Host dispersal shapes the population structure of a tick‐borne bacterial pathogen

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick‐borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies “Candidatus Borrelia aligera” was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick‐borne pathogens are shaped by their host associations and the movement patterns of these hosts.     

Comparison of an oligo-chip based assay with PCR method to measure the prevalence of tick-borne pathogenic bacteria in central Slovakia

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.     

Prevalence and distribution of Borrelia and Babesia species in ticks feeding on dogs in the U.K.

Ticks were collected during March–July 2015 from dogs by veterinarians throughout the U.K. and used to estimate current prevalences and distributions of pathogens. DNA was extracted from 4750 ticks and subjected to polymerase chain reaction and sequence analysis to identify Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) and Babesia (Piroplasmida: Babesiidae) species. Of 4737 ticks [predominantly Ixodes ricinus Linneaus (Ixodida: Ixodidae)], B. burgdorferi s.l. was detected in 94 (2.0%). Four Borrelia genospecies were identified: Borrelia garinii (41.5%); Borrelia afzelli (31.9%); Borrelia burgdorferi sensu stricto (25.5%), and Borrelia spielmanii (1.1%). One Rhipicephalus sanguineus Latreille (Ixodida: Ixodidae), collected from a dog with a history of travel outside the U.K., was positive for B. garinii. Seventy ticks (1.5%) were positive for Babesia spp. Of these, 84.3% were positive for Babesia venatorum, 10.0% for Babesia vulpes sp. nov., 2.9% for Babesia divergens/Babesia capreoli and 1.4% for Babesia microti. One isolate of Babesia canis was detected in a Dermacentor reticulatus (Ixodida: Ixodidae) tick collected from a dog that had recently travelled to France. Prevalences of B. burgdorferi s.l. and Babesia spp. did not differ significantly between different regions of the U.K. The results map the widespread distribution of B. burgdorferi s.l. and Babesia spp. in ticks in the U.K. and highlight the potential for the introduction and establishment of exotic ticks and tick‐borne pathogens.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proposing a new hypothesis: Rickettsia spp. as a mechanism maintaining parapatry between two Australian reptile tick species

This study investigates two parasitic reptile ticks — Bothriocroton hydrosauri and Amblyomma limbatum — of the sleepy lizard (Tiliqua rugosa) that abut at a 1–2 km wide parapatric boundary in South Australia. Long‐term research has investigated potential mechanisms to explain the maintenance of this boundary but has not uncovered why the distribution of A. limbatum does not extend further south. It has been previously hypothesised that pathogens may be responsible for maintaining parapatric boundaries. Rickettsia spp. has previously been reported in B. hydrosauri ticks. This study explored whether Rickettsia spp. occurs in co‐occurring A. limbatum. We observed that Rickettsia spp. was absent from all A. limbatum ticks and that 83% of examined B. hydrosauri were found to be positive with a spotted fever group Rickettsia strain. This study puts forward the hypothesis that Rickettsia spp. could contribute to the maintenance of the Mt Mary parapatric boundary between these two tick species. Further work is required to determine whether Rickettsia spp. can be transmitted from B. hydrosauri to A. limbatum and — if transmission can occur — to explore whether Rickettsia is lethal to A. limbatum ticks.     

Successful care and propagation of the endangered amargosa vole (Microtus californicus scirpensis) in captivity

The Amargosa vole (Microtus californicus scirpensis) is a highlyendangered rodent endemic to a small stretch of the California portion of the Amargosa River basin in Inyo County's Mojave Desert. Although the Amargosa vole has survived in this naturally fragmented ecosystem for thousands of years, recent habitat degradation due to land development, water drainage, and marsh exploitation has further isolated the species and reduced its available habitat. As part of a conservation effort to preserve the species, a captive breeding population was established in 2014 to serve as an insurance colony and as a source of individuals to release into the wild as restored habitat becomes available. As this is the only captive colony for this species, there is little published information about appropriate care and husbandry for the Amargosa vole. Here we provide information about behavior, diet, reproduction, drug sensitivities, and diseases that affect successful captive care. We also provide recommendations for housing and disease management to preserve natural behaviors and defenses in captive‐born animals.     

