TY - JOUR
AU - Klein, Terry, A
AB - Abstract A total of 6,255 ticks belonging to three genera and six species (Haemaphysalis flava Neumann, Haemaphysalis longicornis Neumann, Haemaphysalis phasiana Saito, Ixodes nipponensis Kitaoka & Saito, Ixodes persulcatus Schulze, and Amblyomma testudinarium Koch) collected from May–August, 2013, at four southwestern provinces in the Republic of Korea (ROK) were submitted to the Armed Forces Research Institute of Medical Sciences and assayed for selected tick-borne pathogens. One pool each of H. flava and H. phasiana was positive by PCR and sequencing for a Francisella-like endosymbiont, while all pools were negative for Francisella tularensis, the causative agent of tularemia. Haemaphysalis flava, Haemaphysalis phasiana, Francisella tularensis endosymbiont, tick surveillance, Korea Tick-borne disease surveillance has taken on increasing importance in the Republic of Korea (ROK), as more pathogens affecting human health are discovered (Cho et al. 1991; Park et al. 1992, 2014a; Shim et al. 1993; Kee et al. 1994; Uchida et al. 1995; Sachar 2000; Heo et al. 2002; Chae et al. 2003, 2008; Lee et al. 2003, 2013; Kim et al. 2003, 2005, 2009; Jang et al. 2004; Choi, et al. 2005; Ko et al. 2010; Kang et al. 2013, 2016; Shin et al. 2013; Yun et al. 2014), including the recent discovery of a novel Bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), in 2009 (Yu et al. 2011, Zhang et al. 2012) and subsequent discovery in the ROK in 2013 with 35 reported cases and 16 deaths (45.7% mortality; Kim et al. 2013a, Park et al. 2014b, Yun et al. 2016). To address these and other concerns, the 65th Medical Brigade, Korea, established a tick-borne disease surveillance program using tick drag methods (Chong et al. 2013a) to determine their relative abundance for selected habitats in the southwestern provinces of the ROK and prevalence and distribution of associated tick-borne pathogens. Francisella spp. are gram-negative bacteria that include Francisella tularensis, F. novicida, F. hispaniensis, F. philomiragia, F. noatunensis, and Francisella-like endosymbionts (FLEs). Francisella tularensis, the causative agent of tularemia, is classified as a Tier 1 agent of bioterrorism (Dennis et al. 2001, Federal Register 2012) and is divided into several subspecies, e.g., F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. tularensis subsp. mediasiatica (Sjostedt, 2007). Tularemia infections in humans may be contracted through the consumption of contaminated food or water, contact with infected animals, or mechanical and biological transmission by biting arthropods, especially ticks (Centers for Disease Control and Prevention 2002, Sjostedt 2007). The only culture-confirmed human case of tularemia in Korea was reported in 1998 from Pohang-si, Gyeongsangbuk province, in the ROK (Lim et al. 1998, Ahn et al. 1999). The patient, a 40-yr-old man, found a dead wild rabbit, skinned it, and consumed the cooked rabbit, and 20 d later was admitted with lymph node swelling in both the axillary and upper arm area and furuncles on the forearm and palm. Several tick species capable of transmitting F. tularensis also harbor FLEs that are genetically similar to F. tularensis and therefore pose a challenge for accurate identification, especially when polymerase chain reaction (PCR) is used for detection. Several previously published primers designed for the specific detection of F. tularensis have been shown to cross-react with FLEs in multiple tick species belonging to Genera of Dermacentor, Amblyomma, Ornithodoros, Hyalomma, and Rhipicephalus (Sun et al. 2000, Scoles 2004, Ivanov et al. 2011, Michelet et al. 2013). Therefore, use of molecular assays that can discriminate F. tularensis from FLEs is critical to avoid misidentification and confusion with respect to the presence and prevalence of F. tularensis in animals, vectors, and the environment. As previous studies have also reported a high prevalence of FLEs in tick species that are capable of transmitting F. tularensis (de Carvalho et al. 2011, Michelet et al. 2013), careful evaluation of molecular assays used for F. tularensis detection is important for an accurate medical threat assessment of tularemia in any region. Taken together, it is important to study the prevalence and distribution of FLEs in tick species capable of transmitting F. tularensis, especially in those regions where testing of ticks for F. tularensis has not previously been reported. In this study, we report the finding of a Francisella-like endosymbiont in two species of Haemaphysalis ticks collected from the ROK during the 2013 annual tick surveillance. Materials and Methods Collection Sites and Methods Ticks were collected monthly for 3–5 d by tick drag from April–October, 2013, as described by Chong et al. (2013a, b) that included four provinces and one metropolitan area in the southwestern part of the ROK (Fig. 1) as described by Coburn et al. (2016). Briefly, collection sites were located along primary, secondary, and tertiary roads that included grasses and herbaceous vegetation and hillsides and mountainous areas of deciduous, conifer, and mixed forests. Collection sites were sampled by three to four collectors for a period of 30–90 min, depending on the numbers of ticks collected. Tick drags, consisting of a 1.0 m-wide by 1.5 m-long white flannel cloth attached to a wooden dowel (2.0 cm diameter and 1.1 m long) and a nylon rope attached to each end of the rod, were pulled at a slow to moderate walking pace behind the collector over vegetation and ground cover for ∼10–15 m, then inspecting both sides of the drags for ticks. Adults and nymphs were carefully removed from the cloth using a fine forceps and up to 25 placed live in 2 ml