MALDI-TOF MS Identification of Dromedary Camel Ticks and Detection of Associated Microorganisms, Southern Algeria

This study used MALDI-TOF MS and molecular tools to identify tick species infesting camels from Tamanrasset in southern Algeria and to investigate their associated microorganisms. Ninety-one adult ticks were collected from nine camels and were morphologically identified as Hyalomma spp., Hyalomma dromedarii, Hyalomma excavatum, Hyalomma impeltatum and Hyalomma anatolicum. Next, the legs of all ticks were subjected to MALDI-TOF MS, and 88/91 specimens provided good-quality MS spectra. Our homemade MALDI-TOF MS arthropod spectra database was then updated with the new MS spectra of 14 specimens of molecularly confirmed species in this study. The spectra of the remaining tick specimens not included in the MS database were queried against the upgraded database. All 74 specimens were correctly identified by MALDI-TOF MS, with logarithmic score values ranging from 1.701 to 2.507, with median and mean values of 2.199 and 2.172 ± 0.169, respectively. One H. impeltatum and one H. dromedarii (2/91; 2.20%) tested positive by qPCR for Coxiella burnetii, the agent of Q fever. We also report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a potentially new Ehrlichia sp. in H. impeltatum.


Introduction
Ticks are obligate hematophagous ectoparasites that infest most vertebrates worldwide, from the warmest regions of the globe to the coldest ones [1]. To date, over 900 tick species have been described around the world [2]. Ticks are significant vectors of a wide range of microorganisms, including bacteria, helminths, viruses and protozoa [1,[3][4][5]. They are considered a major global human and veterinary public health problem by being reservoirs and/or vectors of infectious disease agents to susceptible hosts [6].
Dromedary camel (Camelus dromedarius) populations are widespread in the Middle East and North Africa, including Algeria [7][8][9][10]. They play an important role in the culture and economy of some countries. In fact, they represent their main source of milk and meat production because of their very unique adaptative physiological characteristics to arid and semi-arid ecosystems [11,12]. Camel production is negatively impacted by zoonotic diseases [7,13]. Ticks are one of the most predominant ectoparasites that negatively affect their productivity as well as their performance by either transmitting infectious pathogenic agents or causing traumatic lesions and severe anemia due to blood loss [9,14,15].

Ethics Statement
Risk assessment was submitted to and approved by the ethics committee and decision board of the Inspection Vétérinaire (Direction des Services Agricoles) of Tamanrasset province of Algeria. This institution is affiliated with the Directions des Services Veterinaires (Algerian Ministry of Agriculture and Rural Development). To facilitate fieldwork, collaboration was established with the veterinary inspector of Tamanrasset province. The dromedary camels were handled by local veterinarians. Importation authorization was obtained from the relevant entity (DDPP/Prefecture of Bouches-du-Rhône, Marseille, France) in August 2018 under the number ER 11/18.

Study Area and Tick Sampling
In August 2018, nine camels were examined for tick collection at the livestock market in the commune of Tamanrasset, which is located in the province of Tamanrasset, in southernmost Algeria, at the border with Mali and Niger (22 • 47 25.069 N, 5 • 31 9.577 E) ( Figure 1). Ticks were carefully removed using forceps and placed in individually labeled tubes for each camel. The tubes contained 70% ethanol and were stored at room temperature. Three months later, samples were transported to Marseille, France, for subsequent analysis. Tick morphological identification was performed by using standard keys and descriptions with a Leica EZ4 binocular microscope [34,35]. Specimens were codified according to the genus, species and sex. ArcGIS 10.3 software (http://www.esri.com, accessed on 25 October 2021) was used to map the spatial localization of tick sampling in our study region.
All specimens were then subjected to MALDI-TOF MS analyses after being morphologically identified. For some specimens, molecular identification was also carried out as described below.

Sample Preparation for MALDI-TOF MS
Ethanol-preserved ticks were rinsed with distilled water and then dried with filter paper. Four legs from only one side were dissected using a sterile surgical blade used for MALDI-TOF MS analysis. The leg sample preparation was performed using a previously described protocol [27]. One microliter of the supernatant of the protein extract of each sample was spotted in quadruplicate onto a MALDI-TOF plate (Bruker Daltonics, Wissembourg, France). All spots were left to dry and then covered with 1 μL of HCCA matrix composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile, 2.5% trifluoroacetic acid (Aldrich, Dorset, UK) and HPLC grade water [27]. The target plate was left to dry at room temperature and then introduced into a Microflex LT MALDI-TOF Mass Spectrometer device (Bruker Daltonics, Bremen, Germany) for analysis.
The tick's remaining body was longitudinally cut into two halves, one half being used for molecular identification and the detection of microorganisms and the other half being preserved at −20 °C [27].

