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Article

First Molecular Survey and Genetic Characterization of Rickettsia spp. in Haemaphysalis hystricis Ticks Infesting Dogs in Taiwan

by
Chien-Ming Shih
1,2,3,4,
Xing-Ru Huang
2,
Esmeralda Erazo
1 and
Li-Lian Chao
1,2,*
1
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
2
M.Sc. Program in Tropical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
3
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan
4
Center for Tropical Medicine and Infectious Disease Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(2), 424; https://doi.org/10.3390/microorganisms13020424
Submission received: 17 January 2025 / Revised: 13 February 2025 / Accepted: 13 February 2025 / Published: 15 February 2025
(This article belongs to the Section Public Health Microbiology)
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Abstract

Rickettsia infection in Haemaphysalis hystricis ticks infesting dogs was first screened in Taiwan by nested-PCR assay targeting the citrate synthase gene (gltA) of Rickettsia. A general infection rate (3.46%) was detected in a total of 1186 examined ticks, and infection rates of 3.20%, 3.6%, and 4.27% were detected in females, males, and nymphs, respectively. The monthly prevalence of Rickettsia infection was observed from March to November, and the highest infection was detected in April (6.92%) followed by a higher infection in July (5.56%), October (4.72%), September (3.57%), and May (3.54%). The prevalence of Rickettsia infection in ticks infesting stray dogs (4.15%) is significantly higher than ticks infesting domestic dogs (1.11%) (chi-square test, p = 0.015). Genetic analysis based on the gltA gene sequences from 13 Taiwan specimens, compared with 13 genospecies of Rickettsia strains documented in GenBank, revealed that the genetic identities of these Taiwan strains were phylogenetically affiliated with the genospecies of the transitional group (R. felis) and the spotted fever group (R. aeschlimannii and R. raoultii) of Rickettsia. This study demonstrates the first molecular screening of Rickettsia spp. in H. hystricis ticks infesting dogs in Taiwan. The human pathogenic strain of R. aeschlimannii was first discovered in H. hystricis ticks infesting dogs. Because dogs serve as companion animals to humans, the presence of various Rickettsia species existing in H. hystricis ticks may pose a potential threat to human health in Taiwan.

1. Introduction

Ticks are bloodsucking ectoparasitic arachnids which are commonly observed on canine hosts around the world [1,2]. The Haemaphysalis hystricis tick has been recorded in various countries of Southeast Asia including India, Vietnam, Myanmar, Thailand, Laos, Indonesia, Japan, China, and Taiwan [3]. Infested hosts of the H. hystricis tick have been recorded on pangolin, goat, cattle, wild boar, and dogs [4,5,6,7,8]. In our previous report, the H. hystricis tick was described as the second-most dominant tick species infesting dogs in Taiwan [9]. In addition, this tick species has been detected with various tick-borne pathogens including Borrelia and Coxiella in Malaysia [10,11], Rickettsia in China [6], and Babesia in Taiwan [5,12]. Although the H. hystricis tick was suspected as a potential vector tick for various tick-borne pathogens, a molecular survey regarding the existence and monthly prevalence of Rickettsia infection in H. hystricis ticks infesting dogs has never been conducted in Taiwan.
The Rickettsia microorganism is an obligate intracellular bacterium, and four major groups (i.e., ancestral group, typhus group, transitional group, and spotted fever group) have been identified from various hosts [13,14,15]. Tick-borne rickettsial infections have been recognized as a global threat of emerging and re-emerging tick-borne diseases for humans [16,17]. The hard ticks of Ixodidae have been recognized as the major vector for transmitting Rickettsia agents and have served as reservoir hosts for amplifying Rickettsia agents [18]. Indeed, transstadial and transovarial transmission of spotted fever group (SFG) rickettsiae have been described in various species of hard ticks [19,20]. Although most SFG rickettsiae are discovered in a particular geographic location [21,22,23,24,25,26], many SFG rickettsial species have been identified from Central and South America, Australia, and Asia [27,28,29,30,31,32,33]. However, genetic identification of rickettsial agents detected in H. hystricis ticks has never been reported in Taiwan.
Molecular analysis based on the citrate synthase gene (gltA) has been proved to be a useful tool for the phylogenetic analysis of the genetic diversity of Rickettsia species [34]. Indeed, previous investigations based on the molecular marker of the gltA gene have provided reliable information for analyzing the genetic relatedness among the diversity of Rickettsia species isolated from various ticks [35,36,37]. Therefore, molecular screening for Rickettsia infection and genetic identification of Rickettsia species based on the phylogenetic analysis of the gltA gene have made it feasible to discriminate the Rickettsia species within ticks.
The objectives of the present study are to determine the monthly prevalence of Rickettsia infection in H. hystricis ticks infesting dogs in Taiwan and to identify the genetic affiliation of Rickettsia agents detected in these ticks. Phylogenetic analysis of Rickettsia strains detected in H. hystricis ticks from Taiwan was compared with other Rickettsia strains documented in GenBank that have been validated from various biological and geographical origins (Table 1).