cryovials. Larvae were similarly removed from the tick drag with up to 100 placed in 2 ml cryovials containing 100% ethanol (EtOH). Following the collection period, all collectors returned to a central point where data were recorded as described by Coburn et al. (2016) that included a unique collection number, GPS coordinates, site description, and other important information. The vials containing the ticks were labeled with the collection site number, placed in 4-oz Whirl-Pak plastic bags (Nasco, Inc., Fort Atkinson, WI), and then placed in a Styrofoam cooler with refrigerated gel packs to keep the specimens cool. Field data, including photos of the collection sites, were entered into an electronic database (Microsoft Excel) at the end of each collection day. Fig. 1. Open in new tabDownload slide Map of Korea showing the survey area for ticks collected from selected habitats by tick drag at Chungcheongnam (CN), Chungcheongbuk (CB), Jeollanam (JN), and Jeollabuk (JB) provinces, and Daejeon Metropolitan city (DJ) from April–October, 2013. Fig. 1. Open in new tabDownload slide Map of Korea showing the survey area for ticks collected from selected habitats by tick drag at Chungcheongnam (CN), Chungcheongbuk (CB), Jeollanam (JN), and Jeollabuk (JB) provinces, and Daejeon Metropolitan city (DJ) from April–October, 2013. Specimen Identification At the termination of each monthly collection, ticks were transported to the 5th Medical Detachment/65th Medical Brigade, United States Army Garrison (USAG) Yongsan, Seoul, where they were maintained at −80 °C until identified. Ticks were identified using dissecting stereo-microscopes (×100) and keys developed by Yamaguti et al. (1971) and Saito et al. (1974). After the ticks were identified, they were placed, by species, habitat, collector, and life stage (adults [1] and nymphs [1–5] and larvae [1–50 in 80% EtOH]) in 2-ml cryovials. Vials were then labeled with a unique identification + subnumber and returned to the −80 °C freezer. Approximately 50% of the collected ticks were provided to the Armed Forces Research Institute of Medical Science (AFRIMS) on dry ice where they were assayed for bacteria and protozoan pathogens. Voucher specimens were provided to Dr. Richard Robbins, Armed Forces Pest Management Board, for species confirmation and placed in the Peabody Museum of Natural History, New Haven, CT. Genomic DNA Extraction From Tick Specimens Ticks were not dipped in EtOH or other washing agents prior to homogenizing them for DNA extraction. Extraction of total DNA from ticks was performed with Wizard Genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions with some modifications. Approximately 1 ml of liquid nitrogen was poured into a sterile microcentrifuge tube containing ticks. When the liquid nitrogen completely evaporated, the ticks were homogenized fairly completely with a blue pestle rod. A total of 300 μl of nucleic lysis buffer was added into each microcentrifuge tube and then mixed thoroughly by vortex. The homogenate was centrifuged at 12,000 rpm for 10 min and the supernatant transferred to a new sterile microcentrifuge tube and incubated with 20 μl of Proteases K (20 mg/ml) at 55 °C for 1 h, then incubated with 3 μl of RNase A (10 mg/ml) at 37 °C for 15 min. Then 200 μl of protein precipitation solution was added and mixed vigorously by vortex and the mixture maintained on wet ice for 5 min. Insoluble materials were removed by centrifugation at 13,000 rpm for 4 min and the supernatant transferred into a sterile microcentrifuge tube. DNA was precipitated by adding 600 μl of isopropanol and centrifuged at 13,000 rpm for 1 min. The DNA pellet was washed with 70% EtOH, air dried, then resuspended with 50 μl of EB buffer (10 mM Tris Cl, pH 8.5; Promega), and then the DNA solution stored at −20 °C for further analysis. Detection and Characterization of Francisella tularensis Two F. tularensis real-time TaqMan PCR assays that target the fopA and tul4 (lpnA) genes were used for screening (Versage et al. 2003). Primers and probes were synthesized by the ABI Company (Singapore). Real-time PCR conditions were performed following a previously published protocol (Versage et al. 2003). Any positive pool from screening assay was confirmed and characterized by conventional PCR amplifying—1) 250-bp fragment of the tul4 (lpnA) gene of Francisella spp. using primers FT393/FT642 (Long et al. 1993), 2) 838-bp fragment of the tul4 (lpnA) gene of F. tularensis (TUL4) using primers TUL4B-F/TUL4B-R (Sjostedt et al. 1990), 3) 484-bp fragment of the tpiA gene of Francisella spp. using primers tpiA-F/tpiA-R (tpiA-F: 5′-TGG TCA TTC TGA GAG AAG ATC-3′/tpiA-R: 5′-CGT ACA TAT CTT GTT TGC TTG-3′) at an annealing temperature of 52 °C, and 4) 1,128-bp fragment of the 16S rRNA gene using primer Fr153F0.1/Fr1281R0.1 (Fr153F0.1: 5′-GCC CAT TTG AGG GGG ATA CC-3′/Fr1281R0.1: 5′-GGA CTA AGA GTA CCT TTT TGA GT-3′) at an annealing temperature of 60 °C. Amplification products were purified using QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA) following the manufacturer’s instruction. Sequencing was performed with ABI PRISM BigDye terminator chemistry (version 3.1) according to the associated ABI protocol. DNA Sequence and Phylogenetic Analysis Sequence data were assembled using the Sequencher v5.1 software (Gene Code Corporation, Ann Arbor, MI), and the consensus sequences (tpiA = 460 bp and 16S rRNA = 1,125 bp) were used for further analyses. BLAST comparisons were performed using the nucleotide blast search page provided by NCBI for aligning the query sequences against a nonredundant nucleotide collection (nr/nt) database using the MEGABLAST program for obtaining high sequence identities (Altschul et al. 1990). Multiple sequences alignment by