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Article

Molecular Screening and Genetic Identification of Anaplasma platys in Brown Dog Tick (Rhipicephalus sanguineus s. l.) Infested on Stray Dogs in Taiwan

by
Li-Lian Chao
1,
Pei-Yin Ko
2 and
Chien-Ming Shih
1,2,3,*
1
M.Sc. Program in Tropical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807378, Taiwan
2
Graduate Institute of Pathology and Parasitology, National Defense Medical Center, Taipei 114201, Taiwan
3
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807377, Taiwan
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(9), 1779; https://doi.org/10.3390/microorganisms12091779
Submission received: 31 July 2024 / Revised: 15 August 2024 / Accepted: 26 August 2024 / Published: 28 August 2024
(This article belongs to the Special Issue One Health Research on Zoonotic Tick-Borne Pathogens)
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Abstract

:
Anaplasma platys is a tick-borne zoonotic pathogen of canines. In this study, the presence of A. platys was screened for in brown dog ticks (Rhipicephalus sanguineus s. l.) infesting stray dogs in Taiwan to determine overall prevalence. This study represents the first instance of genetic identification of A. platys in brown dog ticks in Taiwan. In total, we examined 324 brown dog ticks for A. platys infection by nested polymerase chain reaction assay targeting the 16S ribosomal RNA gene. The general prevalence of A. platys infection was 3.1%, with 3.6%, 4.0%, and 2.1% in nymph, female, and male ticks, respectively. Monthly prevalence of infection was observed from May to September. Genetic relatedness was determined by comparing the sequences of the 16S rRNA gene obtained from six Taiwan strains and seventeen other strains, representing six genospecies of Anaplasma spp. and three outgroups (Ehrlichia canis, Rickettsia rickettsia, and Escherichia coli). All Taiwan specimens were shown to genetically belong to the A. platys group, and could be clearly discriminated from other Anaplasma spp. Genetic similarities revealed a 100% identity match with various A. platys documented in GenBank. This study highlights the epidemiological importance of geographical transmission of A. platys among dogs and the possible risk for human infections in Taiwan.

1. Introduction

Anaplasma platys (formerly Ehrlichia platys) is a Gram-negative intracellular rickettsiae that resembles a blue intraplatelet organism in a stained blood smear. It is recognized as the etiological agent for cyclic thrombocytopenia in canines [1,2,3]. This canine infection has been demonstrated around the world, distributed across various countries in North America [4,5], Central and South America [6,7,8], Europe [9,10,11,12,13], and Asia [14,15,16,17,18,19]. Human infections with A. platys have also been reported, based on the blood of infected persons [7,20,21,22]. The brown dog tick (Rhipicephalus sanguineus s. l.) has been considered the primary vector for the transmission of A. platys [23,24,25]. Therefore, epidemiological surveying of this zoonotic agent in brown dog ticks is essential to realize the possible risk of emerging tick-borne A. platys infections in the Taiwan area.
The brown dog tick is a bloodsucking arthropod that has emerged as the dominant ectoparasite infesting canines in tropical and subtropical countries [26,27]. In addition, this tick species has been identified as a major vector for various zoonotic tick-borne pathogens, including Babesia spp., Ehrlichia spp., and Rickettsia spp., that are transmitted among vertebrate hosts [28,29,30]. The increasing detection of Babesia and Rickettsia infections in brown dog ticks in Taiwan [31,32,33,34] and the associated medical and veterinary importance of these infections have resulted in increased research attention focusing on investigating zoonotic tick-borne pathogens in brown dog ticks in Taiwan. Although this tick species has been recognized as a primary vector for various zoonotic tick-borne pathogens, the prevalence of A. platys infection and the genetic identity of this pathogen have never been identified in R. sanguineus ticks in Taiwan.
Molecular analysis of genetic relatedness by comparing the sequence variation of individual base-pairs provides direct evidence for determining the genetic divergence between and within species of Rickettsia [35,36]. Indeed, previous studies according to the 16S rRNA gene of the A. platys strains identified from dogs have revealed that it is instructive for the phylogenetic analysis of the genetic relatedness of Anaplasma pathogens from different areas [10,11,12,13,14,15,16,17,18]. Therefore, phylogenetic analysis according to the genetic comparison of the 16S rRNA gene has made it feasible to determine the genetic relatedness of Anaplasma spp. within vector ticks.
The proposed aims of the present study are to screen for Anaplasma infection in brown dog ticks (R. sanguineus s. l.) infesting stray dogs in Taiwan, and to identify the Anaplasma spp. by comparing with the existing common genospecies of Anaplasma recorded from different geographical areas and biological origins in GenBank.

