Molecular Identification of Borrelia spp. from Ticks in Pastures Nearby Livestock Farms in Korea

Simple Summary Lyme borreliosis is caused by a spirochete from the Borrelia burgdorferi sensu lato group. Borrelia afzelii and B. garinii are known to be pathogenic to humans. The main vector for Lyme borreliosis is the Ixodes tick. In this study, Borrelia infection was confirmed in Ixodes, Haemaphysalis, and Amblyomma ticks. To our knowledge Borrelia infection was first confirmed in Amblyomma testudinarium in Korea. Based on phylogenetic analysis, all sequences were aligned with B. afzelii isolates and showed a close relationship with high identity. Considering that B. afzelii causes infectious zoonotic diseases, continuous monitoring and attention are still required (although a low prevalence was recorded in this study). Abstract Ticks are vectors that spread pathogenic bacteria, viruses, and protozoa. As the number of ticks increases due to climate change, the importance of the study of tick-borne pathogens has also increased. This study was conducted to investigate the distribution of the major tick species causing Lyme borreliosis and regional differences in the prevalence of Borrelia spp. by tick species. Borrelia infection was confirmed not only in Ixodes ticks, which are the major vectors of Borrelia spp., but also in Haemaphysalis and Amblyomma ticks. PCR targeting the 5S-23S rRNA intergenic spacer region (rrf-rrl) was performed to confirm Borrelia positivity. A total of 6102 ticks (736 pools) were tested, and the proportion was Haemaphysalis longicornis nymphs and adults at 69.2%, Haemaphysalis flava nymphs and adults at 13.9%, Haemaphysalis spp. larva at 14.3%, Ixodes nipponensis at 0.8%, and Amblyomma testudinarium at 1.9%. Ixodes nipponensis showed the highest minimum infection rate (MIR: 34.00; 17 pools/50 ticks) for Borrelia spp., followed by A. testudinarium (MIR: 0.88), and H. longicornis (MIR: 0.05). In particular, to our knowledge Borrelia infection was first confirmed in A. testudinarium in Korea. As a result of phylogenetic analysis, all sequences were grouped with Borrelia afzelii isolates and showed a close relationship with high identity. Considering that B. afzelii causes infectious zoonotic diseases, continuous monitoring and attention are needed, although it has a low prevalence in this study.


Introduction
Ticks are ectoparasites that feed on humans as well as wild and domestic animals. Ticks are also a vector for spreading viruses and protozoa, as well as bacteria, e.g., Borrelia, Coxiella, or Rickettsiales [1,2]. Lyme borreliosis is also a tick-borne zoonotic disease. The disease is caused by the spirochete (causative agent) and the most common mode of transmission is via tick bite [3,4]. Although the taxonomy of the Borreliaceae family (genus Borrelia) has recently been revised into two genera, Borrelia (causing relapsing fever) and Borreliella (causing Lyme disease), this change has been challenged [5]. The Borrelia burgdorferi s. l. genospecies currently consists of 20 accepted and three proposed genospecies [6], and seven species (Borrelia afzelii, B. garinii, B. japonica, B. tanukii, B. turdi, B. sinica, and B. valaisiana) have been identified in Asia. Of these, B. afzelii and B. garinii are known to be pathogenic to humans. [4]. The main vectors of Lyme borreliosis are the ticks of the genus Ixodes and the main species in Asia are Ixodes persulcatus and I. nipponensis [7,8]. After B. burgdorferi s. l. was first identified in Korea in 1993, a human Lyme borreliosis case also occurred during the same year. The incidence has increased since then [4,7]. In Korea, on average, 15.4 cases per year have been reported in the past decade, with 23 cases reported in 2019 and 18 cases reported in 2020 [9,10]. In Korea, research on Borrelia is currently being carried out on the pathogenicity of various wild animals, including ticks, rodents, and companion animals [3,7,[11][12][13].
In Korea, B. afzelii and B. garinii were first identified in Ixodes ticks and wild rodents. Then, B. bavariensis, B. tanukii, B. turdi, B. valaisiana, and B. yangtzensis were identified in ticks and wild animals [7], whereas B. afzelii and B. garinii were also detected in domestic and wild animals [4,7,13]. B. afzelii (6/329, 1.8%) was detected in ticks collected from mammals such as horses, wild boars, native goats, and Korean water deer [4]. In a study examining the national prevalence of Borrelia in ticks of wild rodents in Korea, regional features of Borrelia spp. were observed; B. afzelii was found in all regions except Jeju Island, and some species were found only in the southern regions (Borrelia valaisiana, B. yangtzensis, and B. tanukii). Furthermore, some were found only in Jeju Island (B. yangtzensis and B. tanukii) [7]. Recently, B. burgdorferi sensu stricto, known to cause Lyme borreliosis in northern America, has been identified for the first time in Korea [10].
This study aimed to identify regional differences of ticks in the distribution and prevalence of Borrelia spp. based on tick species near a farm environment and to also identify Borrelia infections not only in Ixodes ticks, which are the major vectors of Borrelia spp., but also in Haemaphysalis and Amblyomma ticks.

