Molecular Detection and Phylogenetic Analysis of Tick-Borne Pathogens in Ticks Collected from Horses in the Republic of Korea

The horse industry has grown rapidly as a leisure industry in the Republic of Korea (ROK) in parallel with an increased demand for equestrian activities. As a result, there has been an increase in horse breeding and equestrian population and potential exposure to ticks and their associated pathogens. To provide a better understanding of the potential disease risks of veterinary and medical importance, a study was conducted to determine the geographical distribution and diversity of ticks collected from horses and vegetation associated with horse racetracks/ranches throughout the ROK. This included a survey of five associated common pathogens, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia spp., Babesia caballi, and Theileria equi. A total 9220 ticks were collected from horses and associated pastures. Ticks were identified to species, stage of development, and sex. Two species of ticks, Haemaphysalis longicornis (99.9%) and Ixodes nipponensis (0.1%) were identified. Two of the target pathogens, A. phagocytophilum and Borrelia spp., were detected in 5/1409 tick pools (0.35%) and 4/1409 pools (0.28%) of H. longicornis, respectively, both of which are zoonotic pathogens of medical importance. The results of 16S rRNA phylogenetic analysis of A. phagocytophilum showed a close relationship to strains distributed in China, USA, Germany, Italy, Turkey, and Poland. Borrelia spp. showed a close relationship, based on 16S rRNA gene, to the strains reported from the USA (B. burgdorferi and B. americana) and Japan (B. tanukii and B. garinii). These results provide information about the potential risks of veterinary and medical importance and the development of mitigation strategies for disease prevention.


Introduction
The number of horses has grown rapidly following the enactment of the Horse Industry Promotion Act by the Republic of Korea (ROK) government in 2011. The Korean government invested 600 billion won (USD 543,133,883) from 2011-2018 to foster the horse industry [1,2]. By 2019, there were 27,246 horses, 459 horseback riding facilities, and 919,556 riders in the ROK, with increases of 11.4%, 38.7% and 18.1%, respectively, compared to 2013 data [1]. The higher numbers of horses, associated horse facilities, and riders have increased the potential for exposure to ticks and transmission of tick-borne pathogens to both horses and associated equestrian personnel.
In the ROK, Haemaphysalis longicornis harbors various pathogens, e.g., Candidatus Rickettsia longicornii, Ehrlichia canis, and Theileria luwenshuni that have been reported in horses at Jeju Island [17]. However, investigations on the distribution of ticks and associated pathogens that are associated with horses and pasture lands in the ROK have not been conducted. The aims of this study were to identify tick species associated with horses and horse ranches at three metropolitan cities and seven provinces in the ROK, and to detect selected tick-borne pathogens: A. phagocytophilum, E. chaffeensis, Borrelia spp., B. caballi, and T. equi.

Detection of Tick-Borne Pathogens
Only two, A. phagocytophilum and Borrelia spp., of the five pathogens surveyed were detected (Table 3). Anaplasma phagocytophilum was detected in five pools of H. longicornis ticks with a minimum infection rate (MIR) was 0.54%, and the pools of t A. phagocytophilumpositive ticks were all adults (MIR = 3.30%) ( Table 3, Figures 2 and 3). The number of tick