2. Materials and Methods

2.1. Collection and Identification of Tick Species

All R. sanguineus ticks infesting stray dogs were collected monthly from several localities in the Wanhua district of Taipei City, located in northern Taiwan. All partially engorged ticks were collected from infested dogs and subsequently stored in separate glass vials. The tick species was identified according to the pictorial keys of R. sanguineus s. l. [31]. The morphological structures of the R. sanguineus s. l. ticks were observed with a Nikon stereo-microscope (Model SMZ 1500, Tokyo, Japan) and photographed for species identification. Molecular identification by targeting the 16S mitochondrial DNA was also performed for species identification, as described previously [31].

2.2. Genomic DNA Extraction

In this study, each tick specimen was used for extraction of total genomic DNA. Individual tick specimens were cleaned by sonication for 3–5 min in 75% ethanol solution and then washed twice with sterile distilled water. Thereafter, the individual tick specimens were placed in an Eppendorf microtube containing 180 μL lysing buffer supplied from a DNeasy Blood & Tissue Kit (catalogue no. 69506, Qiagen, Taipei, Taiwan) and then homogenized with a TissueLyser II apparatus (catalogue no. 85300, Qiagen, Hilden, Germany). After centrifugation, the supernatant fluid was further processed by a DNeasy Blood & Tissue Kit. Thereafter, the DNA-containing fluid was collected, and the DNA concentration was quantitated with an Epoch spectrophotometer (Biotek, Winooski, VT, USA). All extracted DNA specimens were stored in a freezer (−80 °C) until further assays were due to be performed.

2.3. Molecular Screening for Anaplasma Infection in Collected Ticks

Extracted DNA specimens from each tick were used as a DNA template for screening of Anaplasma infection by PCR assay. According to the 16S rRNA gene of Anaplasma, two primer sets were synthesized and used for nested PCR assays. Firstly, the primary DNA product was amplified by using the initial primer set of ECC (5′-AGAACGAACGCTGGCGGCAAGC-3′) and ECB (5′-CGTATTACCGCGGCTGCTGGCA-3′). Thereafter, the species-specific primer set of EPLAT5 (5′-TTTGTCGTAGCTTGCTATGAT-3′) and EPLAT3 (5′-CTTCTGTGGGTACCGTC-3′) targeting the DNA fragment of Anaplasma platys was used as the secondary primer set for amplifying a DNA product approximately 359 bp in length [37,38]. Each 25 μL reaction mixture contained 2.5 μL 10× PCR buffer (Mg2+), 2 μL dNTP mixture (10 mM each), 1.5 μL each of the forward and reverse primers, 1 unit of Taq DNA polymerase (Takara Shuzo Co., Ltd., Tokyo, Japan), 3 μL DNA template, with the remaining volume made up with an adequate volume of ddH2O. In addition, the reaction mixture without the DNA template was used as a negative control. All PCR amplifications were performed in a thermocycler (Veriti, AppliedBiosystems, Taipei, Taiwan) and the initial PCR assay was performed under a pre-cycling condition of denaturation at 94 °C for 3 min, followed by 35 cycles under the conditions of denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. The nested PCR assay was performed under the same pre-cycling condition and followed by 35 cycles with the same conditions of denaturation and annealing, but extension was thereafter performed at 72 °C for 90 s, and a final extension step at 72 °C for 2 min was also added.
All resulting PCR products were loaded in wells of 1.5% agarose gel and were electrophoresed in Tris-Borate-EDTA buffer. After staining with ethidium bromide, the expected DNA bands were visualized under ultraviolet light. For measuring the molecular size, a 100-bp DNA ladder (GeneRuler, Thermo Scientific, Hsinchu, Taiwan) was used as the standard marker.

2.4. Gene Sequencing and Phylogenetic Analysis of Genetic Relatedness

For gene sequencing, 10 μL of each selected specimen with an expected band on the agarose gel was prepared for gene sequencing. Briefly, the sequencing process was conducted with 25 cycles of nested amplification using the Big Dye Terminator Cycle Sequencing Kit (Mission Biotech Co., Ltd., Taipei, Taiwan) under a DNA Sequencer (ABI Prism 377-96, Applied Biosystems, Foster City, CA, USA), as described previously [34]. For phylogenetic analysis, the initial sequences were edited using BioEdit software (V5.3) and aligned with the CLUSTAL W software (version X) [39]. Therefore, the aligned sequences of six Taiwan strains were compared with available sequences from GenBank, including fourteen strains of Anaplasma spp. and three outgroup strains documented from various geographical areas and biological origins. To estimate the entire alignment, the neighbor-joining (NJ) method was performed and compared with the maximum likelihood (ML) method using the MEGA X software package (http://www.ebi.ac.uk/clustalw/, accessed on 30 July 2024) [40]. The genetic distance values were also analyzed to reveal the inter- and intra-species variations using the Kimura two-parameter model, as described previously [41]. To evaluate the reliability of the construction, all phylogenetic trees were performed with 1000 bootstrap replications [42].

