Introduction

The causative agent for Lyme disease, Borrelia burgdorferi sensu lato, was firstly identified within the gut of vector ticks (Burgdorfer et al. 1982) and the spirochete species can be classified into at least thirteen genospecies based on their genetic differences (Wang et al. 1999; Masuzawa et al. 2001; Richter et al. 2006; Postic et al. 2007). The tick species of Ixodes ricinus complex serve as the main vectors for transmission and perpetuation of B. burgdorferi spirochetes through a natural cycle between vector ticks and rodent hosts in North America and Europe (Spielman 1988; Matuschka et al. 1990). Although I. persulcatus and I. ovatus had been recognized as the principle vector for the transmission of B. burgdorferi spirochetes in the Northeast Asia including the northeastern regions of China, Korea, and Japan (Kawabata et al. 1987; Ai et al. 1988; Nakao et al. 1992; Park et al. 1993), the hard ticks of I. granulatus and Haemaphysalis bispinosa were suggested as the principle vector for the transmission of B. burgdorferi spirochetes in the southern regions (adjacent to Taiwan) of China (Zhang et al. 1997; Wan et al. 1998).

The abundance and widespread of I. granulatus had been recorded for the first time from various countries in Southeast Asia and Taiwan (Wilson 1970). The medical importance with the recent emergence of human babesiosis (Shih et al. 1997) and Lyme borreliosis (Shih et al. 1998b) in Taiwan raises the focus of research attention on I. granulatus tick. In addition, Lyme disease spirochetes (B. burgdorferi sensu lato) had also been isolated from six species of rodent hosts parasitized by various stages of I. granulatus ticks of Taiwan (Shih and Chao 1998) and all these Taiwan isolates were genetically classified into the genospecies of B. burgdorferi sensu stricto, a genospecies firstly identified in Asia (Shih et al. 1998a; Shih and Chao 2002). Although the hard tick of I. granulatus was presumed to be the tick vector for the enzoonotic transmission of Borrelia spirochetes in Taiwan (Shih and Chao 2004), the genetic diversity of Borrelia spirochetes harbored by this tick species remain undefined.

The existence of outer surface protein (Osp) genes in all Borrelia isolates belonging to the major genospecies of B. burgdorferi sensu lato were verified and described (Bergstrom et al. 1989; Jonsson et al. 1992). Genomic similarities of Borrelia spirochetes can be clarified by their differential reactivity with genospecies-specific OspA primer sets and by analyzing the homogeneity of OspA sequences (Zumstein et al. 1992; Wilske et al. 1993; Caporale and Kocher 1994; Demaerschalck et al. 1995). In addition, different genospecies of B. burgdorferi sensu lato are distributed unevently throughout the world and are associated with distinct ecologic features (Wang et al. 1999). It may be that the Borrelia spirochetes exist in I. granulatus ticks of Taiwan are genetically distinct from the Borrelia spirochetes within common vector ticks (I. ricinus complex) in Europe and the United States. The potential of genetic variation in relation to the geographic distribution may also exist among the same Borrelia species detected in variant ticks. Thus, the objective of the present study intends to identify Borrelia spirochetes in I. granulatus ticks by polymerase chain reaction (PCR) assay targeting the OspA gene of B. burgdorferi sensu lato and to clarify the genetic identity of detected spirochetes by analyzing phylogenetic relationships with other Borrelia species.

Materials and methods

Collection and identification of tick specimens

All specimens of adult ticks were removed from rodents captured at various field sites of Taiwan and all field-collected ticks were subsequently stored in separate mesh-covered vials. Adult ticks of male and female I. granulatus collected on Taiwan were identified to species level on the basis of their morphological characteristics, as described and sketched previously (Teng and Jiang 1991). Ultrastructural observations by scanning electron microscope (SEM) were also used to identify the morphological features of I. granulatus ticks of Taiwan, as described previously (Chao et al. 2009).

