Introduction

Hard ticks are a type of external parasite that feeds on the blood of both animals and humans and are known to transmit various viruses, parasites, and bacteria1. In a study on geographical distribution of Ixodid ticks in Korea from 2013 to 2015, Haemaphysalis longicornis accounted for 88.9%, followed by H. flava (10.1%), Ixodes nipponensis (0.5%), I. persulcatus (0.2%), H. japonica (0.2%), Amblyomma testudinarium (0.1%), and I. granulatus (< 0.1%)2. H. longicornis peaked in May to July (with larvae in September, and nymphs in May, and adults in July), while H. flava collected mainly in September to October (with larvae and adults in September, and nymphs in October) based on dry-ice bait trap method.2. Hard ticks mainly found in the southern region of Korea include H. longicornis, H. flava, I. nipponensis, and A. testudinarium3.

The important tick-borne pathogens (TBPs) that are transmitted by hard ticks include the following: Severe Fever with Thrombocytopenia Syndrome (SFTS) virus, Rickettsia spp., Coxiella burnetii, Borrelia spp., Anaplasma phagocytophilum, Ehrlichia spp1,4. SFTS is a vector-borne infectious disease that was first reported in China in 20115. Its incidence has increased in China, Japan, and Korea, and it is currently designated as a category three national notifiable infectious disease in Korea6. It is mainly transmitted by H. longicornis, with H. flava, I. nipponensis, and A. testudinarium also known as vectors for the SFTS virus in Korea7. The prevalence of SFTS virus in ticks8, and wild animals9,10,11 has been determined. Kim et al. also reported a molecular epidemiological correlation between a patient with SFTS and questing ticks collected from the patient's residence12.

Spotted fever group rickettsioses (SFGR) are febrile diseases caused by Rickettsia species associated with chiggers, fleas, and hard ticks13,14. In Korea, R. rickettsii and R. japonica were confirmed in H. longicornis by PCR13. Japanese spotted fever (caused by R. japonica) was first reported in 200514. Moreover, R. monacensis was also isolated from a patient in Korea13. Recently, a study provided the first description of R.raoultii detected in H. longicornis ticks, which were collected from patients with a history of tick bites in Korea15. This pathogen was also shown to have a high prevalence in ticks collected from dogs in Korea16.

Q fever is a globally occurring zoonotic illness caused by Coxiella burnetii17. C. burnetii is known to be transmitted to humans via inhalation of contaminated aerosols from animals and consumption of contaminated milk18. The role of C. burnetii-infected ticks in human Q fever is being disputed, as ticks are not essential vectors for C. burnetii transmission. However, several studies have shown ticks may play an important role in the transmission of coxiellosis between livestock and wildlife, which may lead to human coxiellosis19.

Lyme disease is a tick-borne illness caused by Borrelia burgdorferi sensu lato (s.l.), which comprises approximately 20 genospecies20,21. In Korea, Borrelia burgdorferi s.l. was first detected in Ixodes ticks in 1993, and the first human case of Lyme disease was reported in the same year22. To date, Borrelia afzelii, B. garinii, B. tanukii, B. turdi, B. yangtzensis, B. bavariensis, and B. valaisiana genospecies have been identified in ticks and wild animals in Korea21.

Anaplasma phagocytophilum and Ehrlichia spp. belong to the family Anaplasmataceae and share similar characteristics23. Both A. phagocytophilum and Ehrlichia spp. have been identified in H. longicornis, I. nipponensis, and I. persulcatus in Korea13.

As a result of tropical climate change, the summer season in South Korea is becoming longer and warmer24,25. This leads to an increased risk of tick-borne infectious diseases due to the higher survival rate, increased egg-laying rate, and larger population of ticks26. Moreover, not only agricultural workers but also the general population are facing an increased risk of tick exposure, due to the rising popularity of outdoor activities such as hiking, camping, and the increasing pet population27. The growing risk of tick-borne diseases necessitates research on the distribution of ticks and the presence of the pathogens they carry. Therefore, we investigated the distribution of ticks and tick-borne pathogens in Gwangju city, located in southwestern region of Korea, from 2019 to 2022.