Tick Collection and Species Identification
A total of 6102 ticks were collected once a month each on an area of about 70 m 2 grasslands and mixed forests around livestock farms (horses, deer, goats, and cattle) in three central and southern regions of Korea [Chungcheong (CC), Gyeongsang (GS), and Jeolla (JL) regions] ( Figure 1) from March to July 2021. These regions were chosen due to the fact that a grant was supported to assess the tick prevalence in these areas. For tick collection, flagging and dragging were performed and the collected ticks were stored in 70% ethanol. For morphological identification, a dissecting microscope was used with reference to the previously reported standard keys [14] and species, developmental stage, and sex for adult ticks were confirmed. The sexes of adult ticks were identified based on the size of the scutum on the dorsal side.

DNA Extraction, PCR and Phylogenetic Analysis
For DNA extraction, the adult ticks were extracted individually; 1-13 nymphs were pooled, and up to 50 larvae were pooled. Since the ticks were pooled after identifying them by species, different species were not mixed during the DNA extraction process. However, Haemaphysalis larvae were pooled with Haemaphysalis spp. regardless of species. For DNA extraction, a commercially available DNeasy ® Blood & Tissue Kit (Qiagen, Hilden, Germany) was performed according to the manufacturer's instructions. For PCR, the AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) was used as previously described for Borrelia spp. detection including the primers and PCR conditions for amplifying the 5S-23S rRNA intergenic spacer region (rrf-rrl) [3]. DNA templates of B. afzelii and B. garinii identified in our previous studies [3] were used as positive controls. Sterile distilled water without Borrelia DNA was used as the negative control. All PCRpositive products were sent to Macrogen (Daejeon, Korea) for sequence analysis and each aligned sequence was obtained using MEGA (v. 7.0; Raleigh, NC, USA) and BioEdit (v. 7.2.5; Raleigh, NC, USA). Phylogenetic trees were constructed based on the maximum likelihood method using MEGA (v. 7.0; State College, PA, USA) for the sequences obtained by BLAST search using the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 5 September 2021) and each aligned sequence obtained in this study.

DNA Extraction, PCR and Phylogenetic Analysis
For DNA extraction, the adult ticks were extracted individually; 1-13 nymphs were pooled, and up to 50 larvae were pooled. Since the ticks were pooled after identifying them by species, different species were not mixed during the DNA extraction process. However, Haemaphysalis larvae were pooled with Haemaphysalis spp. regardless of species. For DNA extraction, a commercially available DNeasy ® Blood & Tissue Kit (Qiagen, Hilden, Germany) was performed according to the manufacturer's instructions. For PCR, the AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Korea) was used as previously described for Borrelia spp. detection including the primers and PCR conditions for amplifying the 5S-23S rRNA intergenic spacer region (rrf-rrl) [3]. DNA templates of B. afzelii and B. garinii identified in our previous studies [3] were used as positive controls. Sterile distilled water without Borrelia DNA was used as the negative control. All PCR-positive products were sent to Macrogen (Daejeon, Korea) for sequence analysis and each aligned sequence was obtained using MEGA (v. 7.0; Raleigh, NC, USA) and BioEdit (v. 7.2.5; Raleigh, NC, USA). Phylogenetic trees were constructed based on the maximum likelihood method using MEGA (v. 7.0; State College, PA, USA) for the sequences obtained by BLAST search using the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 5 September 2021) and each aligned sequence obtained in this study.

