Molecular Detection of Anaplasma, Ehrlichia and Rickettsia Pathogens in Ticks Collected from Humans in the Republic of Korea, 2021

Tick-borne pathogens (TBPs), transmitted by the bites of ticks, are of great medical and veterinary importance. They include bacteria, viruses, and protozoan parasites. To provide fundamental data on the risk of tick contact and public health strategies, we aimed to perform a molecular investigation on four tick-borne bacterial pathogens in ticks collected from humans across the Republic of Korea (ROK) in 2021. In total, 117 ticks were collected, including Haemaphysalis longicornis (56.4%), Amblyomma testudinarium (26.5%), Ixodes nipponensis (8.5%), H. flava (5.1%), and I. persulcatus (0.9%). Among the ticks, 20.5% (24/117) contained tick-borne bacterial pathogens, with infection rates of 17.9% for Rickettsia (Candidatus Rickettsia jingxinensis, R. tamurae, R. monacensis, and Candidatus Rickettsia tarasevichiae), 2.5% for Anaplasma (A. phagocytophilum, A. capra, and A. bovis), and 0.9% for Ehrlichia (Ehrlichia sp.). Additionally, the co-detection rate for R. monacensis and A. phagocytophilum was 0.9%. To our knowledge, this is the first report of A. capra and A. bovis detection in ticks collected from humans in the ROK. This study contributes to the understanding of the potential risk of tick contact and provides fundamental data for establishing a public health strategy for tick-borne disease management in the ROK.


Introduction
Ticks are major blood-feeding arthropod vectors distributed worldwide, owing to their ability to adapt to various hosts, environments, and climates [1]. During blood feeding, ticks can transmit bacteria, viruses, and parasites that are of great concern for public health and veterinary medicine [2].
Tick populations have increased recently owing to rising temperatures associated with climate change. An increase in temperature has been observed to accelerate the developmental cycle, increase egg production, and lead to increased tick population densities [3,4]. Additionally, human activities such as leisure, travel and urban development have increased the opportunities for contact with ticks [5,6].
Several TBPs, including Anaplasma, Ehrlichia, Rickettsia, Borrelia, Babesia, Bartonella, and severe fever with thrombocytopenia syndrome virus, have been reported in the Republic of Korea (ROK) [7]. As tick-borne bacterial diseases present with non-specific symptoms, such as fever, headache, nausea, and vomiting, they are difficult to recognize without an accurate diagnosis [8]. Serological tests are commonly used for diagnosis; however, they require careful interpretation because of their reduced specificity for related pathogens or sensitivity to intrinsic factors, such as epitope selection [7,9].
Anaplasma species are zoonotic pathogens with tick vectors and mammalian reservoir hosts. Among them, A. phagocytophilum was the first species of tick reported to cause human

Tick Collection and Identification
Ticks removed from human bodies were collected from hospitals and local public health centers across the ROK from January to October 2021. The tick specimens were transferred to the Korea Disease Control and Prevention Agency (KDCA).
The ticks were identified morphologically under a dissection microscope to the species level, while life stage (larva, nymph, and adult) and sex (female and male) were also identified, as per the study by Yamaguti et al. [37]. Ticks were recorded as engorged (swollen) or unengorged based on differences in external morphology. Individual ticks were placed in a 2 mL tube and frozen at −80 • C until DNA extraction was performed.

DNA Extraction
Genomic DNA was extracted from individual ticks using the Clear-S Quick DNA Extraction Kit (InVirus Tech, Gwangju, Republic of Korea) with 500 µL of lysis buffer. The ticks were homogenized using a Precelly Evolution homogenizer (Bertin Technologies, Bretonneux, France) twice for 30 s at a speed of 4.5 m/s. After homogenization, the tubes were centrifuged for 10 min at 12,000× g, and 150 µL of supernatant was used for DNA extraction, according to the manufacturer's instructions. The extracted DNA was stored at −20 • C until use.

Molecular Detection of TBPs
Individual ticks were tested for the presence of the pathogens Anaplasma, Ehrlichia, Rickettsia, and Borrelia. The primer sets for the target gene fragments used to identify each pathogen are listed in Table 1. As positive controls, the genomic DNA of A. phagocytophilum and R. sibirica provided by the Division of Zoonotic and Vector Borne Disease Research and of E. chaffeensis and B. garinii provided by the Division of Bacterial Disease, KDCA, was used for each PCR.

Pathogens Target Gene Sequence 5 to 3 Amplicon Size (bp) Reference
The primary PCR was performed in 20 µL reaction volumes using the AccuPower PCR PreMix (Bioneer Corp., Daejeon, Republic of Korea). Each PCR mixture was composed of 1 µL of each oligonucleotide primer (10 pmol/µL), 5 µL genomic DNA as a template, and 13 µL distilled water. In the second round, nested PCR was performed using 1 µL of the primary PCR product as a template. Each reaction was performed using the ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA), and the amplified products were subjected to electrophoresis in the automated QIAxcel ® system (QIAgen, Hilden, Germany). The DNA extraction, PCR amplification, and automated electrophoresis were performed in separate rooms to prevent cross-contamination.