Results

Distribution of field collected ticks

During the study period in Gwangju, South Korea, a total of 13,280 ticks were collected. Among the adult and nymph ticks, H. longicornis accounted for 86.1% of the collected ticks, with 349 adults and 4320 nymphs. H. flava comprised 9.4% of the ticks, with 252 adults and 260 nymphs. I. nipponensis made up 3.6% of the ticks, with 169 adults and 27 nymphs. A. testudinarium constituted 0.8% of the ticks, with 3 adults and 42 nymphs. Additionally, there were 7858 larvae that were difficult to differentiate between H. longicornis and H. flava, accounting for 59.2% of the total ticks collected, as shown in Table 1. Ticks were collected throughout the year, with the highest prevalence observed in the spring and fall seasons, as illustrated in Figs. 1 and 2. Specifically, larvae were primarily collected in the spring, while nymphs were predominantly collected in the fall. Interestingly, Ixodes ticks were observed from autumn to spring, while A. testudinarum ticks were observed in early summer (Fig. 3).

Table 1 Prevalence (minimum infection rate, %) of pathogens in field-collected ticks in Korea, 2019–2022.
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Figure 1
figure 1

Seasonal distribution of tick samples collected in Gwangju, Korea.

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Figure 2
figure 2

Seasonal distribution of Haemaphysalis species per life stage (adults and nymph) collected in Gwangju, Korea.

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Figure 3
figure 3

Seasonal distribution of Ixodes nipponensis and Amblyomma testudinarum ticks collected in Gwangju, Korea.

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Detection of tick-borne pathogens

A total of 983 pools were tested, including 460 pools for H. longicornis, 194 pools for H. flava, 135 pools for I. nipponensis, 37 pools for A. testudinarium, and 157 pools for larvae. Adult ticks were analyzed individually or pooled, while nymphs and larvae were pooled according to their species, sex, and life stage to compare the prevalence of pathogens. In total, 2.0% (minimum infection rate, MIR) of the field-collected ticks were found to be pathogen-positive; H. longicornis, H. flava, I. nipponensis, A. testudinarium, and larvae (Haemaphysalis spp.) exhibited detection rates of 2.5%, 2.2%, 29.6%, 13.3%, and 0.9% MIR, respectively. Compared to other tick species, I. nipponensis and A. testudinarium demonstrated significantly higher pathogen prevalence rates (P < 0.001). Specifically, I. nipponensis exhibited a high detection rate for both Borrelia spp. and Rickettsia spp., while A. testudinarium was mainly associated with Rickettsia spp.

Rickettsia spp. were the most prevalent pathogens across all tick species (216 pools, 1.6% MIR, P < 0.001). Notably, Rickettsia spp. exhibited a high MIR in both I. nipponensis (13.8% MIR, P < 0.001) and A. testudinarium (13.3% MIR, P < 0.001).

Borrelia spp. were the second most commonly reported pathogen in this study (28 pools, 0.2% MIR). It was mainly detected in I. nipponensis (27 pools, 13.8% MIR, P < 0.001), with only one positive pool found in H. longicornis ticks.

A. phagocytophilum (15 pools, 0.1% MIR) was found in H. longicornis (11 pools, 0.2% MIR) and I. nipponensis (4 pools, 2.0% MIR), while Ehrlichia spp. (3 pools, 0.02% MIR) were confirmed only in H. longicornis ticks.

Co-infections were detected in I. nipponensis (14 pools) and larvae of Haemaphysalis spp. (6 pools); Borrelia spp. and Rickettsia spp. were found in 12 pools of I. nipponensis; Borrelia spp. and A. phagocytophilum were found in 1 pool of I. nipponensis; Rickettsia spp. and A. phagocytophilum were found in 6 pool of larvae (Haemaphysalis spp.). Additionally, one female I. nipponensis tick was positive for 3 pathogens simultaneously; Borrelia spp. and Rickettsia spp. and A. phagocytophilum. Meanwhile, there were no positive samples for SFTSV and Coxiella burnetii in this study.

Molecular and phylogenetic analysis

Sequencing analysis of groEL gene, obtained from 196 Rickettsia spp. positive samples (196/612), revealed the presence of R. canadensis, R. japonica, and R. monacensis in 5, 160, and 31 pools, respectively. Among the Rickettsia species, R. japonica, the etiological agent of Japanese spotted fever (JSF), exhibited the highest detection frequency, with 160 pools (1.2% MIR) of all ticks testing positive. The majority of sequences of R. japonica were detected in Haemaphysalis spp (159/160 pools); only one adult I. nipponensis tick tested positive for this pathogen. Interestingly, R. japonica was predominantly detected in male H. longicornis ticks (25% MIR, P < 0.001). The nucleotide sequences of R. japonica showed significant similarity to those identified in humans from Japan (AP017595) (Fig. 4). R. monacensis was the second most frequently detected species. Unlike R. japonica, which was primarily detected in Haemaphysalis spp., R monacensis exhibited a remarkably high prevalence in I. nipponensis and A. testudinarium ticks. The nucleotide sequences of R. monacensis showed close similarity to those obtained from Ixodes ricinus ticks collected in Munich, Germany (LN794217) (Fig. 4). R. canadensis, recently recognized as a pathogenic species46, was detected in 4 pools of H. longicornis nymphs and in one pool of A. testudinarium nymphs.

