The presence of Ixodes pavlovskyi and I. pavlovskyi–borne microorganisms in Rishiri Island: an ecological survey

ABSTRACT Rishiri Island, towering toward the Sea of Japan, has been volcanically dormant for approximately 8,000 years. This small inhabited island is free of middle- to large-sized wildlife and a crucial stopover for wild birds migrating along various routes of the East Asian Flyway. A 5-year survey was conducted to explore the biogeography of ticks and tick-borne microorganisms. By flagging vegetation, Ixodes pavlovskyi (Pomerantzev, 1948), distributed in limited spots in the Far East, was predominantly collected (60%–80% collection) throughout the survey period. The I. pavlovskyi consisted of two haplogroups, Asahikawa-type and Rishiri-type, with the prevalence and nucleotide diversity of Rishiri-type being over 90% and 0.068, respectively. A survey of wild animals revealed that red-backed voles and wild birds, including Oriental greenfinches and black-faced buntings, are their hosts for blood feeding. Furthermore, the red-backed voles were infected with tick-borne Candidatus Ehrlichia khabarensis (5/21, 24%). Till date, microorganisms with identical gene sequences have only been reported from Khabarovsk and Vancouver. Ca. E. khabarensis gene has also been detected in host-seeking adult I. pavlovskyi. These results indicated that Rishiri Island is a refuge for both I. pavlovskyi and I. pavlovskyi–borne microorganisms. Additionally, the Babesia microti US lineage, which is vectored by Ixodes persulcatus in the Far East, appears to be maintained between I. pavlovskyi and wild rodents. Various factors have influenced the unique ecosystem of the island. The historical and ecological biogeography of Rishiri Island helps us understand the origin, evolution, and expansion of ticks and associated microorganisms. IMPORTANCE Understanding the ecology of ticks and tick-borne microorganisms is important to assess the risk of emerging tick-borne diseases. Despite the fact that the Ixodes pavlovskyi tick bites humans, we lack information including population genetics and the reason for the inadequate distribution in Japan. A 5-year survey revealed that Rishiri Island, the main stopover in the East Asian Flyway of wild birds in the northern Sea of Japan, was a refuge of I. pavlovskyi. The I. pavlovskyi included two haplogroups, which were supposed to diverge a long time before the island separated from the continent and Hokkaido mainland. The detection of microorganisms from wildlife revealed that wild birds and rodents play a role in diffusion and settlement, respectively, of not only I. pavlovskyi but also I. pavlovskyi–borne microorganisms including Candidatus Ehrlichia khabarensis and Babesia microti US lineage. Various island-specific factors control I. pavlovskyi dominance and tick-borne pathogen maintenance. The results may enable us to explain how tick-borne infectious microorganisms are transported.


Field collection
From 2018 to 2022, unfed, host-seeking ticks were collected in April and May from Rishiri Island and the neighboring Rebun Island, 10 km north of Rishiri Island, by flagging vegetation on the sides of forest paths easily accessible by car and on foot (Fig. 1A).Ticks were also collected from the Hokkaido mainland.Wakkanai is located approximately 40 km northeast of Rishiri Island.The ticks in Shibetsu, Asahikawa, and Sapporo have also been investigated since I. pavlovskyi was first recorded (8,9).The ticks were initially classified morphologically, based on descriptions by Nakao et al. and Takada (8,14).Female and male ticks were kept alive separately in tightly sealed plastic tubes over a moistened filling of solidified plaster with activated charcoal at 4℃ until mating.Other ticks were frozen and stored at −30°C until DNA extraction.Tick collection efficiency was used to determine how often tick-carrying wild animals appeared at the survey site (tick/min/person) (15).Several people collected ticks from Asahikawa and Sapporo in 2022, and the collection efficiency was not calculated.Ticks collected in 2018 on Rishiri Island were originally used in Zamoto-Niikura et al. (13).
Small wild mammals were collected on Rishiri Island in 2020 and 2022 using small snap traps (Panchu PMP, HOGA, Kyoto, Japan) and Sharman live traps, respectively.The animal species were identified morphologically (shrews and rodents) and molecularly (rodents) based on the cytochrome b gene (cyt-b) sequence (16).Ticks feeding on wildlife were manually removed.The ticks almost engorged were reared in the laboratory at 20°C to hatch or lay eggs.DNA was extracted from the resulting ticks and used for the detection of microorganisms by PCR as described below (transstadial transmission).The permissions required for the survey were obtained under the Wildlife Protection, Control, and Hunting Management Act.The ticks in road-killed animals including birds, weasels, and cats were also investigated.