DNA extraction from tick specimens

Total genomic DNA was extracted from individual tick specimen used in this study. Briefly, tick specimens were cleaned by sonication for 3–5 min in 75% ethanol and then washed twice in sterile distilled water. Afterwards, individual tick specimen was dissected into pieces, placed in a microcentrifuge tube filled with 180-μl lysing buffer solution supplied in the DNeasy Tissue Kit (catalogue no. 69506, Qiagen, Taipei, Taiwan) and then homogenized with a sterile tissue grinder (catalogue no. 358103, Wheaton Scientific Products, Millville, NJ, USA). The homogenate was centrifuged at room temperature and the supernatant fluid was further processed using a DNeasy Tissue Kit, as per manufacturer’s instructions. After filtration, the filtrate was collected and the DNA concentration was determined spectrophotometrically with a DNA calculator (GeneQuant II; Pharmacia Biotech, Uppsala, Sweden).

DNA amplification by polymerase chain reaction

DNA samples extracted from the tick specimens were used as template for PCR amplification. A specific primer set, SL-1 (5′-AATAGGTCTAATAATAGCCTTAATAGC-3′) corresponding to the 3′ end of the OspA gene and SL-2 (5′-CTAGTGTTTTGCCATCTTCTTTGAAAA-3′) corresponding to the 5′ end of the OspA gene, were designed to amplify DNA of all major genospecies of B. burgdorferi sensu lato, as described previously (Demaerschalck et al. 1995). All PCR reagents and Taq polymerase were obtained and used as recommended by the supplier (Takara Shuzo Co., Ltd., Japan). Briefly, a total of 0.2-μmol of the appropriate primer set and various amounts of template DNA were used in each 50-μl reaction mixture. The PCR amplification was performed with a Perkin-Elmer Cetus thermocycler (GeneAmp system 9700; Applied Biosystems, Taipei, Taiwan) for 40 cycles with denaturation at 93°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Amplified DNA products were electrophoresed in 2% agarose gels in Tris–Borate-EDTA (TBE) buffer and visualized under ultraviolet (UV) light after staining with ethidium bromide. A 1-kb plus DNA ladder (catalogue no. 10787-018, Gibco BRL, Taipei, Taiwan) was used as the standard marker for comparison.

Sequence alignments and phylogenetic analysis

After purification with a QIAquick PCR purification kit (catalogue no. 28104, Qiagen, Taipei, Taiwan), the nucleotide sequences of 13 strains of Borrelia spirochetes detected in I. granulatus ticks of Taiwan were sequenced using an ABI Prism 377-96 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The resulting sequences were initially aligned with the CLUSTAL W software (Thompson et al. 1994) and further analyzed by neighbour-joining (NJ) compared with maximum parsimony (MP) methods to estimate the phylogeny of the entire alignment using MEGA 4.0 software package (Tamura et al. 2007). A similarity matrix was also constructed using DNASTAR program (Lasergene, version 8.0). The genetic distance values of intra- and inter-specific variations of Borrelia spirochetes were also analyzed by the Kimura two-parameter model (Kimura 1980), as implemented in MEGA 4.0. All phylogenetic trees were constructed and performed with 1,000 bootstrap replications to evaluate the reliability of the constructions, as described previously (Felsenstein 1985).

Nucleotide sequence accession numbers

The nucleotide sequences of PCR-amplified OspA gene of Borrelia spirochetes determined in this study have been registered and assigned the following GenBank accession numbers: strains KH-58 (GU002658), KH-71 (GU002659), KS-18 (GU002660), KS-19 (GU002661), KS-42 (GU002662), KS-48 (GU002663), KS-67 (GU002664), KS-68 (GU002665), KC-44-1 (GU002666), KH-62 (GU002667), KH-74 (GU002668), KH-100 (GU002669), and KH-103 (GU002670). For phylogenetic analysis, nucleotide sequences of OspA from 22 strains of Borrelia spirochetes downloaded from GenBank were included for comparison and their GenBank accession numbers are shown in Table 1.