Molecular Identification of Ticks
Molecular identification was performed in order to validate the morphological identification of specimens whose MS spectra were added to our in-house arthropod spectra MS database. It was also carried out to confirm the identification assigned by MALDI-TOF MS of randomly selected specimens morphologically identified as Hyalomma spp.
We followed the same DNA extraction protocol described elsewhere [27]. DNA obtained from each tick specimen was subjected to standard PCR in an automated DNA thermal cycler (Applied Biosystems, 2720, Foster City, CA, USA) to amplify a 455 base- ArcGIS 10.3 software (http://www.esri.com, accessed on 25 October 2021) was used to map the spatial localization of tick sampling in our study region.
All specimens were then subjected to MALDI-TOF MS analyses after being morphologically identified. For some specimens, molecular identification was also carried out as described below.

Sample Preparation for MALDI-TOF MS
Ethanol-preserved ticks were rinsed with distilled water and then dried with filter paper. Four legs from only one side were dissected using a sterile surgical blade used for MALDI-TOF MS analysis. The leg sample preparation was performed using a previously described protocol [27]. One microliter of the supernatant of the protein extract of each sample was spotted in quadruplicate onto a MALDI-TOF plate (Bruker Daltonics, Wissembourg, France). All spots were left to dry and then covered with 1 µL of HCCA matrix composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile, 2.5% trifluoroacetic acid (Aldrich, Dorset, UK) and HPLC grade water [27]. The target plate was left to dry at room temperature and then introduced into a Microflex LT MALDI-TOF Mass Spectrometer device (Bruker Daltonics, Bremen, Germany) for analysis.
The tick's remaining body was longitudinally cut into two halves, one half being used for molecular identification and the detection of microorganisms and the other half being preserved at −20 • C [27].

Molecular Identification of Ticks
Molecular identification was performed in order to validate the morphological identification of specimens whose MS spectra were added to our in-house arthropod spectra MS database. It was also carried out to confirm the identification assigned by MALDI-TOF MS of randomly selected specimens morphologically identified as Hyalomma spp.
We followed the same DNA extraction protocol described elsewhere [27]. DNA obtained from each tick specimen was subjected to standard PCR in an automated DNA thermal cycler (Applied Biosystems, 2720, Foster City, CA, USA) to amplify a 455 base-pair fragment of the 16S rRNA gene using tick 16S primers (F-CCGGTCTGAACTCAGATCAAGT and 16S-R CTGCTCAATGATTTTTTAAATTGCTGTGG) [36]. The amplified products were purified and sequenced as described previously [32]. The obtained sequences were assembled and corrected by Chromas Pro1.77 (Technelysium Pty. Ltd., Tewantin, Australia) and a BLAST query was performed against the NCBI GenBank database (http: //www.ncbi.nlm.nih.gov/blast/ accessed on 13 May 2021).

Spectral Analysis and Database Creation
The reproducibility of the MS profiles of tick species was assessed using both Flex analysis V 3.3 and ClinProTools v.2.2 software packages (Bruker Daltonics, Germany) [32]. Intra-species reproducibility and inter-species specificity were validated by comparing and analyzing the spectral profiles obtained from the four spots of each individual specimen. Poor-quality spectra were excluded from the analysis (<3000 arbitrary units (a.u.) and background noise). Cluster analysis (MS dendrogram) was performed with MS spectra of specimens of each species using MALDI-Biotyper 3.0 software. The database was then created by adding high-quality MS spectra of each species to our homemade arthropod spectra MS database after molecular confirmation.

Blind Test
All MS spectra obtained from tick leg specimens were subjected to blind test analysis, except for those used to create the MS database (MS reference spectra). The log-score values (LSVs), ranging from 0 to 3, were computed by the MALDI-Biotyper v.3.0 (Bruker Daltonic, Germany). The LSVs refer to the degree of homology between MS reference spectra and MS spectra used for the blind test [47].

