Introduction

Babesiosis is an emerging tick-borne disease of animals and humans caused by intraerythrocytic protozoa of the genus Babesia (Irwin 2009), and one species was firstly identified by Dr. Victor Babes as the cause of bovine haemoglobinuria or red water fever (Uilenberg 2006). Traditionally, Babesia species can be identified on the basis of the morphology of the intra-erythrocytic forms and are classified either as large (3–5 μm) or small (1.5–2.5 μm) piroplasms (Solano-Gallego and Baneth 2011). Canine babesiosis is one of the most important tick-borne infections of dogs and was long thought to be caused by the species of Babesia canis (large Babesia) and B. gibsoni (small Babesia) (Boozer and Macintire 2003). According to the differences in antigenic properties, geographical distribution and vector specificity, large canine piroplasms are further classified into three subspecies, namely B. canis transmitted mainly by Dermacentor reticulatus in Europe, B. vogeli transmitted mainly by Rhipicephalus sanguineus in tropical and subtropical regions, and B. rossi transmitted mainly by Haemaphysalis leachi (now H. elliptica) in South Africa (Hauschild et al. 1995; Zahler et al. 1998; Apanaskevich et al. 2007). In addition, B. vogeli has a global distribution similar to that of its vector Rh. sanguineus throughout tropical, subtropical and Mediterranean areas (Gulander et al. 2006; Criado-Fornelio et al. 2007; Cardoso et al. 2008; Beck et al. 2009). Although the B. gibsoni had been identified from blood specimens of dogs in Taiwan (Lee et al. 2010), no clinical case of B. vogeli from dog and its vector tick have been reported in Taiwan. The vector ticks responsible for its transmission as well as other Babesia species carried by vector ticks have never been identified in Taiwan.

The brown dog tick, Rh. sanguineus, is the most widespread tick species around the world and is recognized as the dominant ectoparasite of dogs that can occasionally parasitize other hosts, including humans (Dantas-Torres 2010). Indeed, cases of human infestation by Rh. sanguineus have also been described in various countries throughout the world (Goddard 1989; Felz et al. 1996; Manfred et al. 1999; Uspensky and Ioffe-Uspensky 2002; Dantas-Torres et al. 2006). In Europe, this tick species is responsible for the transmission of B. vogeli among dogs (Gharbi and Bouattour 2008; René et al. 2012). In Japan, ticks of the species of Haemaphysalis longicornis, H. flava, Rh. sanguineus and Ixodes ovatus were most frequently found on canine hosts, and were responsible for the abundance of tick bites and Babesia infection in canine hosts (Shimada et al. 2003; Inokuma et al. 2003). However, the abundant tick species infested on dogs and the vector competence for Babesia infections have never been thoroughly investigated in Taiwan.

The diagnosis for canine babesiosis has mainly been based on the clinical symptoms and rarely on the detection of Babesia protozoa in Giemsa-stained blood smears. Recent advances in molecular methodologies by PCR and sequence analysis have made possible the detection and phylogenetic analysis of Babesia infection in canine hosts (Birkenheuer et al. 2003; Inokuma et al. 2003; Jefferies et al. 2003; Martin et al. 2006; Gharbi and Bouattour 2008; Beck et al. 2009; René et al. 2012) and in vector ticks (Inokuma et al. 2003; Gharbi and Bouattour 2008; René et al. 2012). Their sensitivity and specificity have contributed to the detection of Babesia infection, and the differentiation of Babesia species and subspecies. Thus, the objectives of the present study are to detect the Babesia infections and to clarify the genospecies of Babesia protozoa in Rh. sanguineus ticks collected from dogs in southern Taiwan by analyzing the sequence similarity of the PCR-amplified 18S ssrRNA genes. In addition, the phylogenetic relationships of these Babesia detected in Taiwan were compared with other Babesia species identified from various biological and geographical sources which have been documented in GenBank.

