Morphological, molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam

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.


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][8][9][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][22][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][28][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.

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.  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.

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 halftick 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 airdried 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.  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.
https://doi.org/10.1371/journal.pntd.0009813.t001 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

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).

MALDI-TOF MS tick identification by blind test
The   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.  (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).

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.
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.
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.
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 tickassociated 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.