Muscle algorithm implemented in Molecular Evolutionary Genetics Analysis (MEGA) 6.0 software (Tamura et al. 2013) was used to align DNA sequences derived from tick samples along with reference sequences. Phylogenetic analysis by MEGA 6.0 software (Tamura et al. 2013) was performed for each gene. Maximum likelihood (ML) trees were constructed from 16S rRNA gene and tpiA gene with the best fit nucleotide substitution model for each sequence data. Bootstrap analysis with 1,000 samplings was performed to test the robustness of the tree branching. Results A total of 6,255 ticks of all life stages (adults 499 [160 males, 369 females], nymphs 2,391, and larvae 3,365) belonging to three genera and six species collected from May–August, 2013, were submitted to AFRIMS and assayed for Francisella spp. (Table 1). Haemaphysalis flava Neumann accounted for 45.9% (91 adults, 619 nymphs, 2,163 larvae) of all ticks assayed, followed by Haemaphysalis longicornis Neumann (44.0%; 314 adults, 1,691 nymphs and 747 larvae), Ixodes nipponensis Kitaoka & Saito (9.5%; 77 adults, 64 nymphs, 455 larvae), Ixodes persulcatus Schulze (0.3%, 16 adults), Amblyomma testudinarium Koch (0.3%; 1 adult, 15 nymphs), and Haemaphysalis phasiana Saito, Hoogstraal and Wassef (<0.1%; 2 nymphs). Table 1. Number of ticks collected monthly from April–August, 2013, and assayed for Francisella spp. by species and for all habitats (forests, forests + grasses, and grasses), Chungcheongnam, Chungcheongbuk, Jeollanam, and Jeollabuk provinces, and Daejeon Metropolitan city, Republic of Korea Species . Total . Larvae . Nymphs . Adults . No. of ticks tested (%)a . No. of poolsb . No. of positive poolsc . MFIRd . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . Amblyomma testudinarium 16 (0.3) 7 0 – 0 0 0 – 15 (0.6) 6 0 – 1 (0.2) 1 0 – Haemaphysalis flava 2,873 (45.9) 352 1 0.03 2,163 (64.3) 79 0 – 619 (25.9) 182 1 0.16 91 (18.2) 91 0 – Haemaphysalis longicornis 2,752 (44.0) 747 0 – 747 (22.2) 33 0 – 1,691 (70.7) 400 0 – 314 (62.9) 314 0 – Haemaphysalis phasiana 2 (<0.1) 2 1 50.00 0 0 0 – 2 (<0.1) 2 1 50.00 0 0 0 – Ixodes nipponensis 596 (9.5) 135 0 – 455 (13.5) 22 0 – 64 (2.7) 36 0 – 77 (15.4) 77 0 – Ixodes persulcatus 16 (0.3) 16 0 – 0 0 0 – 0 0 0 – 16 (3.2) 16 0 – TOTAL 6,255 1,259 2 0.03 3,365 134 0 – 2,391 626 2 0.08 499 499 0 – Species . Total . Larvae . Nymphs . Adults . No. of ticks tested (%)a . No. of poolsb . No. of positive poolsc . MFIRd . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . Amblyomma testudinarium 16 (0.3) 7 0 – 0 0 0 – 15 (0.6) 6 0 – 1 (0.2) 1 0 – Haemaphysalis flava 2,873 (45.9) 352 1 0.03 2,163 (64.3) 79 0 – 619 (25.9) 182 1 0.16 91 (18.2) 91 0 – Haemaphysalis longicornis 2,752 (44.0) 747 0 – 747 (22.2) 33 0 – 1,691 (70.7) 400 0 – 314 (62.9) 314 0 – Haemaphysalis phasiana 2 (<0.1) 2 1 50.00 0 0 0 – 2 (<0.1) 2 1 50.00 0 0 0 – Ixodes nipponensis 596 (9.5) 135 0 – 455 (13.5) 22 0 – 64 (2.7) 36 0 – 77 (15.4) 77 0 – Ixodes persulcatus 16 (0.3) 16 0 – 0 0 0 – 0 0 0 – 16 (3.2) 16 0 – TOTAL 6,255 1,259 2 0.03 3,365 134 0 – 2,391 626 2 0.08 499 499 0 – a Numbers of ticks assayed for F. tularensis, by species and stage of development. b Number of pools of ticks (1, adult; 1–5 nymphs; 1–50 larvae) assayed for F. tularensis, by species and stage of development. c Number of pools positive for F. tularensis using the fopA real-time PCR assay primer set. d MFIR, Minimum field infection rate = (Total number of positive pools/Total number of ticks) × 100. Open in new tab Table 1. Number of ticks collected monthly from April–August, 2013, and assayed for Francisella spp. by species and for all habitats (forests, forests + grasses, and grasses), Chungcheongnam, Chungcheongbuk, Jeollanam, and Jeollabuk provinces, and Daejeon Metropolitan city, Republic of Korea Species . Total . Larvae . Nymphs . Adults . No. of ticks tested (%)a . No. of poolsb . No. of positive poolsc . MFIRd . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . Amblyomma testudinarium 16 (0.3) 7 0 – 0 0 0 – 15 (0.6) 6 0 – 1 (0.2) 1 0 – Haemaphysalis flava 2,873 (45.9) 352 1 0.03 2,163 (64.3) 79 0 – 619 (25.9) 182 1 0.16 91 (18.2) 91 0 – Haemaphysalis longicornis 2,752 (44.0) 747 0 – 747 (22.2) 33 0 – 1,691 (70.7) 400 0 – 314 (62.9) 314 0 – Haemaphysalis phasiana 2 (<0.1) 2 1 50.00 0 0 0 – 2 (<0.1) 2 1 50.00 0 0 0 – Ixodes nipponensis 596 (9.5) 135 0 – 455 (13.5) 22 0 – 64 (2.7) 36 0 – 77 (15.4) 77 0 – Ixodes persulcatus 16 (0.3) 16 0 – 0 0 0 – 0 0 0 – 16 (3.2) 16 0 – TOTAL 6,255 1,259 2 0.03 3,365 134 0 – 2,391 626 2 0.08 499 499 0 – Species . Total . Larvae . Nymphs . Adults . No. of ticks tested (%)a . No. of poolsb . No. of positive poolsc . MFIRd . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . No. of ticks tested (%) . No. of pools . No. of positive pools . MFIR . Amblyomma testudinarium 16 (0.3) 7 0 – 0 0 0 – 15 (0.6) 6 0 – 1 (0.2) 1 0 – Haemaphysalis flava 2,873 (45.9) 352 1 0.03 2,163 (64.3) 79 0 – 619 (25.9) 182 1 0.16 91 (18.2) 91 0 – Haemaphysalis longicornis 2,752 (44.0) 747 0 – 747 (22.2) 33 0 – 1,691 (70.7) 400 0 – 314 (62.9) 314 0 – Haemaphysalis phasiana 2 (<0.1) 2 1 50.00 0 0 0 – 2 (<0.1) 2 1 50.00 0 0 0 – Ixodes nipponensis 596 (9.5) 135 0 – 455 (13.5) 22 0 – 64 (2.7) 36 0 – 77 (15.4) 77 0 – Ixodes persulcatus 16 (0.3) 16 0 – 0 0 0 – 0 0 0 – 16 (3.2) 16 0 – TOTAL 6,255 1,259 2 0.03 3,365 134 0 – 2,391 626 2 0.08 499 499 0 – a Numbers of ticks assayed for F. tularensis, by species and stage of development. b Number of pools of ticks (1, adult; 1–5 nymphs; 1–50 larvae) assayed for F. tularensis, by species and stage of development. c Number of pools positive for F. tularensis using the fopA real-time PCR assay primer set. d MFIR, Minimum field infection rate = (Total number of positive pools/Total number of