Table 1 Genospecies and strains of Borrelia spirochetes analyzed in this study and their GenBank accession numbersa
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Results

Detection of spirochetal infection in Ixodes granulatus ticks

To verify the existence of Borrelia spirochetes in adult I. granulatus ticks removed from rodents of Taiwan. A total of 147 adult ticks (121 female and 26 male) were examined and tested for the evidence of spirochetal infection by PCR using specific primers targeting the OspA gene of B. burgdorferi sensu lato. Borrelia spirochetes were detected in 23 female and 4 male adult I. granulatus ticks with an infection rate of 19.0% (23/121) and 15.4% (4/26), respectively (Table 2). All the positive-infected adult ticks feed on the rodent host of Rattus losea.

Table 2 Spirochetal infection detected in adult Ixodes granulatus ticksa by polymerase chain reaction (PCR) assay targeting the OspA of B. burgdorferi sensu lato
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Sequence alignment and genetic analysis

To clarify the genetic identity of these Borrelia spirochetes detected in adult I. granulatus ticks of Taiwan, sequences of PCR-amplified OspA fragments of 13 strains of Borrelia spirochetes were aligned and compared with the downloaded sequences of 22 strains of Borrelia spirochetes (11 B. valaisiana, 3 B. burgdorferi sensu stricto, 2 B. garinii, 2 B. afzelii, 2 B. bissettii, and 2 B. califoeniensis) from GenBank. The lengths of the aligned sequences were measured from 206 to 230 bp, and the nucleotide components indicate that the OspA of these spirochetes is highly AT-rich with average nucleotide frequencies of thymine (T) = 22.2%, cytosine (C) = 13.7%, adenine (A) = 43.7%, and guanine (G) = 20.4%, respectively (Fig. 1). The nucleotide sequences between the 13 Borrelia spirochetes of Taiwan were highly conserved with only a few point mutations/substitutions (Fig. 1) and the nucleotide variations within these Borrelia spirochetes of Taiwan were measured from 0 to 3.5% (Table 3). In contrast, the nucleotide variations among other genospecies of Borrelia compared with the Taiwan strains were measured from 3 to 19.6%. Inter- and intra-specific variations analyzed by the pairwise comparisons of genetic distance values reveal that all these Borrelia spirochetes of Taiwan were genetically affiliated with the B. valaisiana groups from China and Korea, and can be distinguished from the European group of B. valaisiana as well as other genospecies of B. burgdorferi sensu lato (Table 4). However, intraspecies analysis of B. valaisiana based on the genetic distance values also indicates a lower level of genetic divergence (<0.011) within these Borrelia spirochetes of Taiwan and all these Taiwan strains of Borrelia spirochetes were genetically more distant to the European group of B. valaisiana (>0.024) and other genospecies of B. burgdorferi sensu lato (>0.032) (Table 4).

Fig. 1
figure 1

Nucleotide sequences of the OspA gene of 13 strains of Borrelia spirochetes performed by this study were aligned and compared with the downloaded sequences of other 16 strains of Borrelia spirochetes from GenBank. The sequence for the Borrelia strain named B. valaisiana (VS116) is given as reference. Dots indicate nucleotides that are identical to the sequence of reference strain. Dashes indicate deletions within the sequence

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Table 3 Sequence similarity between OspA gene sequences from Taiwan strains of Borrelia detected in I. granulatus and strains of other genospecies of Borrelia
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Table 4 Inter- and intra-specific analysis of genetic distance valuesa based on the OspA gene sequences between Taiwan strains of Borrelia detected in I. granulatus and strains of other genospecies of Borrelia
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Phylogenetic analysis