Seasonal dynamics of tick burden and associated Borrelia burgdorferi s.l. and Borrelia miyamotoi infections in rodents in a Dutch forest ecosystem

Ixodes ricinus ticks transmit Borrelia burgdorferi sensu lato (s.l.) as well as Borrelia miyamotoi. Larvae become infected when feeding on infected rodents, with horizontal transmission of B. burgdorferi and horizontal and vertical transmission of B. miyamotoi. We studied seasonal dynamics of infection rates of I. ricinus and their rodent hosts, and hence transmission risk of these two distinctly different Borrelia species. Rodents were live-trapped and inspected for ticks from May to November in 2013 and 2014 in a forest in The Netherlands. Trapped rodents were temporarily housed in the laboratory and detached ticks were collected. Borrelia infections were determined from the trapped rodents and collected ticks. Borrelia burgdorferi s.l. and B. miyamotoi were found in ticks as well as in rodents. Rodent density was higher in 2014, whereas tick burden as well as the Borrelia infection rates in rodents were higher in 2013. The density of B. miyamotoi-infected nymphs did not differ between the years. Tick burdens were higher on Apodemus sylvaticus than on Myodes glareolus, and higher on males than on females. Borrelia-infection rate of rodents varied strongly seasonally, peaking in summer. As the larval tick burden also peaked in summer, the generation of infected nymphs was highest in summer. We conclude that the heterogeneity of environmental and host-specific factors affects the seasonal transmission of Borrelia spp., and that these effects act more strongly on horizontally transmitted B. burgdorferi spp. than on the vertically transmitted B. miyamotoi.

     

Borrelia tanukii Sp. Nov. and Borrelia turdae Sp. Nov. Found from Ixodid Ticks in Japan: Rapid Species Identification by 16S rRNA Gene-Targeted PCR Analysis

Based on the results of RFLP-ribotyping, whole DNA/DNA hybridization and phylogenetic analysis of the 16S rRNA gene, we previously defined two genomic groups of spirochetes closely related to Borrelia burgdorferi sensu lato: group Hk501 for strains isolated from Ixodes tanuki ticks and group Ya501 for strains isolated from Ixodes turdus ticks. In this report, we propose that group Hk501 should be classified as Borrelia tanukii sp. nov. and group Ya501 as Borrelia turdae sp. nov. The alignment of previously published Borrelia 16S rRNA gene sequences led us to design species-specific PCR primer sets. The primers allowed the rapid identification of B. tanukii and B. turdae.     

Differences in Borrelia infections in adult Ixodes persulcatus and Ixodes ricinus ticks (Acari: Ixodidae) in populations of north-western Russia

Dark-field microscopy was used to determine the number of Borrelia spirochetes in 630 standard preparations obtained from adult ixodid ticks (344 Ixodes persulcatus and 286 I. ricinus) collected in 1989-92 in the Leningrad region of Russia. The average numbers of Borrelia in I. persulcatus and I. ricinus preparations were 34.7 and 23.3 per 100 microscopic fields, respectively. The maximal individual values registered each year for ticks of both species were several hundred times greater than the minimal values. Ticks carrying relatively small numbers of Borrelia generally predominated. Proportions of more heavily infected ticks varied considerably from year to year. These parameters were significantly higher in foci with predominance of I. persulcatus ticks. As a consequence, risk to acquire Lyme borreliosis in such foci is considered greater than in foci where I. ricinus predominates.     

Tritrophic interactions between a fungal pathogen,a spider predator,and the blacklegged tick

The blacklegged tick Ixodes scapularis is the primary vector for the bacterium causing Lyme disease in eastern North America and for other medically important pathogens. This species is vulnerable to attack by fungal pathogens and arthropod predators, but the impacts of interactions between biocontrol agents have not been examined. The biocontrol agent Met52^®, containing the entomopathogenic fungus Metarhizium brunneum (=M. anisopliae), controls blacklegged ticks with efficacy comparable to chemical acaricides. The brush‐legged wolf spider Schizocosa ocreata is a predator of I. scapularis that reduces their survival under field conditions. We conducted a field microcosm experiment to assess the compatibility of Met52 and S. ocreata as tick biocontrol agents. We compared the fits of alternative models in predicting survival of unfed (flat) and blood‐fed (engorged) nymphs. We found the strongest support for a model that included negative effects of Met52 and S. ocreata on flat nymph survival. We found evidence for interference between biocontrol agents, with Met52 reducing spider survival, but we did not find a significant interaction effect between the two agents on nymph survival. For engorged nymphs, low recovery rates resulted in low statistical power to detect possible effects of biocontrol agents. We found that nymph questing activity was lower when the spider was active above the leaf litter than when the spider was unobserved. This provides the first evidence that predation cues might affect behavior important for tick fitness and pathogen transmission. This study presents field microcosm evidence that the biopesticide Met52 and spider Schizocosa ocreata each reduced survival of blacklegged ticks Ixodes scapularis. Met52 reduced spider survival. Potential interference between Met52 and the spider should be examined at larger scales, where overlap patterns may differ. Ticks were more likely to quest when the spider was inactive, suggesting the ticks changed their behavior to reduce danger.     

