https://orcid.org/0000-0002-1061-1927
Figures
Abstract
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia rustica, Babesia vogeli, Theileria sinensis, and Theileria orientalis were detected in 25 ticks. Co-infection by A. phagocytophilum and T. sinensis was found in one Rh. (B) microplus.
Author summary
Ticks are one of the important vectors and reservoirs of multiple pathogens infecting humans and animals such as bacteria, protozoans, viruses, and helminths. Nevertheless, studies on ticks and tick-borne infections remain limited in Vietnam. That said, serological and molecular evidence of tick infections in animals and humans have been reported on several occasions in Vietnam and Southeast Asia in recent decades. The identification of ticks and tick-associated diseases has an important role to play in epidemiological investigation and in assessing the risks of disease transmission to humans and animals. Recently, MALDI-TOF MS has been used as an innovative tool for the rapid and accurate identification of alcohol-preserved ticks based on proteins from extracted legs. This procedure represents a time-cost saving and does not require expert knowledge. This goal of this study was to assess the efficiency and reliability of MALDI-TOF MS for the identification of alcohol-preserved ticks collected in Vietnam and to determine the presence of their relative pathogens. Our study revealed 97% correspondence between morphological and MALDI-TOF MS identification. The detected microorganisms that were confirmed by sequencing belonged to the Anaplasmataceae and Piroplasmida families. These findings suggested that ticks and tick-associated pathogens are likely to pose challenges to public and veterinary health in Vietnam.
Citation: Huynh LN, Diarra AZ, Pham QL, Le-Viet N, Berenger J-M, Ho VH, et al. (2021) Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam. PLoS Negl Trop Dis 15(9): e0009813. https://doi.org/10.1371/journal.pntd.0009813
Editor: Lynn Soong, University of Texas Medical Branch, UNITED STATES
Received: May 11, 2021; Accepted: September 13, 2021; Published: September 28, 2021
Copyright: © 2021 Huynh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the “Investissements d’avenir” programme, reference ANR-10-IAHU-03, the Région Provence Alpes Côte d’Azur and European ERDF PRIMI funding. LNH received a grant of PhD scholarship from IHU Méditerranée Infection. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ticks have been incriminated as the second most important vectors of human and animal infectious pathogens in the world after mosquitoes [1] and are able to transmit a wide range of pathogens, including bacteria, protozoans, viruses, and helminths [2]. In Southeast Asia (SEA), there are 104 known tick species, representing 12 genera, which is approximately 12% of all recognised and classified species [3]. Among them, Rhipicephalus sanguineus sensu lato (s.l.) are the most common ticks that parasitise dogs in SEA. These ticks are the ectoparasite vectors of bacterial and protozoal pathogens that can be transmitted to animals [4] and humans [5]. Rhipicephalus (Boophilus) microplus is an important vector of livestock pathogens [6]. Amblyomma (formerly Aponomma) varanensis, Dermacentor auratus, and Dermacentor compactus may act as vectors of infectious agents (e.g. Rickettsia spp., Anaplasma spp., Ehrlichia spp., Borrelia spp., Babesia spp. and Theileria spp.) to humans, and to domestic and wild animals in Malaysia, Laos, Thailand, and Vietnam [7–10].
In Vietnam, the agricultural sector makes up one-third of the developing nation’s economy [11], and livestock represents the second biggest contribution to household incomes after crop growing [12]. Despite the perceived food and economic benefits of livestock production, the country is potentially faced with challenges such as the emergence and re-emergence of zoonotic diseases, which can cause huge losses [13, 14]. One such example is the risk of infectious diseases spreading through the large number of dogs that are illegally imported into Vietnam from neighbouring countries for food consumption without any veterinary controls [15, 16]. In 2014, an outbreak of oriental theileriosis, which causes abortion and death, in imported cattle from Australia to Vietnam was associated with Theileria orientalis [17]. The serological detection of both Babesia bovis and Babesia bigemina parasite species transmitted by ticks has also been reported in cattle imported from Thailand [18].
Limited data is available on ticks and tick-associated pathogens in Vietnam. Nevertheless, 48 species of nine tick genera have been reported by Kolonin [19] and recently two new species of ticks of the genus Dermacentor (Dermacentor limbooliati and Dermacentor filippovae) have been described by Apanaskevich [9, 20]. Also in Vietnam, some tick-borne microorganisms have been reported in ticks and animals [19, 21–23], more precisely in Hepatozoon canis, Ehrlichia canis, and Babesia vogeli ticks [24].
In recent years, several studies have focused on acarology in Vietnam [4, 10, 25]. The correct identification of ticks is a crucial step in distinguishing tick vectors from non-vectors. The lack of reference data and standard taxonomic keys specific to Vietnamese tick species makes the morphological identification of Vietnamese ticks difficult or almost impossible. The morphological identification of tick species therefore remains a challenge for Vietnamese researchers [19]. Molecular tools have been used to overcome the limitations of morphological identification [26]. However, there are several drawbacks to these tools, which are time-consuming, expensive, and require primer-specific targeting [27–29].
Recently, the MALDI-TOF MS method has been proposed as an alternative and innovative tool to overcome the limitations of the above two methods in arthropod identification [30]. Since then, studies in several laboratories have demonstrated that MALDI-TOF MS is a remarkably robust tool for identifying many species of arthropod vectors and non-vectors [30]. The aim of this study was to identify tick species collected from domestic and wild animals in Vietnam and their associated pathogens using morphological, MALDI-TOF MS and molecular tools.
Materials and methods
Ethics statement
Ethical approval was obtained from the Institute of Malariology, Parasitology, and Entomology, Quy Nhon (IMPE-QN) on behalf of the Vietnamese Ministry of Health (approval no: 401/VSR-CT-2010, 333/CT-VSR-2018). Permission was obtained from the communal authorities for wild animals that were not listed in the Red Data Book of Vietnam, and agreement was obtained from the owners of cows, goats, and dogs.
Tick collection and morphological identification
Ticks were collected in four provinces: Quynh Luu (19o13’ N; 105o60’ E) District, Nghe An Province; Nam Giang (15o65’ N; 107o50’ E) District, Quang Nam Province; Van Canh (13o37’ N; 108o59’ E) District, Binh Dinh Province; and Khanh Vinh (12o16’ N; 108o53’ E) District, Khanh Hoa Province in Vietnam in September 2010, between April and September 2018. The map of Vietnam showing the collection sites was made with QGIS version 3.10 and the Vietnamese layers were downloaded from DIVA-GIS at the following link: https://www.diva-gis.org/datadown (Fig 1A). All engorged and non-engorged ticks were collected from the skin of domestic animals (cows, goats, and dogs) and wild animals (pangolins, wild pigs) using forceps. Ticks from wild animals were collected in a collaborative manner by rangers and trained care personnel from the Wildlife Rescue, Conservation and Development Center. Ticks were morphologically identified first at species level using dichotomous keys [9, 31] by an entomological team from the Institute of Malariology, Parasitology and Entomology, Quy Nhon, Vietnam (IMPE-QN). Ticks from the same host were counted and placed in the same tube containing 70% v/v alcohol, before being sent to the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection in Marseille, France for MALDI-TOF MS and molecular analysis. In Marseille, the morphological identification of ticks was verified by two specialists in morphological identification of ticks using a magnifying glass (Zeiss Axio Zoom.V16, Zeiss, Marly le Roi, France) and dichotomous keys. Morphological identification was carried out only if all discriminating characters had been observed.
Map of Vietnam showing tick collection sites realised with QGIS version 3.10, the layers have been uploaded to the DIVA-GIS website: https://www.diva-gis.org/datadown (A); Morphologically, the 70% alcohol tick-preserved species were collected in Vietnam over a period of 10 years: Amblyomma varanensis [♀: a, b]; Amblyomma sp. [♀: c, d]; Dermacentor auratus [♂: e, f]; Dermacentor compactus [♂: g, h]; and approximately 2 years: Rhipicephalus (B) microplus [♀: I, k]; Rhipicephalus sanguineus s.l [♂: l, m] (B).