Figure 4
figure 4

Phylogenetic relationship for Rickettsia species, based on the nucleotide sequences of groEL gene. The neighbor-joining method was used for constructing a phylogenetic tree. Sequences identified in this study are indicated by black circles (●) for Haemaphysalis spp, black triangles (▲) for I. nipponensis, and black squares (■) for A. testudinarum. Scale bar indicates sequence distances.

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Borrelia spp., the causative agents of Lyme disease and tick-borne relapsing fever, were predominantly detected in I. nipponensis ticks (13.8% MIR), and in one pool of H. longicornis ticks (Fig. 5). Nucleotide sequence analysis revealed the presence of B. afzelii in 20 adult pools and 1 pool from nymphs of I. nipponensis ticks. The partial flaB sequences of the B. afzelii group showed high identity with that of B. afzelii detected in I. nipponensis tick in Korea (MH102391). Borrelia garinii and Borrelia miyamotoi were each detected in two pools from female I. nipponensis ticks, respectively.

Figure 5
figure 5

Phylogenetic relationship for Borrelia species, based on the nucleotide sequences of flaB gene. The neighbor-joining method was used for constructing a phylogenetic tree. Sequences identified in this study are indicated by black triangles (▲). Scale bar indicates sequence distances.

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A. phagocytophilum was detected in I. nipponensis ticks (2.0% MIR) and H. longicornis ticks (0.24% MIR). Considering the sample size, the detection rate was notably high in I. nipponensis ticks. The nucleotide sequences displayed high similarity to those reported in Korea (OM681329). Additionally, 10 pools from Haemaphysalis spp. nymphs exhibited nucleotide sequences highly similar to those found in deer and ticks in Korea (GU046565, GU556621) (Fig. 6).

Figure 6
figure 6

Phylogenetic relationship for A. phagocytophilum, based on the nucleotide sequences of 16 s rRNA gene. The neighbor-joining method was used for constructing a phylogenetic tree. Sequences identified in this study are indicated by black triangles (▲) for Haemaphysalis spp, and empty triangles (△) for I. nipponensis. Scale bar indicates sequence distances.

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Ehrlichia spp. were exclusively detected in H. longicornis ticks; however, the PCR targeting the 16S rRNA gene did not reveal any distinct Ehrlichia species (Fig. 7).

Figure 7
figure 7

Phylogenetic relationship for Ehrlichia species, based on the nucleotide sequences of 16s rRNA gene. The maximum likelihood method was used for constructing a phylogenetic tree. Sequences identified in this study are indicated by black triangles (▲) for Haemaphysalis spp. Scale bar indicates sequence distances.

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In our study, 0.25% MIR of the field-collected ticks were found positive for more than one tick-borne pathogen, primarily in I. nipponensis ticks and larvae of Haemaphysalis spp. R. monacensis and B. afzelii were identified in I. nipponensis ticks (MIR 6.5%); R. monacensis and B. garinii in I. nipponensis ticks (MIR 1.2%); B. afzelii and A. phagocytophilum in I. nipponensis ticks (MIR 1.2%). Both R. japonica and A. phagocytophilum were detected in 6 pools from larvae (MIR 0.1%). Furthermore, three pathogens were identified in one pool of I. nipponensis; R. monacensis, B.afzelli, and A. phagocytophilum.

Discussion

In South Korea, the incidence rate of tick-borne diseases mediated by hard ticks is relatively low compared to countries such as the United States and Europe. For instance, the reported occurrence of Lyme disease in the United States is 73.3 cases per 100,000 individuals28, while in Finland, it is around 118 cases29. Canada reported 7.0 cases in 201930, and Lyme disease patients are reported annually in China and Japan as well31. In Korea, the total number of confirmed cases over a 10-year period from 2012 to 2021 was exceptionally low, with only 110 cases (domestically)32. Consequently, research on these ticks, which serve as vectors for TBPs, is limited. However, there is an increasing possibility of a higher incidence of tick-borne diseases due to the introduction of exotic tick species and the potential introduction of pathogens facilitated by climate change33,34. Factors such as increased international exchanges and outdoor activities contribute to this possibility34. This study aimed to investigate the distribution of tick species, seasonal variations, and conduct molecular epidemiological analysis of pathogens in field ticks from the southwestern region of Korea between 2019 and 2022.