2.5. Registration of Nucleotide Sequences of Anaplasma platys from Taiwan

The 16S rRNA gene sequences of the six Taiwan strains of A. platys identified from brown dog ticks (R. sanguineus) of Taiwan were registered and assigned the following GenBank accession numbers: 97-TP-WH-08-sd06-EN2 (OP389147), 97-TP-WH-08-sd06-M4 (OP389149), 97-TP-WH-08-sd06-PEA2 (OP392571), 97-TP-WH-08-sd06-M10 (OP392576), 97-TP-WH-08-sd06-F4 (OP392577), and 97-TP-WH-08-sd06-F2 (OP392579). In addition, the 16S rRNA gene sequences from other fourteen strains of Anaplasma spp. and the three outgroup strains were included for comparison (Table 1).

3. Results

3.1. Screening for Anaplasma Platys Infection in Brown Dog Ticks (R. sanguineus) of Taiwan

A. platys was detected in brown dog ticks of Taiwan by nested PCR assays targeting the 16S rRNA gene. In total, 3.09% (10/324) of the examined ticks were determined to be carrying A. platys infection. In addition, A. platys infection was detected at infection rates of 3.64%, 4.03%, and 2.07% in nymph, female, and male brown dog ticks, respectively (Table 2). Monthly prevalence of A. platys infection was observed from May to September, and the prevalence was significantly different between seasons (Table 2).

3.2. Genetic Relatedness of Anaplasma platys Detected in Brown Dog Ticks (R. sanguineus) of Taiwan

The sequences of the 16S rRNA gene of six Anaplasma strains identified from brown dog ticks in Taiwan were analyzed with the 16S rRNA gene sequences from fourteen other Anaplasma strains and three outgroup strains, identified from various geographical areas and biological origins and downloaded from GenBank. Results revealed that all Taiwan strains of Anaplasma spp. detected in brown dog ticks were identified to have 100% sequence similarity with the genospecies of A. platys (Table 3). Based on the genetic distance (GD) values, inter- and intra-species analysis of 16S rRNA genes revealed no genetic variance (GD = 0) among the A. platys strains of Taiwan, as compared with the type strains of A. platys (Table 3).

3.3. Phylogenetic Analysis of Anaplasma platys Detected in Brown Dog Ticks (R. sanguineus s. l.) of Taiwan

Based on the sequence similarity of the 16S rRNA genes, phylogenetic relationship analysis was performed to reveal the genetic relatedness among the 20 strains of Anaplasma and the 3 outgroup strains. Generally, the phylogenetic relationships of the Anaplasma strains were constructed and analyzed using both the neighbor-joining (NJ) and maximum likelihood (ML) methods. Results demonstrated seven major clades of Anaplasma with congruent basal topologies that could be easily discriminated by both NJ (Figure 1) and ML analyses (Figure 2). Briefly, a monophyletic clade was constituted by all Anaplasma strains of Taiwan, revealing a genetic affiliation with the genospecies of A. platys (Figure 1 and Figure 2). In addition, there was no sequence variation within the same genospecies of A. platys detected in R. sanguineus s. l. ticks of Taiwan, but there existed higher sequence divergence from other genospecies of Anaplasma documented from various geographical areas and biological origins (Table 3).