DNA extraction from ticks and rodents
DNA was extracted from ticks by a standard protocol using the phenol/ethanol method (15).The spleens of the wild rodent were shred to 100 mg pieces and homogenized in a tube containing a ceramic ball (Ceramic Sphere, MP Biomedicals; catalog no.6540412), beads (Garnet Matrix A Bulk, MP Biomedicals; catalog no.6540-427), and TNE buffer ], 100 mM NaCl, and 0.1 mM EDTA) by shaking at 4,000 rpm for 30 s (Micro Smash MS-100, TOMY).Proteinase K (0.1 mg/mL) and SDS (0.1%) were added and incubated at 55°C overnight.DNA was extracted using the standard phenol/ethanol method, dissolved in 200 µL TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), and stored at −30°C.

Establishment of a PCR-restriction fragment length polymorphism (RFLP) system for live tick identification
To distinguish live I. pavlovskyi from I. persulcatus and I. ovatus, we developed a PCR-RFLP method based on hints from the hemolymph test (17,18)."Leg II" or "leg III" of one side was amputated using ophthalmic scissors and directly added to the PCR mixture as the template (Fig. 2).All ticks with an amputated leg were reared in moistened tubes at 4°C.All ticks were alive (data not shown).PCR was performed using the Phusion Blood Direct PCR Kit (Thermo Fisher Scientific; catalog no.F547S).The thermal cycling program was set to the annealing temperature at 55°C and extension time of 1 min.The primer set, Ixodes COI F1/Ixodes COI R1 (Supplement S2), was designed to amplify the mitochondrial COI sequence of various Ixodes spp.collected from the Hokkaido mainland and Rishiri Island.Sequence diversity among Ixodes spp. was used to design an RFLP assay (Table 1).

Molecular identification of wild rodents
The mammalian Cyt-b gene (16) (Supplement S2) was amplified from spleen DNA by using Ex Taq (Takara; catalog no.RR001A) according to the manufacturer's instructions.The PCR amplicons were directly sequenced using the primers used for amplification (Eurofins Genetics, Tokyo, Japan).

Population genetics
The COI sequence-based haplotypes (partial 701 bp) were determined using DnaSP version 6.12.03 (http://www.ub.edu/dnasp/), and the network was constructed using the median-joining method in the NETWORK 10.2.0.0 program (https://www.fluxus-engineering.com/).In total, 104 sequences from I. pavlovskyi, 84 sequences from I. persulca tus, and a COI sequence from I. pavlovskyi from Tomsk (accession number JX288763)  were used.The fixation index (FST) and other values related to genetic diversity were calculated using DnaSP version 6.12.03.

Cross-breeding of ticks
Male and female ticks were kept in different containers immediately after collection.After the species and haplogroups were identified, designated males and females were kept in a clear-colored tube at room temperature (20-30°C), and mating was visually confirmed.After the male detached from the female, the female was allowed to feed on a gerbil.Engorged females were reared at 20°C until egg laying.The eggs were aliquoted to small glass tubes and incubated at 20°C.The larvae hatched from the eggs approximately 1 month later.If the larvae did not hatch from the egg, they were kept in a container for 6 months for extended observation.

Whole mitochondrial genome sequencing of I. pavlovskyi
The full-length mitochondrial genome sequence of both haplogroups was determined using a combination of long PCR and Illumina short-read sequencing.Genomes were extracted from eggs laid by I. pavlovskyi females and used as templates.Pri mers were designed to anneal the conserved region of the mitochondrial genome of Ixodes (Supplement S2).Two PCR products were obtained using the primer sets IxoMt_18F/IxoMt_9356R or IxoMt_8326F/IxoMt_3458R and Gflex polymerase (Takara).Both fragments were used for library construction (TruSeq Nano DNA Kit, Illumina).Sequencing was performed by using Novaseq6000 (Macrogen, Japan) (150 bp paired end, 4 GB).The sequences were assembled using Bowtie2 version 2.3.4.1.