During the study period, a total of 13,280 hard ticks were collected, and the distribution of ticks was in accordance with other studies conducted in Korea. According to research by Seo et al.35, which utilized the dry-ice trap method throughout Korea, out of 63,376 hard ticks collected, H. longicornis accounted for 96.5%, H. flava for 2.8%, I. nipponensis for 1.7%, and A. testudinarium for 0.5%. In a study conducted by Lee et al.3, utilizing the flagging and dragging method, H. longicornis accounted for 80.7%, and H. flava accounted for 16.2%. Similarly, in this study, H. longicornis was identified as the dominant species, accounting for 86.1%. A. testudinarium were found very rarely. A. testudinarium was mainly found in southern region of Korea3.

In Korea, adult ticks and nymphs typically peak from May to August, while larvae peak from August to September2,36,37. Following their life cycle, adult ticks lay eggs during the summer, which then hatch into larvae. These larvae predominantly feed on hosts in the autumn and subsequently molt into nymphs, which spend the winter to spring period. Consequently, there is a higher distribution of nymphs in the spring and larvae in the autumn35. In this study, adult ticks and nymphs were primarily collected from April to July, while larvae were collected from July to November. Notably, Ixodes ticks were predominantly collected from autumn to spring in this study. Since there have been no previous reports on the distribution of I. nipponensis ticks, including the winter season, in Korea, this observation represents a novel finding. It underscores the importance of continuous monitoring of I. nipponensis tick distribution throughout Korea.

Meanwhile, there are reports suggesting that climate change can affect tick abundance38,39. When the seasons favored by nymphs and larvae overlap, there is an increased risk of pathogen transmission as they can co-feed on the same host. In cases of tick-borne pathogens with non-systemic infection, larvae have no chance of acquiring the pathogen. However, with climate change leading to simultaneous population peaks of nymphs and larvae, infected nymphs can transmit the pathogen to larvae via the host's blood system, without causing systemic infection38,39. The results of this study also indicate a trend of increasing ambiguity in the timing of peak periods for adult ticks, nymphs, and larvae after 2019. This highlights the need for continuous monitoring of tick distribution and its peak seasons. It should be noted that factors such as the specific method employed for collecting ticks, the timing of the collection, and the type of sampler utilized can introduce some degree of variability in species distribution and seasonal patterns.

The results confirmed that the detection rate of Rickettsia spp. was the highest when examining the pathogens carried by field ticks in southwestern Korea. This result may reflect the characteristics of the pathogen, such as trans-stadial and trans-ovarial infections in ticks40. Similar findings were reported in studies conducted in Latvia41, Spain42, and France43, where the detection rate of Rickettsia spp. was found to be the highest. On the other hand, in a study conducted in Finland, Borrelia burgdorferi, the causative agent of Lyme disease, was predominantly detected44. Meanwhile, information about the prevalence of various tick-borne pathogens, including Rickettsia spp. in Korea is very scarce. Our report confirms the potential risk of Rickettsia spp. to humans.

In the Asian region, Rickettsia spp. have been predominantly detected in Haemaphysalis spp., A. testudinarium, or Dermacentor spp. Among Rickettsia species, R. japonica is commonly reported to be confirmed mainly in Haemaphysalis spp. or Dermacentor spp., while R. monacensis has been reported in Amblyomma dissimile, Dermacentor variabilis, Ixodes boliviensis, I. persulcatus, I. ricinus, I. sinensis, I. pacificus, and Rhipicephalus sanguineus45,46,47. In this study, R. japonica was predominantly confirmed in Haemaphysalis spp., which is consistent with previous findings45,46.

R. monacensis was detected at a very high rate in I. nipponensis. A study was conducted on ticks collected from wild rodents captured in the U.S. military bases and training grounds located in Korea, where I. nipponensis accounted for 99.5% of the ticks collected. Among the 197 pooled samples, Rickettsia spp. was detected in 58.4%, with 87 pools of R. monacensis and 18 pools of R. japonica48. Recent studies conducted in Korea and Japan also reported a high correlation between R. monacensis and I. nipponensis49,50.