Tick dissection and sample preparation
Ticks were individually removed from the alcohol and were rinsed and dissected with a sterile surgical blade, as previously described [32]. The four legs of each tick and the half part without legs were submitted for MALDI-TOF MS and molecular biology analysis, respectively. The remaining parts with legs were frozen and stored as samples for any further research.
DNA extraction and molecular identification of ticks
DNA from each half-tick or legs (for ticks from which we did not obtain sequences with half-tick DNA) was individually extracted using an EZ1 DNA tissue kit (Qiagen), according to the manufacturer’s recommendations, as previously described [33]. DNA was monitored with Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) and either immediately used or stored at -20°C until use.
DNA from ticks was submitted to standard PCR in an automated DNA thermal cycle to amplify a 465-base pair (bp) fragment of the mitochondrial 16S DNA gene, as described previously [34]. The 12S tick gene, amplifying about 405-bp of the mitochondrial DNA fragment, was used for all specimens for which we did not have a sequence with the 16S gene. DNA from Rh. sanguineus s.l., reared in our laboratory, was used as a positive control. Purified PCR products were sequenced as previously described [34]. The obtained sequences were assembled and analysed using the ChromasPro software (version 1.7.7) (Technelysium Pty. Ltd., Tewantin, Australia), and were then blasted against the reference sequences available in GenBank (http://blast.ncbi.nlm.nih.gov/).
MALDI-TOF MS analysis
Sample preparation.
The four legs of each tick were first put into an Eppendorf tube and dried overnight at 37°C and then put into an Eppendorf tube with 40 μL of high-performance liquid chromatography (HPLC) grade water and incubated overnight at 37°C. The legs were then crushed in a mix of 20 μL of 70% (v/v) formic acid (Sigma) and 20 μL of 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland), with glass beads (Sigma, Lyon, France), as described previously [35]. The crushed legs were centrifuged and 1 μL of the supernatant of each sample was deposited in quadruplicate onto a MALDI-TOF MS steel plate (Bruker Daltonics, Wissembourg, France). After drying at room temperature, 1μL of matrix solution composed of a saturated solution of α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile (v/v), 2. 5% trifluoroacetic acid (v/v) (Aldrich, Dorset, United Kingdom), and high performance liquid chromatography (HPLC) grade water was added [36]. The target plate was air-dried one more at room temperature before being introduced into the Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. The quality of the matrix, sample loading, and performance of the MALDI-TOF MS device were controlled using the legs of a Rh. sanguineus s.l. reared in our laboratory as a positive control.
MALDI-TOF MS parameters, spectral analysis and reference database creation.
The spectral profiles obtained from the tick legs were visualised using a Microflex LT MALDI-TOF mass spectrometer with FlexControl software (version 3.3, Bruker Daltonics). The setting parameters of the MALDI-TOF MS apparatus were identical to those previously used [32].
The FlexAnalysis v.3.3 software was used to evaluate spectral quality (smoothing, baseline subtraction, peak intensities). MS spectra reproducibility was assessed by comparing the average spectral profiles (MSP, main spectrum profile) obtained from the four spots of each tick leg, according to species, using MALDI-Biotyper v3.0 software (Bruker Daltonics) [37]. MS spectra reproducibility and specificity were assessed based on a principal component analysis (PCA) and cluster analysis (MSP dendrogram). PCA was performed using ClinProTools v2.2 with the manufacturer’s default settings. Cluster analysis was performed based on a comparison of the MSP given by MALDI-Biotyper v3.0. software with clustering according to protein mass profile (i.e., their mass signals and intensities) [37].
Based on the morphological identification, eight and seven reference spectra of Rh. sanguineus and Rh. (B) microplus, respectively, were added to our MALDI-TOF MS database. However, two, one, and one spectra of D. auratus, Am. varanensis, D. compactus, respectively, which were only identified morphologically by three tick identification specialists, were also added to our MALDI-TOF MS database. To create a database, reference spectra (MSP, Main Spectrum Profile) were created by combining the results of spectra from specimens of each species using the automated function of the MALDI-Biotyper v3.0 software (Bruker Daltonics). MSPs were created based on an unbiased algorithm using peak position, intensity, and frequency data [38]. Four tick species that could not be identified by molecular biology were temporarily added into the MS reference database to identify the remaining specimens from the same species.
Blind test for tick identification.
A blind test was performed with the remaining tick specimens not included in our MALDI-TOF MS database after the database had been upgraded with 19 MS spectra from specimens of the five tick species to determine their identification. The reliability of tick species identification was estimated using the log score values (LSVs) obtained from the MALDI-Biotyper software, which ranged from 0 to 3. These LSVs correspond to the degree of similarity between the MS reference spectra in the database and those submitted to blind tests. An LSV was obtained for each spectrum of the samples tested. According to one previous study [37], an LSV of at least 1.8 should be obtained to be considered reliable for species identification.
Detection of microorganisms.
Quantitative PCR (qPCR) was performed for screening microorganisms using specific primers and probes targeting Anaplasmataceae, Piroplasmida, Borrelia spp., Bartonella spp., Coxiella burnetii, and Rickettsia spp. PCR reactions were performed according to the manufacturer’s instructions, using a CFX96 Touch detection system (Bio-Rad). qPCR amplification was performed using the thermal profile described previously [39]. The DNA of Rickettsia montanensis, Bartonella elizabethae, Anaplasma phagocytophilum, Coxiella burnetii, Borrelia crocidurae, and Babesia vogeli were used as a positive control and DNA from Rh. sanguineus s.l from our laboratory, which were free of bacteria, were used as negative controls. The samples were considered to be positive when the cycle threshold (Ct) was strictly less than 36 [40].
All samples that were positive following qPCR were submitted to standard PCR and sequencing to identify the microorganism species. For the Rickettsia sp. positive sample, we first used a primer targeting a 630-bp fragment of the OmpA gene [35] and then another targeting a 401-bp fragment of the gltA gene [33]. Samples which were Anaplasmataceae positive following qPCR were subjected to amplifying and sequencing of a 520-bp fragment of the 23S rRNA gene [33]. Samples which were Piroplasmidae positive following qPCR were subjected to amplifying and sequencing of a 969-bp fragment of the 18S rRNA [41]. Samples which were Borrelia sp. positive following qPCR was subjected to amplifying and sequencing of a 344-bp fragment of the flaB gene [42]. The primers and probes used in this study are listed in Table 1. The obtained sequences were assembled and analysed using the ChromasPro software (version 1.7.7) (Technelysium Pty. Ltd., Tewantin, Australia), and were then blasted against the reference sequences available in GenBank (http://blast.ncbi.nlm.nih.gov/). The method used for phylogenetic tree analysis was the neighbour-joining (NJ) method with 1,000 replicates. DNA sequences were aligned using MEGA software version 7.0 (https://www.megasoftware.net/). The various statistical analyses were performed using R software version 3.4 (R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria) and ggplot packages were used to perform the graphics.
Results
Tick collection and morphological identification
A total of 1120 ticks including 334 (30%) engorged ticks were collected in four provinces of Vietnam: Nghe An, Quang Nam, Binh Dinh, and Khanh Hoa. Morphologically, ticks were identified as belonging to six species (Fig 1A), including 935 (83.5%) Rh. sanguineus s.l. collected from dogs, 174 (15.5%) Rh. (B) microplus) from cows and goats, seven (0.6%) D. auratus from pangolins, two (0.2%) Am. varanensis from wild pigs, and one (0.1%) D. compactus and one (0.1%) Amblyomma sp. from a pangolin (Table 2). Rhipicephalus sanguineus s.l. and Rh. (B) microplus were collected between April and September 2018. The other ticks were collected in September 2010. The different specimens that could not be identified by molecular biology are shown in the pictures in Fig 1B that we took using a magnifying glass (Zeiss Axio Zoom.V16, Zeiss, Marly le Roi, France).