4. Discussion

In the present study, we report the initial molecular screening of Anaplasma infection in brown dog ticks (R. sanguineus s. l.) infesting stray dogs in Taiwan, and determine the genetic identity of A. platys detected in brown dog ticks in Taiwan. In previous reports, Anaplasma platys was first described as a canine pathogen that infects the host’s platelets, and was identified as the etiological agent for canine cyclic thrombocytopenia [1,2]. This canine infection is often reported subclinically, with only mild clinical signs, throughout the world [4,5,6,8,9,10,12,13,14,15,16,17,18,19]. Severe infections have mostly been found in Europe [10,12,13]. Although the R. sanguineus tick is recognized as the primary vector for the transmission of A. platys, A. platys DNA has also been detected in other tick species, such as Dermacentor auratus from Thailand [15], and Hemaphysalia longicornis and Ixodes persulcatus from Korea [16]. However, the vector competence of these tick species requires further verification. Accordingly, our results provide the first molecular confirmation of A. platys identified in R. sanguineus ticks of Taiwan, and demonstrate the first conclusive sequences of A. platys discovered in R. sanguineus s. l. ticks of Taiwan (Table 1).
Genetic relatedness among A. platys strains can be verified by phylogenetic analysis of the genetic similarity of the 16S rRNA gene sequence. In previous studies, sequence comparison of the 16S rRNA gene of A. platys strains identified from various geographical areas and biological origins has revealed the feasibility of discriminating the phylogenetic relationships of A. platys among other genospecies of Anaplasma [5,6,7,8,12,13,14,15,16,17,18,23,25]. Indeed, Anaplasma can be classified into several genospecies. However, the A. platys species is mainly identified in canine hosts [28,30]. In the present study, genetic analysis based on phylogenetic analysis of the 16S rRNA gene sequences of Anaplasma strains detected in R. sanguineus ticks of Taiwan revealed a 100% sequence similarity with the genospecies of A. platys documented in GenBank (Figure 1 and Figure 2, Table 3). The A. platys strains of Taiwan are mainly affiliated with the A. platys strains identified from Thailand, India, China, and Mexico (Table 3). The genetic discrimination between the A. platys strains in R. sanguineus ticks of Taiwan and other genospecies of Anaplasma identified from various geographic areas and biological origins is strongly supported by phylogenetic trees constructed using either NJ or ML methods (Figure 1 and Figure 2). Therefore, this study revealed a monophyletic group of Taiwan strains, verified genetically as A. platys, detected in brown dog ticks (R. sanguineus s. l.) in Taiwan.
The seasonal variation of A. platys infection in brown dog ticks in Taiwan remains unclear. In this study, monthly prevalence of A. platys infection was observed from May to September in brown dog ticks, and the highest rate of infection was detected during the summer season (June to August) (Table 2). This result is consistent with the seasonal abundance of the tick population surveyed during the summer season, as described in our previous report [43]. Indeed, the hot climate of the summer season may enhance the searching activity of brown dog ticks for feeding opportunities on stray dogs in their natural environment. In addition, a previous study also indicated a higher prevalence of A. platys infection in dogs observed in a heavily tick infested kennel [19]. Thus, the abundance of tick population is highly associated with the prevalence of A. platys infection in either ticks or dogs.
The biological mechanisms for the natural maintenance of A. platys by R. sanguineus ticks are controversial. In a previous study, the R. sanguineus ticks failed to acquire detectable levels of A. platys from laboratory-infected dogs [44]. However, it has also been found that A. platys infection can be maintained transstadially from naturally infected nymphs into the adult stage of R. sanguineus [45]. Although it is generally accepted that transovarial transmission (TOT) by vector ticks is inefficient, persistent TOT infection of A. platys over multiple generations has been described in a laboratory colony of R. sanguineus ticks [46]. In addition, the horizontal transmission of A. platys may also occur between co-feeding ticks. Indeed, uninfected ticks can acquire the A. platys infection through feeding on blood from infected ticks parasitized on the same dog [46]. These investigations may emphasize the possible role of R. sanguineus ticks as vector ticks and reservoir hosts for the transmission of A. platys in nature.

5. Conclusions

This investigation describes the first molecular screening of A. platys detected in brown dog ticks (R. sanguineus s. l.) infesting stray dogs of Taiwan, and represents the first verification of the genetic identity of this pathogen in brown dog ticks in Taiwan. Due to the close contact between dogs and humans, further studies focused on the vectorial capacity of R. sanguineus ticks would be beneficial to realize the possible risk of human infections in Taiwan.