Phylogenetic relationships based on the alignment of OspA sequences were performed to analyze the genetic divergence among 35 Borrelia spirochetes investigated in this study. Bootstrap analysis was used to analyze the repeatability of the clustering of specimens represented in phylogenetic trees. Phylogenetic trees constructed by both NJ and MP analyses showed congruent basal topologies with eight major branch of distinguished clades (Figs. 2, 3). All Borrelia spirochetes detected in adult I. granulatus ticks represent three major groups of Borrelia spirochetes (groups A–C) which constituted a separate clade that can be easily distinguished from the European group of Borrelia spirochetes and other genospecies of Borrelia spirochetes. Within the same clade, 13 strains of Borrelia spirochetes from Taiwan represent three groups (groups A–C) and can be easily distinguished from the European group of Borrelia spirochetes with a bootstrap value of 86 in NJ analysis (Fig. 2). The phylogenetic tree of MP analysis was identical to the NJ tree and strongly support the separation of different lineages between the Borrelia spirochetes from Taiwan and Europe with a bootstrap value of 82 (Fig. 3). These results reveal a lower genetic divergence within the same genospecies of Borrelia spirochetes from Taiwan, but a higher genetic variations among different genospecies or group of Borrelia spirochetes.

Fig. 2
figure 2

Phylogenetic relationships of 13 OspA genes of Borrelia spirochetes detected in I. granulatus were compared with the sequences of six genospecies (i.e., B. v., B. valaisiana; B. g., B. garinii; B. a., B. afzelii; B. b., B. burgdorferi sensu stricto; B. bis., B. bissettii; B. ca., B. californiensis) of Borrelia spirochetes. The tree was constructed and analyzed with the neighbour-joining method with 1,000 bootstrap replicates. Numbers at the nodes indicate the percentages of reliability of each branch of the tree. Branch lengths are drawn proportional to the estimated sequence divergence

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Fig. 3
figure 3

Phylogenetic relationships of 13 OspA genes of Borrelia spirochetes detected in I. granulatus were compared with the sequences of six genospecies (i.e., B. v., B. valaisiana; B. g., B. garinii; B. a., B. afzelii; B. b., B. burgdorferi sensu stricto; B. bis., B. bissettii; B. ca., B. californiensis) of Borrelia spirochetes. The tree was constructed and analyzed with the maximum parsimony method with 1,000 bootstrap replicates. Bootstrap percentage values from 1,000 replicates are indicated for relevant clades

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Discussion

This report describes the first detection of B. valaisiana-related spirochetes in adult I. granulatus ticks collected in Taiwan. In our previous investigations, B. burgdorferi sensu stricto was isolated from six species of rodent hosts captured at various locations in Taiwan and I. granulatus ticks were observed on these highly infected rodent hosts (Shih and Chao 1998). Because of the high prevalence of Borrelia infection among captured rodents in Taiwan, the existence of zoonotic transmission of Borrelia spirochetes was suggested and the identification of Borrelia spirochetes in possible vector ticks is required to clarify the natural transmission cycle in Taiwan. Indeed, results from the present study confirm the existence of B. valaisiana-related spirochetes in adult I. granulatus ticks and indicate that the R. losea serves as the principle reservoir host for perpetuation of Borrelia spirochetes in nature. Further studies focused on the seasonal abundance of reservoir hosts and the prevalence of spirochetal infection among vector ticks would help to illustrate the ecologic feature regarding the transmission cycle of Borrelia spirochetes in Taiwan.