2. Materials and Methods

2.1. Tick Collection and Species Identification

All tick specimens were collected from May 2012 to April 2013 and were carefully removed from stray/domestic dogs from various localities in six districts of Taipei city in northern Taiwan (Figure 1). A total of 1186 ticks including 461 females, 444 males, and 281 nymphs were collected. All collected ticks were subsequently stored in separate vials filled with 75% ethanol solution, and tick species were further identified based on their morphological features [3,38]. In general, the external characteristics of the H. hystricis tick were photographed for species identification for ticks collected from dogs using a dissecting microscope (SMZ 1500, Nikon, Tokyo, Japan), as described previously [39].

2.2. Genomic DNA Extraction from Tick Specimens

Tick specimens were immersed in 75% ethanol solution and cleaned for 3–5 min by sonication. After being washed twice in sterile distilled water, the total genomic DNA was extracted from the individual tick specimen. In general, each tick specimen was homogenized with a TissueLyser II apparatus (Qiagen, Hilden, Germany) using a microcentrifuge vial containing 180 μL lysing buffer solution supplied by the commercial kit (DNeasy Blood & Tissue Kit, Qiagen, Taipei, Taiwan). After centrifugation of homogenate, the supernatant fluid was further processed by a DNeasy Blood & Tissue Kit, as instructed by the manufacturer. The eluted fluid was collected for quantifying the DNA concentration with an Epoch spectrophotometer (Biotek, Winooski, VT, USA). The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of DNA, and a ratio of 1.6~1.8 was accepted for PCR analysis. The extracted DNA samples were stored at −80 °C until further assays.

2.3. Rickettsia Detection by Nested Polymerase Chain Reaction (PCR)

Rickettsia detection was performed by a nested-PCR assay using each tick extraction as a template, and two primer sets based on the citrate synthase gene (gltA) of Rickettsia were used for PCR assay. Firstly, the primer set of RpCS.877p/RpCs.1258n was used as the forward/reverse primer for amplifying the initial product of gltA. Afterward, the species-specific primer set of RpCS.896p/RpCS.1233n was performed for nested-PCR to amplify a Rickettsia-specific DNA product approximately 338 bp, as described previously [21]. The Taq polymerase and all PCR reagents were used following the instruction of the supplier (Takara Shuzo Co., Ltd., Kyoto, Japan). In general, each 25 μL reaction mixture contained 1.5 μL of forward and reverse primers, 2.5 μL of 10X PCR buffer (Mg2+), 2 μL of dNTP mixture (10 mM each), 3 μL of DNA template, and 1 unit of Taq DNA polymerase, and was filled up with ddH2O. The negative control was added with an adequate amount of sterile distilled water, and the positive control was used for our internal check. PCR amplification was conducted with a thermocycler (Veriti, Applied Biosystems, Taipei, Taiwan), and the initial PCR amplification was performed with a denaturation at 95 °C for 5 min and amplified for 35 cycles under the following conditions: denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension step at 72 °C for 3 min. For the nested-PCR assay, the following conditions were used: denaturation at 95 °C for 5 min, followed by amplification for 40 cycles under the same conditions as the initial PCR, except for annealing at 50 °C for 30 s. All amplified PCR products were electrophoresed on 1.5% agarose gels and then stained with ethidium bromide. Thereafter, the DNA bands on gels were observed in a UV box. Molecular size was determined by comparing with a standard marker of 100 bp DNA ladder (GeneRuler, Thermo Scientific, Taiwan). In parallel with each amplification, a negative control of distilled water was included.