Bacterial detection
Anaplasmataceae were detected in the DNA extracted from rodents and ticks using nested PCR targeting 16S rRNA and groEL (19) (Supplement S2).Primers for Bartonella taylorii gltA amplification were designed in this study (Supplement S2).Ex Taq polymer ase (Takara Bio, Tokyo, Japan) was used according to the manufacturer's instructions.All PCR amplicons of the expected size were directly sequenced (Eurofins Genetics, Tokyo, Japan).

Sample preparation for scanning electron microscopy (SEM)
The ticks were fixed with 70% ethanol.After dehydration using an ascending series of ethanol solutions, the specimens were air-dried.After mounting on aluminum stubs with carbon paste, the dried specimens were coated with osmium using an osmium plasma coater (Neoc-ST; Meiwafosis, Tokyo, Japan) and observed under a scanning electron microscope (SU6600; Hitachi, Tokyo, Japan).

Experimental animals
Pathogen-free Mongolian gerbils (MON/Jms/Gbs) were purchased from SLC (Shizuoka, Japan).The animal experiments were performed in accordance with the Laboratory Animal Control Guidelines of the National Institute of Infectious Diseases (215038).

Identification of I. pavlovskyi by PCR-RFLP
The distribution, habitat, and active season of I. pavlovskyi overlapped with those of I. persulcatus and I. ovatus, both of which are predominantly found in Hokkaido (mainland).Furthermore, they are morphologically similar (Fig. 2A).To avoid misclassification, we developed a PCR-RFLP method based on the COI gene of Ixodes.The various restriction enzyme recognition sites and the expected fragment sizes after treatment are summar ized in Table 1. Figure 2B shows the PCR procedure using a tick leg as the template.PCR amplification and EcoRV digestion (Fig. 2C) distinguished I. pavlovskyi from I. persulcatus and I. ovatus (Fig. 2D), regardless of the sequence type (13) (Supplement S1, phyloge netic tree of I. pavlovskyi and I. persulcatus).Both male and female ticks survived after removing the legs individually, and some were subsequently mated.We also screened the specimens collected previously from the eastern part of Hokkaido (15,23) and did not detect I. pavlovskyi (data not shown).Hybrid of I. pavlovskyi/I.persulcatus was recently described from Siberia (24) and the Far East (3).The molecular identification of I. pavlovskyi based on a chromosomal gene, toll (25) (Supplement S8), did not detect the hybrids, although species identification should be carefully performed on the ticks collected in sympatric areas (24,25).

SEM
The internal spur of coxa I and the spiracular plate of adult I. pavlovskyi (Rishiri-type) are shown in Fig. 3.The internal spur of coxa I in both males and females was shorter than that of I. persulcatus.The spiracular plates of I. pavlovskyi are oval.These keys correspond to those proposed by Nakano et al.The apices of the hypostomes of I. pavlovskyi and I. persulcatus females were similar, although they were described to be acute and round at the apex, respectively (Supplement S5).Other organs are shown in Supplement S5.

Haplotype network of I. pavlovskyi revealed a structured population
A haplotype network based on COI sequences of I. pavlovskyi collected from five areas (Fig. 1B) (n = 114) and a reference from Tomsk is shown in Fig. 4. Two dis tinct haplogroups, Rishiri and Asahikawa types, were clearly identified, regardless of the geographic origin of the ticks (Fig. 1A).These haplogroups correspond to the two lineages in the phylogenetic tree (13) (Supplement S1).Among the 114 I. pavlov skyi examined, 88 (77%) were of Rishiri-type and 97% (85/88) were identical (H_20

Rishiri-type and Asahikawa-type ticks are genetically distinct
To investigate the degree of genetic differentiation between the Rishiri and Asahikawa types, the genetic differentiation index (FST) was determined based on all COI sequences used in the haplotype network (Table 2).In all comparisons, the FST values were higher than 0.95.In particular, the FST value between the Rishiri and Asahikawa types was 0.95316, suggesting substantial genetic differentiation.
The genetic diversity of all tick populations is shown in Table 3.The haplotype diversity (Hd) of the Asahikawa-type and I. persulcatus was higher than 0.5, whereas that of the Rishiri-type was 0.067.The nucleotide diversity (Pi) of the Rishiri-type (approxi mately 0.0001) was one-tenth of that of the Asahikawa-type and I. persulcatus, suggest ing that the Rishiri-type has been structured recently and that genetic variation among the Rishiri-type populations has not yet accumulated.