Sequencing and Phylogenetic Analysis
PCR-positive samples were purified and sequenced using a commercial sequencing service (BIOFACT, Daejeon, Republic of Korea). The nucleotide sequences were compared with reference sequences obtained from GenBank using nucleotide BLAST (National Center for Biotechnology Information, NCBI) and aligned using CLUSTAL Omega (v.1.2.1). A phylogenetic tree was constructed using the MEGA 11.0 program based on the maximum likelihood method, and bootstrap analysis (1000 replicates) was performed according to the Kimura two-parameter method.

Statistical Analyses
Statistical analyses were performed using the GraphPad software (GraphPad Software, Inc.; https://www.graphpad.com/quickcalcs/chisquared1/ San Diego, CA, USA). A chisquare test was performed to analyze the significant differences among the pathogens for each tick species and life stage. p < 0.05 was considered statistically significant.

Molecular Detection of TBPs
In total, 20.5% (24/117) of the ticks were positive for TBPs (Table 2). Three ticks (2.6%) tested positive for the 16S rRNA genes of Anaplasma. Among them, the I. nipponensis specimen was positive for ankA and msp4, A. phagocytophilum-specific genes. One H. longicornis specimen was positive for groEL, an A. bovis-specific gene, and another H. longicornis specimen was negative for groEL and gltA, A. capra-specific genes.
When detecting Ehrlichia, one H. flava specimen was positive for 16S rRNA, groEL, and gltA.
For Rickettsia, 21 ticks (17.9%) were positive for the 17 kDa, ompA, and gltA genes. Candidatus R. jingxinensis was identified in 11 H. longicornis specimens. Rickettsia tamurae was detected in five A. testudinarium specimens, and R. monacensis was identified in three I. nipponensis specimens and one Ixodes spp. specimen. Ixodes persulcatus tested positive for Candidatus R. tarasevichiae. Co-detection with A. phagocytophilum and R. monacensis was identified in I. nipponensis. Borrelia was not observed in this study.

Discussion
A total of 117 ticks were collected from humans across the ROK in 2021, and molecular detection of tick-borne bacterial pathogens, including Anaplasma, Ehrlichia, Rickettsia, and Borrelia, was performed in this study.
In this study, ticks collected from humans had a high TBP infection rate of 20.5% (24/117). In particular, the infection rate in adult ticks (26.5%) was 2.2-times higher than that in nymphs (12.0%) (p > 0.05). This result is similar to that of a previous study that showed that the infection rate of tick-borne bacterial pathogens, including Anaplasma, Ehrlichia, and Rickettsia, was 1.8 times higher in adults (69.6%) than that in nymphs (39.2%) collected from water deer in the ROK [50]. Klitgaard et al. [51] reported that the infection rates of Anaplasma, Rickettsia, and Borrelia in adults (52.2%) were 2.7 times higher than those of nymphs (19.1%) collected by flagging in Denmark. Considering the transstadial transmission of various TBPs throughout the life stages of ticks, this is a reasonable result, as adults have one to two times more opportunities to acquire pathogens by means of blood feeding from different hosts infected with pathogens than nymphs [52,53].
The genus Rickettsia is an obligate intracellular bacterium responsible for tick-borne rickettsiosis. Rickettsia is among the oldest known vector-borne pathogens, and its public health importance has been re-evaluated [54,55]. It was initially identified in ticks from 1930 to 1960, and much later (after 1990) in human specimens [56]. Several species of tick-