2.4. Genetic Relatedness Determined by Phylogenetic Analysis

Each 10 μL of selected samples was submitted for DNA sequencing (Mission Biotech Co., Ltd., Taiwan). After further purification, sequencing reaction was conducted for 25 cycles (same primer set and conditions of nested-PCR assay) using the dye-deoxy terminator reaction method by the Sequencing Kit with a DNA Sequencer (ABI Prism 377, Applied Biosystems, Foster City, CA, USA). The resulting gltA sequences were first edited by BioEdit software (V5.3), and BLAST analysis was conducted to compare the GenBank nucleotide in NCBI, and then aligned with the CLUSTAL W software (Version 2.0) [40]. Thereafter, the aligned sequences of Rickettsia gltA genes from 13 Taiwan specimens were compared with other 16 Rickettsia sequences documented in GenBank (Table 1). Phylogenetic analysis was completed by using the neighbor-joining (NJ) and maximum likelihood (ML) methods to estimate the entire alignment using MEGA X software [41]. The intra- and inter-species variations in genetic distance values were analyzed by the Kimura two-parameter model [42]. The reliability of the phylogenetic trees was analyzed with 1000 bootstrap replications [43].

2.5. GenBank Accession Numbers of Submitted Nucleotide Sequences

The nucleotide sequences of the gltA genes of 13 Rickettsia strains identified from the H. hystricis ticks of Taiwan were assigned with the GenBank accession numbers (PQ212712-24) indicated in Table 1. For phylogenetic analysis, the nucleotide sequences of gltA genes from other 16 Rickettsia strains documented in GenBank were also included for comparison (Table 1).

2.6. Statistical Analysis

The percentage of Rickettsia infection between the stray dogs and domestic dogs was compared and analyzed by Pearson’s chi-square test.

3. Results

3.1. Molecular Detection of Rickettsia Infection in H. hystricis Ticks of Taiwan

Rickettsia infection was detected in H. hystricis ticks by nested-PCR assay targeting the gltA gene. In general, Rickettsia infection was detected in 3.46% (41/1186) of H. hystricis ticks, and it was detected in females, males, and nymphs of H. hystricis ticks with infection rates of 3.2% (13/461), 3.6% (16/444), and 4.27% (12/281), respectively (Table 2). The prevalence of Rickettsia infection in H. hystricis ticks infesting stray dogs (4.15%) is significantly higher than ticks infesting domestic dogs (1.11%) (Table 2). In addition, the monthly prevalence of Rickettsia infection was observed from March to November, and the highest infection rate was detected in April (6.92%) followed by a higher infection rate in July (5.56%), October (4.72%), September (3.57%), and May (3.54%) (Figure 2).

3.2. Genetic Relatedness of Rickettsia spp. Detected in H. hystricis Ticks

In this study, the aligned sequences of gltA gene fragments from 13 Rickettsia specimens of Taiwan were compared with the downloaded sequences of 16 other Rickettsia strains documented in GenBank (Table 1). Our results reveal that 11 Rickettsia strains detected in H. hystricis ticks of Taiwan were genetically affiliated with the genospecies of R. felis with a high sequence similarity of 99.7–100%, and one Rickettsia strain was affiliated with the genospecies of R. aeschlimannii and R. raoultii with sequence similarities of 100% and 99.7%, respectively (Table 3). Based on the genetic distance (GD) values of the gltA gene, the intra- and inter-species analysis revealed lower levels (GD < 0.003, <0.0, and <0.003) of genetic divergence within the Rickettsia strains of Taiwan, as compared with the type strain of R. felis, R. aeschlimannii, and R. raoultii, respectively (Table 3). However, a higher level (GD > 0.038) of genetic divergence was observed compared to other Rickettsia strains.