MS Identification of Dromedary Camel Ticks
A total of 91 tick leg samples were subjected to MALDI-TOF MS. The visualization of all MS spectra using Flex analysis v3.4 software showed that 96.70% (88/91) of MS spectra were of excellent quality. Representative MS profiles of the three tick species are presented in Figure 2A. Cluster analysis ( Figure 2B) revealed inter-species specificity, as all specimens of the same species were clustered on the same branch.

MS Identification of Dromedary Camel Ticks
A total of 91 tick leg samples were subjected to MALDI-TOF MS. The visualization of all MS spectra using Flex analysis v3.4 software showed that 96.70% (88/91) of MS spectra were of excellent quality. Representative MS profiles of the three tick species are presented in Figure 2A. Cluster analysis ( Figure 2B) revealed inter-species specificity, as all specimens of the same species were clustered on the same branch. This was confirmed by a principal component analysis (PCA) diagram generated by ClinProTools software, which revealed a visible distinction between the three species ( Figure 3A). This was confirmed by a principal component analysis (PCA) diagram generated by Clin-ProTools software, which revealed a visible distinction between the three species ( Figure 3A).
Our homemade MALDI-TOF arthropod spectra database (MALDI-Biotyper 3.0) was upgraded with the MS reference spectra of 14 specimens, 12 H. dromedarii, 1 H. excavatum and 1 H. impeltatum, all of which were molecularly identified. The remaining 74 goodquality spectra from tick legs were subjected to a blind test against our upgraded database ( Figure 4). The results showed that of the 36 specimens of ticks morphologically identified as Hyalomma spp., 34   Our homemade MALDI-TOF arthropod spectra database (MALDI-Biotyper 3.0) wa upgraded with the MS reference spectra of 14 specimens, 12 H. dromedarii, 1 H. excavatum and 1 H. impeltatum, all of which were molecularly identified. The remaining 74 good quality spectra from tick legs were subjected to a blind test against our upgraded databas ( Figure 4). The results showed that of the 36 specimens of ticks morphologically identified as Hyalomma spp., 34 Figure 4).  Our homemade MALDI-TOF arthropod spectra database (MALDI-Biotyper 3.0) was upgraded with the MS reference spectra of 14 specimens, 12 H. dromedarii, 1 H. excavatum and 1 H. impeltatum, all of which were molecularly identified. The remaining 74 goodquality spectra from tick legs were subjected to a blind test against our upgraded database ( Figure 4). The results showed that of the 36 specimens of ticks morphologically identified as Hyalomma spp., 34 (Figure 4). The tick whose morphological identification was corrected by sequencing was identified by MS as H. dromedarii [LSV: 2.342], in agreement with the molecular identification ( Table 1). The median of all log-score values was 2.199, and the mean LSV was 2.172 ± 0.169. All of the obtained score values of each species, H. dromedarii, H. impeltatum and H. excavatum, are presented in Figure 3B. Our camel tick MALDI-TOF MS
Ten tick specimens morphologically identified as Hyalomma spp., of which eight were identified by MALDI-TOF MS as H. dromedarii and two were identified as H. excavatum, were also submitted to molecular analysis to verify the MS identifications ( Figure 4). The sequencing results revealed that eight specimens were 99.73-100% identical to H. dromedarii (MN960589), and two specimens were 100% identical to H. excavatum (MK601704). Molecular tools confirmed the identification of ticks that were unable to be morphologically identified to the species level. The 16S sequences of the ticks obtained in this study have been deposited in the GenBank database under the following accession numbers: OL672219, OL672220, OL672221, OL672222, OL672223 and OL672224 for H. dromedarii, OL672225 for H. impeltatum and OL672226 for H. excavatum.
The sequencing of a fragment of the Anaplasmataceae 16rRNA gene was only successful for three out of eight specimens. BLAST analysis revealed that the two sequences obtained from two positive H. dromedarii ticks showed 100% similarity to Candidatus Anaplasma camelii (MT510533) and A. platys (MN630836), respectively. One sequence detected in the H. impeltatum tick revealed 99.63% similarity to Candidatus Midichloria mitochondrii (endosymbiotic bacterium), which was previously detected in an H. dromedarii tick from Tunisia (MK416236) ( Table 2). The maximum likelihood (ML) phylogenetic tree based on the 16S rRNA gene ( Figure 5) showed that Anaplasma detected in H. dromedarii belonged to the cluster of Ca. A. camelii and A. platys. Table 2. Molecular characterization of Anaplasmataceae identified in ticks.