Interestingly, R. monacensis, known to cause a Mediterranean spotted fever-like illness51,52, was detected at a very high rate in A. testudinarium, as well. To the best of our knowledge, this study represents the first report of this pathogen being detected in A. testudinarium. In a previous study conducted from 2014 to 2018 in Korea4, no pathogens were detected in A. testudinarium. A. testudinarium is primalrily reported to inhabit southern regions of Korea, and it is necessary to monitor whether their habitats are changing due to climate change.

Ixodes ticks are recognized as major vectors of Borrelia bacteria, with I. persulcatus or I. nipponensis being identified in Asia. In a study of ticks collected from pasture around livestock farms in Korea, the detection rate of Borrelia spp. was found to be 34.0% MIR in I. nipponensis3. Another study in Korea also reported that the MIR of Borrelia spp. in I. nipponensis was 2.1%, while in H. longicornis and H. flava, it was 0.09%, and 0.1%, respectively53. The incidence of Lyme disease is associated with Borrelia genotypes, yet there are few reports on genotypes in field ticks in Korea. B. afzelii and B. miyamotoi were previously detected in 3 pools (12.0%), and 1 pool (4%), respectively in I. nipponensis adults in Korea54. Lee et al. reported that all Borrelia species, detected in I. nipponensis collected from Korean water deer and by tick drag were identified as B. afzelii by the ospA gene sequences55. During our survey, B. afzelii, B.garinii, and B. miyamotoi were detected in 10.7%, 1.0%, and 1.0%, respectively. These results indicate B. afzelii is common, while B.garinii, and B. miyamotoi are rarely detected in I. nipponensis in Korea.

A. phagocytophilum is reported to be transmitted by Ixodes ticks. In the United States, representative vectors include Ixodes scapularis or Ixodes pacificus. In Western Europe, transmission is primarily associated with I. ricinus, while in Asia, I. persulcatus is known as a vector56. In this study, I. nipponensis exhibited high detection rates of A. phagocytophilum.

Research on the genus Ehrlichia has been limited in Korea. In a study investigating TBPs in ticks from grazing cattle in Korea, E. canis was detected with the highest rate, followed by E. chaffeensis, E. ewingii, and E. muris57. Another study found E. chaffeensis was the most frequently detected species in H. longicornis ticks collected in northern Korea58. However, in this study, the PCR targeting the 16S rRNA gene did not reveal any distinct Ehrlichia species. Three samples belonging to the genus Ehrlichia clustered with Ehrlichia spp. detected in H. longcornis in Japan (MT258399) . Since the 16S rRNA gene is known to be more conserved, further study is warranted by comparing more divergent gene sequences, such as the groEL gene.

Ixodes ticks are commonly co-infected with other tick-borne pathogens such as Borrelia spp., Babesia spp., Ehrlichia spp., Rickettsia spp., and Powassan virus59. In this study, co-infections were observed in a total of 14 pools of I. nipponensis, mostly consisting of Borrelia spp. and Rickettsia spp. These findings corroborate previous research suggesting that co-infection is common among Ixodes ticks41,59. Co-infection may lead to increased diversity, severity, and duration of symptoms60. Therefore, raising awareness of potential co-infections is crucial, and further studies are warranted.

Meanwhile, Coxiella burnetii was not detected in this study. Its primary route of transmission is through the inhalation of contaminated aerosols18. Although C. burnetii has been detected in various tick species, tick-borne transmission is considered to be low19. A case report described an 8-year-old Korean girl who was co-infected with the SFTS virus and C. burnetii after playing with a dog and being bitten by a tick. In a study of 816 horses in Korea, six samples (0.7%) tested positive by PCR, suggesting the potential for C burnetii transmission to humans during horseback riding. Additionally, other studies provided evidence of ticks harboring C burnetii in Korea61,62. Given these reports, further investigation into the epidemiology of Q fever is required.

There are several limitations to this study. Firstly, our research was focused solely on the Gwangju city, southwestern Korea, which may not be fully representative of the entire tick population in Korea. Secondly, our tick collection methods primarily relied on dragging and flagging, which could introduce sampling bias. /these method might not accurately capture the overall tick population and their habitats. Thirdly, our pooling strategy for tick samples may raise question about accuracy. Some research has shown that pools with more than 20 nymphs did not significantly improve the detection probability of Rickettsia species63. Additionally, the calculation of the minimum infection rate (MIR) assumes that only one infected individual exists in a positive pool, potentially underestimating the actual prevalence of infection. Therefore, our reported prevalence rates may represent a lower bound of the infection rate64. Lastly, our analysis of Rickettsia species was based on the groEL gene. While this gene provides valuable information, a more comprehensive analysis could have been achieved by analyzing additional sequences targeting genes such as rrs, gltA, and ompA. Despite these limitations, our study offers valuable insight into the distribution and pathogen characteristics of hard ticks in the natural environments of Korea. This is particularly relevant for less-studied species like I. nipponensis and A. testudinarium, which have been underrepresented in research due to their smaller collection scale compared to other tick species. Future research incorporating a broader range of molecular markers and expanding the geographic scope of sampling could further enhance our understanding of tick-borne diseases in Korea.

