A comparative analysis of molecular markers for the detection and identification of Borrelia spirochaetes in Ixodes ricinus

Abstract

Borrelia burgdorferi sensu lato, carried by Ixodes ticks, is one of the most significant human pathogens, causing Lyme disease. As there is no standardized PCR method for detection and identification of spirochaete DNA, we carried out a comparative analysis using a set of complementary primers for three regions in the genomic DNA of these bacteria (genes fla and rrs and the non-coding rrs-rrlA region). DNA extracted from 579 Ixodes ricinus ticks was subjected to nested PCR. DNA of the examined spirochaetes was detected in 43 (7.4 %) lysates when the fla gene was used as a molecular marker, in 7 (1.2 %) lysates when using primers complementary to the rrs gene, and in 12 (2.1 %) lysates using primers complementary to the non-coding rrs-rrlA sequence. RFLP analysis based on the fla gene helped identify species from the B. burgdorferi sensu lato complex (B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana), detect co-infections, and also identify Borrelia miyamotoi. Therefore, the fla gene is the most sensitive and specific molecular marker for the detection and identification of Borrelia spirochaetes in I. ricinus.

Borrelia miyamotoi: a widespread tick-borne relapsing fever spirochete

“…Sites and studies were excluded if a PCR was performed only in culture and/or direct fluorescence assay-positive ticks, and if accurate establishment of prevalence was impossible due to a bias (i.e., only some of the B. burgdorferi s.l.-positive ticks were tested for B. miyamotoi) or lack of clarification (i.e., the total sampled tick population was unknown or no distinction was made between host-derived and questing ticks). Data were collected from[12,14,[21][22][23][24][25]27,[29][30][31][32][33][34][35]37,38,40,[42][43][44][45].…”

First Detection of Borrelia burgdorferi sensu lato DNA in Serum of the Wild Boar (Sus scrofa) in Northern Portugal by Nested-PCR

“…Detection of Borrelia DNA in sera of wild boar was accomplished using a nested-PCR protocol that targets the flagellin-encoding gene fla (outer primers: 132f/905r; inner primers: 220f/823r) (Wodecka 2007, as cited in Wodecka et al 2010), a molecular target chosen in other studies (Skotarczak et al 2008;Wodecka et al 2009Wodecka et al , 2014Kiewra and Zaleśny 2013) and considered genus specific for Borrelia (Situm et al 2000). PCR assays targeting the fla gene have a significantly higher sensitivity in relation to other DNA markers both for detection of B. burgdorferi s.l.…”

“…PCR assays targeting the fla gene have a significantly higher sensitivity in relation to other DNA markers both for detection of B. burgdorferi s.l. in I. ricinus (Wodecka et al 2010) and in clinical samples (Situm et al 2000).…”

flaB Gene as a Molecular Marker for Distinct Identification of Borrelia Species in Environmental Samples by the PCR-Restriction Fragment Length Polymorphism Method

“…The examined material consisted of 52 Borrelia DNA isolates that had been obtained in previous studies from I. ricinus ticks collected from vegetation and removed from birds in west Poland and that were divided into 6 species of Borrelia (7,(17)(18)(19)(20)22).…”

“…The PCR-RFLP protocol with a single restriction enzyme, HpyF3I (Fermentas, Lituania), recognizing the CTNAG sequence inside a flaB gene fragment had been used previously for identification of Borrelia species (20,21). This study constitutes an extension to that protocol with the use of an additional restriction enzyme, Ecl136II (Fermentas), which recognizes the GAGCTC sequence, allowing the identification of all Borrelia species vectored by I. ricinus.…”