Homologous but not heterologous pairs produced offspring
Genetic differentiation between the two types was high (Table 2), and both types were collected sympatrically during the field examination (Fig. 1).These data indicate that reproductive isolation may occur.To test the biological difference, we tried to conduct a cross-breeding experiment by mating homologous and heterologous pairs (Fig. 5).The feeding period (approximately 7 d) and initiation of egg laying (approximately 1 month) did not differ among females tested (data not shown).However, larvae hatched from eggs within 3 months only when the homologous pairs were mated.The experiment (four pairs) was repeated once and resulted in similar observations.The number of Asahikawa-type was not sufficiently collected, and paired ticks were not always mated.More tests are required to confirm the sterility.

I. pavlovskyi fed on wild rodents and migrating birds in spring
The ticks collected from wild animals are shown in Table 4. I. pavlovskyi of all stages fed on wild birds, including the Oriental Greenfinch (Chloris sinica) and black-faced bunting (Emberiza spodocephala) (Supplement S7).Immature I. pavlovskyi was also found on redbacked voles captured in May.I. pavlovskyi was completely absent from wild rodents captured in June and October.During these months, I. angustus and I. tanuki were major ticks.

Transstadial transmission of Bab. microti US lineage in I. pavlovskyi
From the rodents captured in May 2022, 15 engorged ticks, including I. pavlovskyi larvae and nymph and I. angustus larva and females, were collected from three rodents (Table 6).All rodents tested positive for Bab.microti, and one M. rex (RIS22-2) was positive for Bar.taylorii (mixed infection), as determined by PCR.Larvae successfully hatched from the eggs of all females (n = 5).PCR and direct sequencing on resulting ticks revealed that three nymphs and one male contained Bab.microti US lineage (Table 6), indicating transstadial transmission.The Ca. E. khabarensis or Bartonella spp. was not detected.

Host-seeking I. pavlovskyi Rishiri-type carried Ca. E. khabarensis and Babesia venatorum genes
The presence of tick-borne microorganisms in host-seeking ticks was investigated using PCR (Table 7).Three and one I. pavlovskyi Rishiri-type ticks were positive for Anaplasmata ceae groEL and Piroplasmida 18S rRNA, respectively.Sequencing following a BLAST search revealed that three groEL sequences (329 nt) were identical to each other and those from Ca. E. khabarensis strain m3 (KR063139) and rodents on Rishiri Island in this study (Table 5) (27).
Additional investigation of I. persulcatus DNA revealed the presence of Anaplasma phagocytophilum groEL, which was identical to those reported from I. persulcatus in Khabarovsk and Hokkaido (HM366578 and JQ622144, respectively) and Homo sapiens in South Korea (CP035303).