Author Contributions

Conceptualization, C.-M.S. and L.-L.C.; investigation, L.-L.C. and P.-Y.K.; 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 (NSTC113-2320-B-037-010; NSTC113-2923-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 the National Defense Medical Center (IACUC-11-169) and Kaohsiung Medical University (IACUC-106142).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to express our sincere appreciation for the veterinary practitioners and pet clinics of Taipei City, Taiwan for their help in the collection of ticks for this study.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Harvey, J.W.; Simpson, C.P.; Gaskin, J. Cyclic thrombocytopenia induced by a Rickettsia-like agent in dogs. J. Infect. Dis. 1978, 137, 182–188. [Google Scholar] [CrossRef] [PubMed]
  2. Dumler, J.S.; Barbet, A.F.; Bekker, C.P.; Dasch, G.A.; Palmer, G.H.; Ray, S.C.; Rikihisa, Y.; Rurangirwa, F.R. Reorganization of genera in the families Rickettsiaceae and Anaplasmaceae in the order Rickettsiales. Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and HGE agent as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 2001, 51, 2145–2165. [Google Scholar]
  3. Cohn, L.A. Ehrlichiosis and related infections. Vet. Clin. N. Am. Small Anim. Pract. 2003, 33, 863–884. [Google Scholar] [CrossRef] [PubMed]
  4. Baker, D.C.; Simpson, H.; Gaunt, S.D.; Corstvet, R.E. Acute Ehrlichia platys infection in the dog. Vet. Pathol. 1987, 24, 449–453. [Google Scholar] [CrossRef]
  5. Almazán, C.; González-Alvarez, V.H.; Fernández de Mera, I.G.; Cabezas-Cruz, A.; Rodriguez-Martinez, R.; de la Fuente, J. Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico. Ticks Tick Borne Dis. 2016, 7, 276–283. [Google Scholar] [CrossRef] [PubMed]
  6. Huang, H.; Unver, A.; Perez, M.J.; Orellana, N.G.; Rikihisa, Y. Prevalence and molecular analysis of Anaplasma platys in dogs in lara, Venezuela. Braz. J. Micrpbiol. 2005, 36, 211–216. [Google Scholar] [CrossRef]
  7. Arraga-Alvarado, C.M.; Qurollo, B.A.; Parra, O.C.; Berrueta, M.A.; Hegarty, B.C.; Breitschwerdt, E.B. Molecular evidence of Anaplasma platys infection in two women from Venezuela. Am. J. Trop. Med. Hyg. 2014, 91, 1161–1165. [Google Scholar] [CrossRef]
  8. Pesapane, R.; Foley, J.; Thomas, R.; Castro, L.R. Molecular detection and characterization of Anaplasma platys and Ehrlicia canis in dogs from northern Colombia. Vet. Microbiol. 2019, 233, 184–189. [Google Scholar] [CrossRef]
  9. Kontos, V.I.; Papadopoulos, O.; French, T.W. Naturnal and experimental canine infections with a Greek strain of Ehrlichia platys. Vet. Clin. Pathol. 1991, 20, 101–105. [Google Scholar] [CrossRef]
  10. Beaufils, J.P.; Inokuma, H.; Martin-Granel, J.; Jumelle, P.; Barbault-Jumelle, M.; Brouqui, P. Anaplasma platys (Ehrlichia platys) infection in a dog in France: Description of the case and characterization of the agent. Rev. Med. Vet 2002, 153, 85–90. [Google Scholar]
  11. Sparagano, O.A.; de Vos, A.P.; Paoletti, B.; Camma, C.; de Santis, P.; Otranto, D.; Giangaspero, A. Molecular detection of Anaplasma platys in dogs using polymerase chain reaction and reverse line blot hybridization. J. Vet. Diagn. Investig. 2003, 15, 527–534. [Google Scholar] [CrossRef]
  12. Aguirre, E.; Tesouro, M.A.; Ruiz, L.; Amusategui, I.; Sainz, A. Genetic characterization of Anaplasma (Ehrlichia) platys in dogs in Spain. J. Vet. Med. B 2006, 53, 197–200. [Google Scholar] [CrossRef] [PubMed]
  13. Cardoso, L.; Tuna, J.; Vieira, L.; Yisaschar-Mekuzas, Y.; Baneth, G. Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from the North of Portugal. Vet. J. 2010, 183, 232–233. [Google Scholar] [CrossRef] [PubMed]
  14. Motoi, Y.; Satoh, H.; Inokuma, H.; Kiyuuna, T.; Muramatsu, Y.; Ueno, H.; Morita, C. First detection of Ehrlichia platys in dogs and ticks in Okinawa, Japan. Microbiol. Immunol. 2001, 45, 89–91. [Google Scholar] [CrossRef]
  15. Parola, P.; Cornet, J.P.; Sanogo, Y.O.; Miller, R.S.; Thien, H.V.; Conzalez, J.P.; Raoult, D.; Telford III, S.R.; Wongsrichanalai, C. Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp. and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J. Clin. Microbiol. 2003, 41, 1600–1608. [Google Scholar] [CrossRef]
  16. Kim, C.M.; Yi, Y.H.; Yu, D.H.; Lee, M.J.; Cho, M.R.; Desai, A.R.; Shringi, S.; Klein, T.A.; Kim, H.C.; Song, J.W.; et al. Tick-borne rickettsial pathogens in ticks and small mammals in Korea. Appl. Environ. Microbiol. 2006, 72, 5766–5776. [Google Scholar] [CrossRef]