ticks) × 100. Open in new tab Francisella Spp. Detection and Analysis When extracted DNA samples from the tick pools were tested, neither of the two positive samples of H. flava or H. phasiana was positive for both F. tularensis fopA and tul4 multitarget real-time PCR assays. One pool each of H. flava (5 nymphs) and H. phasiana (1 nymph) was positive by the fopA real-time PCR assay and negative by the tul4 real-time PCR assay (Table 1). Both pools were also negative by conventional F. tularensis PCR assays utilizing two different sets of primers (FT393/FT642 and TUL4B-F/TUL4B-R) for amplification of the tul4 gene. PCR products were amplified from both tick pools by conventional PCR using Francisella spp. primers for the tpiA gene. Additionally, a fragment of the 16S rRNA gene was amplified from one pool (H. flava) using Francisella spp. primers. Sequencing and BLAST comparisons of the tpiA nucleotide sequences amplified from the two pools showed the greatest similarity to the Francisella persica tpiA gene (AY794497; Larson et al. 2016). The alignment and nucleotide identity of the tpiA gene showed that the sequences in the two tick pools displayed 100% nucleotide identity with each other and 94.7% nucleotide identity to F. persica, followed by 88.6% identity to the F. tularensis Group and 79.5% identity to other Francisella species (Fig. 2). Phylogenetic analysis of the tpiA sequences showed that the detected agent clustered in a clade with the FLE, F. persica, and was distinct from strains in the F. tularensis subspecies group and other Francisella species (Fig. 2). Fig. 2. Open in new tabDownload slide The phylogenetic tree of tpiA gene constructed by the ML method based on the Tamura 3-parameter (T92 + G) model. GenBank accession numbers are noted after each sequence. Only bootstrap values of 70% or greater are shown. Scale bar represents substitutions per site. Fig. 2. Open in new tabDownload slide The phylogenetic tree of tpiA gene constructed by the ML method based on the Tamura 3-parameter (T92 + G) model. GenBank accession numbers are noted after each sequence. Only bootstrap values of 70% or greater are shown. Scale bar represents substitutions per site. BLAST comparison of the 16S rRNA gene sequence amplified from the H. flava pool showed the highest sequence similarity to the FLEs in Dermacentor auratus (JQ764629) from Thailand and Ornithodoros moubata B (AB001522) from Japan (99.4% identity). The nucleotide sequence identities ranged from 98.4–98.9% and 97.1–97.4% for the 16S rRNA sequence from H. flava to F. tularensis subspecies group and to other Francisella species, respectively. The phylogenetic analysis (Fig. 3) showed that the sequence grouped into the FLE group and was distinctively separated from F. tularensis subspecies and other Francisella species. Fig. 3. Open in new tabDownload slide The phylogenetic analysis of the 16S rRNA gene of Francisella spp. constructed using the ML method based on the Kimura 2-parameter (K2 + G) model. Only bootstrap values of 70% or greater are shown. Scale bar represents substitutions per site. Fig. 3. Open in new tabDownload slide The phylogenetic analysis of the 16S rRNA gene of Francisella spp. constructed using the ML method based on the Kimura 2-parameter (K2 + G) model. Only bootstrap values of 70% or greater are shown. Scale bar represents substitutions per site. Discussion As a result of increased interest in tick-borne diseases in the ROK, the 65th Medical Brigade conducted tick-borne disease surveillance to determine potential tick-borne disease threats to U.S. military populations associated with military operations and recreational activities in the southwestern part of the ROK. The primary purpose was to identify the prevalence of tick-borne pathogens (e.g., F. tularensis, SFTS virus, and spotted fever group Rickettsia). While there was one human case of tularemia in a patient associated with skinning and consuming a dead wild rabbit, there have not been any additional reported cases and no previously reported detection of F. tularensis in ticks in the ROK. Similarly, in this study, F. tularensis was not detected in any of the tested ticks from the southwestern provinces of the ROK. There are a number of possibilities for this finding including that the distribution of F. tularensis may be focal (i.e., the ticks tested were collected in different provinces from where the human case was reported), the primary tick species collected (H. longicornis, H. flava, and I. nipponensis) may not play a role in transmission of F. tularensis, or maintenance of F. tularensis in the environment either does not involve arthropods or involves other tick species or arthropod vectors. Further work is needed to understand the ecology of F. tularensis in the ROK and whether tick vectors play a role. A Francisella-like endosymbiont in H. flava and H. phasiana was reported for the first time, adding onto the growing list of FLEs reported in other tick genera throughout the world (de Carvalho et al. 2011, Ivanov et al. 2011, Larson et al. 2016). The FLE-positive tick pools were positive only by the fopA assay and not by the tul4 PCR. This result is not surprising since it has been described previously that fopA PCR, but not the tul4 PCR, could detect bacteria closely related to F. tularensis, including tick endosymbionts (Barns et al. 2005, Michelet et al. 2013). By 16S rRNA gene sequencing and phylogenetic analysis, the agent detected in the H. flava ticks was confirmed as a Francisella-like endosymbiont. The results from tpiA gene sequences were consistent with this finding, as the FLE, F. persica, was shown to be the most closely related Francisella spp. (94.7% identity) to the FLE detected in the Haemaphysalis ticks from