Molecular Investigation of Tick-Borne Pathogens in Ticks removed from Tick-Bitten Humans in the Republic of Korea

Background Tick-borne infections are continuously increasing due to climate change, increased outdoor activities and increased travel between countries. This study was to investigate the presence of tick-borne pathogens in ticks removed from tick-bitten humans in southwest provinces of Republic of Korea (ROK). Methods Ticks were obtained from those tick-bitten humans between May 2014 and September 2017 in Jeollanam provinces and Gwangju metropolitan city in ROK. The presence of the tick-borne pathogens in ticks removed from tick-bitten humans was analyzed using pathogen-specific polymerase chain reaction (PCR). Results We identified 33 ticks from three tick species, namely Amblyomma testudinarium (60.6%), Haemaphysalis longicornis (27.3%), and Ixodes nipponensis (12.1%) in order of occurrence by morphology and 16S rDNA-targeting PCR. Tick-borne pathogens were found in 16 ticks using pathogen-specific PCR. From the results, 12 ticks (36.4%) tested positive for spotted fever group (SFG) Rickettsia: Rickettsia monacensis (1/12), R. tamurae (8/12), and Candidatus Rickettsia jingxinensis (3/12). Three ticks (9.1%) were positive for Anaplasma phagocytophilum . In addition, three ticks (9.1%) tested positive for Babesia gibsoni (1/3) and B. microti (2/3). Conclusions In conclusion, we identified three tick species; the most common species was A. testudinarium followed by H. longicornis and I. nipponensis . SFG Rickettsia , A. phagocytophilum , and Babesia spp. were the most frequently detected pathogens in ticks removed from tick-bitten humans. R. tamurae and Ca. R. jingxinensis were firstly detected in Korea. The present results will contribute to the understanding of tick-borne infections in animals and humans in the ROK. determine The present study aimed to investigate the presence of tick-borne pathogens in ticks removed from humans in the southwest provinces of the ROK. Our study detected the DNA of tick-borne pathogens from ticks using pathogen-specific nested PCR. The results of this study will contribute to the understanding of the interaction between ticks and pathogens that Molecular detection humans We examined 33 ticks for the detection of tick-borne pathogens using pathogen-specific nested PCR. The presence of tick-borne pathogens was detected in 16 ticks. From the results, 12 ticks (36.4%) tested positive for spotted fever Rickettsia, namely R. monacensis (1 of 33, 3.0%), R. tamurae (8 of 33, 24.2%), and Candidatus Rickettsia jingxinensis (3 of 33, 9.1%). Three ticks (9.1%) were positive for A. phagocytophilum, while another three ticks (9.1%) were positive for either B. gibsoni (1 of 33, 3.0%) or B. microti (2 of 33, 6.0%) (Table 3). All ticks were negative for Borrelia spp., Bartonella spp., and C. burnetii. Previous studies that investigated the prevalence of tick-borne infectious agents in ticks collected by dragging and flagging grass vegetation in the ROK showed that A. phagocytophilum was detected in 1.9% of H. longicornis ticks (15) and 0.1% of I. persulcatus ticks, and Rickettsia spp. were detected in 1.7% of H. longicornis ticks (16). One study reported that a pool of H. longicornis, H. flava, and I. nipponensis ticks collected by dragging vegetation in the ROK were positive for the Rickettsia spp. 17 kDa antigen (60/311, 19.3%) and ompA gene (53/311, 17.04%) (17). In the present study, the infection prevalence of Rickettsia species (R. monacensis, R. tamurae, and Ca. R. jingxinensis) and A. phagocytophilum in the ticks collected from humans was higher than that of ticks collected from the vegetation. Thus, we suggest that further study is needed to compare the infection prevalence of tick-borne pathogens, including Rickettsia spp., A. phagocytophilum, and Babesia between ticks isolated from humans and ticks collected from grass vegetation. in the ROK reported that H. longicornis was the most common tick species infected with Babesia (16, 19). Our results showed that B. microti was found in both H. longicornis and A. testudinarium. In the USA, the primary vector for the transmission of B. microti to humans is the tick Ixodes scapularis in the nymphal stage (32). The present results suggest that further study is needed to determine the type of ticks that are the vectors for the transmission of B. microti to humans in the ROK.