Molecular identification of ticks
To confirm our morphological identification, 25 tick specimens were submitted to molecular analysis using the 16S rDNA gene, including eight specimens of Rh. sanguineus s.l., seven Rh. (B) microplus, seven D. auratus, one Am. varanensis, one D. compactus and one Amblyomma sp. Sequences were obtained only for the specimens of Rh. sanguineus s.l. and Rh. (B) microplus. BLAST analysis indicated that obtained sequences from Rh. sanguineus s.l. were 99.75 to 100% identical to the corresponding sequences of Rh. sanguineus s.l. (Genbank: MG651947, MG793434, KX632154) and those obtained from Rh. (B) microplus were 100% identical to the corresponding sequences of Rh. (B) microplus (Genbank: MN880401, MT462222, EU918187). Unfortunately, for the specimens morphologically identified as D. auratus, Am. varanensis, Amblyomma sp. and D. compactus, we were unable to amplify any DNA from the half-tick or legs of these tick species with PCR targeting part of the two genes (16S and 12S rDNA), despite the fact that the nanodrop had indicated that the amount of DNA contained in these samples was 7.8 to 19.4 ng/μl.
MS reference spectra analysis
The legs of 361 specimens, including 251 morphologically identified as Rh. sanguineus s.l., 99 Rh. (B) microplus, seven D. auratus, two Am. varanensis, one Amblyomma sp. and one D. compactus were randomly selected and subjected to MALDI-TOF MS analysis. Visualisation of MS spectra from all specimens using FlexAnalysis v.3.3 software showed that 91% (329) of specimens had excellent quality spectra (peak intensity > 3,000 a.u., no background noise and baseline subtraction correct) (Figs 2A and S1 and Table 2). The MS spectra of different specimens showed intra-species reproducibility and inter-species specificity, as confirmed by PCA (Figs 2B and 3B) and dendrogram (Fig 3A) analysis. PCA and dendrogram analysis showed that all specimens of the same species were grouped together or were on the same branches. Additionally, at the genus level, all specimens from the same genus were also gathered in the same part of dendrogram (Fig 3A).
The MS spectra revealed intra-species reproducibility and inter-species specificity (A); The MS spectra were compared by Principal Component Analysis (B); a.u., arbitrary units; m/z, mass-to-charge ratio.
The dendrogram was built using between one and eight representative MS spectra from six distinct tick species (A). The MS spectra of different specimens showed intra-species reproducibility and inter-species specificity as confirmed by PCA (B).
MALDI-TOF MS tick identification by blind test
The 310 MS remaining spectra of excellent quality, including 233 Rh. sanguineus s.l, 71 Rh. (B) microplus, five D. auratus and one Amblyomma sp. were queried against our reference spectra database upgraded with eight Rh. sanguineus s.l. and seven Rh. (B) microplus which were morphologically and molecularly identified, and two D. auratus, one Am. varanensis and one D. compactus identified only morphologically. The spectra of the ticks introduced in the MALDI-TOF MS database have been deposited on the website of the University Hospital Institute (UHI) under the following DOI: https://doi.org/10.35088/rbqp-g648. The blind test revealed that 100% (233) of Rh. sanguineus s.l. specimens were correctly identified as Rh. sanguineus s.l. with LSVs ranging from 1.7–2.351 with a mean of 1.976 ± 0.137, 100% (71) of Rh. (B) microplus identified with LSVs ranging from 1.705–2.346 with a mean of 1.989 ± 0.148 and 100% (five) D. auratus with LSVs of 1.949–2.396 with a mean of 2.164 ± 0.149 (Table 2). The tick identified morphologically as Amblyomma sp. was identified by MALDI-TOF MS as Am. varanensis (LSV = 1.857) (Table 2). All our specimens were identified with LSVs ranging from 1.7–2.396 with a mean of 1.982 ± 0.142 and a median of 1.971, and 97% (301) had LSVs >1.8, which is considered the threshold for identification (Fig 3B). No blind test was performed for D. compactus because of the low number of specimens.
Detection of microorganisms in ticks
A total of 361 ticks, including 260 (72%) non-engorged and 101 (28%) engorged ticks, were examined for the DNA of six microorganisms using qPCR. Thirty-nine (10.8%) were positive for at least one of the microorganisms, including Anaplasmataceae, Rickettsia spp, Borrelia spp. and Piroplasmida (Table 3). Notably, two Rh. (B) microplus specimens were co-infected with both Anaplasmataceae and Piroplasmida. No samples were positive for C. burnetii or Bartonella spp.
DNA from bacteria of the Anaplasmataceae family were detected in 18/361 (5%) of ticks by qPCR. The DNA of bacteria belonging to the Anaplasmataceae family was found in 13 (72%) Rh. (B) microplus and five (28%) Rh. sanguineus s.l. We successfully obtained seven (40%) sequences all from Rh. (B) microplus by standard PCR and sequencing using the 23S Anaplasmataceae gene amplifying a 520-pb fragment of rRNA (Table 3). A BLAST analysis showed that four of the sequences obtained were 100% identical to the corresponding sequence of Anaplasma marginale (Genbank: CP023731), one of sequences obtained was 100% identical to the corresponding sequence of Ehrlichia rustica (Genbank: KT364330), one was 99.13% identical to the corresponding sequence of Anaplasma phagocytophilum (Genbank: CP015376) and one was 100% identical to the corresponding sequence of Anaplasma platys (Genbank: CP046391).
DNA of Piroplasmida was detected in 19/361 (5.3%) of ticks by qPCR using the 5.8S rRNA gene. Of these, ten (53%) were found in Rh. sanguineus s.l. and nine (47%) were found in Rh. (B) microplus. We successfully obtained 18 (95%) sequences by standard PCR and sequencing using the 18S rRNA gene amplifying a 969-pb fragment of rRNA. The BLAST analysis of nine sequences obtained from Rh. sanguineus s.l. revealed that they were between 99.75% and 100% identical to the corresponding sequence of Babesia vogeli (GenBank: MN067709), six sequences obtained from Rh. (B) microplus were between 99.82% and 100% identical to the corresponding sequences of Theileria sinensis (GenBank: KF559355, MT271911, AB000270) and three sequences obtained from Rh. (B) microplus were between 99.88 and 100% identical to the corresponding sequences of Theileria orientalis (GenBank: MG599099) (Table 3).
Rickettsia and Borrelia sp. were detected by qPCR in one tick of Amblyomma sp. and one of Rh. sanguineus s.l., respectively. However, all the standard PCR procedures for the identification of Rickettsia and Borrelia species failed. Of the 25 ticks for which we obtained sequences of microorganisms, 16 (64%) came from engorged ticks and one tick (4%) was co-infected with A. phagocytophilum and T. sinensis. The species of microorganism, the species of tick and the state of engorgement of the ticks in which the microorganisms were detected are listed in S1 Table.
Two phylogenetic trees of Anaplasmataceae and Piroplasmida were built from the 23S rRNA and 18S rRNA genes sequences of our amplicons, respectively. These phylogenetic trees showed that the microorganisms detected in this study are close to their homologues available in GenBank (Fig 4A and 4B).
Phylogenetic tree highlighting the position of A. phagocytophilum, A. marginale, A. platys, and E. rustica identified in our study are close to their homologues available in GenBank (A). 18S rRNA gene-based phylogenetic analysis of strains identified in the present study. Phylogenetic tree highlighting the position of B. vogeli, T. sinensis, and T. orientalis relative to their correspondence available in GenBank (B).