  17. Pinyoowong, D.; Jittapalapong, S.; Sukasawat, F.; Stich, R.W.; Thamchaipenet, A. Molecular characterization of Thai Ehrlichia canis and Anaplasma platys strains detected in dogs. Infect. Genet. Evol. 2008, 8, 433–438. [Google Scholar] [CrossRef] [PubMed]
  18. Ybañez, A.P.; Ybañez, R.H.D.; Yokoyama, N.; Inokuma, H. Multiple infections of Anaplasma platys variants in Philippine dogs. Int. J. Syst. Bacteriol. 2016, 9, 1456–1460. [Google Scholar] [CrossRef]
  19. Chang, A.C.H.; Chang, W.L.; Lin, C.T.; Pan, M.J.; Lee, S.C. Canine infectious cyclic thrombocytopenia found in Taiwan. J. Vet. Med. Sci. 1996, 58, 473–476. [Google Scholar] [CrossRef]
  20. Arraga-Alvarado, C.; Palmar, M.; Parra, O.; Salas, P. Fine structural characterization of a Rickettsia-like organism in human platelets from patients with symptoms of ehrlichiosis. J. Med. Microbiol. 1999, 48, 991–997. [Google Scholar] [CrossRef]
  21. Maggi, R.G.; Mascarelli, P.E.; Havenga, L.N.; Naidoo, V.; Breitschwerdt, E.B. Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum in a veterinarian. Parasit. Vectors 2013, 6, 103. [Google Scholar] [CrossRef] [PubMed]
  22. Breitschwerdt, E.B.; Hegarty, B.C.; Qurollo, B.A.; Saito, T.B.; Maggi, R.G.; Blanton, L.S.; Bouyer, D.H. Intravascular persistence of Anaplasma platys, Ehrlichia chaffensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasit. Vectors 2014, 7, 298. [Google Scholar] [CrossRef]
  23. Inokuma, H.; Raoult, D.; Brouqui, P. Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. J. Clin. Microbiol. 2000, 38, 4219–4221. [Google Scholar] [CrossRef] [PubMed]
  24. Shaw, S.E.; Day, M.J.; Birtles, R.J.; Breitschwerdt, E.B. Tick-borne infectious diseases of dogs. Trends Parasitol. 2001, 17, 74–80. [Google Scholar] [CrossRef]
  25. Ybanez, A.P.; Perez, Z.O.; Gabotero, S.R.; Yandug, R.T.; Matsumoto, K.; Inokuma, H. First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from dogs in Cebu, Phillipines. Ticks Tick Borne Dis. 2012, 3, 288–293. [Google Scholar] [CrossRef] [PubMed]
  26. Dantas-Torres, F. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 2010, 3, 26. [Google Scholar] [CrossRef]
  27. Walker, J.B.; Keirans, J.E.; Horak, I.G. The Genus Rhipicephalus (Acari, Ixodidae): A Guide to the Brown Ticks of the World; Cambridge University Press: Cambridge, UK, 2000. [Google Scholar]
  28. Otranto, D.; Dantas-Torres, F.; Breitschwerdt, E.B. Managing canine vector-borne diseases of zoonotic concern: Part one. Trends Parasitol. 2009, 25, 157–163. [Google Scholar] [CrossRef]
  29. Eremeeva, M.E.; Zambrano, M.L.; Anaya, L.; Beati, L.; Karpath, S.E.; Santos-Silva, M.M.; Salceda, B.; Macbeth, D.; Olguin, H.; Dasch, G.A.; et al. Rickettsia rickettsii in Rhipicephalus ticks, Mexicali, Mexico. J. Med. Entomol. 2011, 48, 418–421. [Google Scholar] [CrossRef]
  30. Dantas-Torres, F.; Chomel, B.B.; Otranto, D. Ticks and tick-borne diseases: A One Health perspective. Trends Parasitol. 2012, 28, 437–446. [Google Scholar] [CrossRef]
  31. Chao, L.L.; Shih, C.M. Molecular analysis of Rhipicephalus sanguineus (Acari: Ixodidae), an incriminated vector tick for Babesia vogeli in Taiwan. Exp. Appl. Acarol. 2016, 70, 469–481. [Google Scholar] [CrossRef]
  32. Chao, L.L.; Liao, H.T.; Ho, T.Y.; Shih, C.M. First detection and molecular identification of Babesia gibsoni from Rhipicephalus sanguineus ticks. Acta Trop. 2017, 166, 356–362. [Google Scholar] [CrossRef] [PubMed]
  33. Shih, C.M.; Chao, L.L. Molecular Detection and Genetic Identification of Rickettsia Infection in Rhipicephalus sanguineus (Acari: Ixodidae) ticks collected from Southern Taiwan. Exp. Appl. Acarol. 2021, 85, 291–304. [Google Scholar] [CrossRef]
  34. Chao, L.L.; Robinson, M.; Liang, Y.F.; Shih, C.M. First detection and molecular identification of Rickettsia massiliae, a human pathogen, in Rhipicephalus sanguineus ticks collected from Southern Taiwan. PLoS Negl. Trop. Dis. 2022, 16, e0010917. [Google Scholar] [CrossRef]
  35. Yu, X.; Jin, Y.; Fan, M.; Xu, G.; Liu, Q.; Raoult, D. Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China. J. Clin. Microbiol. 1993, 31, 83–88. [Google Scholar] [CrossRef] [PubMed]
  36. Roux, V.; Rydkina, E.; Eremeeva, M.; Raoult, D. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int. J. Syst. Bacteriol. 1997, 47, 252–261. [Google Scholar] [CrossRef] [PubMed]
  37. Mathew, J.S.; Ewing, S.A.; Murphy, G.L.; Kocan, K.C.; Cortvet, R.E.; Fox, J.C. Characterization of a new isolate of Ehrlichia platys (order Rickettsiales) using electron microscopy and polymerase chain reaction. Vet. Parasitol. 1997, 68, 1–10. [Google Scholar] [CrossRef]