Background
Ticks are major vectors of pathogens such as bacteria, viruses, and protozoans. These arthropods can transmit a variety of diseases to humans and animals (1). Tick-borne diseases are caused by viral or bacterial pathogens transmitted through tick bites. Several tick-borne diseases such as Lyme disease (caused by Borrelia species), spotted fever group rickettsioses (caused by Rickettsia spp.), anaplasmosis (caused by Anaplasma phagocytophilum), bartonellosis (caused by Bartonella spp.), Q fever (caused by Coxiella burnetii), and babesiosis (caused by Babesia spp.) have been reported in the Republic of Korea (ROK) (2).
The incidence of tick-borne diseases in the ROK is increasing due to global warming, increased outdoor activities, and increased international travel. The growing number of tick bites each year poses an escalating risk of tick-borne diseases (2). Few studies have investigated the prevalence of tick-borne pathogens in ticks removed from tick-bitten humans in the ROK. However, it is necessary to determine the extent of tick-borne pathogens in the ROK, and to characterize them.
The present study aimed to investigate the presence of tick-borne pathogens in ticks removed from humans in the southwest provinces of the ROK. Our study detected the DNA of tick-borne pathogens from ticks using pathogen-specific nested PCR. The results of this study will contribute to the understanding of the interaction between ticks and pathogens that cause diseases in humans.   For N-PCR, the reaction mixture was identical to that used in C-PCR, except that the first PCR product was used as template DNA, and the N-PCR primers were included. With each PCR run, a positive and a negative control (molecular grade water) were included.

Methods
All amplifications were performed in an AB thermal cycler (Applied Biosystem, Foster City, CA, USA).
The amplified products were separated by electrophoresis on a 1.2% agarose gel, and stained with ethidium bromide for visualization.

Sequencing and phylogenetic analysis
The amplified PCR products were purified using QIAquick PCR purification kits (QIAGEN, Hilden, Germany) and sequenced with the PCR primers at Solgent Inc. (Daejeon, Korea). The sequences obtained in this study were compared for similarity with the GenBank sequences using BLAST. Gene sequences, excluding the primer regions, were aligned using the multisequence alignment program in

Tick identification
We obtained 33 ticks from 30 tick-bitten humans. Out of these, 15 ticks (45.5%) were adults, namely 12 females and 3 males, and 18 ticks (54.5%) were nymphs. Based on morphological examination using a microscope for tick identification, the ticks were identified as Amblyomma testudinarium (20, 60.6%; 7 adults and 13 nymphs), Haemaphysalis longicornis (9, 27.3%; 5 adults and 4 nymphs), and Ixodes nipponensis (4, 12.1%; 3 adults and 1 nymph), as described in Table 2. Tick identification using 16S rDNA C-PCR and DNA sequencing yielded the same results as the microscopic examination with the exception of four samples without tick DNA (shown in Table 4).  monacensis. In addition, co-infections of R. tamurae and Babesia spp. were identified in A.
testudinarium (adult male) that presented in Table 4.

Sequencing and phylogenetic analysis
The positive PCR products were sequenced and the sequencing results were aligned with the sequences obtained from the GenBank database to identify known sequences with a high degree of similarity using ClustalW. The neighbor-joining tree was constructed using the Kimura 2-parameter model (1,000 bootstrap replicates). JX962780, 100% bootstrap support, Fig. 1D) and Babesia spp. from a tick in Japan (accession no. LC169083, 96% bootstrap support, Fig. 1D).