Materials and methods

Collection and identification of tick specimen

All specimens of adult, nymphal and larval ticks of Rh. sanguineus were carefully removed from dogs captured at various residential sites of Dashu district in Kaohsiung City of southern Taiwan (22°43′N, 120°25′E). A total of 578 ticks (134 partially engorged females, 153 flat male and 291 partially engorged nymphs) were collected from 30 dogs, and all these ticks were subsequently stored in separate mesh-covered and plaster-bottomed vials. All tick specimens of Rh. sanguineus were identified to species level on the basis of their morphological characteristics, as described previously (Chao et al. 2009a, 2013). Ultrastructural observations by stereomicroscope were used to delineate the morphological features of all stages of Rh. sanguineus ticks in Taiwan. Briefly, tick specimens were cleaned by sonication in 70 % ethanol solution for 5–10 min and then washed twice in sterile distilled water. Afterwards, each stage of tick specimen was placed on a glass slide and photographed using a stereo-microscope (SMZ 1500, Nikon, Tokyo, Japan) equipped with a fiber lamp. The external features of the Rh. sanguineus ticks were recorded for species identification.

DNA extraction from tick specimen

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 ethanol solution and then washed twice in sterile distilled water. Afterwards, the individual tick specimen dissected into pieces was placed in a microcentrifuge tube filled with 180-μl lysing buffer solution supplied with a DNeasy Tissue Kit (Catalogue No. 69506, Qiagen, Taipei, Taiwan) and then homogenized with a TissueLyser II (Catalogue No. 85300, Qiagen, Germany), instructed by the manufacturer. The homogenate was centrifuged at room temperature and the supernatant fluid was further processed by a DNeasy Tissue Kit, as instructed by the manufacturer. After filtration, the filtrate was collected and the DNA concentration was determined spectrophotometrically with a DNA calculator (GeneQuant II; Pharmacia Biotech, Uppsala, Sweden), as described previously (Chao et al. 2009b).

DNA amplification by Babesia-specific nested polymerase chain reaction (nested-PCR)

DNA samples extracted from the tick specimens were used as a template for PCR amplification. For screening of Babesia infection, a nested PCR was performed with primers designed to amplify the specific fragment encoding the nuclear small subunit ribosomal RNA (18S ssrRNA) gene of Babesia parasites. A specific primer set of 5-22F (5′-GTTGATCCTGCCAGTAGT-3′) and 1661R (5′-AACCTTGTTACGACTTCTC-3′) was designed and applied for the primary amplification, as described previously (Birkenheuer et al. 2003). In the nested PCR, a primer set of 455-479F (5′-GTCTTGTAATTGGAATGATGGTGAC-3′) and 793-772R (5′-ATGCCCCCAACCGTTCCTATTA-3′) was used and expected to yield approximately a 340 bp fragment depending on the Babesia strain (Birkenheuer et al. 2003). All PCR reagents and Taq polymerase were obtained and used as recommended by the supplier (Takara Shuzo, Japan). Briefly, a total of 0.2-μmol of the appropriate primer set and adequate 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), and the primary amplification included 5 min denaturation at 95 °C followed by 35 cycles of the following conditions: denaturation at 95 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min. The nested amplification included 5 min denaturation at 95 °C followed by 40 cycles of the following conditions: denaturation at 95 °C for 45 s, annealing at 58 °C for 45 s, and extension at 72 °C for 45 s, as described previously (Birkenheuer et al. 2003). Thereafter, amplified DNA products were electrophoresed on 2 % agarose gels in Tris–Borate-EDTA (TBE) buffer and visualized under ultraviolet (UV) light after staining with ethidium bromide. A DNA ladder (1-kb plus, Catalogue No. 10787-018, Invitrogen, Taipei, Taiwan) was used as the standard marker for comparison. A negative control of distilled water was included in parallel with each amplification.

Sequence alignments and phylogenetic analysis

After purification (QIAquick PCR Purification Kit, Catalog No. 28104), sequencing reaction was performed with 25 cycles under the same conditions and same primer set (primer 455-479F and 793-772R) of nested amplification by dye-deoxy terminator reaction method using the Big Dye Terminator Cycle Sequencing Kit in an ABI Prism 377-96 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The resulting sequence was initially edited by BioEdit software (V5.3) and aligned with the CLUSTAL W software (Thompson et al. 1994). Thereafter, the aligned sequence was further analyzed by comparing with other Babesia sequences based on the type-strain of different genospecies and different geographical/biological origin of Babesia protozoa that are available in GenBank. Phylogenetic analysis was performed by neighbour-joining (NJ) compared with maximum parsimony (MP) methods to estimate the phylogeny of the entire alignment using MEGA 6.0 software package (Tamura et al. 2013). The genetic distance values of inter- and intra-species variations of Babesia protozoa were also analyzed by the Kimura two-parameter model (Kimura 1980). All phylogenetic trees were constructed and performed with 1000 bootstrap replications to evaluate the reliability of the construction, as described previously (Felsenstein 1985).

