in questing larvae (Fingerle et al 2008, Kjelland et al 2010, Strube et al 2010, Tappe et al 2014). Borrelia miyamotoi, on the other hand, can be transmitted transovarially from female tick to larva (Scoles et al 2001) but is only shortly maintained in rodents (Taylor et al 2013a).…”