3.3. Phylogenetic Analysis of Rickettsia spp. Detected in H. hystricis Ticks

Based on the aligned sequences of gltA genes among 13 Taiwan strains and 16 other Rickettsia strains, phylogenetic trees were constructed by neighbor-joining (NJ) and maximum likelihood (ML) methods. The results demonstrate congruent basal topologies with eight major clades of Rickettsia that can be easily distinguished by ML analysis (Figure 3), which were also supported by NJ analysis (Supplementary Figure S1). Briefly, 11 Rickettsia strains from Taiwan constitute a monophyletic clade genetically affiliated with the genospecies of R. felis, and one Rickettsia strain of Taiwan was genetically affiliated with the genospecies of R. aeschlimannii and R. raoultii, respectively (Figure 3). Our results reveal a lower genetic divergence within the same genospecies of Rickettsia detected in H. hystricis ticks from Taiwan, but higher genetic variation among other Rickettsia groups from different biological and geographical origins.

4. Discussion

Our investigation provides the first molecular survey and genetic identification of Rickettsia species detected in H. hystricis ticks of Taiwan. In general, the Rickettsia species detected in H. hystricis ticks are genetically affiliated with the genospecies of R. felis, R. aeschlimannii, and R. raoultii (Figure 3 and Figure S1, which are different from other tick-borne pathogens discovered in previous reports described in Malaysia [9,10], China, [5] and Taiwan [4,11]. Although R. felis, R. rhipicephali, and R. massiliae have been detected in Ixodes granulatus and Rhipicephalus haemaphysaloides ticks from Taiwan [44,45,46], this study described the initial detection of R. aeschlimannii and R. raoultii in H. hystricis ticks of Taiwan. In previous reports, R. aeschlimannii has been recognized as a human pathogen found in tourists returning from Morocco and South Africa, and identified from Hyalomma marginatus and Rhipicephalus appendiculatus ticks [47,48]. Thus, this study reveals the first confirmed evidence regarding the existence of various Rickettsia species in H. hystricis ticks of Taiwan.
The genetic identity of Rickettsia spp. detected in H. hystricis ticks can be determined by the phylogenetic analysis of genetic relatedness among Rickettsia. In previous studies, sequence analysis based on the gltA gene of Rickettsia strains identified from different origins has been proved as a feasible method for determining the genetic identity of Rickettsia detected in various geographical and biological sources [13,34,35,36,37,44,45,46]. In the present study, sequence similarity based on the gltA gene of Rickettsia strains detected in H. hystricis ticks of Taiwan revealed a highly genetic homogeneity affiliated with the genospecies of R. felis (99.7–100% similarity), R. aeschlimannii (100% similarity), and R. raoultii (99.7%) (Figure 3, Table 3). The R. felis strains are mainly affiliated with the Rickettsia strain identified in the Rhipicephalus sanguineus tick of Taiwan (GenBank accession no. MT847616). However, R. aeschlimannii and R. raoultii are mainly affiliated with the Rickettsia strains identified in the Dermacentor reticulatus tick of Russia (GenBank accession no. OR687097) and the Hyalomma marginatum tick of Portugal (GenBank accession no. LC229628), respectively. The phylogenetic analysis constructed by both NJ and ML trees strongly supports the distinction between Rickettsia strains in H. hystricis ticks of Taiwan and other Rickettsia genospecies identified from various geographic and biological sources. Thus, our findings reveal the first evidence for the genetic identity of Rickettsia strains detected in H. hystricis ticks of Taiwan.
The factors accounting for the monthly prevalence of Rickettsia infection in H. hystricis ticks of Taiwan remain unclear. In this study, Rickettsia infection in H. hystricis ticks seems highly associated with the seasonal abundance of the tick population. Indeed, the adult ticks were mostly collected from the infested dogs during early spring (March) to late autumn (November) and Rickettsia infection was detected during these months (Figure 2). In addition, a significant difference in Rickettsia infection in H. hystricis ticks was observed between stray (4.15%) and domestic (1.11%) dogs (Table 2). A possible factor responsible for this variation is the total number of collected ticks from stray/domestic (915/271) dogs. Indeed, stray dogs tend to live in groups, and they usually have 5–7 dogs in a single herd, which provides an ideal contact situation for cross-infection of Rickettsia pathogens. Another possible factor described by the previous study indicates that antibiotic treatment reduces the Rickettsia and Coxiella endosymbionts within ticks [49]. In Taiwan, domestic dogs usually have routine vaccination and various treatments, including antibiotic therapy, and this process may also reduce Rickettsia infection in domestic dogs. Thus, there is an essential need for developing control strategies for reducing the population of stray dogs in Taiwan.
The actual mechanism responsible for the transmission of the Rickettsia pathogen in H. hystricis ticks remains elusive. In this study, our results revealed a higher prevalence of Rickettsia infection in male H. hystricis ticks, and this high prevalence of Rickettsia infection in male ticks may have been inherited from infected nymphs (Table 2). Indeed, previous reports have verified that Rickettsia pathogens can be preserved transstadially/transovarialy within vector ticks [19,20,50]. The co-feeding mechanism may also contribute to another possible mode of transmission, in which ticks feeding in close proximity to another infected tick on the same host may facilitate Rickettsia transmission between these ticks [51,52]. Because of the close contact between dogs and humans, our results may highlight the role of dogs serving as carrier/reservoir hosts for Rickettsia transmission in nature. Nevertheless, further studies focusing on vector competence and seasonal variation in H. hystricis ticks for various tick-borne pathogens would help to illustrate their epidemiological significance for human infection in Taiwan.