Detection of Tick-Borne Pathogens
Only two, A. phagocytophilum and Borrelia spp., of the five pathogens surveyed were detected (Table 3). Anaplasma phagocytophilum was detected in five pools of H. longicornis ticks with a minimum infection rate (MIR) was 0.54%, and the pools of t A. phagocytophilumpositive ticks were all adults (MIR = 3.30%) ( Table 3, Figures 2 and 3). The number of tick pools positive for A. phagocytophilum collected from grasses/herbaceous vegetation associated with horse ranches and directly from horses was 3/5 (60%) and 2/5 (40%), respectively. The distributions of the infected ticks included 1/5 pools (20%) from LHR (Gyeonggi province), while the other four pools (80%) were from LHR (3)     . PCR analysis of A. phagocytophilum from ticks collected from horses and associated vegetation in the Republic of Korea (ROK). Positive detection of A. phagocytophilum in five tick pools (378, 513, 517, 523, and 736 ticks) was confirmed with an expected 511 bp band observed using electrophoresis. "P" and "N" represent a positive control using recombinant A. phagocytophilum DNA and a negative control without a DNA template, respectively. "M" represents a 100 bp DNA marker.  Borrelia spp. were detected by real time PCR in four pools of ticks (MIR = 0.43%) (Table 3, Figure 3). Borrelia spp. was only detected in H. longicornis with an MIR of 0.43‰, in which three (MIR = 0.55%) and one pool (MIR = 0.66‰) were nymphs and adults, respectively. All the Borrelia spp. infected ticks were collected in May and June 2017, in which three pools were collected from grasses/herbaceous vegetation associated with RHA (1; Jeju Island), LHR (2; Jeollanam province (1) and Busan metropolitan city (1)), while one positive pool was collected directly from a horse at Jeju Island (Table 3).

Sequencing and Phylogenetic Analysis
Sequencing analysis of the 16S rRNA gene of A. phagocytophilum from five tick samples showed 100% amino acid (aa) homology with each other and 99.78 to 100% nucleotide (nt) identity with A. phagocytophilum sequences deposited in the NCBI. Phylogenetic analysis based on the 16S rRNA gene (511 bp) showed that all the detected A. phagocytophilum had the same genotype and shared a close relationship with A. phagocytophilum distributed in China, USA, Canada, and Russia ( Figure 4). The phylogenetic analysis showed a close relationship between Borrelia sp. in this study and strains reported from the USA (B. burgdorferi and B. americana) and Japan (B. tanukii and B. garinii) ( Figure 5). The derived sequences of pathogens were submitted to the Gen-Bank database under the accession numbers MW715063 -MW715067 (A. phagocytophilum) and MW715293 (Borrelia sp.)

Discussion
Haemaphysalis longicornis was the predominant tick species collected from horses in the ROK, which is consistent with previous reports that horses in the ROK are primarily infested with H. longicornis ticks [18][19][20]. The prevalence of ticks in the ROK was identified with a predominance of H. longicornis, followed by H. flava, and other less abundant species, such as I. nipponensis, I. persulcatus, H. japonica, Amblyomma testudinarium, and I. granulatus [21]. However, only two tick species, H. longicornis and I. nipponensis were detected in horses in this study. This implies that the habitat of tick species might have been affected by land use and the presence of animal reservoirs [22][23][24][25][26].
Detection of E. chaffeensis, T. equi, and B. caballi from cattle grazing in ROK during 2010 and 2011 showed that 19.4%, 7.2%, and 0.35%, respectively, of the tick pools were positive for the three pathogens [27], and T. equi infections in horses have been serologically confirmed [28]. However, the three pathogens were not detected in ticks collected from horses and associated vegetation in the ROK during 2016 and 2017 in this study. The collection of ticks and detection of associated pathogens provide information for disease