DISCUSSION
This study has four major findings.First, I. pavlovskyi, a rare species in Japan, is predomi nantly distributed on Rishiri Island.Second, the tick population included two hap logroups: the Rishiri-type and Asahikawa-type, which are genetically distinct from a representative gene record from Tomsk.Third, I. pavlovskyi feeds on wild birds and rodents, with the rodents carrying Ca.E. khabarensis.Since this microorganism has been reported only in Khabarovsk, wild birds and were suggested to play a role in Ca. khabarensis diffusion and settlement, respectively.Fourth, we identified new foci of Babesia microti US lineage, one of the major tick-borne protozoa in the Northern Hemisphere.This organism is maintained between wild rodents and I. pavlovskyi, though I. persulcatus is a principal vector in the Far East.The results of this study suggest that Rishiri Island has a suitable climate, hosts, and vegetation for I. pavlovskyi colonization and I. pavlovskyi-borne microorganism introduction/habitat.
Wildlife on Rishiri Island is unique as it does not include large-to medium-sized predators.Mustela itatsi is the largest and most common medium-sized wild species.This animal is an alien species introduced between 1933 and 1935 to eliminate wild rodents (33).Additionally, snakes and hares, which damage birds and woodlands, respectively, are not present.In contrast, wild birds are abundant on the island.Rishiri Island is located within the migration routes of many birds and serves as an important location for stopovers (rest and nourishment) and breeding.A total of 326 species were recorded, with the percentage of birds breeding, wintering, residing, and traveling being 30%, 10%, 10%, and 50%, respectively (34).These specific biological features are advantageous for the life cycle of I. pavlovskyi, as I. pavlovskyi feeds on birds that collect food on the ground (1,35).The generalist tick, I. persulcatus, has similar seasonal activity and geographical distribution (Fig. 1).However, it feeds on large-to-medium-sized animals (females), and expanding its population on the island is hard.In contrast, I. pavlovskyi cannot colonize the Hokkaido mainland, which is overpopulated by the wild sika deer, Cervus nippon.It has increased almost twice since 2002, and approximately 690,000 deer inhabit the northern, eastern, and central areas (36).In these areas, I. persulcatus and other tick species that favor wild sika deer were densely distributed (Fig. 1) (23).We conclude that Rishiri Island has specific ecological factors required for the dominant colonization of I. pavlovskyi, including abundant wild birds, cold climate, negative pressure on sympatric I. persulcatus, and isolated woodlands.I. pavlovskyi is also dominant in parks in the urban areas of Tomsk.Tick species in city parks have recently been transferred from their original habitats to the Altai Mountains.Similarly, the absence of middle-to-large-sized wildlife and the abundance of wild birds are important factors (37).
Ticks are carried by migrating birds over short and long distances (35,38).Detection of Ca.E. khabarensis gene sequence, which is identical to that reported from Khabarovsk, indicates that the microorganism was carried by birds migrating via the Crossing Sea of Japan route.Rishiri Island, as well as the Hokkaido mainland, is located on the East Asian Flyway, which includes the most diverse flyways worldwide.Although there are    Japan.In contrast, I. pavlovskyi-infested black-faced banting (Table 4) were bred in Japan and to Sakhalin, though I. pavlovskyi have not been collected in Sakhalin by flagging vegetation (43).
When the Rishiri and Asahikawa types diverged is unknown as ticks do not have a molecular clock.The evolutionary rate of mitochondrial COI in insects is 2.3%/mya (44).This clock indicates that the Rishiri and Asahikawa types separated approximately 0.45 mya since the nucleotide difference in the complete I. pavlovskyi COI gene between Rishiri and Asahikawa types was 1.04% (16/1,359 nt) (Supplement S6).The invertebrate model (1.76 ± 0.66-1.22± 0.27/mya) reported by Wilke et al. (45) is another molecular clock.This clock indicates that the haplogroups separated approximately 0.85-0.59mya.The estimated separation of the haplogroups occurred before Hokkaido, including Rishiri Island, separated from the continent (approximately 12,000 years ago) (46).We assume that the haplogroups did not diverge on the Hokkaido mainland or Rihsiri Island; instead, their ancestral populations separated elsewhere on the continent and then arrived at different times and/or by different routes.This is consistent with the haplotype network (Fig. 4) and the nucleotide difference between the Rishiri and Asahikawa types (16/1,539 nt) being higher than those between the Tomsk and Japanese haplogroups (7/1,539 and 13/1,539 nt for the Rishiri and Asahikawa types, respectively).Further understanding of the trails and population structure of I. pavlovskyi in the Far East relies on extensive field surveys of the Eurasian Continent, including that of Primorsky Krai, where a previous study indicated the presence of I. pavlovskyi (1,3).
A survey of wild rodents indicated that M. rex and A. speciosus (Table 5) were infected with Anaplasmataceae spp.whose 16S rRNA and groEL sequences were identical to those of Ca.E. khabarensis (Fig. 6).The Ca. Ehrlichia khabarensis (taxonomy ID 430555), initially referred to as Ehrlichia sp., is a rodent-borne bacterium reported in the Khekhtsir Nature Reserve near Khabarovsk, Russia (27, 28) (Fig. 1).One specific feature of Ca.E. khabarensis in rodents is limited to the Khekhtsir Nature Reserve (48°15′N, 135°01′E), and it is absent from other areas in Khabarovsk, as well as various spots including Irkutsk, Novodibirsk, and Sverdlovsk (27).Regardless of extensive field surveys of ticks by flagging vegetation in the Khekhtsir Nature Reserve (28), no tick has been identified as a vector for Ca.E. Khabarensis.The survey included predominant I. persulcatus adults.The tick species not included in the study, such as less than 3% of the tick population (I.pavlovskyi, I. angustus, and Ixodes maslovi) and/or premature ticks, may participate in the transmission of Ca.E. khabarensis.In our study, microorganisms were detected in the host-seeking adult I. pavlovskyi (Table 7).Recently, a gene sequence identical to that of Ca.E. khabarensis was found in a Great Basin pocket mouse (Perognathus parvus)captured in Okanagan, approximately 200 km east of Vancouver, Canada (47).We assume Ca.E. khabarensis is possibly transported by migrating birds across not only the Sea of Japan but also the Pacific Ocean.I. pavlovskyi has not been reported from the American continent.Thus, I. pavlovskyi may only play an important role as a disseminator of tick-borne microorganisms.As emerging infectious diseases caused by Ehrlichia spp.have been increasingly reported (48,49), the transmission dynamics of ticks and birds should be quantified globally.
PCR and sequencing analyses indicated that wild small mammals (9/41) were infected with Babesia microti U.S. lineage (Table 5).The lineage is widely distributed in the Northern Hemisphere and genetically further classified into three sub-lineages: North America, Europe-Central Asia, and East Asia, based on marker genes such as CCT7 and beta-tubulin (15,50).The CCT7 sequences of the Asian sub-lineage distributed in Korea, Vladivostok, Irkutsk, and eastern Hokkaido (around Nemuro, Fig. 1) varied, whereas CCT7 from Rishiri Island was identical to those reported in eastern Hokkaido, from Myodes rutilus, Myodes rufocanus, and I. persulcatus (Fig. 6).In an endemic area of eastern Hokkaido, I. pavlovskyi was not collected in our previous studies (15,51).Examination of molted nymphs and adults (Table 6) showed that I. pavlovskyi transstadially transmitted to the US lineage.Previous transmission studies using US lineage and I. persulcatus larvae showed that the maximum transmission rate in resulting nymphs was 31.5% (51).
Although few ticks were tested in this study, four of nine ticks were positive (44.4%), indicating that I. pavlovskyi has high vectorial capacity.