Associations Between Coinfection Prevalence of <Emphasis Type="Italic">Borrelia lusitaniae</Emphasis>, <Emphasis Type="Italic">Anaplasma</Emphasis> sp., and <Emphasis Type="Italic">Rickettsia</Emphasis> sp. in Hard Ticks Feeding on Reptile Hosts

An increasing number of studies reveal that ticks and their hosts are infected with multiple pathogens, suggesting that coinfection might be frequent for both vectors and wild reservoir hosts. Whereas the examination of associations between coinfecting pathogen agents in natural host–vector–pathogen systems is a prerequisite for a better understanding of disease maintenance and transmission, the associations between pathogens within vectors or hosts are seldom explicitly examined. We examined the prevalence of pathogen agents and the patterns of associations between them under natural conditions, using a previously unexamined host–vector–pathogen system—green lizards Lacerta viridis, hard ticks Ixodes ricinus, and Borrelia, Anaplasma, and Rickettsia pathogens. We found that immature ticks infesting a temperate lizard species in Central Europe were infected with multiple pathogens. Considering I. ricinus nymphs and larvae, the prevalence of Anaplasma, Borrelia, and Rickettsia was 13.1% and 8.7%, 12.8% and 1.3%, and 4.5% and 2.7%, respectively. The patterns of pathogen prevalence and observed coinfection rates suggest that the risk of tick infection with one pathogen is not independent of other pathogens. Our results indicate that Anaplasma can play a role in suppressing the transmission of Borrelia to tick vectors. Overall, however, positive effects of Borrelia on Anaplasma seem to prevail as judged by higher-than-expected Borrelia–Anaplasma coinfection rates.     

Effects of invasive plants on fire regimes and postfire vegetation diversity in an arid ecosystem

We assessed the impacts of co‐occurring invasive plant species on fire regimes and postfire native communities in the Mojave Desert, western USA. We analyzed the distribution and co‐occurrence patterns of three invasive annual grasses (Bromus rubens, Bromus tectorum, and Schismus spp.) known to alter fuel conditions and community structure, and an invasive forb (Erodium cicutarium) which dominates postfire sites. We developed species distribution models (SDMs) for each of the four taxa and analyzed field plot data to assess the relationship between invasives and fire frequency, years postfire, and the impacts on postfire native herbaceous diversity. Most of the Mojave Desert is highly suitable for at least one of the four invasive species, and 76% of the ecoregion is predicted to have high or very high suitability for the joint occurrence of B. rubens and B. tectorum and 42% high or very high suitability for the joint occurrence of the two Bromus species and E. cicutarium. Analysis of cover from plot data indicated two or more of the species occurred in 77% of the plots, with their cover doubling with each additional species. We found invasive cover in burned plots increased for the first 20 years postfire and recorded two to five times more cover in burned than unburned plots. Analysis also indicated that native species diversity and evenness as negatively associated with higher levels of relative cover of the four invasive taxa. Our findings revealed overlapping distributions of the four invasives; a strong relationship between the invasives and fire frequency; and significant negative impacts of invasives on native herbaceous diversity in the Mojave. This suggests predicting the distributions of co‐occurring invasive species, especially transformer species, will provide a better understanding of where native‐dominated communities are most vulnerable to transformations following fire or other disturbances.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Recent discovery of widespread Ixodes affinis (Acari: Ixodidae) distribution in North Carolina with implications for Lyme disease studies

Ixodes affinis, which is similar morphologically to Ixodes scapularis, is widely distributed in North Carolina. Collections have documented this species in 32 of 41 coastal plain counties, but no piedmont or mountain counties. This coastal plain distribution is similar to its distribution in Georgia and South Carolina, where it is considered an enzootic vector of Borrelia burgdorferi sensu stricto. An updated list of hosts for I. affinis in the U.S.A. is included, increasing the number to 15 mammal and one bird species. The presence of questing adults of I. affinis from April to November reinforces the need for confirmed identifications of suspected tick vectors of Borrelia spirochetes collected during warm months.     

