Tick Populations and Molecular Analysis of Anaplasma Species in Ticks from the Republic of Korea

The present study was performed to survey the dominant tick populations and molecularly determine the pathogenic agents of anaplasmosis in ticks from Gyeongsang, Republic of Korea. A total of 3825 questing ticks were collected by the flagging method from 12 sites near animal farms in Gyeongsang from March to October 2021. A molecular genomic study was performed with ticks stored in 70% ethanol to detect Anaplasma genes by the previously described method. The monthly incidence of ticks varied by developmental stages, i.e., nymphs, adults, and larvae, and each of their populations peaked in May, March, and October, respectively. The predominant tick species were Haemaphysalis longicornis, Haemaphysalis sp., Haemaphysalis flava, Ixodes nipponensis, and Amblyomma testudinarium in order. To determine the Anaplasma infection rate, collected ticks were pooled into 395 groups. The minimum infection rate (MIR) of Anaplasma was 0.7% (27 pools). That of A. phagocytophilum was highest (23 pools, MIR 0.6%), followed by A. phagocytophilum-like Anaplasma spp. clade B (2 pools, MIR 0.1%), A. bovis (1 pool, MIR 0.1%), and A. capra (1 pool, MIR 0.1%), respectively. In this study, five species of ticks, including unidentified Haemaphysalis species, were collected in 12 survey sites in Gyeongsang, but their prevalence was somewhat different according to the tick species and survey sites. Further, the incidence rate (6.8%) of 4 Anaplasma spp. was not as high in tick pools. However, the results of this study may offer a basis for future epidemiological research and risk assessment of tick-borne diseases.


Introduction
Numerous emerging tick-borne diseases were present in ticks, humans, and animals before they were recognized as the causal agents of clinical illnesses [1]. Numerous factors, such as tick incidence, human activity, duration of tick attachment, climatic and geographic factors, and the biological stage, affect the ability of ticks to attach to humans and transmit pathogens [2]. Owing to the gradual changes in temperature in the Republic of Korea from a temperate to a subtropical region, the Korean Peninsula may soon experience increases in tick populations. Ticks depend on environmental factors to survive, and climate change provides conducive conditions [3].
Diseases associated with Anaplasma species have affected the health and production of animals for more than a century and are currently posing a serious threat to livestock [4]. 2 of 12 Ticks transmit Anaplasma in nature, and there are numerous vertebrate hosts and infection sources for ticks, humans, and animals [4]. The sporozoan parasites of the genus Anaplasma have been taxonomically known as several verified species, i.e., A. phagocytophilum, A. marginale, A. centrale, A. bovis, A. platys, A. ovis, and Candidatus Anaplasma species. These species have a somewhat different host cell tropism. The predilection site of A. phagocytophilum is neutrophils [5], A. marginale, A. centrale, and A. ovis prefer erythrocytes [6], A. bovis and A. platys specifically parasitize in monocytes [7] and platelets [8], respectively. A recently recognized Anaplasma species, Anaplasma capra, may infect endothelial cells [9]. Additionally, it is crucial to distinguish between pathogenic A. phagocytophilum and closely related A. phagocytophilum-like Anaplasma spp. (APL), which are currently thought to be nonpathogenic and do not cause clinical symptoms in infected animals [10].
Numerous Anaplasma spp. in tick populations have been detected in the Republic of Korea, such as A. capra and A. bovis from ticks parasitizing water deer (Hydropotes inermis argyropus) [11]; A. capra, A. bovis, APL clade A, and APL clade B from ticks parasitizing cattle [12]; A. bovis from ticks parasitizing native Korean goats [13]; and A. phagocytophilum and A. platys from small wild-caught mammals or by dragging/flagging [14]. In other countries, several Anaplasma spp. have also been identified in tick populations, such as A. marginale, A. platys, and A. capra from ticks parasitizing cattle and goats in China [15]; A. phagocytophilum by flagging in Austria [16]; and A. bovis by flagging in Canada [17].
To predict the origin of infections and offer risk assessments for tick-borne diseases, the present study was performed to study the prevalence of tick populations and the incidence rates and risk factors for pathogenic Anaplasma species in ticks, which were collected from 12 sites near animal farms in Gyeongsang (six in Gyeongnam and six in Gyeongbuk), Republic of Korea.