Nucleotide sequence accession numbers

The nucleotide sequences of PCR-amplified nuclear small subunit ribosomal RNA (18S ssrRNA) genes from six Babesia strains of Taiwan determined in this study have been registered and assigned the following GenBank accession numbers: strains 98KH-DS11-F (KT438553), 98KH-DS11-M (KT438554), 98KH-DS12-F (KT438555), 99KH-DS01-EN (KT438556), 99KH-DS01-EN2 (KT438557) and 99KH-DS01-M (KT438558), respectively. For phylogenetic analysis, the nucleotide sequences of 18S small subunit ribosomal RNA genes from other 18 strains of Babesia and 1 strain of other protozoa (i.e. Toxoplasma gondii) were included for comparison and their GenBank accession numbers are shown in Table 1.

Table 1 Babesia and protozoa strains used for phylogenetic analysis in this study
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Results

Detection of Babesia infection by nested-PCR in various stages of Rhipicephalus sanguineus ticks

To verify the existence of Babesia infection in Rh. sanguineus ticks collected from dogs of southern Taiwan, a total of 578 ticks (134 partially engorged females, 153 flat males and 291 partially engorged nymphs) were collected and examined for the evidence of Babesia infection by nested-PCR using specific primers targeting the nuclear small subunit ribosomal RNA (18S ssrRNA) genes of Babesia parasites. Results indicate that Babesia infections were evidenced in male, female and nymphal stage of Rh. sanguineus ticks with an infection rate of 1.31 % (2/153), 1.49 % (2/134) and 0.69 % (2/291), respectively (Table 2). The overall infection rate was detected in 1.04 % (6/578) of Rh. sanguineus ticks.

Table 2 Detection of Babesia infection within various life-cycle stages of Rhipicephalus sanguineus ticks by nested-PCR assay targeting the 18S ssrRNA gene of Babesia protozoa
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Sequence analysis and genetic identification of detected Babesia protozoa

To clarify the genetic identity of Babesia parasites detected in Rh. sanguineus ticks collected from dogs of southern Taiwan, sequences of PCR-amplified 18S ssrRNA gene fragments of 6 strains of detected Babesia from Kaohsiung City of Taiwan were aligned and compared with the downloaded sequences of known genospecies of 18 strains of Babesia species and 1 strain of other protozoa. Results indicate that these detected Babesia strains of Taiwan were genetically affiliated to the genospecies of B. vogeli and were verified with a high sequence homology (97.3–99.4 % similarity) within the genospecies of B. vogeli, and can be discriminated from other genospecies of Babesia protozoa (<88.1 % similarity) (data not shown). Intraspecies analysis based on the genetic distance values indicates a lower level (<0.008) of genetic divergence (GD) within the genospecies of B. vogeli detected in various geographical areas, and all these Taiwan strains were genetically affiliated but more distant (GD 0.008–0.027) to the same genospecies of B. vogeli. Interspecies analysis also reveals that these Taiwan strains have a higher heterogeneity (GD > 0.079) distinguished from B. canis, other genospecies of Babesia (GD > 0.094) and outergroup protozoa (GD > 0.236) (Table 3).

Table 3 Inter- and intra-species analysis of genetic distance valuesa based on the 18S ssrRNA gene sequences among Babesia strains detected in Rhipicephalus sanguineus ticks from dogs in Taiwan, other sources and genospecies of Babesia strains around the world, and Toxoplasma protozoa
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Phylogenetic analysis of detected Babesia protozoa

Phylogenetic relationships based on the alignment of 18S ssrRNA gene sequences were also performed to analyze the genetic divergence among 24 Babesia strains and 1 protozoa strain investigated in this study. Phylogenetic trees constructed by both NJ (Fig. 1) and MP (Fig. 2) analyses showed congruent basal topologies with eight major branches of distinguished clades. All Babesia strains detected in Kaohsiung of Taiwan represent one monophyletic group closely affiliated to the genospecies of B. vogeli, and can be distinguished clearly from other Babesia genospecies by both neighbour-joining and maximum parsimony methods (Figs. 1, 2). The phylogenetic analysis of NJ and MP trees strongly support the separation of different lineages between the Babesia strains from Taiwan and other genospecies of Babesia with a bootstrap value of 100 and 99, respectively.