Discussion
Haemaphysalis longicornis was the predominant tick species collected from horses in the ROK, which is consistent with previous reports that horses in the ROK are primarily infested with H. longicornis ticks [18][19][20]. The prevalence of ticks in the ROK was identified with a predominance of H. longicornis, followed by H. flava, and other less abundant species, such as I. nipponensis, I. persulcatus, H. japonica, Amblyomma testudinarium, and I. granulatus [21]. However, only two tick species, H. longicornis and I. nipponensis were detected in horses in this study. This implies that the habitat of tick species might have been affected by land use and the presence of animal reservoirs [22][23][24][25][26].
Detection of E. chaffeensis, T. equi, and B. caballi from cattle grazing in ROK during 2010 and 2011 showed that 19.4%, 7.2%, and 0.35%, respectively, of the tick pools were positive for the three pathogens [27], and T. equi infections in horses have been serologically confirmed [28]. However, the three pathogens were not detected in ticks collected from horses and associated vegetation in the ROK during 2016 and 2017 in this study. The collection of ticks and detection of associated pathogens provide information for disease risks of veterinary and medical importance and are critical for assessing disease risks and development of tick-borne disease mitigation strategies [29]. However, more rapid detection methods should be developed, such as point-of-care diagnostics from ticks Pathogens 2021, 10, 1069 9 of 15 collected from horses and associated vegetation, for early detection and instituting early control measure of tick-borne diseases in the future [30].
The two tick-borne pathogens, Anaplasma phagocytophilum and Borrelia spp., detected from ticks collected from horses and associated vegetation are causative agents of anaplasmosis and Lyme borreliosis in humans [31,32]. Even though the positive rate of A. phagocytophilum-positive ticks was very low (MIR = 0.54‰) from horse ticks in this study compared to 9.9% of ticks collected from different domestic and wild animals reported in the ROK in 2003 [33], the potential risk of transmission of this zoonotic pathogen to humans was identified [34]. Analyzing the relationship of the outbreak of human granulocytic anaplasmosis (HGA) and the collection areas of A. phagocytophilum-positive ticks has not been conducted due to lack of data of HGA outbreaks at areas, such as Hwasong in Gyeonggi province, and Jeju and Seoguipo cities at Jeju Island.
Borrelia spp. were detected in ticks associated with horses in the ROK for the first time. While the seroprevalence of B. burgdorferi in horses in the ROK was 5.2% during 2009 through 2013 [35], its prevalence in ticks collected from horses and associated vegetation during 2016-2017 was very low (MIR = 0.43%). Borrelia burgdorferi, the causative agent of Lyme disease, is the most prevalent zoonotic TBD worldwide. Domestic animals that are susceptible to B. burgdorferi infections include various species, e.g., dogs, cats, horses, and ruminants [36]. In this study, Borrelia spp.-positive ticks were collected from vegetation associated with leisure horseback riding ranches and horse racing parks, in addition to directly from horses. However, Borrelia spp.-infections in horses has not been determined in this study, while human cases of Lyme disease are reported annually in Korea [26]. Therefore, there is a need to investigate the horse infectious status of Borrelia spp. for regions of Borrelia spp.-infected ticks to control and prevent zoonotic TBD in the future.
The high homology of A. phagocytophilum from different areas (100%) demonstrates the low variation of A. phagocytophilum distributed throughout the ROK. In addition, the A. phagocytophilum sequences detected in ticks during this study demonstrated 100% similarity to those previously detected in infected horses [37]. Various gene fragments have been used for identification of A. phagocytophilum [38]. However, other genes, e.g., groEL and msp2, were shown not to be helpful for the detection of A. phagocytophilum in the ROK [37].
Borrelia spp. were detected by specific probe-based real-time PCR and then confirmed based on sequencing analysis of the 16S rRNA gene. However, sequence results of 16S rRNA gene was not useful for phylogenetic identification of B. burgdorferi sensu lato because the sequence also shared 100% identity to B. tanukii. Therefore, further analysis using various primer sets of alternate gene fragments [39,40], and specific primers for each species detection are necessary. Unfortunately, the nucleic acids extracted from the positive samples were exhausted. Therefore, we could not conduct further analysis for the detected Borrelia spp. in this study.
A nationwide surveillance of tick prevalence and tick-borne pathogens harbored by horse ticks was conducted in this study for the first time. The result revealed that horses in the ROK are infested by two tick species, H. longicornis and I. nipponensis, with majority of H. longicornis (99%). These ticks are vectors of two important tick-borne pathogens, A. phagocytophilum and Borrelia spp., among the selected five targets for detection (A. phagocytophilum, E. chaffeensis, Borrelia spp., B. caballi, and T. equi). The survey of tick-borne pathogens harbored by horse ticks should be further extended for other important pathogens, such as Rickettsia, by which a strategy for diagnosis and prevention of the related diseases could be established.