5. Conclusions

This study conducts the first molecular screening and genetic characterization of the Rickettsia agents present in H. hystricis ticks infesting dogs of Taiwan. The genetic relatedness based on the phylogenetic analyses of the gltA gene reveals its affiliation with the genospecies of R. felis, R. aeschlimannii, and R. raoultii. The human pathogenic strain of R. aeschlimannii was firstly discovered in H. hystricis ticks. Because dogs serve as companion animals to humans, this discovery of various Rickettsia agents in H. hystricis ticks may highlight the potential threat for human infections in Taiwan. Further studies for reducing the population of stray dogs in Taiwan as well as the preventive treatment with acaricide for domestic dogs will facilitate the control of ticks and tick-borne pathogens in Taiwan.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13020424/s1.

Author Contributions

Conceptualization, C.-M.S. and L.-L.C.; investigation, X.-R.H. and E.E.; formal analysis, C.-M.S. and L.-L.C.; writing of manuscript, C.-M.S. and L.-L.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in part by grants from the National Science and Technology Council (NSTC 113-2320-B-037-010; NSTC 114-2923-B-037-001), Taipei, Taiwan, Republic of China.

Institutional Review Board Statement

The collection of ticks from dogs was assisted by veterinary practitioners and approved by the Institutional Animal Care and Use Committee (IACUC) of National Defense Medical Center (IACUC-11-169) and Kaohsiung Medical University (IACUC-106142).

Data Availability Statement

The GenBank accession numbers (PQ212712-24) submitted by this study are available in GenBank after publication.

