Detection of Borrelia miyamotoi in Ixodes nipponensis in Korea

Background This study investigated Borrelia species prevalence in ticks from vegetation, through a molecular method, in Gwangju Metropolitan City, South Korea. Methodology/Principal findings A total of 484 ticks were collected through flagging and dragging in a suburban area of Gwangju Metropolitan City, South Korea, in 2014. These ticks were morphologically identified and subjected to nested PCR, targeting Borrelia–specific CTP synthase (pyrG), outer surface protein A (ospA) and flagellin (flaB) genes. Molecular biological species identification of Borrelia-positive ticks was conducted via 16S rRNA PCR assays. Of the 484 ticks collected, 417 (86.2%) were identified as Haemaphysalis longicornis, 42 (8.7%) as H. flava, and 25 (5.2%) as Ixodes nipponensis. All the ixodid ticks containing Borrelia species bacteria were confirmed to be I. nipponensis adults, by both morphological and molecular methods. Of the 25 I. nipponensis ticks collected, four (16%) were positive for Borrelia species, three of which were B. afzelii and one B. miyamotoi. Conclusions/Significance Our study has shown the harboring of B. miyamotoi by I. nipponensis in South Korea. Morphological and molecular genetic analyses revealed that, in South Korea, I. nipponensis could potentially transmit B. miyamotoi to humans.


Introduction
Vector-borne, infectious diseases can be transmitted via mosquitoes, ticks, fleas, and other vectors, and account for more than 17% of all infectious diseases [1]. Five genera and 27 species of ixodid ticks have been reported in South Korea [2]. Lyme borreliosis is a vector-borne disease, PLOS [3,4]. Of the tick species capable of transmitting Lyme borreliosis pathogens, I. ricinus is typically found in Europe, I. scapularis and I. pacificus are found in North America, and I. persulcatus and I. nipponensis in Asia [5][6][7]. I. scapularis is the primary vector of B. burgdorferi (the Lyme disease spirochete), B. miyamotoi (the B. miyamotoi disease spirochete), Anaplasma phagocytophilum (the causative agents of anaplasmosis), Babesia microti (the causative agents of babesiosis), and Powassan virus (the causative agents of Powassan virus infection) [8]. B. garinii, B. afzelii, B. valaisiana, B. japonica, B. tanukii, B. turdi, and B. sinica have all been detected in Asia [3]. In 2011, the genotypes of 102 B. burgdorferi sensu lato isolates, from 11 provinces in China, were investigated. Most B. garinii strains were isolated from I. persulcatus ticks collected in northern China; however, B. afzelii strains were found in I. persulcatus ticks collected in northern China and H. bispinosa ticks collected in southern China [9]. B. miyamotoi is also transmitted by Ixodes spp. ticks, and unlike the species causing Lyme borreliosis, it has been reported that larvae are able to transmit the pathogen. B. miyamotoi is transovarially transmitted from adult females to larval offspring, via their eggs [10]. Thus, nymphal, adult ticks, and previously unfed larval Ixodes ticks, can transmit the pathogen.
Since its discovery in I. persulcatus in Japan in 1994 [11], B. miyamotoi has been detected in various Ixodes spp. in North America, Europe, and Russia [4,12,13]. In South Korea, B. burgdorferi sensu lato was detected in I. persulcatus ticks for the first time in 1993 [14].B. miyamotoi has, however, never been detected in South Korea and there have been no previous studies of the vector for B. miyamotoi. In the present study, 484 ticks, collected in a suburban area of Gwangju Metropolitan City, South Korea, in 2014, were morphologically identified and subjected to nested-PCR(N-PCR) targeting the Borrelia-specific CTP synthase (pyrG) gene and the outer surface protein A (ospA) gene. To verify the result using an additional target gene, N-PCR testing using the Borrelia-specific flagellin gene (flaB) was performed. In addition, mitochondrial 16S rRNA PCR was performed for species identification of Borrelia-positive tick species. This is the report of B. miyamotoi in ixodid ticks in South Korea. Morphological and molecular genetic analyses revealed that, in South Korea, I. nipponensis could be involved in B. miyamotoi infections by functioning as a vector.

Collection and identification of ticks
From April to September 2014, five sites within Gwangju Metropolitan City (Mudeung mountain (35˚08 0 07.0@N 126˚59 0 19.1@E), Buk-gu Konkuk-dong (35˚13 0 59.9@N 126˚53 0 11.6@E), Bunjeok mountain (35˚06 0 28.7@N 126˚55 0 12.3@E), Eodeung mountain (35˚09 0 19.2@ N, 1264 5 0 05.4@ E) and Seo-gu Central Park (35˚08 0 10.9@N 126˚51 0 53.8@E)) in South Korea were selected and ticks were collected by flagging and dragging methods. No specific permissions were required for the collection of ticks; however, permission from landowners was attained as the collections were conducted on private property. The field studies did not involve endangered or protected species. Recovered ticks were kept in containers labeled with the location, date, and collection time. Life cycle stages and species of all collected ticks were morphologically identified using microscopy and standard taxonomic keys [15].