  38. Murphy, G.L.; Ewing, S.A.; Whitwirth, L.C.; Fox, J.C.; Kocan, A.A. A molecular and serological syrvey of Ehrlichia canis, E. chafeensis, and E. ewingiin dogs and ticks from Oklahoma. Vet. Parasitol. 1998, 79, 325–339. [Google Scholar] [CrossRef] [PubMed]
  39. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef]
  40. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  41. Kimura, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980, 16, 111–120. [Google Scholar] [CrossRef]
  42. Felsenstein, J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985, 52, 1119–1134. [Google Scholar]
  43. Chao, L.L.; Hsieh, C.K.; Ho, T.Y.; Shih, C.M. First zootiological survey of hard ticks (Acari: Ixodidae) infesting dogs in northern Taiwan. Exp. Appl. Acarol. 2019, 77, 105–115. [Google Scholar] [CrossRef] [PubMed]
  44. Simpson, R.M.; Gaunt, S.D.; Hair, J.A.; Kocan, K.M.; Henk, W.G.; Casey, H.W. Evaluation of Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys. Am. J. Vet. Res. 1991, 52, 1537–1541. [Google Scholar] [CrossRef] [PubMed]
  45. Aktas, M.; Ozubek, S. Molecular evidence for trans-stadial transmission of Anaplasma platys by Rhipicephalus sanguineus sensu lato under field condition. Med. Vet. Entomol. 2018, 32, 78–83. [Google Scholar] [CrossRef]
  46. Snellgrove, A.N.; Krapiunaya, I.; Ford, S.L.; Stanley, H.M.; Wickson, A.G.; Hartzer, K.L.; Levin, M.L. Vector competence of Rhipicephalus sanguineus sensu lato for Anaplasma platys. Ticks Tick Borne Dis. 2020, 11, 101517. [Google Scholar] [CrossRef]
Microorganisms 12 01779 g001
Figure 1. Phylogenetic analysis based on the 16S rRNA gene. The aligned sequences of 6 Taiwan strains (indicated as ●) identified in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including 14 strains of Anaplasma spp. and 3 outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed using the neighbor-joining (NJ) method using 1000 bootstrap replicates. Numbers at the nodes indicate the percentages of reliability of the tree. Branch length is drawn proportional to the estimated sequence divergence.
Figure 1. Phylogenetic analysis based on the 16S rRNA gene. The aligned sequences of 6 Taiwan strains (indicated as ●) identified in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including 14 strains of Anaplasma spp. and 3 outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed using the neighbor-joining (NJ) method using 1000 bootstrap replicates. Numbers at the nodes indicate the percentages of reliability of the tree. Branch length is drawn proportional to the estimated sequence divergence.
Microorganisms 12 01779 g001
Microorganisms 12 01779 g002
Figure 2. Phylogenetic analysis based on the 16S rRNA gene. The aligned sequences of 6 Taiwan strains (indicated as ●) identified in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including 14 strains of Anaplasma spp. and 3 outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed using the maximum likelihood (ML) method using 1000 bootstrap replicates. Numbers at the nodes indicate the percentages of reliability of the tree. Branch length is drawn proportional to the estimated sequence divergence.
Figure 2. Phylogenetic analysis based on the 16S rRNA gene. The aligned sequences of 6 Taiwan strains (indicated as ●) identified in R. sanguineus ticks of Taiwan were compared with available sequences from GenBank, including 14 strains of Anaplasma spp. and 3 outgroup strains identified from different biological and geographical origins. The constructed tree was analyzed using the maximum likelihood (ML) method using 1000 bootstrap replicates. Numbers at the nodes indicate the percentages of reliability of the tree. Branch length is drawn proportional to the estimated sequence divergence.
Microorganisms 12 01779 g002
Table 1. Phylogenetic analysis of Anaplasma strains of Taiwan with other documented strains from GenBank.
Table 1. Phylogenetic analysis of Anaplasma strains of Taiwan with other documented strains from GenBank.
StrainOrigin of Bacterial Strain16S Gene
Accession Number a
BiologicalGeographic
Taiwan strain
97-TP-WH-08-sd06-EN2Rhipicephalus sanguineus s. l.TaiwanOP389147
97-TP-WH-08-sd06-M4Rhipicephalus sanguineus s. l.TaiwanOP389149
97-TP-WH-08-sd06-PEA2Rhipicephalus sanguineus s. l.TaiwanOP392571
97-TP-WH-08-sd06-M10Rhipicephalus sanguineus s. l.TaiwanOP392576
97-TP-WH-08-sd06-F4Rhipicephalus sanguineus s. l.TaiwanOP392577
97-TP-WH-08-sd06-F2Rhipicephalus sanguineus s. l.TaiwanOP392579
Anaplasma platysDog bloodThailandMT180539
Anaplasma platysDog bloodSaudi ArabiaMW199129