Ethical Approval
Approval from Kyungpook National University's Institutional Animal Care and Use Committee was not required for the present study, which was conducted in 2021. The collected samples from questing ticks in the environment in this study did not cause hazard to any animals. The collected ticks did not include endangered species. Specific approval for each collection site was not needed because the sites were not located within national parks or protected regions.

Tick Collection
Questing ticks were collected by the flagging method, in which the vegetation is swept with a flannel cloth [18], from 12 sites near animal farms in Gyeongsang from March to October 2021. The survey sites were administratively in Gyeongbuk (GB), Gyeongnam (GN), and Ulsan Metropolitan City (UMC), Republic of Korea. The six sites in GB were Bonghwa (BH), Chilgok (CG), Gumi (GM), Uiseong (US,), Yeongcheon (YC), and Yeongdeok (YD). The four in GN were Geochang (GC), two in Goseong (GS), and Haman (HA), and the remaining two were UMC regions (Table 1 and Figure 1). Unfed ticks were collected once per month. In the two provinces, the GS and ULS regions had two collection sites each, and each of the other eight regions was used as a collection site, totaling 12 tick collection sites close to livestock farms (cattle, deer, horse, and goat farms). The collected ticks were stored in 70% ethanol.
Tick species and developmental stages were identified based on morphological characteristics using a microscope according to taxonomic key [19]. Each identified tick was pooled according to species, developmental stage, survey period, and collection site. Adults and nymphs were classified at the species level, whereas larvae were identified to the genus level because of morphological similarities. The number of ticks in the pools was one to 10 nymphs and one to 50 larvae, and the adults were individually examined.   Tick species and developmental stages were identified based on morphological ch acteristics using a microscope according to taxonomic key [19]. Each identified tick w pooled according to species, developmental stage, survey period, and collection s Adults and nymphs were classified at the species level, whereas larvae were identified the genus level because of morphological similarities. The number of ticks in the po was one to 10 nymphs and one to 50 larvae, and the adults were individually examine

DNA Extraction and PCR detection
Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hild Germany) according to the manufacturer's instructions. PCR was performed using an cuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Republic of Korea). Nested PCR w performed to detect the genus Anaplasma by amplifying the 16S rRNA gene, as previou

DNA Extraction and PCR Detection
Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR was performed using an AccuPower HotStart PCR Premix Kit (Bioneer, Daejeon, Republic of Korea). Nested PCR was performed to detect the genus Anaplasma by amplifying the 16S rRNA gene, as previously described [20] using the primer pairs EE1/EE2 and EE3/EE4, which produced an amplicon of 924-926 bp. A sample of A. phagocytophilum detected in cattle in the Republic of Korea [21] was included as a positive control, and a sample without a DNA template was used as a negative control.

Sequencing and Phylogenetic Analyses
All the PCR-positive products with EE3/EE4 primers were sent to Macrogen (Daejeon, Republic of Korea) for Sanger sequencing. The sequences obtained in the present study and previously reported in GenBank were aligned and analyzed using the multiple sequence alignment program CLUSTAL Omega (v. 1.2.1, Bioweb, Ferndale, WA, USA). Among all the long-aligned nucleotide sequences, unnecessary sequences in the front and back were deleted based on the sequences detected in this study using BioEdit (v. 7.2.5, Bioedit, Manchester, UK). Sites containing gaps or having ambiguous alignment were also removed prior to phylogenetic analysis. Phylogenetic analysis was performed using the maximum likelihood method with the Kimura two-parameter distance model in molecular evolutionary genetics analysis (v. 7.0, Mega software solutions, Madhurawadha, India). The aligned sequences of Anaplasma 16S rRNA were pairwise compared to determine homology. The stability of the obtained trees was estimated using bootstrap analysis with 1000 replicates.