Acknowledgments

We would like to appreciate the sincere help in the collection of ticks from the veterinary practitioners and pet clinics of Taipei City, Taiwan.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microorganisms 13 00424 g001
Figure 1. Map of Taipei city in Taiwan showing the various collection sites (indicated as ★) for ticks removed from dogs. The different shades of blue color indicate the tick collection rate (■, 20–40%; ■, 5–10%; ■, <5%).
Figure 1. Map of Taipei city in Taiwan showing the various collection sites (indicated as ★) for ticks removed from dogs. The different shades of blue color indicate the tick collection rate (■, 20–40%; ■, 5–10%; ■, <5%).
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Microorganisms 13 00424 g002
Figure 2. Monthly prevalence of Rickettsia infection and number of H. hystricis ticks collected from dogs (May 2012–April 2013) in Taipei city of Taiwan.
Figure 2. Monthly prevalence of Rickettsia infection and number of H. hystricis ticks collected from dogs (May 2012–April 2013) in Taipei city of Taiwan.
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Microorganisms 13 00424 g003
Figure 3. Genospecies identification based on the citrate synthase gene (gltA) sequences of Rickettsia among 13 specimens identified from Haemaphysalis hystricis ticks of Taiwan and 16 other Rickettsia strains validated in GenBank. The Taiwan strains were affiliated with the genospecies of R. felis (indicated as ■), R. aeschlimannii (indicated as ■), and R. raoultii (indicated as ■). The phylogenetic tree was constructed by the maximum likelihood (ML) method and analyzed with 1000 bootstrap replicates. Numbers at the nodes represent the reliability of each branch in the tree. Branch length is drawn proportional to the estimated sequence divergence.
Figure 3. Genospecies identification based on the citrate synthase gene (gltA) sequences of Rickettsia among 13 specimens identified from Haemaphysalis hystricis ticks of Taiwan and 16 other Rickettsia strains validated in GenBank. The Taiwan strains were affiliated with the genospecies of R. felis (indicated as ■), R. aeschlimannii (indicated as ■), and R. raoultii (indicated as ■). The phylogenetic tree was constructed by the maximum likelihood (ML) method and analyzed with 1000 bootstrap replicates. Numbers at the nodes represent the reliability of each branch in the tree. Branch length is drawn proportional to the estimated sequence divergence.
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Table 1. Rickettsia strains from Taiwan compared with other Rickettsia species documented in GenBank.
Table 1. Rickettsia strains from Taiwan compared with other Rickettsia species documented in GenBank.
Strain/SpeciesOrigin of Rickettsia StraingltA Gene
Accession Number a
BiologicalGeographic
Taiwan strain
97-TP-SL-04-SD1-PEA5Haemaphysalis hystricisTaiwanPQ212712
97-TP-WH-08-SD2-FHaemaphysalis hystricisTaiwanPQ212713
97-NTC-XZ-08-SD1-M1-HhHaemaphysalis hystricisTaiwanPQ212714
97-NTC-XZ-08-SD2-M12-HhHaemaphysalis hystricisTaiwanPQ212715
97-TP-NH-05-SD1-PEA1-HhHaemaphysalis hystricisTaiwanPQ212716
97-TP-NH-10-SD15-PEN24-HhHaemaphysalis hystricisTaiwanPQ212717
97-TP-SL-07-SD14-PEN10-HhHaemaphysalis hystricisTaiwanPQ212718
98-TP-NH-03-SD2-M1-HhHaemaphysalis hystricisTaiwanPQ212719
98-TP-NH-09-SD2-M1-HhHaemaphysalis hystricisTaiwanPQ212720
98-TP-SL-04-SD1-PEA1Haemaphysalis hystricisTaiwanPQ212721
99-TP-BT-08-SD3-M2-HhHaemaphysalis hystricisTaiwanPQ212722
99-TP-ZS-08-SD1-M2Haemaphysalis hystricisTaiwanPQ212723
100-TP-ZS-09-SD2-PEA2-HhHaemaphysalis hystricisTaiwanPQ212724
Rickettsia felisCtenocephalides felisAustriaMF374381
Rickettsia felisBooklice from herbalsChinaMG818715
Rickettsia felisRhipicephalus sanguineusTaiwanMT847616
Rickettsia australisHumanAustraliaU59718
Rickettsia akariHumanUSAU59717
Rickettsia aeschlimanniiDermacentor reticulatusRussiaOR687097
Rickettsia raoultiiHyalomma marginatumPortugalLC229628
Rickettsia rickettsiiDermacentor andersoniUSAU59729
Rickettsia parkeriAmblyomma ovaleMexicoMK814825
Rickettsia africaeDiatomItalyMK938655