Tick Species
Host 16SrRNA Gene (n = Sequence; % ID)  BLAST of the 23S rRNA gene sequences obtained from these three Anaplasmataceaepositive samples showed that two sequences from H. dromedarii ticks were 99.78-100% identical to an uncultured Anaplasma sp. (MN626401), and the sequence obtained from H. impeltatum was 97.28% identical to Candidatus Ehrlichia rustica (KT364330) ( Table 2). The 23S sequence of Ca. A. camelii was not available in GenBank. The ML phylogenetic tree based on the 23S rRNA gene ( Figure 6) revealed that the two sequences from the Anaplasma-positive samples (H. dromedarii) belonged to the main cluster of A. platys. More specifically, the obtained sequences formed a separate and well-supported subcluster with the uncultured Anaplasma (referred to as A. platys-like) isolated from the blood of camels from Egypt (MN626401) but closely related to the subcluster of A. platys. One sequence of Ehrlichia sp. (n = 1; 1.10%) obtained from H. impeltatum formed an independent subcluster. However, it was close to the subcluster of E. canis. BLAST of the 23S rRNA gene sequences obtained from these three Anaplasmataceaepositive samples showed that two sequences from H. dromedarii ticks were 99.78-100% identical to an uncultured Anaplasma sp. (MN626401), and the sequence obtained from H. impeltatum was 97.28% identical to Candidatus Ehrlichia rustica (KT364330) ( Table 2). The 23S sequence of Ca. A. camelii was not available in GenBank. The ML phylogenetic tree based on the 23S rRNA gene ( Figure 6) revealed that the two sequences from the Anaplasma-positive samples (H. dromedarii) belonged to the main cluster of A. platys. More specifically, the obtained sequences formed a separate and well-supported subcluster with the uncultured Anaplasma (referred to as A. platys-like) isolated from the blood of camels from Egypt (MN626401) but closely related to the subcluster of A. platys. One sequence of Ehrlichia sp. (n = 1; 1.10%) obtained from H. impeltatum formed an independent subcluster. However, it was close to the subcluster of E. canis.
The H. impeltatum specimen positive for Anaplasmataceae, whose sequence was close to "Candidatus Ehrlichia rustica" for the 23S rRNA gene, was then subjected to sequencing with the groEL gene for confirmation. BLAST analysis showed that the obtained groEL sequence was 94.59% identical to E. canis ( Table 2). The groEL ML phylogenetic analysis (Figure 7) revealed that the sequence of Ehrlichia sp. (H. impeltatum) formed an independent and well-supported (bootstrap support ≈ 100%) branch separated from the other Ehrlichia spp., showing that it may potentially be a novel species of Ehrlichia. The H. impeltatum specimen positive for Anaplasmataceae, whose sequence was close to "Candidatus Ehrlichia rustica" for the 23S rRNA gene, was then subjected to sequencing with the groEL gene for confirmation. BLAST analysis showed that the obtained groEL sequence was 94.59% identical to E. canis ( Table 2). The groEL ML phylogenetic analysis (Figure 7) revealed that the sequence of Ehrlichia sp. (H. impeltatum) formed an independent and well-supported (bootstrap support ≈ 100%) branch separated from the other Ehrlichia spp., showing that it may potentially be a novel species of Ehrlichia. In summary, two ticks, one H. impeltatum and one H. dromedarii, were positive for C. burnetii using both IS30A and IS1111 primers, confirming the presence of C. burnetii DNA. In two other H. dromedarii (2.20%), we identified an Anaplasma sp. closely related to A. platys using the 16S and 23S genes and a potentially new Ehrlichia sp. using 23S and groEL in H. impeltatum. The sequences of the ticks and microorganisms have been deposited in Figure 7. Phylogenetic tree based on groEL sequences aligned with MEGA 7 using Tamura 3-parameter model. Statistical support for internal branches of the trees was evaluated by bootstrapping with 500 iterations. Rickettsia typhi was added as an outgroup.
In summary, two ticks, one H. impeltatum and one H. dromedarii, were positive for C. burnetii using both IS30A and IS1111 primers, confirming the presence of C. burnetii DNA. In two other H. dromedarii (2.20%), we identified an Anaplasma sp. closely related to A. platys using the 16S and 23S genes and a potentially new Ehrlichia sp. using 23S and groEL in H. impeltatum. The sequences of the ticks and microorganisms have been deposited in GenBank, and the accession numbers of these sequences are OL672236, OL672237 and OL672238 for the 16SrRNA gene, OL672216, OL672217 and OL672218 for the 23S rRNA gene and OL757650 for the groEL gene.