Discussion
The correct identification of tick species and associated pathogens can contribute to improving vector control efforts adapted to the surveillance and prevention of outbreaks of tick-borne diseases. In this study, our ticks were identified using traditional methods (morphological) and then confirmed by molecular methods and MALDI-TOF MS, and the associated pathogens were researched using molecular tools. In this study, we combined these three tools to identify ticks and to search for microorganisms associated with these ticks collected in Vietnam.
In this study, the morphological identification of ticks collected in Vietnam revealed six species, including Rh. sanguineus s.l., Rh. (B) microplus, Am. varanensis, Amblyomma sp., D. auratus and D. compactus. All these species had already been reported in Vietnam [3, 19, 25] and neighbouring countries including Laos, Malaysia, Cambodia, and Thailand [3, 23, 43]. Among the Rh. sanguineus s.l. were the species most commonly found on dogs in Vietnam. This tick species is the most widely distributed worldwide and is known to be a vector of several pathogens such as Anaplasma, Rickettsia, Ehrlichia, and Babesia spp. [44, 45]. Rhipicephalus (Boophilus) microplus was collected from both cows and goats and is responsible for the transmission of livestock pathogens [6, 24]. There have been several reports of tick-borne livestock pathogens such as Anaplasma spp., Ehrlichia ruminantium, Babesia bigemina, Babesia bovis, and Theileria spp. [46–48]. However, this tick rarely bites humans [22]. Other tick species were collected from wild animals (pangolins and pigs). Several species of ticks of the genus Amblyomma have been collected from almost all species of pangolins [49, 50] and are vectors of Rickettsia, Ehrlichia spp. [51]. Recently, several studies reported Amblyomma javanense detected from pangolins in Singapore [52] and China [53], and Amblyomma compressum ticks on pangolins from Congo [54]. Our study is the first to observe Am. varanensis, Amblyomma sp. on pangolins from Vietnam. Dermacentor auratus, D. compactus are widely distributed across Sri Lanka, Bangladesh, India, and SEA including Vietnam [55, 56], and are well known vectors of Rickettsia, Coxiella burnetii, Borrelia, and Anaplasma spp. [57, 58].
Molecular techniques were used to confirm our morphological identification of tick species by amplifying a portion sequence of a 465-bp fragment 16S rRNA gene. The choice of the 16S rRNA gene was based on previous studies that reported that this gene was a reliable tool for tick identification [29, 59]. Interrogating the GenBank database with 16S rDNA sequences from Rh. sanguineus s.l and Rh. (B) microplus showed similarity with the reference sequences available in Genbank for these species that were stored in 70% alcohol for approximately two years. Conversely, we were unable to obtain sequences for all specimens that had been preserved for more than 10 years in alcohol (i.e., Am. varanensis, Amblyomma sp., D. auratus, and D. compactus) with the 16S and 12S rDNA genes. This might be due to the fact that the alcohol was not completely eliminated during extraction [60] and/or to the fact that these ticks contained blood from their host, which includes several factors that can inhibit the PCR reaction, as already reported [61].
In this study, MALDI-TOF MS was used to identify ticks collected in Vietnam from domestic and wild animals. Among the spectra of tick legs that were subjected to MS analysis, the correct identification rates (LSVs >1.8) were 97%, almost identical to the identification rate reported in other studies [32, 33, 62]. Interestingly, specimens that were not able to be identified by molecular biology were identified by MALDI-TOF MS. This confirms that the tool is reliable and accurate for the identification of ticks. Despite these numerous advantages, this technique is limited by the high cost of the device, although it can be used for clinical microbiology and mycology in addition to entomology, with no additional cost. Maintenance may be another limitation but this can be compensated for by the low cost of reagents once the device is acquired [30]. Secondly, the development of protocols, the choice of the arthropod compartment to be used, the spectra for the creation of the database and, finally, the methods and time of conservation of the arthropods can influence the performance of MALDI-TOF MS [30, 37, 63].
In this study, 10.8% of the ticks were positive for at least one of the microorganisms by qPCR, of which 16/25 (64%) of the ticks carrying DNA of microorganisms by sequencing were engorged ticks. The detection of microorganisms in engorged ticks doesn’t have the same epidemiological meaning as when detected in a questing or non-engorged attached tick. Such ticks may potentially have fed on hosts with bacteraemia, thus biasing the estimate of the actual rate of tick infestation.
The microorganisms detected in this study and confirmed by sequencing belong to the Anaplasmataceae family (A. phagocytophilum, A. marginale, A. platys, and E. rustica), which are known aetiologies of zoonotic diseases [8, 13, 64, 65]. The Piroplasmida family (B. vogeli, T. sinensis, and T. orientalis) was mainly known as the potential zoonotic pathogens [66].
Anaplasma marginale is responsible for bovine anaplasmosis and is an intracellular bacterium transmitted by tick species mainly belonging to the Rhipicephalus and Dermacentor genera [67]. The DNA and specific antibodies against A. marginale were previously reported in the blood of cattle and cows from Vietnam [23, 24]. This study is the first report of A. marginale in Rh. (B) microplus and Rh. sanguineus s.l ticks collected in Vietnam. However, A. marginale had previously been reported in cattle and cattle Rh. (B) microplus ticks in China [68], the Philippines [69] which is a neighbouring country to Vietnam, in cattle and cattle ticks in Malaysia [70], and many African countries [71].
Anaplasma platys, the aetiological agent of infectious canine cyclic thrombocytopenia and which can be transmitted by Rh. sanguineus s.l., A. platys has been recorded in China [48], Colombia [72], and detected on various ectoparasites such as Rh. (B) microplus [48] and Hyalomma dromedarii [73]. Anaplasma platys is one of the most significant tick-borne zoonotic pathogens [24, 74] and several cases of human infections have been described in Venezuela [75], Chicago [76], and South Africa [77]. Anaplasma platys has already been detected from blood specimens of cattle and dogs in Vietnam [24], but it was the first discovery in Rh. sanguineus s.l. ticks from Vietnam in our study. It had been previously detected in Rh. sanguineus s.l. in SEA [25], including in the Philippines [78], Thailand, and Malaysia [79, 80].
The pathogen A. phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) and tick-borne fever in ruminants [81]. It is rarely found in Rh. (B) microplus and is known to be transmitted by the Ixodes tick genus [82]. Of the detected tick-borne diseases, A. phagocytophilum is the most important bacterium due to its wide distribution across Europe, Asia, and North America [83, 84], with several reports of human infections [85, 86]. This is the first study reporting the detection of A. phagocytophilum in Rh. (B) microplus ticks using the molecular method in Vietnam. It has also been described in the same tick species in China [87] and Malaysia [70].
We found Candidatus Ehrlichia rustica in the Ehrlichia chaffeensis group, the agent of human monocytic ehrlichiosis [88]. Canine ehrlichiosis was first recorded in a serological study in US military dogs serving in the Vietnam war [89]. The vectors of this pathogen are Rhipicephalus, Amblyomma, Dermacentor spp. [90]. Another study from 2003 reported that Ehrlichia spp., which gathered with E. chaffeensis, was also discovered in other species, such as Haemaphysalis hystricis from wild pigs in Vietnam [22], and Ixodes sinensis in China [91].
Babesia vogeli, the agent of canine babesiosis in North and South America, is transmitted by Rh. sanguineus s.l. and is the less pathogenic species. It is a protozoan found mainly in tropical or subtropical areas of northern, eastern and southern Africa, Asia, and northern and central Australia [92]. In SEA, B. vogeli has been described in Malaysia [93] and in the Philippines [94]. The molecular evidence of B. vogeli in Rh. sanguineus s.l. collected from dogs has been reported in Vietnam [4] and in ticks collected from East and Southeast Asia [25]. The DNA of B. vogeli was detected in this study in Rh. sanguineus s.l. ticks, confirming the presence of the protozoan in Vietnam.