the ROK. Francisella-like endosymbionts are not considered to be pathogenic, as their genomes are reduced as compared to F. tularensis. Their presence as symbionts in a number of different tick genera is believed to provide a fitness advantage to the tick. In a recent study of the FLE in Haemaphysalis doenitzi, the presence of the symbiont was associated with ovary development (Liu et al. 2016). However, F. tularensis is a considered a biological threat agent and has been detected in some of the species tested here, including H. flava and I. persulcatus (Suzuki et al. 2016). Thus, the identification of FLEs is necessary so as not to lead to false assumptions regarding F. tularensis. Haemaphysalis flava is commonly associated with forest habitats and where forests interface with grasses (e.g., gravesites and agriculture) are uncommonly found on small rodents and birds. Haemaphysalis longicornis is collected mostly from open grasses and herbaceous vegetation and is uncommonly collected on birds and has never been collected from >10,000 small mammals (rodents and shrews) during hantavirus surveys in the ROK, but is commonly found on larger animals, e.g., deer and domestic cattle and horses (Kim et al. 2013b, 2014b, Kang et al. 2016). Ixodes nipponensis larvae and some nymphs are frequently collected from small mammals (rodents and shrews), with the nymphs and adults feeding on larger animals. There are few reports, if any, for ticks found on rabbits, but it might be assumed that all three species feed on rabbits during some stage of their life cycle. As families tend to their crops are exposed to bites from all developmental stages of ticks, primarily H. longicornis and H. flava. Similarly, during Chuseok, family members that traverse forest habitats to attend gravesites are exposed to questing H. flava, I. nipponensis, H. longicornis, I. persulcatus, H. phasiana, and A. testudinarium ticks and their associated pathogens (e.g., SFG rickettsiae, SFTS and TBE viruses, and Ehrlichia, Borrelia, Bartonella, Anaplasma, and Babesia spp.) that they are known to harbor in the ROK. Despite habitat modification of gravesites (caretaking) and narrow to broad trails connecting adjoining gravesites, H. longicornis larvae and H. flava and I. nipponensis nymphs and adults were frequently collected the week following Chuseok. Haemaphysalis longicornis, H. flava, I. nipponensis, I. persulcatus, and A. testudinarium have been reported to bite man in the ROK (Cho et al. 1995; Ryu et al. 1998; Chae et al. 2000; Ko et al. 2002; Suh et al. 2008; Kim et al. 2010, 2014a). There have been more reports of I. nipponensis and A. testudinarium bites, perhaps due to dermatological reactions and a broader range of hosts and higher propensity to bite man, even though fewer numbers were collected. From 2013–2015, there were 413 (annual mean 137.7) tick bites reported by Korea Centers for Disease Control and Prevention (KCDC) (Yang et al. 2016), with many unreported based on the low estimated SFTS infection rate of <1% in ticks and the increased annual numbers of SFTS infections reported from 2013–2016 (Yun et al. 2014, KCDC 2016). In summary, tick-borne disease risks are underestimated, as tick bites often go unreported, based on the number of tick bites reported annually and the number of SFTS virus cases reported from 2013–2016 (35, 55, 79, and 159 cases, respectively) (KCDC 2016). In addition, only selected tick-borne diseases (Lyme disease and tick-borne encephalitis [TBE], and SFTS viruses) are reportable events by KCDC, limiting our knowledge of the prevalence of many tick-borne diseases present in the ROK. Therefore, comprehensive tick-borne disease surveillance programs should be established to better understand the geographical, seasonal, habitat distributions, and life histories of ticks and the prevalence of associated pathogens (e.g., Rickettsia, Ehrlichia, Anaplasma, Borrelia, Bartonella, Francisella, and Babesia spp. and TBE and SFTS viruses). In addition, pathogen discovery should be employed to identify previously unknown or new records of pathogens that affect veterinary and human health in the ROK. In addition, it is important to accurately distinguish pathogenic from nonpathogenic bacteria, viruses, and protozoa to avoid misidentification and confusion, especially when performing disease threat analyses. In conclusion, a Francisella-like endosymbiont was detected in two Haemaphysalis spp. collected from vegetation by tick drag surveillance in the ROK. These results aid our understanding of the epidemiology of a Francisella-like endosymbiont and emphasize the need for continuous tick-based pathogen surveillance to monitor the geographical distribution, presence, and emergence of tick-borne diseases in the ROK. Additional studies, including larger scale surveys of ticks collected from vegetation, domestic and wild animals, and rodent-borne disease surveys, to identify potential zoonotic hosts and transovarial and transmission studies should be conducted to determine the prevalence of tick-borne pathogens and the risks they pose to human populations in the ROK. Acknowledgments We thank CPT Theodore Snyder, MAJ Lewis Long, and MAJ Richard McNemee, United States Army Public Health Command Region-Pacific (USAPHCR-PAC), and COL Hee-Choon (Sam) Lee and Mr. Larry Pazyra, 65th Medical Brigade (MED BDE), for their support. We also thank MAJ Yeun-Dae Kim, ROK Army Staff Officer, and Korean Augmentation to the U.S. Army (KATUSA) soldiers (Jae-Kyung Lee, Dong-Sam Kam, and Hyo-Geon Lee) of the 5th and 154th Medical Detachments (MED DET), 168th Multifunctional Medical Battalion (MMB), and 65th MED BDE, for their support and assistance with collection efforts. We also thank Suk-Hee Yi, Force Health Protection and Preventive Medicine (FHP&PM), 65th MED BDE, for assisting with statistical analysis and graphics. Funding for this work was provided by the Armed Forces Health Surveillance Branch, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), Silver Spring, Maryland, USA, the 65th Medical Brigade, Yongsan U.S. Army Garrison, Seoul, Korea, and the Public Health Command Region-Pacific, Camp Zama, Japan. The opinions expressed herein are those of the authors and are not to be construed as official or reflecting the views of the U.S. Department of the Army, Department of Defense, Centers for Disease Control and Prevention or the U.S. Government. References Ahn W. S. , Oh M. G., Lee J. H. 1999 . A case of ulceroglandular tularemia . J. Korea Surg. Soc . 