Statistical Analysis
To evaluate the correlation between Anaplasma prevalence and five different tick species, data were statistically analyzed using the chi-square test using the GraphPad Prism analytical software package (v. 5.04, GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at p < 0.05.

Incidence of Anaplasma Genes from Tick Pools
Among 3825 collected ticks, 395 pools were tested using PCR, and Anaplasma-positive ticks were detected in 27 pools (6.8%). The minimum infection rate (MIR) was 0.7 (Table 3).     (Figure 2a). Temporally, the highest peak in tick incidence was observed in May (20.2%, 771/3825), when nymphs were highly prevalent; a second peak was observed in August (15.3%, 584/3825) when larvae were beginning to be prevalent.

Molecular and Phylogenetic Analyses
Phylogenetic analysis revealed that Anaplasma spp. identified in the present study were A. phagocytophilum, APL clade B, A. bovis, and A. capra (Table 4 and Figure 3). Of the 27 positive pools, the A. phagocytophilum incidence was the highest (23 pools, 85.2%), followed by APL clade B (2 pools, 7.5%), A. bovis (1 pool, 3.7%), and A. capra (1 pool, 3.7%).   The two representative sequences of A. phagocytophilum 16S rRNA sequences in the present study shared 99.3% identity with each other. They were also 98.3-100% identical to the 16S rRNA sequences of previously reported A. phagocytophilum isolates. We determined that the two sequences of the APL clade B 16S rRNA sequences shared 99.7% identity with each other and 98.8-100% identity with the 16S rRNA sequences of previously reported APL clade B isolates. One A. capra sequence in the present study shared 99.5-99.8% identity with the 16S rRNA sequences of previously reported A. capra isolates. Similarly, one A. bovis 16S rRNA sequence shared 99.5-99.8% identity with other A. bovis isolates. Among the sequences obtained in the present study, representative sequences used in the phylogenetic analysis were submitted to the GenBank database (accession numbers: OP535541-OP535545).
Questing ticks near animal farms were surveyed monthly between March and October 2021. We evaluated the geographical distribution of tick species and found that most ticks were collected in the HA (13.2%), GS (2 sites with an average of 10.1%), and CG (9.5%) regions. Compared with other regions, the HA and GS regions are located at low latitudes in the Republic of Korea with higher temperatures and more precipitation, which are ideal conditions for tick survival [23]; thus, these regions had a higher distribution of tick populations than that in other regions. Haemaphysalis spp. were widely distributed in the surveyed regions, and I. nipponensis was distributed in five regions (four in GB and one in GN). However, A. testudinarium was only distributed in the GS region, located at the lowest latitude in the surveyed regions. Therefore, ecological and environmental factors may impact the regional variations in tick distribution.
Ticks spend most of their lives in the natural environment, except when feeding; hence, environmental factors impact their biology. Changes in the environment and season, particularly humidity and temperature, affect tick development, survival, distribution, and, subsequently, the tick-borne disease risk [24]. According to a previous study, peaks in adult, nymph, and larval tick populations were generally observed from June to August, May to June, and August to September in the Republic of Korea [18,22], consistent with the findings of the present study, in which the highest peaks in nymphs, adults, and larval populations were observed in May, March, and October, respectively.
We performed molecular detection and phylogenetic analysis of Anaplasma (6.8%) in ticks. Normally, Anaplasma spp. are more prevalent in adult ticks than those in nymphs and larvae because adults have a higher probability of coming into contact with an Anaplasmainfected host. In the present study, nymphs (48.2%) were more prevalent in Anaplasma infections than adults (22.2%) and larvae (29.6%). Temporally, the number of positive tick pools in adults was highest in July. The number of positive tick pools in nymphs was highest in July. Infected larval ticks were detected between August and October. The MIR of Anaplasma in ticks was relatively high in March (0.13) and July (0.18), probably due to the high risk of Anaplasma infection in those months. Therefore, caution is needed when engaging in outdoor activities around this period.