Geographical distribution of B. valaisiana-related spirochetes in Asia remains undefined. It is assumed that different genospecies of B. burgdorferi sensu lato are associated with distinct reservoir hosts and vector ticks (Wang et al. 1999). Indeed, B. valaisiana has been isolated or detected from I. ricinus ticks and avain reservoirs from at least eight European countries (Rijpkema et al. 1996; Postic et al. 1997; Wang et al. 1997; Kirstein et al. 1997; Kurtenbach et al. 1998; Clinco et al. 1998). However, B. valaisiana-related spirochetes were isolated mainly from rodent hosts (R. losea, R. norvegicus, Mus formosanus, Niviventer fulvescens, and Apodemus agrarius) and detected in various hard ticks (I. nipponensis, I. columnae, I. granulatus, and Haemaphysalis longicornis) in Northeast Asia and Southwestern China (Masuzawa et al. 1999, 2001, 2004; Chu et al. 2008). Results from this study also verify the existence of B. valaisiana-related spirochetes detected in I. granulatus ticks removed from the rodent host of R. losea in Taiwan. These findings suggested that B. valaisiana-related spirochetes may persist in a zoonotic cycle between their rodent reservoir hosts and tick vectors in Eastern Asia.

The genetic identity of Borrelia spirochetes can be clarified by their differential reactivities with genospecies-specific PCR primers. In previous investigations, sequence analysis of OspA gene had been used to distinguish closely related Borrelia spirochetes and to assess the phylogenetic relationships of diverse B. burgdorferi sensu lato spirochetes by comparing their nucleotide variations of the OspA gene (Zumstein et al. 1992; Wilske et al. 1993; Caporale and Kocher 1994; Demaerschalck et al. 1995; Masuzawa et al. 1999; Wang et al. 2000; Shih and Chao 2002; Chu et al. 2008). Results from this study demonstrate that the nucleotide composition of OspA gene derived from these I. granulatus ticks of Taiwan is highly A-T rich (~65.9%) and that is similar to the nucleotide frequency of other Borrelia spirochetes either analyzed in this study (Fig. 1) or described in previous investigations (Masuzawa et al. 1999; Chu et al. 2008). This sequence feature observed in this study may imply a recent genetic evolution among these B. valaisiana-related spirochetes in Eastern Asia. Furthermore, the genetic divergence of these B. valaisiana-related spirochetes of Taiwan can be easily distinguished from the European group of B. valaisiana spirochetes and other genospecies of B. burgdorferi spirochetes by their differential nucleotide variations existed in the OspA gene sequences (Tables 3 and 4). Thus, these observations suggest that the genetic identity of B. valaisiana-related spirochetes of Taiwan can be determined either interspecies or intraspecies among Borrelia spirochetes by analyzing their genetic divergence of the nucleotide sequences of OspA gene. Although intraspecific variation within these B. valaisiana-related spirochetes of Taiwan averaged less than 3.5% sequence variations may not fully represent a new genomospecies, interspecific variation between these B. valaisiana of Taiwan and other genospecies of B. burgdorferi spirochetes averaged more than 10.9% sequence variations are much greater than that analyzed by previous studies for distinguishing the sequence variations between the distinct genospecies from different geographical origins (Wang et al. 2000; Masuzawa et al. 1999, 2001, 2004; Chu et al. 2008). Further investigation on the sequence divergence based on various targets of Osp genes of Borrelia spirochetes collected from different localities of Taiwan and its adjacent areas would be required to clarify the genetic divergence as well as the evolutionally origin among Borrelia spirochetes from Taiwan and its adjacent areas.