57 : 304 – 310 . OpenURL Placeholder Text WorldCat Altschul S. F. , Gish W., Miller W., Myers E. W., Lipman D. J. 1990 . Basic local alignment search tool . J. Mol. Biol . 215 : 403 – 410 . Google Scholar Crossref Search ADS PubMed WorldCat Barns S. M. , Grow C. C., Okinaka R. T., Keim P., Kuske C. R. 2005 . Detection of diverse new Francisella-like bacteria in environmental samples . Appl. Environ. Microbiol . 71 : 5494 – 5500 . Google Scholar Crossref Search ADS PubMed WorldCat Centers for Disease Control and Prevention 2002 . Tularemia-United States, 1990-2000 . MMWR 51 : 181 – 184 . PubMed OpenURL Placeholder Text WorldCat Chae K. S. , Gang H., Lee D. W., Byun D. G., Cho B. K., Park C. W., Suh J. K., Lee K. B., Kim H. J. 2000 . Tick bites . Korean J. Dermatol . 38 : 111 – 116 . OpenURL Placeholder Text WorldCat Chae J. S. , Kim C. M., Kim E. H., Hur E. J., Klein T. A., Kang T. K., Lee H. C., Song J. W. 2003 . Molecular epidemiological study for tick-borne disease (Ehrlichia and Anaplasma spp.) surveillance at selected U.S. military training sites/installations in Korea . Ann. N.Y. Acad. Sci . 990 : 118 – 125 . Google Scholar Crossref Search ADS PubMed WorldCat Chae J. S. , Yu D. H., Shringi S., Klein T. A., Kim H. C., Chong S. T., Lee I. Y., Foley J. 2008 . Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, Korea . J. Vet. Sci . 9 : 285 – 293 . Google Scholar Crossref Search ADS PubMed WorldCat Cho S. N. , Lee T. Y., Lee M. K., Kim D. S., Kim J. D. 1991 . Immunoblotting analysis of antibodies against Borrelia burgdorferi, the Lyme disease agent, in sera from the Korean residents . J. Korean Soc. Microbiol . 26 : 263 – 272 . OpenURL Placeholder Text WorldCat Cho B. K. , Na H. W., Cho S. Y., Lee W. K. 1995 . A case of tick bite by a spontaneously retreated Ixodes nipponensis . Korean J. Parasitol . 33 : 239 – 242 . Google Scholar Crossref Search ADS PubMed WorldCat Choi Y. J. , Jang W. J., Kim J. H., Ryu J. S., Lee S. H., Park K. H., Paik H. S., Koh Y. S., Choi M. S., Kim I. S. 2005 . Spotted fever group and typhus group rickettsioses in humans, South Korea . Emerg. Infect. Dis . 11 : 237 – 244 . Google Scholar Crossref Search ADS PubMed WorldCat Chong S. T. , Kim H. C., Lee I. Y., Kollars T. M. Jr., Sancho A. R., Sames W. J., Klein T. A. 2013a . Comparison of dragging and sweeping methods for collecting ticks and determining their seasonal distributions for various habitats, Gyeonggi Province, Republic of Korea . J. Med. Entomol . 50 : 611 – 618 . Google Scholar Crossref Search ADS WorldCat Chong S. T. , Kim H. C., Lee I. Y., Kollars T. M. Jr., Sancho A. R., Sames W. J., Klein T. A. 2013b . Seasonal distribution of ticks in four habitats near the demilitarized zone, Gyeonggi-do (Province), Republic of Korea . Korean J. Parasitol . 51 : 319 – 325 . Google Scholar Crossref Search ADS WorldCat Coburn J. M. , Chong S. T., Kim H. C., Chang N. W., Calix L. C., Resto K., Lee D. J., Johnson J. L., Robbins R. G., Klein T. A. 2016 . Tick surveillance in four southwestern provinces of the Republic of Korea during 2013 . Syst. Applied Acarol . 21 : 147 – 165 . OpenURL Placeholder Text WorldCat de Carvalho I. L. , Santos N., Soares T., Zé-Zé L., Núncio M. S. 2011 . Francisella-like endosymbiont in Dermacentor reticulatus collected in Portugal . Vector-Borne Zoonotic Dis . 11 : 185 – 188 . Google Scholar Crossref Search ADS PubMed WorldCat Dennis D. T. , Inglesby T. V., Henderson D. A., Bartlett J. G., Ascher M. S., Eitzen E., Fine A. D., Friedlander A. M., Hauer J., Layton M., et al. 2001 . Tularemia as a biological weapon: Medical and public health management . JAMA 285 : 2763 – 2773 . Google Scholar Crossref Search ADS PubMed WorldCat Federal Register 2012 . Possession, use, and transfer of select agents and toxins 61083-61115. (https://federalregister.gov/a/2012-24389) (accessed 25 March 2017). Heo E. J. , Park J. H., Koo J. R., Park M. S., Park M. Y., Dumler J. S., Chae J. S. 2002 . Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients . J. Clin. Microbiol . 40 : 3082 – 3085 . Google Scholar Crossref Search ADS PubMed WorldCat Ivanov I. , Mitkova N. N., Reye A. L., Hubschen J. M., Vatcheva-Dobrevska R. S., Dobreva E. G., Kantardjiev T. V., Muller C. P. 2011 . Detection of new Francisella-like tick endosymbionts in Hyalomma spp. and Rhipicephalus spp. (Acari: Ixodidae) from Bulgaria . Appl. Environ. Microbiol . 77 : 5562 – 5565 . Google Scholar Crossref Search ADS PubMed WorldCat Jang W. J. , Kim J. H., Choi Y. J., Jung K. D., Kim Y. G., Lee S. H., Choi M. S., Kim I. S., Walker D. H., Park K. H. 2004 . First serologic evidence of human spotted fever group rickettsiosis in Korea . J. Clin. Microbiol . 42 : 2310 – 2313 . Google Scholar Crossref Search ADS PubMed WorldCat Kang J. G. , Kim H. C., Choi C. Y., Nam H. Y., Chae H. Y., Chong S. T., Klein T. A., Ko S., Chae J. S. 2013 . Molecular detection of Anaplasma, Bartonella, and Borrelia species in ticks collected from migratory birds from Hong-do Island, Republic of Korea . Vector-Borne Zoonotic Dis . 