Borrelia Species Detected in Ticks Feeding on Wild Korean Water Deer (Hydropotes inermis) Using Molecular and Genotypic Analyses

Abstract

Lyme borreliosis is a vector-borne disease transmitted through the bite of ticks infected by Borrelia burgdorferi sensu lato group, including B. burgdorferi sensu stricto, B. afzelii, and B. garinii. The goal of the present study was to detect Borrelia species in ticks infesting wild Korean water deer (KWD; Hydropotes inermis Swinhoe), using molecular and genotypic analyses. In total, 48 ticks were collected from KWD, all of which were morphologically identified as Haemaphysalis longicornis Neumann that is dominant in Korea. Nested PCR was performed to detect the Borrelia-specific 5S (rrf)-23S (rrl) intergenic spacer region and the outer surface protein A (ospA) genes in ticks. Both rrf-rrl and ospA were amplified from one of the 48 ticks (2.1%) and were identified as B. afzelii. To our knowledge, this study constitutes the first molecular detection of B. afzelii in Haemaphysalis ticks in Korea. Because B. afzelii is a zoonotic tick-borne pathogen, understanding the molecular characteristics of this bacterium is important for preventing the transmission of Borrelia from ticks to other animals and humans.

Molecular Detection and Characterization of Borrelia garinii (Spirochaetales: Borreliaceae) in Ixodes nipponensis (Ixodida: Ixodidae) Parasitizing a Dog in Korea

“…Consistently, Borrelia spp. have been identified in Ixodes and Haemaphysalis ticks in Korea and Japan [40,41]. Pal et al [23] revealed that tick receptor for ospA (TROSPA) acts as a receptor for Borrelia spp.…”

“…owing to its conserved characteristics among Borrelia spp. [31,40,43]. However, as suggested by De Michelis et al [6], it is difficult to construct a reliable molecular phylogeny based on only rrf-rrl owing to the fact that the fragment is short and consists of highly conserved and variable regions.…”

“…The primer sets for the detection of Borrelia spp. were Bb23S/Bb23Sa and Bb23SnF/Bb23SanR targeting the rrf-rrl, as previously described [40]. B. afzelii, which was previously confirmed in our laboratory, and distilled water were used as positive and negative controls, respectively [40].…”

“…For molecular characterization, rrf-rrl-positive sample was submitted to amplify the ospA and flagellin genes, as previously described [40,51]. All the amplicons were directly sequenced by Solgent (Daejeon, Korea) bidirectionally.…”

Molecular detection and phylogenetic analysis of tick-borne pathogens in wild Korean water deer and farmed elk in Gyeongbuk and Gangwon Provinces of Korea

“…was amplified by PCR using the genus-specific primer set Rr17.61p/Rr17.492n with the following program: pre-denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec and extension at 72°C for 1 min; and a final post-extension step at 72°C for 7 min [ 24 ]. The 5S ( rrf )–23S ( rrl ) intergenic spacer gene of Borrelia was amplified by nested PCR (nPCR) using two primer sets, Bb23S3/Bb23Sa and Bb23SnF/Bb23SanR with the following program: pre-denaturation at 96°C for 2 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 40 sec; and a final post-extension step at 72°C for 5 min, and the outer surface protein A gene fragment of B. burgdorferi was amplified by nPCR using two primer sets, N1/C1c and N2/C2c with the following program: pre-denaturation at 95°C for 3 min; 20 cycles of denaturation at 95°C for 30 sec, annealing at 40°C for 1 min and extension at 72°C for 1 min, which was then followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 1 min and extension at 72°C for 1 min; and a final post-extension step at 72°C for 7 min [ 32 ]. Multiple primer sets were used to amplify the 16S rRNA of the genus Coxiella , including C. burnetii and Coxiella -like bacteria with the following program: pre-denaturation at 93°C for 3 min; 30 cycles of denaturation at 93°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 1 min; and a final post-extension step at 72°C for 5 min [ 26 ].…”