In the present study, H. longicornis (13 pools, three female adults, and 10 nymphs) was the most prevalent tick species host to Anaplasma, followed by I. nipponensis (six pools, three male adults, and three nymphs), and Haemaphysalis spp. (eight pools and eight larvae). The prevalence of Anaplasma was significantly different among the tick species (p < 0.0001). However, H. flava and A. testudinarium were not the main vector for Anaplasma spp. As the detection of Anaplasma in ticks does not indicate the capacity of the ticks to act as competent vectors, further studies are needed to establish whether these species are Anaplasma vectors. Tick-borne diseases are transmitted through transstadial or transovarial routes. In the present study, 31 pools (1176 larvae) were tested for Anaplasma spp., and eight pools (MIR: 0.7) were positive. Thus, the larvae generally transmitted Anaplasma transovarially ( [28]; and A. phagocytophilum in patients and biting ticks [29]. Analyses of the nucleotide sequences from a variety of genes have been used to report the genetic diversity of the genus Anaplasma [4]. For example, 16S rRNA gene analysis are well supported by comparable groESL clades, as well as biological and antigenic features. The groESL sequences provide support for the divisions shown by the 16S rRNA gene sequences and provide evidence of polymorphisms that may be accidental or may show subtleties of evolutionary selection. Therefore, the current analysis of the 16S rRNA gene may be supported by additional sequence analyses of conserved and semi-conserved genes (such as gltA), whole genome analysis, and analysis of new strains [30]. Because only the 16S rRNA gene was used in this study, there are limitations to the molecular identification of Anaplasma species. In the present study, we detected four Anaplasma species from questing ticks by amplifying the 16S rRNA gene, including A. phagocytophilum (0.6% MIR), APL clade B (0.1% MIR), A. capra (0.1% MIR), and A. bovis (0.1% MIR). Among them, A. phagocytophilum (85.2%) was the most prevalent; seven Haemaphysalis spp. larvae, one H. longicornis adult, nine H. longicornis nymphs, three I. nipponensis adults, and three I. nipponensis nymphs were detected. A. phagocytophilum is a serious zoonotic pathogen in humans and animals [29]. A. capra was detected in one adult H. longicornis. In 2018, A. capra was detected for the first time in Korean cattle in the GN province [21]. A. capra is a potential zoonotic pathogen from ticks parasitizing animals in the Republic of Korea. However, it remains unclear whether A. capra is pathogenic to humans and animals; therefore, additional research is needed to clarify the pathogenicity of this emerging Anaplasma species. A. bovis (one H. longicornis adult) and APL clade B (one H. longicornis nymph and one Haemaphysalis spp. larva) were also detected but were not considered zoonotic pathogens. Further studies are needed to determine the pathogenicity of the Anaplasma species.
Our study reveals the geographical and temporal distribution of several tick species and their Anaplasma infections. In this study, five species of ticks including unidentified Haemaphysalis species were collected in 12 survey sites of Gyeongsang, but their prevalence was somewhat different according to the tick species and survey sites. As mentioned above, four different Anaplasma species were previously detected in several animals, humans, and animal blood-feeding ticks. To the best of our knowledge, this study is the first to investigate the presence of A. phagocytophilum, APL clade B, A. bovis, and A. capra in questing unfed ticks in an environment near animal farms in the Republic of Korea. Further, the incidence rate (6.8%) of four Anaplasma spp. was not as high in tick pools. However, the results of this study may offer a basis for future epidemiological research and risk assessment of tick-borne diseases. Further studies in larger regions with livestock, ticks, and wild animals are also needed to understand biology of tick-pathogen infections and to prevent the transmission of tick-borne diseases and establish effective control strategies.