13 : 215 – 225 . Google Scholar Crossref Search ADS PubMed WorldCat Kang J. G. , Ko S., Kim H. C., Chong S. T., Klein T. A., Chae J. B., Jo Y. S., Choi K. S., Yu D. H., Park B. K., et al. 2016 . Prevalence of Anaplasma and Bartonella spp. in ticks collected from Korean water deer (Hydropotes inermis argyropus) . Korean J. Parasitol . 54 : 87 – 91 . Google Scholar Crossref Search ADS PubMed WorldCat (KCDC) Korea Centers for Disease Control and Prevention 2016 . Current status of selected infectious diseases . Public Health Wkly. Rep. (PHWR) 9 : 1084 – 1091 . OpenURL Placeholder Text WorldCat Kee S. , Hwang K. J., Oh H. B., Park K. S. 1994 . Molecular Identification of Borrelia burgdorferi isolated in Korea using outer surface protein A (Osp A) serotyping system . Microbiol. Immunol . 38 : 989 – 993 . Google Scholar Crossref Search ADS PubMed WorldCat Kim C. M. , Kim M. S., Park M. S., Park J. H., Chae J. S. 2003 . Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea . Vector-Borne Zoonotic Dis . 3 : 17 – 26 . Google Scholar Crossref Search ADS PubMed WorldCat Kim C. M. , Kim J. Y., Yi Y. H., Lee M. J., Cho M. R., Shah D. H., Klein T. A., Kim H. C., Song J. W., Chong S. T., et al. 2005 . Detection of Bartonella species from ticks, mites and small mammals in Korea . J. Vet. Sci . 6 : 327 – 334 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Kim S. Y. , Jeong Y. E., Yun S. M., Lee I. Y., Han M. G., Ju Y. R. 2009 . Molecular evidence for tick-borne encephalitis virus in ticks in South Korea . Med. Vet. Entomol . 23 : 15 – 20 . Google Scholar Crossref Search ADS PubMed WorldCat Kim J. , Joo H. S., Moon H. J., Lee Y. J. 2010 . A case of Amblyomma testudinarium tick bite in a Korean woman . Korean J. Parasitol . 48 : 313 – 317 . Google Scholar Crossref Search ADS PubMed WorldCat Kim H. C. , Chong S. T., Nunn P. V., Jang W. J., Klein T. A., Robbins R. G. 2013a . Seasonal abundance of ticks collected from live-captured small mammals in Gyeonggi province, Republic of Korea, during 2009 . Syst. Appl. Acarol . 18 : 201 – 211 . Google Scholar Crossref Search ADS WorldCat Kim K. H. , Yi J. Y., Kim G. Y., Choi S. J., Jun K. I., Kim N. H., Choe P. G., Kim N. J., Lee J. K., Oh M. D. 2013b . Severe fever with thrombocytopenia syndrome, South Korea, 2012 . Emerg. Infect. Dis . 19 : 1892 – 1894 . Google Scholar Crossref Search ADS WorldCat Kim B. J. , Kim H., Won S., Kim H. C., Chong S. T., Klein T. A., Kim K. G., Seo H. Y., Chae J. S. 2014a . Ticks collected from wild and domestic animals and natural habitats in the Republic of Korea . Korean J. Parasitol . 52 : 281 – 285 . Google Scholar Crossref Search ADS WorldCat Kim J. , Kang H. A., Kim S. S., Joo H. S., Chong W. S. 2014b . Perianal tick-bite lesion caused by a fully engorged female Amblyomma testudinarium . Korean J. Parasitol . 52 : 685 – 690 . Google Scholar Crossref Search ADS WorldCat Ko J. H. , Cho D. Y., Chung B. S., Kim S. I. 2002 . Two human cases of tick bite caused by Ixodes nipponensis . Korean J. Parasitol . 40 : 199 – 203 . Google Scholar Crossref Search ADS PubMed WorldCat Ko S. , Kang J. G., Kim S. Y., Kim H. C., Klein T. A., Chong S. T., Sames W. J., Yun S. M., Ju Y. R., Chae J. S. 2010 . Prevalence of tick-borne encephalitis virus in ticks from southern Korea . J. Vet. Sci . 11 : 197 – 203 . Google Scholar Crossref Search ADS PubMed WorldCat Larson M. A. , Nalbantoglu U., Sayood K., Zentz E. B., Cer R. Z., Iwen P. C., Francesconi S. C., Bishop-Lilly K. A., Mokashi V. B., Sjöstedt A., Hinrichs S. H. 2016 . Reclassification of Wolbachia persica as Francisella persica comb. nov. and emended description of the family Francisellaceae . Int. J. Syst. Evol. Microbiol . 66 : 1200 – 1205 . Google Scholar Crossref Search ADS PubMed WorldCat Lee J. H. , Park H. S., Jung K. D., Jang W. J., Koh S. E., Kang S. S., Lee I. Y., Lee W. J., Kim B. J., Kook Y. H., et al. 2003 . Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea . Microbiol. Immunol . 47 : 301 – 304 . Google Scholar Crossref Search ADS PubMed WorldCat Lee K., M. , Choi Y. J., Shin S. H., Choi M. K., Song H. J., Kim H. C., Klein T. A., Richards A. L., Park K. H., Jang W. J. 2013 . Spotted fever group Rickettsia closely related to Rickettsia monacensis isolated from ticks in South Jeolla province, Korea . Microbiol. Immunol . 57 : 487 – 495 . Google Scholar Crossref Search ADS PubMed WorldCat Lim H. S. , Cheong H. K., Ahn W. S., Kim M. Y., Kim D. H. 1998 . A case of ulceroglandular tularemia occurred in Korea . Korean J. Epidemiol . 20 : 32 – 38 . OpenURL Placeholder Text WorldCat Liu J. N. , Yu Z. J., Liu L. M., Li N. X., Wang R. R., Zhang C. M., Liu J. Z. 2016 . Identification, distribution, and population dynamics of Francisella-like endosymbiont in Haemaphysalis doenitzi (Acari: Ixodidae) . Sci. Rep . 6 : 35178 . Google Scholar Crossref Search ADS PubMed WorldCat Long G. W. , Oprandy J. J., Narayanan R. B., Fortier A. H., Porter K. P., Nacy C. A. 1993 . Detection of Francisella tularensis in blood by polymerase chain reaction . J. Clin. Microbiol . 31 : 152 – 154 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Michelet L. , Bonnet S., Madani N., Moutailler S. 2013 . Discriminating Francisella tularensis and Francisella-like endosymbionts in Dermacentor reticulatus ticks: evaluation of current molecular techniques . Vet. Microbiol . 163 : 399 – 403 . Google Scholar Crossref Search ADS PubMed WorldCat Park K. H. , Lee S. H., Won W. J., Jang W. J., Chang W. H. 1992 . Isolation of Borrelia burgdorferi, the causative agent of Lyme disease, from Ixodes ticks in Korea . J. Korean Soc. Microbiol . 