Theileria sinensis, the causative agent of bovine theileriosis, causes economic losses and threats to the cattle industry. Theileria sinensis is primarily distributed throughout Asia (including China, the Korean Peninsula, Japan, and Malaysia [95–97]. It was identified in Haemaphysalis qinghaiensis ticks collected from cattle and yaks in China [98]. Theileria spp. were then detected in Haemaphysalis longicornis, Hyalomma (i.e., Hy. detritum, Hy. dromedarii, Hy. a. anatolicum, Hy.a asiaticum, Hy. rufipes), and Rhipicephalus sp. [99, 100]. Besides ticks, Theileria spp. were also detected in sheep, goat, and ruminant blood samples [101]. This is the first report of T. sinensis DNA in Rh. (B) microplus in Vietnam.
Similarly, Theileria orientalis, the causative agent of oriental theileriosis, is an economically significant protozoan which infects cattle [95]. Theileria orientalis is widely distributed in countries such as Japan [102], China [103], Indonesia [104], Australia [105], and New Zealand [95]. The Theileria orientalis species has been identified in Vietnam from blood samples from cattle, water buffalo, sheep, goats and Rh. (B) microplus ticks collected from these hosts [46]. Here, we showed the presence of 3% T. orientalis in Rh. (B) microplus collected from cows. Although Rh. (B) microplus is not recorded as a vector of T. orientalis, none of the common vectors Amblyomma, Dermacentor, and Haemaphysalis spp. [106] were detected in our work.
Rickettsia spp. and Borrelia spp. detected by qPCR in this study were not amplified and sequenced to confirm their species. As previously reported, this could be caused by the higher sensitivity of qPCR than standard PCR [107].
Co-infections in ticks usually occur after a blood meal from a host co-infected with different microorganisms. In this study, we reported for the first time the co-infection by A. phagocytophilum and T. sinensis in Rh. (B) microplus ticks. The coinfection rate of 0.3% (1/361) in this study is lower those that have been reported in the Côte d’Ivoire [71], and in Mali [33].
Conclusion
Our work indicates that MALDI-TOF MS is a useful and reliable tool for the identification of alcohol-preserved tick species which have undergone different storage periods collected in Vietnam. Our database demonstrates, for the first time, the prevalence of A. platys, A. phagocytophilum, A. marginale, E. rustica, and T. sinensis pathogens in ticks collected in Vietnam. Our finding should prompt further investigation to evaluate the potential risks of ticks and tick-associated pathogens in Vietnam. Furthermore, it shows that MALDI-TOF MS may be used as an alternative tool for identifying ticks infected or uninfected by pathogens in future studies.
Supporting information
S1 Fig. Flow diagram of tick specimens which were included and analysed using MALDI-TOF MS and molecular tools.
https://doi.org/10.1371/journal.pntd.0009813.s001
(TIF)
S1 Table. The number of microorganisms were detecetd in engorged/non-engored ticks.
*: Tick was co-infections by two microoganisms.
https://doi.org/10.1371/journal.pntd.0009813.s002
(DOCX)
Acknowledgments
We are sincerely grateful to the staff of the Entomological Department of the Institute of Malariology, Parasitology and Entomology, Quy Nhon (IMPE-QN), Vietnam, especially to the staff in the experimental entomology group, for their support with specimen collection and transportation. We would also thank the insectarium team of the Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France, for their assistance and the provision of the MALDI-TOF MS and molecular biology materials.
References
- 1. Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis. 2001; 32(6): 897–928. pmid:11247714
- 2. Otranto D, Dantas-Torres F, Breitschwerdt EB. Managing canine vector-borne diseases of zoonotic concern: part one. Trends Parasitol. 2009; 25(4): 157–63. pmid:19269898
- 3. Petney TN, Kolonin GV, Robbins RG. Southeast Asian ticks (Acari: Ixodida): a historical perspective. Parasitol Res. 2007; 101: S201–05. pmid:17823829
- 4. Nguyen VL, Colella V, Iatta R, Bui KL, Dantas-Torres F, Otranto D. Ticks and associated pathogens from dogs in northern Vietnam. Parasitol Res. 2019; 118(1): 139–142. pmid:30421349
- 5. Dantas-Torres F. The brown dog tick, Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): from taxonomy to control. Vet Parasitol. 2008; 152(3–4): 173–185. pmid:18280045
- 6. Sungirai M, Moyo DZ, Clercq PD, Madder M, Vanwambeke SO, Clercq EMD. Modelling the distribution of Rhipicephalus microplus and R. decoloratus in Zimbabwe. Vet Parasitol Reg Stud Reports. 2018; 14: 41–49. pmid:31014735
- 7. Kaewmongkol G, Lukkana N, Yangtara S, Sirinarumitr T, Jittapalapong S, Fenwick SG, et al. Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand. Vet Microbiol. 2017; 201: 195–200. pmid:28284610
- 8. Koh FX, Panchadcharam C, Sitam FT, Tay ST. Molecular investigation of Anaplasma spp. in domestic and wildlife animals in Peninsular Malaysia. Vet Parasitol Reg Stud Reports. 2018; 13: 141–147. pmid:31014863
- 9. Apanaskevich MA, Apanaskevich DA. Description of New Dermacentor (Acari: Ixodidae) Species from Malaysia and Vietnam. J Med Entomol. 2015; 52(2): 156–62. pmid:26336300
- 10. Petney T, Saijuntha W, Boulanger N, Muders SV, Petney DA, Robbins RG, et al. Ticks (Argasidae, Ixodidae) and tick-borne diseases of continental Southeast Asia. Zootaxa. 2019; 4558:1. pmid:30790915
- 11.
Statista. Agriculture in Vietnam—Statistics & Facts.[cited 23 2021]. Available from: https://www.statista.com/topics/5653/agriculture-in-vietnam.
- 12.
FAO. Small family farms country factsheet,” 2018. [cited 2 Oct 2020]. Available from: http://www.fao.org/3/I8358EN/i8358en.pdf.
- 13. Dantas-Torres F, Chomel BB, Otranto D. Ticks and tick-borne diseases: a One Health perspective. Trends Parasitol. 2012; 28(10): 437–46. pmid:22902521
- 14. Kwaghe A, Teru V, Ndahi M, Usman J, Abubakar A, Iwar V. Veterinary Services as a Panacea for Agricultural Development and Increase in Nigeria’s Gross Domestic Product (GDP): A Review. International Journal of Life Sciences. 2015; 4(2): 134–46.
- 15.
Chin S. Thriving dog meat trade | The ASEAN Post. [cited 21 Mar 2021]. Available from: https://theaseanpost.com/article/thriving-dog-meat-trade.
- 16. Ngo TC, Nguyen DT, Tran HH, Morita K, Nguyen TH, Ehara M, et al. Imported Dogs as Possible Vehicles of Vibrio Cholerae O1 Causing Cholera Outbreaks in Northern Vietnam. The Open Infectious Diseases Journal. 2011; 5: 127–34.
- 17. Gebrekidan H, Nelson LG. Smith RB. Gasser, Jabbar A. An outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Parasitology. 2017; 144(6): 738–46. pmid:27938442
- 18. Sivakumar T, Dinh TBL, Phung TL, Keisuke S, Ikuo I, Naoaki Y, et al. Serological and molecular surveys of Babesia bovis and Babesia bigemina among native cattle and cattle imported from Thailand in Hue, Vietnam. J Vet Med Sci. 2018; 80(2): 333–6. pmid:29249730
- 19. Kolonin GV. Review of the Ixodid tick fauna (Acari: Ixodidae) of Vietnam. J Med Entomol. 1995; 32(3): 276–82. pmid:7616517
- 20. Apanaskevich DA, Apanaskevich MA. Description of a New Dermacentor (Acari: Ixodidae) Species from Thailand and Vietnam. J Med Entomol. 2015b; 52(5): 806–12. pmid:26336207
- 21. Petney TN, Keirans JE. Ticks of the genera Boophilus, Dermacentor, Nosomma and Rhipicephalus (Acari: Ixodidae) in South-east Asia. Tropical Biomedicine. 1996a; 13: 73–84. [cited 30 Mar 2021]. Available from: https://www.scienceopen.com/document?vid=e77f85d6-9946-4217-87e4-0dc7e1c850dd.