Statistics
Statistical analysis was performed with chi-square test using SPSS V. 26.0 (SPSS Inc., Chicago, IL, USA); p < 0.05 were considered significant.

Distribution of Tick Species
A total of 6102 ticks were collected for three genera and four species (Haemaphysalis longicornis, Haemaphysalis flava, Ixodes nipponensis, and Amblyomma testudinarium) ( Table 1)

Prevalence of Borrelia sp.
A total of 6102 ticks (736 pools) were tested and Borrelia-positive ticks were detected in 20 pools (2.7% of the pools). The prevalence [minimum infection rate (MIR)] was 0.33% ( Table 2). Out of 43 pools containing 50 I. nipponensis ticks, 17 pools (34.0% MIR) were positive. Out of 11 pools containing 113 A. testudinarium ticks, only one pool (0.88% MIR) was positive. Out of 526 pools containing 4222 H. longicornis ticks, 2 pools (0.05% MIR) were positive. Regional prevalence were 4.6%, 1.6%, and 0.9% of the GS, JL, and CC region pools, respectively. Borrelia infection was confirmed in all three regions of Ixodes ticks: 65.0% in the GS region, 20.0% in the JL region, and 15.4% in the CC region. In sexes, the prevalences were 10% and 3.2% in the adult males and females, respectively.

Molecular and Phylogenetic Analyses
In this study, seven representative sequences (without duplicate sequences) were identified. All seven sequences were grouped with B. afzelii isolates when compared to GenBank registered reference sequences. When the obtained nucleotide sequences were compared, they showed 96.7-99.5% identity. The seven sequences were submitted to GenBank (OK274218-OK274224) (Figure 2).

Discussion
In the present study, Borrelia spp. of 5S-23S gene sequences were detected in the tick pools (20/736, 2.7%). In Korea, H. longicornis was confirmed to be the dominant species [13,15]. However, Ixodes ticks are the main vector for Borrelia. Therefore, although the population was small (50 ticks), the Borrelia MIR was 34.00, which was higher compared with that of other ticks (H. longicornis: 0.05; A. testudinarium: 0.88). This is consistent with the results of a previous study of tick-borne pathogens in Daejeon and its surrounding areas in South Korea [8]. The values were H. longicornis (1.5%) and H. flava (0.7%), whereas I. nipponensis was the highest at 20.9% [8]. Of the 20 tick pools, only two were H. longicornis and A. testudinarium was detected in only one pool. To our knowledge, this is the first study in Korea to document the presence of B. afzelii in A. testudinarium ticks. In previous studies, B. garinii of I. nipponensis is found on domestic dogs and B. afzelii of Haemaphysalis ticks, which infects wild Korean water deer, were detected [3,13]. Since previous studies have shown that H. longicornis can carry, but cannot transmit Borrelia [7,16], it has been suggested that Ixodes ticks are the primary vehicle for Borrelia transmission in Korea [7]. However, this is the first identification of Borrelia sp. in A. testudinarium, suggesting that further studies are needed to determine whether A. testudinarium may also serve as a vector.
In the present study, most of the ticks collected were in the nymph stage (4948 ticks,