PCR detection and molecular identification of Borrelia species and ticks
The ticks were washed with 70% ethanol for 2-5 min, washed again with sterilized distilled water for 3 min, dried on sterile filter paper and pooled after morphological classification depending on the species and life cycle stage. Collected adult ticks were identified as H. longicornis, H. flava, and I. nipponensis, and they were stored as one tick per tube. Collected nymphs were identified as H. flava and H. longicornis and were pooled from between 1 to 32 ticks per tube, by species. The only collected larvae species was H. longicornis, and 24 larvae were pooled in one tube. A total of 91 individual tick tubes and 28 pooled tick tubes were prepared. Genomic DNA was extracted from these samples, followed by N-PCR, targeting Borrelia-specific genes.
Classified ticks were added to a hard tissue grinding, MK28 tube (Bertin Technology, Rockville, MD, USA) containing 600 μL phosphate-buffered saline, with 10% fetal bovine serum and 5% penicillin/streptomycin, and ground using a FastPrep-24 Classic instrument (MP Biomedicals, Solon, OH, USA). Genomic DNA from the ground ticks was extracted with a G-spin Total DNA Extraction Kit (iNTRON Biotechnology, Sungnam-si, Korea) according to the manufacturer's instructions.
N-PCR, targeting Borrelia-specific pyrG and ospA genes, was performed using genomic DNA from the tick samples, to detect Borrelia species. For the N-PCR targeting pyrG, pyrG-1F/pyrG-1R primers (primers for first PCR) and pyrG-2F/pyrG-2R primers (primers for N-PCR) were used [16]. For the N-PCR targeting ospA, Borrel-ospAF1/Borrel-ospAR1 primers (primers for first PCR) and Borrel-ospAF2/Borrel-ospAR2 primers (primers for N-PCR) were used [3]. To verify the PCR result, we conducted additional flaB N-PCRs, using primers Bmfla-1 st -F/R (for the first PCR) and Bmfla-2 nd -F/R (for the N-PCR) for B. miyamotoi identification, and primers BOR-fla-1F/1R (for the first PCR) and BOR-fla-2F/2R (for the N-PCR) for Borrelia spp. identification. All primer sequences and PCR cycling conditions are shown in Table 1. The PCR with AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA, USA) was performed in a Veriti 96-Well Thermal Cycler (Applied Biosystems). The 20 μL 45 801 [16] pyrG nested PCR (internal primer) pyrG-2F (5'GATATGGAAAATATTTTATTTATTG-3') pyrG-2R (5'-AAACCAAGACAAATTCCAAG-3') 49 707 [16] ospA nested PCR (external primer) This study This study reaction mixture for the first PCR was composed of 1 μL each of 5 μM forward and reverse primers, 10 μL of Master Mix, 2 μL GC enhancer, 2 μL genomic DNA, and 4 μL distilled water. For N-PCR, the same reaction mixture as used for first PCR was used, except that the first PCR product was used as template DNA and the N-PCR primers were included. The PCR was performed using gene-specific PCR primers and annealing temperature, under the following conditions: 10 min at 94˚C for pre-denaturation, 30 cycles of 20 s at 94˚C, 30 s at each annealing temperature, 30 s~1 min at 72˚C and a final extension step of 7 min at 72˚C. In each PCR run, a negative control (reaction mixture without the template DNA) was included. Genomic DNA of B. burgdorferi B31 Clone 5A1served as a positive control. PCR primers, annealing temperatures, and the amplicon sizes are shown in Table 1. The PCR products were examined by electrophoresis in a 1.2% agarose gel containing ethidium bromide.
To identify tick species, conventional PCR (C-PCR), targeting mitochondrial 16S rRNA genes, was performed using the genomic DNA of ticks in which Borrelia spp. was detected [17]. C-PCR was performed with 16s+1-F/16S-1-R primers, AmpliTaq Gold 360 Master Mix (Applied Biosystems, CA, USA), and a Veriti 96-Well Thermal Cycler (Applied Biosystems, CA, USA), under the same condition as above. Primer sequences and PCR cycling conditions are presented in Table 1.

Nucleotide sequencing
For DNA sequencing, the PCR products were purified using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The purified DNA fragments were then sequenced in both directions, using the PCR primers and an automatic sequencer (ABI Prism 3730XL DNA analyzer, Applied Biosystems) at Solgent (Deajeon, Korea). To identify the bacteria present, the sequencing results were analyzed using the BlastN program from the National Center for Biotechnology Information (NIH, Bethesda, MD, USA) website.

Phylogenetic analysis
DNA sequences were identified and analyzed for homology comparisons using DNASTAR--Lasergene v6 software (DNASTAR, Madison, WI, USA) and the NCBI BlastN network service. The concatenating sequences of pyrG and flaB were aligned using Clustal W. A phylogenetic tree was constructed based on the DNA sequences of pyrG and flaB from tick samples with a Borrelia infection, and from various Borrelia strains in GenBank, using the neighbor joining (N-J) method on Clustal X and the Tree Explorer program (DNASTAR, Madison, WI). Bootstrap analysis was conducted using 1000 replicates to improve the confidence level of the phylogenetic tree. All sequence data generated in this study have been submitted to the NCBI GenBank (accession numbers MF948168 to MF948177 and MH102390 to MH102393).  Table 2).