Tick Collection and Identification
Ticks were collected directly from horses and associated vegetation at 72 sites, including horse racetracks (2) and stud farms (3) operated by the Racing Horse Authority, PHF (11), and LHR (56) in the ROK. Twenty four of the ranches were located in Gyeonggi and Gangwon provinces in Northern ROK, while the other 48 ranches were located in central (2) and southern (46) provinces and metropolitan cities ( Figure 6). Ticks on horses were removed by securing the mouthparts with fine forceps as close to the skin as possible and gently pulling the tick away to avoid breaking off the mouthparts, while ticks were collected from the vegetation by the dragging/flagging method. Ticks were placed in 15 mL or 50 mL plastic vials with screw tops. At the end of each collection, the ticks were placed in a cooler where they were transported to the Parasitic and Honeybee Disease Laboratory, the Animal and Plant Quarantine Agency and stored at −80 • C until further identified.
placed in a cooler where they were transported to the Parasitic boratory, the Animal and Plant Quarantine Agency and sto identified.
Sex determination, identification of species and develop out using morphological keys [41][42][43] under a light for all tick ticks was identified using electron microscopy. After identificatio to 1.5 mL cryovials according to species, stage of development, a to the −80 °C freezer until they were processed for the dete agents.

Extraction of Nucleic Acids
Ticks were pooled based on the collection date, locati stage, and sex. Each pool consisted of adults (1)(2)(3)(4)(5), nymphs (1 from each pool and 300 µL of PBS solution were added in with steel beads (SNC, Hanam, Korea), the sample was homo Tissue Homogenizer (Bertin Instruments, Montigny-le-Bre RSC Viral Total Nucleic Acid Purification Kit (Promega, Mad total nucleic acids extraction. The homogenate, 300 µL of lys teinase K solution were added in a new 1.5 mL microcentrifu Sex determination, identification of species and developmental stages were carried out using morphological keys [41][42][43] under a light for all tick individuals, and a subset of ticks was identified using electron microscopy. After identification, the ticks were transferred to 1.5 mL cryovials according to species, stage of development, and sex (adults) and returned to the −80 • C freezer until they were processed for the detection of selected tick-borne agents.

Extraction of Nucleic Acids
Ticks were pooled based on the collection date, location, species, developmental stage, and sex. Each pool consisted of adults (1)(2)(3)(4)(5), nymphs , or larvae . Ticks from each pool and 300 µL of PBS solution were added in a tissue-homogenizing tube with steel beads (SNC, Hanam, Korea), the sample was homogenized using a Precellys 24 Tissue Homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega, Madison, WI, USA) was used for total nucleic acids extraction. The homogenate, 300 µL of lysis buffer, and 30 µL of proteinase K solution were added in a new 1.5 mL microcentrifuge tube. After incubating at 56°C for 10 min purification of nucleic acids was done by using an automated Maxwell RSC Instrument (Promega, Madison, WI, USA). Isolated material was stored at −80 • C until further molecular analysis.
Results of positive detections were expressed as a minimum infection rate (MIR) that assumed that every positive pool contains only one infected tick. The MIR was calculated using the formula: MIR = number of positive pools/total number of tested ticks × 1000 [44,45].

Phylogenetic Analysis
Positive samples of Borrelia spp. detected by real-time PCR were analyzed phylogenetically using the 16S rRNA gene and PCR products (622 bp; Table 4) amplified and sequenced. Phylogenetic analysis of A. phagocytophilum was performed using the 16S rRNA gene and PCR products of positive samples purified using a QIA Quick Purification Kit (Qiagen, Hilden, Germany) and Macrogen (Seoul, Korea) sequenced the PCR products. The homologies of the generated sequences were analysed using the BLASTn tool of the National Center for Biotechnology Information (NCBI) GenBank database. The sequences were aligned using the Clustal W with MegAlign software version 7.1 (DNA-STAR, Madison, WI, USA) and phylogenetic trees generated using the neighbor-joining algorithm in MEGA-6 software [46] with 1000 bootstrap replications. Table 4. PCR primer sets and conditions used for detecting tick-borne pathogens in ticks collected from horses and associated vegetation.

Pathogens
Primers Sequences ( Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.