Discussion
In the present study, Borrelia spp. of 5S-23S gene sequences were detected in the tick pools (20/736, 2.7%). In Korea, H. longicornis was confirmed to be the dominant species [13,15]. However, Ixodes ticks are the main vector for Borrelia. Therefore, although the population was small (50 ticks), the Borrelia MIR was 34.00, which was higher compared with that of other ticks (H. longicornis: 0.05; A. testudinarium: 0.88). This is consistent with the results of a previous study of tick-borne pathogens in Daejeon and its surrounding areas in South Korea [8]. The values were H. longicornis (1.5%) and H. flava (0.7%), whereas I. nipponensis was the highest at 20.9% [8]. Of the 20 tick pools, only two were H. longicornis and A. testudinarium was detected in only one pool. To our knowledge, this is the first study in Korea to document the presence of B. afzelii in A. testudinarium ticks. In previous studies, B. garinii of I. nipponensis is found on domestic dogs and B. afzelii of Haemaphysalis ticks, which infects wild Korean water deer, were detected [3,13]. Since previous studies have shown that H. longicornis can carry, but cannot transmit Borrelia [7,16], it has been suggested that Ixodes ticks are the primary vehicle for Borrelia transmission in Korea [7]. However, this is the first identification of Borrelia sp. in A. testudinarium, suggesting that further studies are needed to determine whether A. testudinarium may also serve as a vector.
In the present study, most of the ticks collected were in the nymph stage (4948 ticks, 81.1%). This is related to the developmental stage of the ticks and seasonal factors, which is consistent with that of previous results [17] of tick collection from March to July. In a previous study investigating the number of ticks by developmental stage, the peak populations of nymphs, adults, and larvae were observed in April-June, June-July, and August-September, respectively [17,18]. With respect to the distribution of ticks, H. longicornis was found abundantly in all three regions (CC: 1374 ticks; GS: 2110 ticks; JL: 765 ticks). In Korea, H. longicornis was confirmed as the dominant tick species [8]. Haemaphysalis flava and Haemaphysalis spp. larvae accounted for the next highest proportion, with Haemaphysalis ticks accounted for 97.3% of the total. However, Haemaphysalis spp. larvae were not found in the GS region in this study. This may have occurred due to the fact that the size of the larva was too small when transferring the tick after flagging and dragging. Ixodes nipponensis and A. testudinarium were not prevalent (2.7%). I. nipponensis were identified in all regions, whereas A. testudinarium were not identified in the CC region. This is consistent with previous results. A. testudinarium was primarily found in southern regions of Korea and very rarely in other regions [8]. Finally, A. testudinarium was identified only at some areas south of the JL and GS regions. We compared regional distributions between tick populations and Borrelia prevalence. Ticks positive with Borrelia were confirmed in all regions, but the GS region showed a three to five times higher prevalence than other regions (p = 0.016). In addition, I. nipponensis was detected in all three regions, and the GS region also showed a higher prevalence than other regions (p = 0.006). In I. nipponensis, the regional difference in the prevalence was significant (p = 0.006). However, since no historical data can be compared, it should be confirmed through continuous research.
From the results of phylogenetic analysis, we confirmed that all sequences were B. afzelii. In particular, the two Borrelia-positive sequences identified in H. longicornis were identical to OK274219 and OK274222 detected in I. nipponensis in this study, respectively. The OK274219 sequence was 100% identical to the Borrelia sequences of H. longicornis (KU596574) and H. flava (MT225121) previously detected in Korea. The Borrelia sequence of A. testudinarium detected in this study (identical to OK274222 sequence) was 100% identical to the sequence detected in voles in France (KY273113). The OK274224 sequence was 100% identical to the B. afzelii sequence (CP018262) identified in Germany.
In this study, only B. afzelii was detected, but previous studies confirmed B. garinii, which is known to be pathogenic to humans, similar to B. afzelii. A recent study in which B. afzelii and B. garinii was detected in wild rodent tissues showed that the Borrelia-positive rate was 29.8% and 25.9% for B. afzelii and 3.7% for B. garinii [10]. Another study identified Borrelia genospecies in various sampling areas in Korea, which were identified as B. afzelii (62.5%) and B. garinii (1.6%) [7]. As such, B. afzelii is more common and B. garinii is rare in Korea. In particular, only 100% B. afzelii was identified in certain areas. However, this may be due to differences in the sampling method and host, survey area, and seasonal factors, thus continuous research is needed.
In conclusion, we found that 20 out of 736 pools (6102 ticks) in central and southern Korea were positive for Borrelia (0.33% MIR). Among them, 17 pools were I. nipponensis, two pools were H. longicornis, and one pool was A. testudinarium. In Ixodes ticks, we found significant regional differences in Borrelia detection (p = 0.006), but this should be confirmed through further research with more ticks in a nationwide study. All positive samples were identified as B. afzelii. To our knowledge, this is the first study to report the presence of B. afzelii in A. testudinarium. Since B. afzelii is a zoonotic pathogen, it is important to gain insight into the role of A. testudinarium as a vector and through continuous surveillance and monitoring.