Discussion
Recently, the risk of tick-borne disease has been associated with exposure to ticks from increasing outdoor activity. This study was performed to detect and identify the tick-borne pathogens in ticks removed from tick-bitten humans. We classified 33 ticks into three species: A. testudinarium (20, 60.6%; 7 adults and 13 nymphs) was the most common followed by H. longicornis (9,27 phagocytophilum in the ticks collected from humans was higher than that of ticks collected from the vegetation. Thus, we suggest that further study is needed to compare the infection prevalence of tickborne pathogens, including Rickettsia spp., A. phagocytophilum, and Babesia between ticks isolated from humans and ticks collected from grass vegetation. A. phagocytophilum infection was first reported with serological evidence from humans in 2002, and it is currently the most frequently reported tick-borne bacterial infection in the ROK (18). The detection of Anaplasma spp. in ticks from grazing cattle collected from all ROK provinces has been reported (19). Another study confirmed a human granulocytic anaplasmosis (HGA) with A. phagocytophilum in a patient from the ROK who had a history of tick bites, clinical symptoms, and positive laboratory findings (20). The present results showed that A. phagocytophilum was detected in A. testudinarium and I. nipponensis ticks. The amplicon sequences of the partial ankA gene in A. testudinarium (Tick 1) and I. nipponensis (Tick 29 and Tick 30) demonstrated more than 99% similarity. In the phylogenetic analysis, the sequences of the ankA gene from different types of ticks clustered together, showed > 99% similarity with A. phagocytophilum strains isolated from humans in the ROK (Fig. 1A).
The first isolation of R. monacensis from ticks in the ROK was reported in 2013 (21 R. tamurae was first isolated from A. testudinarium ticks collected in Japan in 1993. R. tamurae was formally identified as a novel species by genetic and phylogenetic analyses in 2006 (22). In 2011, the first case of human infection was confirmed using molecular and serological analyses in Japan (23).
The presence of SFG Rickettsia including R. tamurae was found in Amblyomma and Dermacentor ticks in Thailand (24) and in Haemaphysalis ticks in Peninsular Malaysia (25). In addition, R. tamurae was found in Amblyomma ticks from an area endemic for Brazilian spotted fever in Brazil (26). Supporting these previous studies, our results showed the presence of R. tamurae in A. testudinarium ticks.
The presence of a potentially novel species of Ca. R. jingxinensis was proposed in H. longicornis nymphs from Jingxin in Northeastern China in 2016 (27) and was detected in H. longicornis ticks in Xi'an, China in 2017 (28). In the ROK, the pathogenicity of Ca. R. jingxinensis is not clear. Therefore, a further assessment of the potential pathogenicity in humans and animals is needed.
There have been no previous reports of R. tamurae or Ca. R. jingxinensis from ticks in the ROK; here, we report the first identification of R. tamurae and Ca. R. jingxinensis in ticks obtained from tick-bitten humans.
Babesia was first discovered in animals by Babes in 1988, and more than 100 species have been  (12). Based on the phylogenetic analysis of the 18S rDNA gene in our study, the pathogen clustered with a group of Babesia spp., isolated from a tick in Japan, which was diverged from the KO1 strain (Fig. 1D). The present results indicate that Babesia spp. may vary based on their geographical distributions.
Further investigation is needed to determine the difference between pathogens found in ticks isolated from humans and ticks collected from grass vegetation. In addition, transmission studies should be conducted to determine whether the pathogens found in ticks are the same as those found in humans bitten by those ticks. To confirm the transmission of pathogens from ticks to humans, serological testing on the blood of tick-bitten patients and their ticks will be necessary. Further experiments and correlation analysis using the blood samples of tick-bitten humans and ticks isolated from them may help predict the transmission of tick-borne diseases.

Conclusions
In conclusion, we confirmed three tick species carrying tick-borne pathogens; the most common species was A. testudinarium followed by H. longicornis and I. nipponensis. These ticks were positive for SFG Rickettsia, A. phagocytophilum, and Babesia. This was the first report of the presence of R.
tamurae and Ca. R. jingxinensis in ticks removed from tick-bitten humans in the ROK.  CHOSUN NON2019-001). The IRB has been approved without written consent for the use of ticks and not human subject.

Consent for publication
Not applicable

Availability of data and materials
The data supporting the conclusions of this article are included within the article. Figure 1 Phylogenetic trees based on partial nucleotide sequences obtained from A.