In conclusion, this study comprehensively investigated the distribution of hard ticks and characteristics of TBPs using 13,280 specimens collected from the southwestern region of Korea between 2019 and 2022. Rickettsia spp. was the most commonly detected pathogen, with R. japonica and R. monacensis being frequently detected in I. nipponensis and Haemaphysalis spp., respectively. Notably, our study identified R. monacensis for the first time in A. testudinarium in South Korea. These findings underscore the imperative for continuous research on indigenous hard ticks and associated pathogens in South Korea. Given the impact of factors such as climate change, increased international exchange, and alterations in wildlife behavior on the dynamics of tick-borne diseases, continuous monitoring is essential to detect and manage the potential introduction of exotic ticks and pathogens, which could pose new challenges for human health.

Materials and methods

Tick sampling and classification

Ticks were collected from the field on a monthly basis for 1–2 days by using the dragging and flagging methods. A 1 m × 1 m white flannel cloth attached to a wooden bar was utilized for this purpose. The collection was conducted in Gwangju city, situated in the southwestern region of the Republic of Korea. Collection sites comprised 3–5 locations in hills and mountainous areas surrounding Gwangju city, chosen based on accessibility or suitablility for crop cultivation. The collection process involved three to four collectors, each spending 15–20 min. Ticks were carefully removed from the flannel cloth using fine forceps and transferred to 50 mL tubes. Subsequently, the collected ticks were stored at − 80 °C until further processing. Identification of ticks was performed using an Axio Zoom.V16 microscope, following the guidelines provided by Yamaguti et al.65.

DNA/RNA extraction

Ticks were pooled in Precellys® 2 mL tubes along with 2 mm ceramic bead and 700 μL of sterile phosphate-buffered saline. Depending on the species, sex, collection date, and stage of development, 1–2 adults, 1–30 nymphs, and 1–50 larvae were pooled from each collection site for comparison in the prevalence of pathogens. The pooled ticks were homogenized for 1 min at 8000 rpm using Precellys® 2000 homogenizer and then centrifuged for 5 min at 10,000 rpm. The resulting supernatants were subjected to DNA/RNA extractions using the Maxwell® RSC viral total nucleic acid purification kit (Promega, Wisconsin, USA), following the manufacturer’s instruction.

Detection and characterization of pathogens

The nucleic acids were analyzed using the Applied Biosystems QuantStudio™ 5 real-time polymerase chain reaction (qPCR) machines, while the remaining samples were stored at 4℃ for further analysis. Molecular identification of SFTSV, Rickettsia spp., C. burnetii, Borrelia spp., A. phagocytophilum, and Ehrlichia spp. was carried out using qPCR assays with Popgen® pathogen detection kits (PostBio, Gyeonggi-do, Korea), following the manufacturer’s instructions. Briefly, the PCR assay was performed in 20 μL reaction mixtures consisting of 15 μL of Popgen® qPCR reaction Mix (aliquot) and 5 μL of template DNA. The reaction conditions included an initial denaturation step at 95 °C for 5 min, followed by 45cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s.

Any positive pools identified in the screening assay were subsequently confirmed by the ProFlex™ PCR machine using primer sets as listed in Table 266,66,67,68,69,70,71,73. The amplified PCR products were sent to Bionics (Daejeon, Korea), for sequencing using an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, USA). The nucleotide sequences obtained were aligned using ClustalW within MEGA-X software and compared with GenBank database using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI).

Table 2 Primers for the detection of tick-borne pathogens.
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Phylogenetic analyses were conducted using MEGA-X software (v.6.4), and the phylogenetic trees were constructed using the neighbor-joining method based on the Kimura 2-parameter model, with 1000 bootstrap replicates.

The prevalence of pathogens was calculated as minimum infection rate (MIR). The MIR for pooled ticks was determined by dividing the number of positive pools by the total number of ticks. The significance of the difference in prevalence for each pathogen among species, sexual, and developmental stages was evaluated using the chi-square test in Excel 2016.