Results
For the accurate genetic identification of ticks, conventional PCR (C-PCR), targeting 16S rRNA, was performed using genomic DNA from the five Ixodes ticks with Borrelia-positive results, then further investigated by DNA sequencing analyses. Molecular detection confirmed that all five ticks were I. nipponensis. A phylogenetic tree was constructed using DNA sequences of 16S rRNA gene fragments from five Borrelia-positive I. nipponensis tick samples (Chosun T32, 35, 37, 41, and 5-30) and using the 16S rRNA gene sequences of various ticks (I. nipponensis, I. persulcatus, I. pavlovskyi, H. flava, H. longicornis, A. maculatum) from GenBank as reference sequences. All sequences of Chosun T32, 35, 37, 41, and 5-30 ticks formed a cluster with I. nipponensis (Fig 1).
Homology testing showed that the 16S rRNA sequences of Chosun T5-30 and T32 ticks had a 99.8% and 98.4% homology with the 16S rRNA partial sequence of I. nipponensis mitochondrial DNA from adult males isolated in Japan, respectively, while all sequences from Chosun T35, 37, and 41 ticks showed a 99.1% homology.
Another phylogenetic tree was constructed by concatenating DNA sequences of the pyrG (675 bp) and flaB (660 bp) genes from Borrelia-positive I. nipponensis tick samples, using various Borrelia strains (B. burgdorferi B31, B. garinii Pbi, B. miyamotoi FR64b, B. afzelii HLJ01) from GenBank as reference sequences. The phylogenetic trees generated from the pyrG and flaB gene sequences showed similar topologies with the sequences from Chosun T32, 35, 37,

Discussion
After B. miyamotoi was first identified in I. persulcatus ticks and Apodemus mice, collected in Hokkaido, Japan [18], it was also found in several other tick species, such as I. scapularis and I.   [21]. According to a report on ticks from Hokkaido, Japan, which is geographically close to South Korea, overall tick infection rates of B. miyamotoi, the relapsing fever spirochete, were approximately 2% (71/3532) for I. persulcatus, 4.3% (5/117) for I. pavlovskyi, and 0.1% (1/676) for I. ovatus [22]. In addition to ticks, a retrospective surveillance of Lyme borreliosis in Japan detected B. miyamotoi DNA in the serum of two patients [23]. Moreover, emerging cases of relapsing fever, caused by B. miyamotoi, were reported in Russia, North America, and Europe, and in particular, B. miyamotoi-related meningoencephalitis was reported in a US patient [24]. The four Korean isolates of B. burgdorferi sensu lato, were first identified in I. persulcatus ticks and Apodemus agrarius rodent [14]. According to a report on ticks collected from humans, of the 261 ticks identified, the most abundant tick was H. longicornis (81.2%), followed by Amblyomma testudinarium (6.5%) and I. nipponensis (5.7%) [25]. Because I. nipponensis is the most frequently collected tick from wild rodents in South Korea, it is possible that patients bitten by I. nipponensis ticks are at a higher risk of infection by Borrelia spp. [26]. B. miyamotoi has, however, never been identified in South Korea nor its vector been reported; thus, the present study was conducted to investigate Borrelia infection in ticks collected in grasses, in South Korea. Of the 484 ticks, 417 (86.2%) were H. longicornis, 42 (8.7%) were H. flava, and 25 (5.2%) were I. nipponensis. All ticks that were positive for harboring Borrelia spp. were I. nipponensis adults, with infection rates of 4.0% for B. miyamotoi and 12.0% for B. afzelii being detected. The most common Borrelia spp. detected was B. afzelii, while B. miyamotoi was identified in South Korea. Prior to the identification of B. miyamotoi, only B. burgdorferi B31 Clone 5A1 was housed in our laboratory, which we included as a positive control, negating the chance of PCR contamination. Because B. miyamotoi has now been detected in South Korea, its reservoirs, rodents and other natural reservoirs, require further study. Because infection by B. miyamotoi results in non-specific fever symptoms, it is difficult to clinically differentiate this infection from anaplasmosis, Lyme diseases, and babesiosis. These three infectious diseases have been confirmed to occur in South Korea [27], and the present study confirmed the presence of B. miyamotoi in ixodid ticks. The detection of a pathogen in an individual, blood-feeding arthropod by no means proves or even suggests its role as a vector and a series of specific requirements would need to be examined to prove vector suitability. Although no patients have been reported to be infected by B. miyamotoi specifically, in South Korea, these results suggest that additional clinical studies should be undertaken to detect human infection cases of B. miyamotoi.
In conclusion, the present study detected B. afzelii and B. miyamotoi in I. nipponensis ticks, collected using a flagging and dragging method, in South Korea. Although only one I. nipponensis tick was positive for B. miyamotoi, our result suggests that I. nipponensis, confirmed by morphological and molecular biological methods, could potentially transmit B. miyamotoi to people in South Korea. Further collections in other areas of South Korea are necessary to establish whether I. nipponensis could viably serve as a vector of B. miyamotoi infections.