Discussion
A total of 117 ticks were collected from humans across the ROK in 2021, and molecular detection of tick-borne bacterial pathogens, including Anaplasma, Ehrlichia, Rickettsia, and Borrelia, was performed in this study.
In this study, ticks collected from humans had a high TBP infection rate of 20.5% (24/117). In particular, the infection rate in adult ticks (26.5%) was 2.2-times higher than that in nymphs (12.0%) (p > 0.05). This result is similar to that of a previous study that showed that the infection rate of tick-borne bacterial pathogens, including Anaplasma, Ehrlichia, and Rickettsia, was 1.8 times higher in adults (69.6%) than that in nymphs (39.2%) collected from water deer in the ROK [50]. Klitgaard et al. [51] reported that the infection rates of Anaplasma, Rickettsia, and Borrelia in adults (52.2%) were 2.7 times higher than those of nymphs (19.1%) collected by flagging in Denmark. Considering the transstadial transmission of various TBPs throughout the life stages of ticks, this is a reasonable result, as adults have one to two times more opportunities to acquire pathogens by means of blood feeding from different hosts infected with pathogens than nymphs [52,53].
The genus Rickettsia is an obligate intracellular bacterium responsible for tick-borne rickettsiosis. Rickettsia is among the oldest known vector-borne pathogens, and its public health importance has been re-evaluated [54,55]. It was initially identified in ticks from 1930 to 1960, and much later (after 1990) in human specimens [56]. Several species of tick-borne Rickettsiae previously considered non-pathogenic are now associated with human infections. Novel Rickettsia species with undetermined pathogenicity have been detected in ticks worldwide [54]. In this study, the genus Rickettsia was the most frequently detected pathogen, specifically Candidatus R. jingxinensis (9.4%, n = 11), R. tamurae (4.2%, n = 5), R. monacensis (3.4%, n = 4), and Candidatus R. tarasevichiae (0.9%, n = 1). These results are consistent with those of a previous study that found R. tamurae (24.2%), Candidatus R. jingxinensis (9.1%), and R. monacensis (3.0%) in the southwestern ROK [35]. In particular, Candidatus R. tarasevichiae is an emerging human pathogen [57] that widely infects I. persulcatus ticks in Russia and China [58,59]. In the ROK, Candidatus R. tarasevichiae was first detected in 2019 in a tick collected from a human [60]. It is necessary to conduct a monitoring survey of the spread and distribution of infections caused by Candidatus R. tarasevichiae in wild rodent and tick species.
Anaplasma and Ehrlichia are obligate intracellular bacteria belonging to the family Anaplasmataceae. The genus Anaplasma includes A. phagocytophilum, A. bovis, A. centrale, A. marginale, A. platy, A. ovis, and A. capra [61]. Of these, A. phagocytophilum is distributed worldwide because of its wide host range, which includes humans, carnivores, ruminants, rodents, insectivores, and birds [62]. Additionally, A. capra has recently been reported in small ruminants, as well as being a human pathogen [63], and A. bovis has been identified in ruminants in numerous countries [64]. In this study, A. phagocytophilum, A. capra, and A. bovis were identified in I. nipponensis and H. longicornis. In the ROK, A. phagocytophilum has been detected in ticks (2.9%, I. nipponensis and I. persulcatus) from humans [36], and another study confirmed its presence in I. nipponensis and patients bitten by it [65]. Additionally, A. bovis and A. capra have been reported in tick-infested cattle in the ROK [66]. In this study, A. phagocytophilum and A. bovis were confirmed with the 16S rRNA as well as specific genes of ankA, msp4, and groEL, whereas A. capra cannot be confirmed by other specific genes such as groEL and gltA. However, a previous study [66] reported the initial molecular detection of A. capra using a partial 16S rRNA sequence obtained from ticks infesting cattle and the 16S rRNA sequence of A. capra showed a higher identity of 99.0% compared to other Anaplasma species, A. ovis (97.6%) and A. phagocytophilum (95.9-96.2%) in this study. Based upon these results, it was considered as A. capra.
The genus Ehrlichia is a pathogenic agent of ehrlichiosis that affects both humans and animals, such as dogs and domestic ruminants [61]. Ehrlichia chaffeensis and E. ewingii are representative species that cause human monocytic and granulocytic ehrlichiosis, respectively. In the ROK, E. chaffeensis was detected in ticks (15.0%, 63/420 tick pools, H. longicornis and I. nipponensis) from grass vegetation and wild rodents in 2004-2005 [67]. E. ewingii was detected in ticks (0.1%, 2/1638 pools, H. longicornis) from grass vegetation in 2001-2003 [18] and in dogs in 2018 [68]. Based on the phylogenetic analysis of 16S rRNA for Ehrlichia in this study, one positive sequence was identified as Ehrlichia sp., whereas groEL and gltA sequences were 95.6% and 84.7% identical to E. ewingii, respectively. As it appears somewhat distinct from E. ewingii, it is suggested to be a new species of Ehrlichia. Therefore, further studies are required to identify and characterize this novel Ehrlichia species in relation to E. ewingii in ticks and their hosts.
The co-detection of R. monacensis and A. phagocytophilum in I. nipponensis was consistent with the results of previous studies on co-detection in ticks [69,70]. Co-detection can result from cofeeding with infected ticks on one host, blood meals on one host carrying several pathogens, or blood meals on different hosts [71,72].
To our knowledge, this is the first study to investigate various tick-borne bacterial pathogens, including Anaplasma, Ehrlichia, Rickettsia, and Borrelia, in ticks collected from humans across the ROK. However, there is no detection of Borrelia. Additionally, this is the first study to detect A. capra and A. bovis in ticks collected from humans in the ROK. Unfortunately, no pathogen analysis was conducted for tick-bitten humans. Because anaplasmosis, ehrlichiosis, and rickettsiosis are not designated as a national notifiable infectious disease, infected patients are not reported. For this reason, we cannot conduct analysis for tick-bitten humans. Therefore, further studies are needed to obtain direct evidence of pathogen transmission in both ticks and bitten humans. This study may be helpful in establishing a potential risk assessment for tick contact with TBPs and public health strategies for ticks in the ROK. Data Availability Statement: Data supporting the conclusions of this article are included within the article. The newly generated sequences were submitted to the GenBank database under the accession numbers OQ552617-OQ552620 and OQ581073-OQ581086. The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.