Fig. 1
figure 1

Phylogenetic relationships based on the 18S small subunit-ribosomal RNA (18S ssrDNA) gene sequences between six Babesia strains detected in Rhipicephalus sanguineus ticks of southern Taiwan and 18 other strains belonging to seven genospecies of Babesia species, and 1 strain of T. gondii served as outgroup comparison. The trees were constructed and analyzed by neighbour-joining (NJ) method using 1000 bootstraps 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. 2
figure 2

Phylogenetic relationships based on the 18S small subunit-ribosomal RNA (18S ssrDNA) gene sequences between six Babesia strains detected from Rhipicephalus sanguineus ticks of southern Taiwan and 18 other strains belonging to seven genospecies of Babesia species, and 1 strain of T. gondii served as outgroup comparison. The trees were constructed and analyzed by maximum parsimony (MP) method using 1000 bootstraps 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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Discussion

This report conducts the first survey for detecting of Babesia infection and provides the first convincing evidence of B. vogeli identified from various stages of Rh. sanguineus ticks removed from dogs in southern Taiwan. In previous investigations, B. vogeli has been detected and identified from dogs of Europe, Africa, Asia, Australia and South America (Jefferies et al. 2003; Oyamada et al. 2005; Passos et al. 2005; Gulander et al. 2006; Martin et al. 2006; Criado-Fornelio et al. 2007; Sasaki et al. 2007; Cardoso et al. 2008; Solano-Gallego et al. 2008; Gharbi and Bouattour 2008; Beck et al. 2009; René et al. 2012). Although Rh. sanguineus tick was recognized as the common vector tick ectoparasitizing on dogs, detection and identification of B. vogeli from Rh. sanguineus ticks was only described in southern France and Tunisia (Gharbi and Bouattour 2008; René et al. 2012). In Japan, B. vogeli was said to be detected by PCR in various tick species including larval tick of Haemaphysalis sp., larval and nymphal ticks of H. longicornis, and female ticks of H. flava and Rh. sanguineus (Inokuma et al. 2003). However, there is no evidence indicating the vector competence of Rh. sanguineus for B. vogeli and no documented GenBank sequences of B. vogeli from Rh. sanguineus tick of Japan were available for verification. Thus, our study demonstrates the first molecular evidence confirming B. vogeli within Rh. sanguineus ticks collected in southern Taiwan and provides the first convincing sequences (GenBank accession numbers: KT438553–KT438558) of B. vogeli detected from Rh. sanguineus ticks in Asia.

Rhipicephalus sanguineus is considered as an effective vector of B. vogeli to animals or humans. The geographical prevalence of Babesia parasites is believed to be consistent with the distribution of its vector tick. Although Rh. sanguineus has been incriminated as the vector for the transmission of B. canis and B. vogeli to dogs (Inokuma et al. 2003; Gulander et al. 2006; Criado-Fornelio et al. 2007; Cardoso et al. 2008; Gharbi and Bouattour 2008; Beck et al. 2009; René et al. 2012), there is only little evidence of the genetic identity of B. vogeli in Rh. sanguineus ticks (Gharbi and Bouattour 2008; René et al. 2012). Results from the previous studies also revealed the differential prevalence of B. vogeli with an infection rate of 0.6 % (1/160) and 9.6 % (8/83) in adult Rh. sanguineus collected from Tunisia and southern France, respectively (Gharbi and Bouattour 2008; René et al. 2012). In the present study, B. vogeli can be detected and identified in various stages (i.e. nymph and adult) of Rh. sanguineus ticks with an overall infection rate of 1.04 % (6/578) collected from stray dogs in southern Taiwan (Table 2). However, no direct evidence of B. vogeli detected in domestic or stray dogs has been reported in Taiwan. Accordingly, the vector competence of Rh. sanguineus tick for the transmission of Babesia parasites to animals or humans remains undetermined in Taiwan and needs to be further identified.