- 22. Parola P, Cornet JP, Sanogo YO, Raoult D, Telford SR III, Wongsrichanalai C, et al. Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol. 2003; 4(4): 1600–08.
- 23. Geurden T, Somers R, Thanh NTG, Dorny P, Giao HK, Vercruysse J, et al. Parasitic infections in dairy cattle around Hanoi, northern Vietnam. Vet Parasitol. 2008; 153(3–4): 384–88. pmid:18328629
- 24. Chien NTH, Nguyen TL, Bui KL, Nguyen VT, Le TH. Anaplasma marginale and A. platys Characterized from Dairy and Indigenous Cattle and Dogs in Northern Vietnam. Korean J Parasitol. 2019; 57(1): 43–47. pmid:30840799
- 25. Nguyen VL, Colella V, Greco G, Hadi UK, Venturina V, Tong KBY, et al. Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia. Parasit Vectors. 2020; 13(1): 420. pmid:32799914
- 26. Mediannikov O and Fenollar F. Looking in ticks for human bacterial pathogens. Microb Pathog. 2014; 77: 142–8. pmid:25229617
- 27. Lauri A, Mariani PO. Potentials and limitations of molecular diagnostic methods in food safety. Genes Nutr. 2009; 4(1): 1–12. pmid:19067016
- 28. Favrot C. Polymerase chain reaction: advantages and drawbacks. The Zurich Open Repository and Archive, University of Zurich. 2015. [cited 18 Dec 2015]. Available from:
- 29. Yssouf A, Almeras L, Raoult D, Parola P. Emerging tools for identification of arthropod vectors. Future Microbiol. 2016; 11(4): 549–66. pmid:27070074
- 30. Sevestre J, Diarra AZ, Laroche M, Almeras L, Parola P. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: an emerging tool for studying the vectors of human infectious diseases. Future Microbiol. 2021; 16: 323–40. pmid:33733821
- 31.
Berry CM. Resolution of the taxonomic status of Rhipicephalus (Boophilus) microplus. Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences University of Glasgow. 2017; 268.
- 32. Boyer PH, Almeras L, Plantard O, McCoy K, Jaulhac B, Boulanger N, et al. Identification of closely related Ixodes species by protein profiling with MALDI-TOF mass spectrometry. PLoS One. 2019; 14(10): e0223735. pmid:31622384
- 33. Diarra AZ, Almeras L, Laroche M, Doumbo O, Raoult D, Parola P, et al. Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali. PLoS Negl Trop Dis. 2017; 11(7). e0005762. pmid:28742123
- 34. Kumsa B, Laroche M, Almeras L, Mediannikov O, Raoult D, Parola P. Morphological, molecular and MALDI-TOF mass spectrometry identification of ixodid tick species collected in Oromia, Ethiopia. Parasitol. Res. 2016; 115(11): 4199–210. pmid:27469536
- 35. Boucheikhchoukh M, Laroche M, Aouadi A, Benakhla A, Raoult D, Parola P, et al. MALDI-TOF MS identification of ticks of domestic and wild animals in Algeria and molecular detection of associated microorganisms. Comp Immunol Microbiol Infect Dis. 2018; 57: 39–49. pmid:30017077
- 36. Yssouf A, Socolovschi C, Flaudrops C, Sokhna CS, Raoult D, Parola P, et al. Matrix-Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry: An Emerging Tool for the Rapid Identification of Mosquito Vectors. PLoS One. 2013; 8(8). pmid:23977292
- 37. Nebbak A, Willcox AC, Bitam I, Raoult D, Parola P, Almeras L Standardization of sample homogenization for mosquito identification using an innovative proteomic tool based on protein profiling. Proteomics. 2016; 16(24): 3148–60. pmid:27862981
- 38. Yssouf A, Parola P, Lindström A, Berenger JM, Raoult D, Almeras L, et al. Identification of European mosquito species by MALDI-TOF MS. Parasitol. Res. 2014; 113(6): 2375–78. pmid:24737398
- 39. Dahmana H, Amanzougaghene N, Davoust B, Chik M, Fenollar F, Mediannikov O, et al. Great diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species. Vet Parasitol Reg Stud Reports. 2019; 18: 100332. pmid:31796173
- 40. Lafri I, Hamzaoui BE, Bitam I, Karakellah M, Raoult D, Parola P, et al. Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria. PLoS Negl Trop Dis. 2017; 11(11): e0006064. pmid:29145396
- 41. Dahmani M, Loudahi A, Mediannikov O, Fenollar F, Raoult D, Davoust B. Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from Kabylie, Algeria. Ticks Tick Borne Dis. 2015; 6(2): 198–203. pmid:25583345
- 42. Vial L, Diatta G, Tall A, Rogier C, Renaud F, Trape JF, et al. Incidence of tick-borne relapsing fever in west Africa: longitudinal study. Lancet. 2006; 368(9529): 37–43. pmid:16815378
- 43. Vongphayloth K, Hertz JC, Lakeomany K, Robbins RG, Sutherland IW, Brey PT, et al. The Genus Dermacentor (Acari: Ixodidae) in Laos: A Review and Update of Species Records. J Med Entomol. 2018; 55(4): 1047–50. pmid:29590396
- 44. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005; 18(4): 719–56. pmid:16223955
- 45. Dantas-Torres F, Otranto D. Further thoughts on the taxonomy and vector role of Rhipicephalus sanguineus group ticks. Vet Parasitol. 2015; 208(1–2): 9–13. pmid:25579394
- 46. Khukhuu A, Dinh TBL, Phung TL, Igarashi I, Xuan X, Yokoyama N, et al. Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam. J Vet Med Sci. 2011; 73(5): 701–5. pmid:21187678
- 47. Adjou Moumouni PF, Aplogan GL, Katahira H,Wang G, Mingming Liu M, Xuan X, et al. Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin. Ticks Tick Borne Dis. 2018; 9(3): 450–64. pmid:29307783
- 48. Guo WP, Zhang B, Wang YH, Xu G, Wang X, Ni X, et al. Molecular identification and characterization of Anaplasma capra and Anaplasma platys-like in Rhipicephalus microplus in Ankang, Northwest China. BMC Infect Dis. 2019; 19(1): 434. pmid:31101084
- 49. Hassan M, Sulaiman MH, Lian CJ. The prevalence and intensity of Amblyomma javanense infestation on Malayan pangolins (Manis javanica Desmarest) from Peninsular Malaysia. Acta Trop. 2013; 126(2): 142–5. pmid:23416121
- 50. Khatri-Chhetri R, Wang HC, Chen CC, Khatri-Chhetri N, Wu HY, Pei KJC, et al. Surveillance of ticks and associated pathogens in free-ranging Formosan pangolins (Manis pentadactyla pentadactyla). Ticks Tick Borne Dis. 2016; 7(6): 1238–44. pmid:27426438