Phylogenetic relationships among Borrelia spirochetes can be determined by analyzing their sequence heterogeneity of the OspA gene. Indeed, the sequence analysis of OspA gene among various genospecies of Borrelia spirochetes had been shown to be useful for evaluating the genetic relatedness of Borrelia spirochetes isolated from various biological and geographical origins (Bergstrom et al. 1989; Jonsson et al. 1992; Zumstein et al. 1992; Wilske et al. 1993; Caporale and Kocher 1994; Demaerschalck et al. 1995; Will et al. 1995; Masuzawa et al. 1999; Wang et al. 2000; Shih and Chao 2002; Chu et al. 2008). In previous study, two distinct subgroups of B. valaisiana spirochetes are evident by comparing their OspA gene sequences between two closely related B. valaisiana isolated from the I. ricinus ticks of Europe (Wang et al. 2000). Phylogenetic analysis of Borrelia spirochetes related to the members of B. valaisiana also revealed intraspecific variation between different biological and geographical origins (Masuzawa et al. 1999, 2001, 2004; Wang et al. 2000; Chu et al. 2008). In this study, phylogenetic analysis based on the OspA gene sequences among various Borrelia genospecies demonstrated a high genetic heterogeneity between B. valaisiana-related spirochetes and other genospecies of Borrelia (Fig. 2). Although a low intraspecific variation was observed among the same genospecies of B. valaisiana, all the 13 strains of B. valaisiana from Taiwan represented as a separate clade that can be distinguished from the European group of B. valaisiana (Figs. 2 and 3). The phylogenetic trees constructed by either NJ or MP analysis strongly support the discrimination recognizing the separation of different lineages between the B. valaisiana from Taiwan and Europe. Within the same clade, geographical variation was also observed among a sister group C (strain KH-74) affiliated to the B. valaisiana from Korea (strains 5MT and 10MT), group A (strains KH-71, KH-100, KH-103, KS-18, KS-19, and KC-44-1) affiliated to the B. valaisiana from Southwestern China (strains QLZSP1, QSYSP3, and QTMP2), and group B (strains KH-58, KH-62, KS-42, KS-48, KS-67, and KS-68) adjacent to the Southeastern China. Accordingly, these observations reveal that all these B. valaisiana-related spirochetes detected in I. granulatus ticks from Taiwan represent three major groups constructed a unique clade distincted from the genospecies of B. valaisiana from Europe.

The pathogenecity of B. valaisiana-related spirochetes to humans remains to be determined. Although B. valaisiana has been recognized as the predominant Borrelia species detected in field-collected I. ricinus ticks and the I. ricinus ticks attached to human skin (Kirstein et al. 1997; Liebisch et al. 1998), the B. valaisiana-related spirochetes has never been isolated from human patients. Indeed, B. valaisiana DNA has been detected in cerebrospinal fluid (CSF) of an European patient (Diza et al. 2004) and B. valaisiana infection was reported in a Japanese man associated with a suspected bite by an I. persulcatus tick in which the DNA of B. valaisiana was detected. However, there is no confirmed evidence for the existence of B. valaisiana spirochetes in the patient’s tissue (Saito et al. 2007). Moreover, the host-associated selection of genetic diversity of Borrelia spirochetes was proposed (Kurtenbach et al. 2002) and enzoonotic transmission by tick species that rarely feed on human hosts had also been suggested as the possible factors responsible for the under estimation of human cases (Maupin et al. 1994; Peavey et al. 2000). Indeed, a total of 13 Borrelia genospecies within the B. burgdorferi sensu lato complex have been described worldwide and only three genospecies (i.e., B. burgdorferi sensu stricto, B. garinii, and B. afzelii) are highly pathogenic to humans (Aguero-Rosenfeld et al. 2005). Thus, the B. valaisiana-related spirochetes to cause a disease in humans in Asia still ambiguity.

In conclusion, our report provides the first evidence regarding the existence of B. valaisiana-related spirochetes within I. granulatus ticks collected in Taiwan. The genetic identity of these spirochetes was confirmed by analyzing sequence homology of OspA and indicated that all these spirochetes detected in I. granulatus ticks of Taiwan were genetically affiliated to the genospecies of B. valaisiana and constituted a separate clade representing three major groups distinguished from the European group of B. valaisiana transmitted by the common vector ticks (I. ricinus complex) for B. burgdorferi sensu lato. Further application of this molecular tool to investigate the genetic variability of OspA and other target genes among Borrelia spirochetes detected in different vector ticks and reservoir hosts may help to clarify the genetic diversity of Borrelia spirochetes in relation to the epidemiological features as well as their pathogenecity for human infection in Taiwan.