Rickettsia honeiHumanAustraliaAF022817
Rickettsia sibiricaDermacentor nuttalliUSSRU59734
Rickettsia typhiHumanUSAU59714
Rickettsia prowazekiHumanPolandU59715
Rickettsia belliiAmblyomma pseudoconcolorBrazilKX020408
Rickettsia belliiDermacentor variabilisUSAU59716
a Bold accession numbers of GenBank were submitted by this study.
Table 2. Molecular detection of Rickettsia infection in Haemaphysalis hystricis ticks infested stray/domestic dogs by nested-PCR assay targeting the gltA gene of the Rickettsia pathogen.
Table 2. Molecular detection of Rickettsia infection in Haemaphysalis hystricis ticks infested stray/domestic dogs by nested-PCR assay targeting the gltA gene of the Rickettsia pathogen.
Parasitized HostRickettsia spp. Detected in Various Life Stages of TickTotal
Partially Engorged
Female
P/E a (%)		Flat
Male
P/E a (%)		Partially Engorged Nymph
P/E a (%)		No. Positive/
No. Examined (%)
Stray dogs11/302 (3.64)15/358 (4.19)12/255 (4.71)38/915 (4.15) b
Domestic dogs2/159 (1.26)1/86 (1.16)0/26 (0)3/271 (1.11) b
Total (%)13/461 (3.2)16/444 (3.6)12/281 (4.27)41/1186 (3.46)
a P/E = No. of positive tick/no. of tick examined; b chi-square test, p = 0.015.
Table 3. Intra- and inter-group analysis of genetic distance values a based on the gltA gene sequences between the Rickettsia strains from Taiwan and other Rickettsia strains documented in GenBank.
Table 3. Intra- and inter-group analysis of genetic distance values a based on the gltA gene sequences between the Rickettsia strains from Taiwan and other Rickettsia strains documented in GenBank.
Rickettsia Strains b123456789101112131415
1. Rickettsia felis (MF374381)—
2. 100-TP-ZS-09-SD2-PEA2-Hh (Taiwan)0.000—
3. 97-NTC-XZ-08-SD2-M12-Hh (Taiwan)0.0000.000—
4. 98-TP-SL-04-SD1-PEA1 (Taiwan)0.0000.0000.000—
5. 98-TP-NH-09-SD2-M1-Hh (Taiwan)0.0000.0000.0000.000—
6. 99-TP-BT-08-SD3-M2-Hh (Taiwan)0.0030.0030.0030.0030.003—
7. Rickettsia aeschlimannii (OR687097)0.0380.0380.0380.0390.0390.042—
8. 97-TP-WH-08-SD2-F (Taiwan)0.0380.0380.0380.0390.0390.0420.000—
9. Rickettsia raoultii (LC229628)0.0380.0380.0380.0390.0390.0420.0060.006—
10. 97-TP-SL-04-SD1-PEA5 (Taiwan)0.0410.0420.0420.0420.0420.0460.0100.0100.003—
11. Rickettsia parkeri (MK814825)0.0410.0420.0420.0420.0420.0460.0030.0030.0030.007—
12. Rickettsia sibirica (U59734)0.0410.0420.0420.0420.0420.0460.0030.0030.0030.0070.000—
13. Rickettsia rickettsii (U59729)0.0530.0530.0530.0540.0540.0570.0170.0170.0170.0200.0130.013—
14. Rickettsia typhi (U59714)0.0890.0900.0900.0910.0910.0940.0820.0820.0820.0860.0860.0860.090—
15. Rickettsia bellii (KX020408)0.1940.1960.1960.1930.1930.1970.1680.1680.1680.1730.1730.1730.1690.213—
a The pairwise distance calculation was performed by the method of Kimura 2-parameter, as implemented in MEGA X (Kumar et al., 2018 [41]). b Strains 1, 7, 9, and 11–15 indicate the various Rickettsia species documented in GenBank.
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Shih, C.-M.; Huang, X.-R.; Erazo, E.; Chao, L.-L. First Molecular Survey and Genetic Characterization of Rickettsia spp. in Haemaphysalis hystricis Ticks Infesting Dogs in Taiwan. Microorganisms 2025, 13, 424. https://doi.org/10.3390/microorganisms13020424

AMA Style

Shih C-M, Huang X-R, Erazo E, Chao L-L. First Molecular Survey and Genetic Characterization of Rickettsia spp. in Haemaphysalis hystricis Ticks Infesting Dogs in Taiwan. Microorganisms. 2025; 13(2):424. https://doi.org/10.3390/microorganisms13020424

Chicago/Turabian Style

Shih, Chien-Ming, Xing-Ru Huang, Esmeralda Erazo, and Li-Lian Chao. 2025. "First Molecular Survey and Genetic Characterization of Rickettsia spp. in Haemaphysalis hystricis Ticks Infesting Dogs in Taiwan" Microorganisms 13, no. 2: 424. https://doi.org/10.3390/microorganisms13020424

APA Style

Shih, C.-M., Huang, X.-R., Erazo, E., & Chao, L.-L. (2025). First Molecular Survey and Genetic Characterization of Rickettsia spp. in Haemaphysalis hystricis Ticks Infesting Dogs in Taiwan. Microorganisms, 13(2), 424. https://doi.org/10.3390/microorganisms13020424

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