- 51. Qiu Y, Kidera N, Hayashi M, Fujishima K, Tamura H. Rickettsia spp. and Ehrlichia spp. in Amblyomma ticks parasitizing wild amphibious sea kraits and yellow-margined box turtles in Okinawa, Japan. Ticks Tick Borne Dis. 2021; 12(2): 101636. pmid:33360921
- 52. Kwak ML, Hsu CD, Douay G, Ahmad AA. The first authenticated record of the pangolin tick Amblyomma javanense (Acari: Ixodidae) in Singapore, with notes on its biology and conservation. Exp Appl Acarol. 2018; 76(4): 551–7. pmid:30298232
- 53. Jabin G, Dewan Y, Khatri H, Singh SK, Chandra K, Thakur M. Identifying the tick Amblyomma javanense (Acari: Ixodidae) from Chinese pangolin: generating species barcode, phylogenetic status and its implication in wildlife forensics. Exp Appl Acarol. 2019; 78(3): 461–7. pmid:31168752
- 54. Mediannikov O, Davoust B, Socolovschi C, Tshilolo L, Raoult D, Parola P. Spotted fever group rickettsiae in ticks and fleas from the Democratic Republic of the Congo. Ticks Tick Borne Dis. 2012; 3(5–6): 371–3. pmid:23137572
- 55. Hoogstraal G, Wassef HY. Dermacentor (Indocentor) auratus (Acari: Ixodoidea: Ixodidae): hosts, distribution, and medical importance in tropical Asia. J Med Entomol. 1985; 22(2): 170–7. pmid:3838555
- 56. Chen Z, Yang X, Bu F, Yang X, Yang X, Liu J. Ticks (acari: ixodoidea: argasidae, ixodidae) of China. Exp Appl Acarol. 2010; 51(4): 393–404. pmid:20101443
- 57. Sumrandee C, Baimai V, Trinachartvanit W, Ahantarig A. Molecular detection of Rickettsia, Anaplasma, Coxiella and Francisella bacteria in ticks collected from Artiodactyla in Thailand. Ticks Tick Borne Dis. 2016; 7(5): 678–89. pmid:26934997
- 58. Nooroong P, Trinachartvanit W, Baimai V, Ahantarig A. Phylogenetic studies of bacteria (Rickettsia, Coxiella, and Anaplasma) in Amblyomma and Dermacentor ticks in Thailand and their co-infection. Ticks Tick Borne Dis. 2018; 9(4): 963–71. pmid:29610046
- 59. Dantas-Torres F, Latrofa MS, Annoscia G, Giannelli A, Parisi A, Otranto D. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasit Vectors. 2013; 6: 213. pmid:23880226
- 60. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012; 113(5): 1014–26. pmid:22747964
- 61. Rodríguez González I, Fraga J, Noda AA, Duarte Y, Echevarria E, Fernández C, et al. An Alternative and Rapid Method for the Extraction of Nucleic Acids from Ixodid Ticks by Potassium Acetate Procedure. Braz. Arch. Biol. Technol. 2014; 57: 542–7.
- 62. Tran NHB, Nguyen HH. Tinh hinh nhiem ngoai ky sinh trung tren cho tai thanh pho Can Tho. Can Tho Junior of Science. 2014; 2: 69–73.
- 63. Nebbak A, Hamzaoui BE, Berenger JM, Raoult D, Almeras L, Parola P, et al. Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI-TOF MS. Med Vet Entomol. 2017; 31(4): 438–48. pmid:28722283
- 64. Dumler JS, Barbet AF, Bekker CP, Ray SC, Rikihisa Y, Rurangirwa FR, et al. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae 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(Pt 6): 2145–65. pmid:11760958
- 65. Heppner DG, Wongsrichanalai C, Walsh DS, Eamsila C, Hanson B, Paxton H, et al. Human ehrlichiosis in Thailand. Lancet. 1997; 350(9080): 785–6. pmid:9298007
- 66. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: a world emerging. Infect Genet Evol. 2012; 12(8): 1788–809. pmid:22871652
- 67. Battilani M, Balboni DAS A, Dondi F. Genetic diversity and molecular epidemiology of Anaplasma. Infect Genet Evol. 2017; 49: 195–211. pmid:28122249
- 68. Wen B, Jian R, Zhang Y, Chen R. Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet. J Clin Microbiol. 2002; 40(9): 3286–90. pmid:12202567
- 69. Ybañez AP, Sivakumar T, Ybañez RHD, Matsumoto K, Yokoyama N, Inokuma H, et al. First molecular characterization of Anaplasma marginale in cattle and Rhipicephalus (Boophilus) microplus ticks in Cebu, Philippines. J Vet Med Sci. 2013; 75(1): 27–36. pmid:22878542
- 70. Tay ST, Koh FX, Kho KL, Ong BL. Molecular survey and sequence analysis of Anaplasma spp. in cattle and ticks in a Malaysian farm. Trop Biomed. 2014; 31(4): 769–76. pmid:25776603
- 71. Ehounoud CB, Yao KP, Dahmani M, Raoult D, Fenollar F, Mediannikov O, et al. Multiple Pathogens Including Potential New Species in Tick Vectors in Côte d’Ivoire. PLoS Negl Trop Dis. 2016; 10(1): e0004367. pmid:26771308
- 72. Pesapane R, Foley J, Thomas R, Castro LR. Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from northern Colombia. Vet Microbiol. 2019; 233: 184–9. pmid:31176406
- 73. Selmi R, Ben Said M, Dhibi M, Ben Yahia H, Messadi L. Improving specific detection and updating phylogenetic data related to Anaplasma platys-like strains infecting camels (Camelus dromedarius) and their ticks. Ticks Tick Borne Dis. 2019; 10(6): 101260. pmid:31327747
- 74. Wei W, Li J, Wang YW, Cui XM, Li LF, Yuan TT, et al. Anaplasma platys-Like Infection in Goats, Beijing, China. Vector Borne Zoonotic Dis. 2020; 20(10): 755–62. pmid:32679008
- 75. Arraga-Alvarado CM, Qurollo BA, Parra OC, Berrueta MA, Hegarty BC, and Breitschwerdt EB. Molecular Evidence of Anaplasma platys Infection in Two Women from Venezuela. The American Journal of Tropical Medicine and Hygiene. 2014; 91(6): 1161–65. pmid:25266347
- 76. Breitschwerdt EB, Hegarty BC, Qurollo BA, Maggi RG, Blanton LS, Bouyer DH, et al. Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasites Vectors. 2014; 7(1): 298. pmid:24984562
- 77. Maggi RG, Mascarelli PE, Havenga LN, Naidoo V, Breitschwerdt EB. Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum in a veterinarian. Parasit Vectors. 2013; 6: 103. pmid:23587235
- 78. Ybanez A. First report on Anaplasma platys infection in a dog in the Philippines. Israel Journal of Veterinary Medicine. 2013; 7: 227–231.