The natural transmission of Babesia parasites to vertebrate hosts occurs mainly through the bite of an infective tick. Previous investigations have indicated that B. gibsoni (a small form of Babesia) is highly prevalent in canine hosts of Southeast Asia, Australia and USA, and the infection can be transmitted by tick bites or directly from dog to dog (Birkenheuer et al. 2005; Jefferies et al. 2007; Konishi et al. 2008; Yeagley et al. 2009). In Southeast Asia, B. gibsoni infection can be commonly observed in both fighting and non-fighting dog breeds, and tick bites appear to be the most common route of transmission (Konishi et al. 2008). However, dogs with history or evidence of fighting bites are much more likely to be infected with B. gibsoni in Australia and USA (Birkenheuer et al. 2005; Jefferies et al. 2007; Yeagley et al. 2009). In Taiwan, B. gibsoni infection is observed in domestic dogs and no evidence of fighting bites occurred among dogs (Lee et al. 2010). In this study, the canine hosts are commonly infested by Rh. sanguineus ticks and the infection of B. vogeli was detected in various stages of Rh. sanguineus ticks. These evidences indicate the high possibility of transmission by an infective tick-bite in Taiwan. In addition, detection of B. vogeli in flat male ticks may imply the possible mechanism of transstadial transmission of B. vogeli from nymphal stage in the tick life-cycle to adult. Nevertheless, the origin of Babesia infection to dogs is mainly attributed to the infective bite by a suitable vector tick.

Genetic identiity and phylogenetic relationships among Babesia species can be determined by analyzing their sequence homogeneity of the nuclear small subunit-ribosomal RNA gene (18S ssrDNA). Genetic heterogeneity of B. canis strains was first demonstrated on the basis of chromosomal profiles of two laboratory strains (Depoix et al. 2002). Recently, PCR-based molecular analysis is particularly useful for detection of infection in dogs with a low parasitemia levels and for speciation of Babesia species. Indeed, PCR amplification of the 18S ssrDNA gene had been proved useful for the detection of Babesia infection and to evaluate the taxonomic relationship of Babesia species from canine hosts (Birkenheuer et al. 2003; Inokuma et al. 2003; Jefferies et al. 2003; Martin et al. 2006; Gharbi and Bouattour 2008; Beck et al. 2009; René et al. 2012) and from vector ticks (Inokuma et al. 2003; Jefferies et al. 2003; Gharbi and Bouattour 2008; René et al. 2012). Based on the genetic and phylogenetic heterogeneity, B. canis was reclassified into three sub-species and was recognized as the separate species belonging to the genera Babesia (Irwin 2009; Solano-Gallego and Baneth 2011). Results from the present study also verify that the genetic identity of Babesia strains detected in Rh. sanguineus ticks of Southern Taiwan is highly homogeneous within the genospecies of B. vogeli, and was clearly distinguished from other species of Babesia parasites. Intraspecies analysis based on the genetic distance (GD) values reveal that B. vogeli detected in Rh. sanguineus ticks of Southern Taiwan was genetically more distant (GD > 0.008) to the same species of B. vogeli identified in dogs from various countries (Table 3). The phylogenetic trees constructed by either NJ or MP analysis also strongly support the discrimination recognizing the separation of different lineages between the Babesia strains from Taiwan and other genospecies of Babesia detected from various biological and geographical sources (Figs. 1, 2). Accordingly, these observations demonstrate that the genetic identities of Babesia strains detected within Rh. sanguineus ticks collected from Southern Taiwan were verified as the genospecies of B. vogeli.

In conclusion, this report provides the first molecular evidence regarding the existence of B. vogeli in Rh. sanguineus ticks collected from dogs in southern Taiwan. The genetic identity of the detected Babesiae in Taiwan reveals a high sequence homology with B. vogeli. Because of the ability of dogs serving as carriers for vector ticks, determination of the prevalence of Babesia infection in infested Rh. sanguineus ticks ectoparasitized on dogs is a pre-requisite for the prevention of canine babesiosis. Further investigations focusing on the detection of Babesia species within different vector ticks parasitizing stray and domestic dogs would help to clarify the significance of genetic diversity of Babesia species in Taiwan.