27 : 307 – 312 . OpenURL Placeholder Text WorldCat Park S. W. , Song B. G., Shin E. H., Yun S. M., Han M. G., Park M. Y., Park C., Ryou J. 2014a . Prevalence of severe fever with thrombocytopenia syndrome virus in Haemaphysalis longicornis ticks in South Korea . Ticks Tick-Borne Dis . 5 : 975 – 977 . Google Scholar Crossref Search ADS WorldCat Park S. W. , Han M. G., Yun S. M., Park C., Lee W. J., Ryou J. 2014b . Severe fever with thrombocytopenia syndrome virus, South Korea, 2013 . Emerg. Infect. Dis . 20 : 1880 – 1882 . Google Scholar Crossref Search ADS WorldCat Ryu J. S. , Lee J. V., Ahn M. H., Min D. Y., Ree H. I. 1998 . A human case of tick bite by Ixodes nipponensis . Korean J. Parasitol . 36 : 59 – 61 . Google Scholar Crossref Search ADS PubMed WorldCat Sachar D. S. 2000 . Ehrlichiosis chaffeensis infection in an active duty soldier, Korea . MSMR . 6 : 9 – 11 . OpenURL Placeholder Text WorldCat Saito Y. , Hoogstraal H., Wassef H. Y. 1974 . The Haemaphysalis ticks (Ixodoidea: Ixodidae) of birds. 4. H. (Ornithophysalis) phasiana sp. n. from Japan . J. Parasitol . 60 : 198 – 208 . Google Scholar Crossref Search ADS PubMed WorldCat Scoles G. A. 2004 . Phylogenetic analysis of the Francisella-like endosymbionts of Dermacentor ticks . J. Med. Entomol . 41 : 277 – 286 . Google Scholar Crossref Search ADS PubMed WorldCat Shim J. C. , Yoon Y. H., Kim C. L., Cho Y. B., Lee J. Y., Shin E. H., Yang Y. C., Baik M. K., Yu W. H., Yu H. S., et al. 1993 . Studies on vector potential of ticks (Ixodidae) in transmitting of Lyme disease (Borrelia burgdorferi) (II) - Vector incrimination and seasonal occurrence of Ixodes granulatus . Rep. Nat. Inst. Health 30 : 131 – 136 . OpenURL Placeholder Text WorldCat Shin S. H. , Seo H. J., Choi Y. J., Choi M. K., Kim H. C., Klein T. A., Chong S. T., Richards A. L., Park K. Y., Jang W. J. 2013 . Detection of Rickettsia monacensis from I. nipponensis collected from rodents in Gyeonggi and Gangwon provinces, Republic of Korea . Exp. Appl. Acarol . 61 : 337 – 347 . Google Scholar Crossref Search ADS PubMed WorldCat Sjostedt A. , Sandstrom G., Tarnvik A. 1990 . Several membrane polypeptides of the live vaccine strain Francisella tularensis LVS stimulate T cells from naturally infected individuals . J. Clin. Mimicrobiol . 28 : 43 – 48 . OpenURL Placeholder Text WorldCat Sjostedt A. 2007 . Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations . Ann. N.Y. Acad. Sci . 1105 : 1 – 29 . Google Scholar Crossref Search ADS PubMed WorldCat Suh K. S. , Park J. B., Han S. H., Lee I. Y., Cho B. K., Kim S. T., Jang M. S. 2008 . Tick bite on glans penis: the role of dermoscopy . Ann. Dermatol . 25 : 528 – 530 . Google Scholar Crossref Search ADS WorldCat Sun L. V. , Scoles G. A., Fish D., O'Neill S. L. 2000 . Francisella-like endosymbionts of ticks . J. Invertebr. Pathol . 76 : 301 – 303 . Google Scholar Crossref Search ADS PubMed WorldCat Suzuki J. , Hashino M., Matsumoto S., Takano A., Kawabata H., Takada N., Andoh M., Oikawa Y., Kajita H., Uda A., et al. 2016 . Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan . Lett. Appl. Microbiol . 63 : 240 – 246 . Google Scholar Crossref Search ADS PubMed WorldCat Tamura K. , Stecher G., Peterson D., Filipski A., Kumar S. 2013 . MEGA6: Molecular evolutionary genetics analysis version 6.0 . Mol. Biol. Evol . 30 : 2725 – 2729 . Google Scholar Crossref Search ADS PubMed WorldCat Uchida T. , Yan Y., Kitaoka S. 1995 . Detection of Rickettsia japonica in Haemaphysalis longicornis ticks by restriction fragment length polymorphism of PCR product . J. Clin. Microbiol . 33 : 824 – 828 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Versage J. L. , Severin D. D., Chu M. C., Petersen J. M. 2003 . Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens . J. Clin. Microbiol . 41 : 5492 – 5499 . Google Scholar Crossref Search ADS PubMed WorldCat Yamaguti N. , Tipton V. J., Keegan H. L., Toshioka S. 1971 . Ticks of Japan, Korea, and the Ryukyu islands . Brigham Young Univ. Sci. Bull. Biol. Ser . 15 : 1 – 226 . OpenURL Placeholder Text WorldCat Yang S. C. , Lee W. G., Ju Y. R. 2016 . Hard tick bite cases and distribution in the Republic of Korea (2013–2015) . Public Health Wkly Rep. (PHWR) 9 : 1054 – 1059 . OpenURL Placeholder Text WorldCat Yu X. J. , Liang M. F., Zhang S. Y., Liu Y., Li J. D., Sun Y. L., Zhang L., Zhang Q. F., Popov V. L., Li C., et al. 2011 . Fever with thrombocytopenia associated with a novel bunyavirus in China . N Engl. J. Med . 364 : 1523 – 1532 . Google Scholar Crossref Search ADS PubMed WorldCat Yun S.M.W.G. , Lee J., Ryou S. C., Yang S. W., Park J. Y., Roh Y. J., Lee C., Park C., Han M. G. 2014 . Severe fever with thrombocytopenia syndrome virus in ticks collected from humans, South Korea, 2013 . Emerg. Infect. Dis . 20 : 1358 – 1361 . Google Scholar Crossref Search ADS PubMed WorldCat Yun S. M. , Lee Y. J., Choi W. Y., Kim H. C., Chong S. T., Chang K. S., Coburn J. M., Klein T. A., Lee W. J. 2016 . Molecular detection of severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses in ixodid ticks collected from vegetation, Republic of Korea, 2014 . Ticks Tick-Borne Dis . 7 : 970 – 978 . Google Scholar Crossref Search ADS PubMed WorldCat Zhang Y.-Z. , Zhou D.-J., Qin X.-C., Tian J.-H., Xiong Y., Wang J.-B., Chen X.-P., Gao D.-Y., He Y.-W., Jin D., et al. 2012 . The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China . J. Virol . 86 : 2864 – 2868 . Google Scholar Crossref Search ADS PubMed WorldCat Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.
TI - Francisella-Like Endosymbiont Detected in Haemaphysalis Ticks (Acari: Ixodidae) From the Republic of Korea
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjx123
DA - 2017-11-07
UR - https://www.deepdyve.com/lp/oxford-university-press/francisella-like-endosymbiont-detected-in-haemaphysalis-ticks-acari-60WqT0vR0t
SP - 1735
VL - 54
IS - 6
DP - DeepDyve
ER -