- 79. Low VL, Prakash BK, Lim YAL, Vinnie-Siow WY, Sofian-Azirun M, AbuBakar S, et al. Detection of Anaplasmataceae agents and co-infection with other tick-borne protozoa in dogs and Rhipicephalus sanguineus sensu lato ticks. Exp Appl Acarol. 2018; 75(4): 429–35. pmid:30073430
- 80. Piratae S, Senawong P, Chalermchat P, Harnarsa W, Sae-chue B. Molecular evidence of Ehrlichia canis and Anaplasma platys and the association of infections with hematological responses in naturally infected dogs in Kalasin, Thailand. Vet World. 2019; 12(1): 131–5. pmid:30936666
- 81. Stuen S, Granquist EG, Silaghi C. Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies. Front Cell Infect Microbiol. 2013; 3(31). pmid:23885337
- 82. Ekner A, Dudek K, Sajkowska Z, Majláthová V, Majláth I, Tryjanowski P. Anaplasmataceae and Borrelia burgdorferi sensu lato in the sand lizard Lacerta agilis and co-infection of these bacteria in hosted Ixodes ricinus ticks. Parasit Vectors. 2011; 4: 182. pmid:21933412
- 83. Silaghi C, Santos AS, Gomes J, Oteo JA, Fuente JDL, Dumler JS, et al. Guidelines for the Direct Detection of Anaplasma spp. in Diagnosis and Epidemiological Studies. Vector Borne Zoonotic Dis. 2017; 17(1): 12–22. pmid:28055579
- 84. Mukhacheva TA, Shaikhova DR, Kovalev SY. Asian isolates of Anaplasma phagocytophilum: Multilocus sequence typing. Ticks Tick Borne Dis. 2019; 10(4): 775–80. pmid:30904539
- 85. Dumler JS, Choi KS, Garcia-Garcia JC, Garyu JW, Grab DJ, Bakken JS, et al. Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum. Emerg Infect Dis. 2005; 11(12): 1828–34. pmid:16485466
- 86. Jin H, Wei F, Liu Q, Qian J. Epidemiology and control of human granulocytic anaplasmosis: a systematic review. Vector Borne Zoonotic Dis. 2012; 12(4): 269–74. pmid:22217177
- 87. Zhang L, Liu H, Xu B, Fan D, Li G, Jin Y, et al. Anaplasma phagocytophilum infection in domestic animals in ten provinces/cities of China. Am J Trop Med Hyg. 2012; 87(1): 185–9. pmid:22764312
- 88. Rar V, Golovljova I. Anaplasma, Ehrlichia, and ‘Candidatus Neoehrlichia’ bacteria: pathogenicity, biodiversity, and molecular genetic characteristics, a review. Infect Genet Evol. 2011; 11(8): 1842–61. pmid:21983560
- 89. Kelch WJ. The canine ehrlichiosis (tropical canine pancytopenia) epizootic in Vietnam and its implications for the veterinary care of military working dogs. Mil Med. 1984; 149(6): 327–31. pmid:6429572
- 90. Fuente J, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: Ticks as vectors of pathogens that cause disease in humans and animals. Front Biosci. 2008; 13: 6938–46, May 2008, pmid:18508706
- 91. Sun J, Liu Q, Lu L, Li G, Liu J, Lin H, et al. Coinfection with four genera of bacteria (Borrelia, Bartonella, Anaplasma, and Ehrlichia) in Haemaphysalis longicornis and Ixodes sinensis ticks from China. Vector Borne Zoonotic Dis. 2008; 8(6): 791–5. pmid:18637722
- 92. Beugnet F, Moreau Y. Babesiosis. Rev Sci Tech. 2015; 34(2): 627–39. pmid:26601462
- 93. Prakash BK, Low VL, Vinnie-Siow WY, Morvarid AR, AbuBakar S, Sofian-Azirun M, et al. Detection of Babesia spp. in Dogs and Their Ticks From Peninsular Malaysia: Emphasis on Babesia gibsoni and Babesia vogeli Infections in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae). J Med Entomol. 2018; 55(5): 1337–40. pmid:29762747
- 94. Ybañez AP, Ybañez RHD, Talle MG, Liu M, Moumouni PFA, Xuan X. First report on Babesia vogeli infection in dogs in the Philippines. Parasitol Int. 2017; 66(1): 813–5. pmid:27713098
- 95. Watts JG, Playford MC, Hickey KL. Theileria orientalis: A review. New Zealand veterinary journal. 2015; 64(1): 1–21. [cited 18 Apr 2021]. Available from: https://www.researchgate.net/publication/279864314. pmid:26440501
- 96. Fujisaki K, Ito Y, Kamio T, Kitaoka S. The presence of Theileria sergenti in Haemaphysalis longicornis overwintering in pasture in Japan. Ann Trop Med Parasitol. 1985; 79(5): 519–24. pmid:3936424
- 97. Kho KL, Amarajothi ADG, Koh FX, Panchadcharam C, Hassan Nizam QN, Tay ST, “The first molecular survey of theileriosis in Malaysian cattle, sheep and goats. Vet Parasitol Reg Stud Reports. 2017; 10: 149–53. pmid:31014589
- 98. Bai Q, Liu G, Yin H, Liu D, Ren J, Li X, et al. Theileria sinensis sp nov: a new species of Bovine Theileria-classical taxonomic studies. Acta Veterinaria et Zootechnica Sinica. 2002a; 33(2): 73–7.
- 99. Tian Z, Du J, Yang J, Liu X, Liu G, Yin H, et al. A PCR-RFLP Assay targeting RPS8 gene for the discrimination between bovine Babesia and Theileria species in China. Parasit Vectors. 2015; 8. pmid:26382041
- 100. Li Y, Li X, Liu J, Liu G, Luo J, Yin H, el at. First Report of Theileria Infection of Bactrian Camels (Camelus bactrianus) in Xinjiang, China. Acta Parasitol. 2019; 64(4): 923–6. pmid:31165983
- 101. Yin H, Luo J, Guan G, Lu C, Yuan Z, Guo S, et al. Transmission of an unidentified Theileria species to small ruminants by Haemaphysalis qinghaiensis ticks collected in the field. Parasitol Res. 2002; 88(13): S25–27. pmid:12051602
- 102. Kim JY, Naoaki Y, Sanjay K, Sachiko S, Kozo F, Chihiro S, et al. Molecular epidemiological survey of benign Theileria parasites of cattle in Japan: detection of a new type of major piroplasm surface protein gene. J Vet Med Sci. 2004; 66(3):251–6. pmid:15107552
- 103. Liu A, Guan G, Liu Z, Gao J, Ma M, Niu Q, et al. Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP). Exp Parasitol. 2010; 126(4): 476–81. pmid:20685208
- 104. Govaerts M, Verhaert P, Jongejan F, Goddeeris BM. Characterisation of the 33kDa piroplasm surface antigen of Theileria orientalis/sergenti/buffeli isolates from West Java, Indonesia. Vet Parasitol. 2002; 104(2): 103–17. pmid:11809330
- 105. Izzo MM, Poe I, Horadagoda N, De Vos AJ, and House JK. Haemolytic anaemia in cattle in NSW associated with Theileria infections. Aust Vet J. 2010; 88(1–2): 45–51. pmid:20148827
- 106.
Sugimoto C, Fujisaki K. Non-Transforming Theileria Parasites of Ruminants. Springer US. 2002; 3: 93–106.
- 107. Kidd L, Maggi R, Diniz PPVP, Hegarty B, Tucker M, Breitschwerdt E. Evaluation of conventional and real-time PCR assays for detection and differentiation of Spotted Fever Group Rickettsia in dog blood. Vet Microbiol. 2008; 129(3–4): 294–303. pmid:18226476
- 108. Djiba ML Mediannikov O, Mbengue M, Fenollar F, Raoult D, Ndiaye M, et al. Survey of Anaplasmataceae bacteria in sheep from Senegal. Trop Anim Health Prod. 2013; 45(7): 1557–61. pmid:23553260
- 109. Rolain JM, Stuhl L, Maurin M, Raoult D. Evaluation of antibiotic susceptibilities of three rickettsial species including Rickettsia felis by a quantitative PCR DNA assay. Antimicrob Agents Chemother. 2002; 46(9): 2747–51. pmid:12183224
- 110. Mura Aet al. Molecular detection of spotted fever group rickettsiae in ticks from Ethiopia and Chad. Trans R Soc Trop Med Hyg. 2008; 102(9): 945–9. pmid:18440576
- 111. Mediannikov O, Trape JF, Diatta G, Parola P, Fournier PE, Raoult D. Rickettsia africae, Western Africa. Emerg Infect Dis. 2010; 16(3): 571–3. pmid:20202453
- 112. Rolain JM, Franc M, Davoust B, Raoult D. Molecular Detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in Cat Fleas, France. Emerg Infect Dis. 2003; 9(3): 339–42. pmid:12643829
- 113. Rolain JM, Raoult D. Molecular detection of Coxiella burnetii in blood and sera during Q fever. QJM. 2005; 98(8): 615–7. pmid:16027172