Development of genomic resources for the tick Ixodes ricinus: isolation and characterization of single nucleotide polymorphisms

Assessing the genetic variability of the tick Ixodes ricinus—an important vector of pathogens in Europe—is an essential step for setting up antitick control methods. Here, we report the first identification of a set of SNPs isolated from the genome of I. ricinus, by applying a reduction in genomic complexity, pyrosequencing and new bioinformatics tools. Almost 1.4 million of reads (average length: 528 nt) were generated with a full Roche 454 GS FLX run on two reduced representation libraries of I. ricinus. A newly developed bioinformatics tool (DiscoSnp), which isolates SNPs without requiring any reference genome, was used to obtain 321 088 putative SNPs. Stringent selection criteria were applied in a bioinformatics pipeline to select 1768 SNPs for the development of specific primers. Among 384 randomly SNPs tested by Fluidigm genotyping technology on 464 individuals ticks, 368 SNPs loci (96%) exhibited the presence of the two expected alleles. Hardy–Weinberg equilibrium tests conducted on six natural populations of ticks have shown that from 26 to 46 of the 384 loci exhibited significant heterozygote deficiency.     

Feeding by Scolytus bark beetles to test for differently susceptible elm varieties

Dutch elm disease (DED), caused by the fungi Ophiostoma ulmi and O. novo‐ulmi, has reduced elm populations severely in Europe and North America. Breeding programmes are in action to find less susceptible elm varieties suitable for re‐establishing elm stands. Bark beetles, mainly Scolytus spp., are the only known natural vectors of DED. During twig feeding, beetles transfer Ophiostoma spores to healthy elms. Thus, less palatable elms should run a lower risk of DED infections. In feeding preference bioassays, we offered twigs from elms exhibiting different degree of susceptibility to O. novo‐ulmi, together with non‐host trees to Scolytus beetles. Scolytus multistriatus preferred wych elm, Ulmus glabra, to 100% in two‐choice tests, whereas S. laevis did not discriminate between a tolerant and a susceptible variety of field elm, U. minor. We suggest that the feeding assay is useful as a low‐tech method in breeding programmes for evaluating the suitability of promising elm genotypes to vector insects.     

Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia

One-hundred and fifty-seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S-23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species-specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but no Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.     

Infestation of taiga ticks with borrelias in the territory of Novosibirsk Scientific Center (Siberian Branch,Russian Academy of Sciences)

Borrelia specimens were revealed in taiga ticks Ixodes persulcatus collected in the wild by flagging and also in ticks provided by the Vaccination section of the Novosibirsk Scientific Center, Siberian Branch of the Russian Academy of Sciences (NSC); these ticks were obtained from patients attacked by ticks. Isolation of borrelias in the BSK-H medium had demonstrated the presence of B. garinii, B. afzelii, and B. miyamotoi in the territory of NCS. B. miyamotoi isolates were unstable, loosing their growth ability during subsequent cultivation. DNA of the three above species was detected by PCR in tick samples collected by flagging and obtained from humans. DNA of B. garinii was recorded in ticks more often; DNA of B. afzelii was found less frequently; B. miyamotoi DNA was detected in the smallest number of ticks. In ticks collected by flagging, DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 38.6%, 9.9%, and in 3.9% of specimens, respectively. In ticks collected from attacked humans, the number of positive tests was lower; e.g., DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 24.2%, 6.9%, and in 5.6% of samples, respectively. Mixed infection of ticks with two Borrelia species was also detected; DNA of B. miyamotoi and of B. garinii was detected in mixed infections more frequently.     

Molecular detection of <Emphasis Type="Italic">Borrelia valaisiana</Emphasis>-related spirochetes from <Emphasis Type="Italic">Ixodes granulatus</Emphasis> ticks in Taiwan

Borrelia valaisiana-related spirochetes were detected for the first time in Ixodes granulatus ticks collected in Taiwan. The genetic identities of these detected spirochetes were determined by analyzing the gene sequences amplified by a genospecies-specific polymerase chain reaction assay based on the outer surface protein A (OspA) gene of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of OspA gene obtained from 35 strains of Borrelia spirochetes representing six genospecies of Borrelia. Eight major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Except one strain (KH-74), all these Borrelia spirochetes of Taiwan were genetically affiliated to the same clade with highly homogeneous sequences (97.8–100% similarity), and can be discriminated from other groups of B. valaisiana and other genospecies of Borrelia spirochetes with a sequence divergence ranging from 3 to 19.6%. Moreover, intraspecific analysis also revealed that three distinct groups are evident between the same species of B. valaisiana spirochetes detected in Taiwan. Our results provide the first evidence of B. valaisiana spirochetes detected in I. granulatus ticks collected in Taiwan and demonstrate that all these B. valaisiana spirochetes of Taiwan represent three major groups distinct from the European group of B. valaisiana spirochetes.