FIG 2
FIG 2 Identification of Ixodes pavlovskyi by PCR-RFLP.(A) Images of I. persulcatus, I. pavlovskyi, and I. ovatus (left to right).Upper and lower panels show males and females, respectively.(B) Direct PCR using a tick leg cut and directly submerged into PCR mixture.(C) Schematic diagram of the amplification primers and EcoRV recognition site.(D) Electrophoresis after PCR-RFLP of I. pavlovskyi, I. persulcatus, and I. ovatus.Two haplogroups, Rishiri-type and Asahikawa-type, of I. pavlovskyi are used.M, DNA size markers.

FIG 5
FIG 5 Cross-breeding Ixodes pavlovskyi Rishiri-type and Asahikawa-type.The images on the upper panel show females laying eggs and larvae hatched from the eggs.Female ticks of both types mated with the same or different types of male ticks are shown.The lower panel shows the summary of the cross-breeding experiment.
individual varieties, most migrating birds that stop over Island migrate along two major routes: the Sakhalin-Kuril and the Crossing Sea of Japan routes(39).Passerines including Stejneger's stonechat (Saxicola stejnegeri)(40) and rooks (Corvus frugilegus)(41) use the Crossing route.To cross the Sea of Japan in the shortest way, the rooks fly from their wintering sites up to the Hokkaido mainland along the Japanese archipelago, turn west-northwest and cross the sea, and breed on the Eurasian continent.Bird banding surveys by the Yamashina Institute (42) also show that the Daurian redstart (Phoenicurus auroreus) and rustic bunting (Emberiza rustica) migrate between Primorsky Krai and

FIG 6
FIG6 Evolutionary analysis of various microorganisms detected from ticks and rodents.The evolutionary history was inferred using the maximum likelihood method and Tamura-Nei model.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Evolutionary analyses were conducted in MEGA X. Representative samples determined in this study were included in the phylogenetic analyses.

TABLE 2
Genetic diversity between I. pavlovskyi Rishiri-type and Asahikawa-type and I. persulcatus

TABLE 5
Detection of vector-borne microorganisms in wild small mammals a Confirmed by CCT7 and 18S rRNA sequences.b Confirmed by groEL and 16S rRNA sequences.c Confirmed by gltA and 16S rRNA sequences.d A mixed infection of Bab.microti (US) and Ca.E. khabarensis was included.e A mixed infection of Bab.microti (US) and Bar.taylorii was included.

TABLE 6
Detection of Bab.microti in resulting ticks

TABLE 7
Detection of tick-borne microorganism in host-seeking ticks on Rishiri Island a Only adult ticks were examined.b Determined by 18S rRNA sequence.c Determined by 16S rRNA and groEL sequences.