Anaplasma platysDog bloodIndiaMW440530
Anaplasma platysRhipicephalus sanguineusMexicoMW599734
Anaplasma platysTickChinaOK639189
Anaplasma platysDog bloodThailandMT211318
Anaplasma phagocytophilumDog bloodAustriaJX173652
Anaplasma phagocytophilumTickUSAAB196721
Anaplasma phagocytophilumGoatChinaHQ872464
Anaplasma capraHomo sapiensChinaKM206273
Anaplasma marginaleBoophilus microplusPhilippinesJQ839012
Anaplasma marginaleCattleChinaAJ633048
Anaplasma ovisSheep bloodChinaAY262124
Anaplasma centraleVaccine strainNetherlandAF318944
Ehrlichia canisDog bloodItalyEU439944
Rickettsia rickettsiaUnknownFranceL36217
Escherichia coliNeotype strainFranceX80725
a Bold accession numbers of GenBank were submitted by this study.
Table 2. Molecular detection of Anaplasma platys in Rhipicephalus sanguineus s. l. ticks infesting stray dogs of Taiwan by nested-PCR assay targeting the 16S ribosomal RNA gene.
Table 2. Molecular detection of Anaplasma platys in Rhipicephalus sanguineus s. l. ticks infesting stray dogs of Taiwan by nested-PCR assay targeting the 16S ribosomal RNA gene.
Collection
Month		Anaplasma platys Detected in Various Life-Stage of TickTotal
Nymph
(P/E) a		Female
(P/E) a		Male
(P/E) a		No. Positive/
No. Examined (%)
January0/20/40/40/10 (0.00)
February0/20/50/50/12 (0.00)
March0/20/100/150/27 (0.00)
April0/30/100/120/25 (0.00)
May0/21/120/101/24 (4.17)
June1/51/120/142/31 (6.45)
July1/81/121/163/36 (8.33)
August0/142/231/333/70 (4.29)
September0/60/151/161/37 (2.70)
October0/40/70/50/16 (0.00)
November0/40/60/50/15 (0.00)
December0/30/80/100/21 (0.00)
Total (%)2/55 (3.64)5/124 (4.03)3/145 (2.07)10/324 (3.09)
a P/E = No. positive/no. examined.
Table 3. Intra- and inter-group analysis of genetic distance values a based on the 16S rRNA gene sequences between the Anaplasma strains of Taiwan and other Anaplasma, Ehrlichia, Rickettsia, and E. coli strains documented in GenBank.
Table 3. Intra- and inter-group analysis of genetic distance values a based on the 16S rRNA gene sequences between the Anaplasma strains of Taiwan and other Anaplasma, Ehrlichia, Rickettsia, and E. coli strains documented in GenBank.
Bacterial Strains b1234567891011121314151617
1. 97-TP-WH-08-sd06-M4 (Taiwan)–
2. 97-TP-WH-08-sd06-EN2 (Taiwan)0.00–
3. 97-TP-WH-08-sd06-F2 (Taiwan)0.000.00–
4. 97-TP-WH-08-sd06-M10 (Taiwan)0.000.000.00–
5. 97-TP-WH-08-sd06-PRA2 (Taiwan)0.000.000.000.00–
6. 98-TP-WH-08-sd06-F4 (Taiwan)0.000.000.000.000.00–
7. Anaplasma platys (MT180539)0.000.000.000.000.000.00–
8. Ana. platys (OK639189)0.000.000.000.000.000.000.00–
9. Ana. platys (MW599734)0.000.000.000.000.000.000.000.00–
10. Ana. platys (MW440530)0.000.000.000.000.000.000.000.000.00–
11. Ana. phagocytophilum (AB1967212)0.020.020.020.020.020.020.020.020.020.02–
12. Ana. marginale (JQ839012)0.020.020.020.020.020.020.020.020.020.020.02–
13. Ana. ovis (AY262124)0.030.030.030.030.030.030.030.030.030.030.030.04–
14. Ana. capra (KM206273)0.030.030.030.030.030.030.030.030.030.030.030.050.02–
15. Ehrlichia canis (EU439944)0.060.060.060.060.060.060.060.060.060.060.060.070.060.07–
16. Rickettsia rickettsii (L36217)0.200.190.190.190.190.190.200.190.190.190.190.190.160.180.17 –
17. Escherichia coli (X80725)0.290.290.290.290.290.290.290.290.290.290.290.290.280.270.28 0.33–
a The pairwise distance calculation was performed using the Kimura 2-parameter method, as implemented in MEGA X (Kumar et al., 2018 [40]). b Strains 7–14, 15, 16, and 17 indicate the Anaplasma, Ehrlichia, Rickettsia, and E. coli strains documented in GenBank, respectively.
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Chao, L.-L.; Ko, P.-Y.; Shih, C.-M. Molecular Screening and Genetic Identification of Anaplasma platys in Brown Dog Tick (Rhipicephalus sanguineus s. l.) Infested on Stray Dogs in Taiwan. Microorganisms 2024, 12, 1779. https://doi.org/10.3390/microorganisms12091779

AMA Style

Chao L-L, Ko P-Y, Shih C-M. Molecular Screening and Genetic Identification of Anaplasma platys in Brown Dog Tick (Rhipicephalus sanguineus s. l.) Infested on Stray Dogs in Taiwan. Microorganisms. 2024; 12(9):1779. https://doi.org/10.3390/microorganisms12091779

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Chao, Li-Lian, Pei-Yin Ko, and Chien-Ming Shih. 2024. "Molecular Screening and Genetic Identification of Anaplasma platys in Brown Dog Tick (Rhipicephalus sanguineus s. l.) Infested on Stray Dogs in Taiwan" Microorganisms 12, no. 9: 1779. https://doi.org/10.3390/microorganisms12091779

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