Discussion
In recent years, MALDI-TOF MS has been used to accurately and rapidly diagnose bacterial species and identify fungi [48,49]. This method is now incorporated into diagnostic routines in many clinical laboratories [50]. MALDI-TOF MS has also been used in medical entomology to identify several species of arthropods, such as ticks, mosquitoes, bed bugs, fleas, sandflies and others, using protein extracts from different body parts [28,31,33,51,52]. More recently, MALDI-TOF MS has also been used in malacology [53]. Once the device has been purchased for a technological platform, there is no additional cost for its use because this technique is inexpensive, simple and fast in comparison to conventional methods [26]. As previously mentioned, the robustness of MALDI-TOF MS for arthropod identification has been evaluated for several years using laboratory-reared and field-collected samples. MALDI-TOF MS profiling uses a highly standardized protein extraction method coupled with a spectrum acquisition approach to generate reference spectra that will be specific and reproducible enough to identify specimens of the same species [54]. MALDI-TOF MS has especially shown its reliability and usefulness in field studies by significantly decreasing the costs and time of identification but also allowing the detection of low-occurrence species [55] Camels can be infested with different tick species of various genera [9,10,14,15]. In our study, the ticks belonged to the genus Hyalomma. The morphological identification of engorged females was only possible at the genus level because some morphological criteria were distorted and not visible, making their identification challenging. In our study, three species, namely, H. dromedarii, H. impeltatum and H. excavatum, were identified using molecular, morphological and proteomic tools. Similar observations have been reported by other researchers on these tick species infesting camels in Algeria, Tunisia and other parts of the world [10,18,[56][57][58]. The camel tick H. dromedarii was the most frequent tick species found on camels. This species is known to have a host preference for dromedary camels but can also infest sheep, goats and horses, and it adapts well to extreme dryness and desert climates [34]. However, another study in Tunisia showed that H. impeltatum was the most abundant in Camelus dromedarius, followed by H. dromedarii, as described in [59].
In order to upgrade our database with MS spectra, as well as to randomly verify MS identifications, we used the 16S rRNA tick gene for the sequencing and definitive identification of ticks. We obtained a discrepancy between morphological and molecular/MALDI-TOF MS identifications for one tick specimen, with the MS identification in agreement with molecular identification. As previously stated, the taxonomy of the genus Hyalomma includes several species with important within-species morphological variability, which makes their morphological identification challenging, even for tick experts [60,61]. The morphological error may also be due to the invisible characters of this specimen. We therefore relied on molecular tools as well as MALDI-TOF MS to overcome morphological errors and ambiguous identification.
The MALDI-TOF MS results were shown to be in agreement with all molecular identifications. As for the ten specimens morphologically designated as Hyalomma spp., MALDI-TOF MS enabled the correct identification of those ticks at the species level. All ticks were identified by MALDI-TOF MS with high LSVs. This study highlights the ability of MALDI-TOF MS to overcome the limitations related to ticks' taxonomical identification, such as the identification of damaged or engorged specimens and the lack of taxonomy keys, suitable documentation and entomological expertise [26,32]. Walker et al. (2003) reported that the accurate identification of H. dromedarii and H. impeltatum is confusing, especially those in the engorged state [34]. Here, the MS dendrogram and PCA results show that MALDI-TOF MS can discriminate between these two species, regardless of the engorged state.
Molecular tools, including polymerase chain reaction and sequencing, have enabled a major advance in the study of zoonotic pathogens and their reservoirs. In this work, we were able to detect C. burnetii in ticks. This intracellular Gram-negative bacterium is the causative zoonotic agent of Q fever. However, certain ruminants are considered the principal reservoirs for the transmission of C. burnetii to humans. Coxiella burnetii transmission to humans can be through aerosol inhalation or the ingestion of infected animal products, such as unpasteurized or insufficiently pasteurized milk products or derivatives [7]. Transmission can occur through birth products, including the placenta and amniotic liquid. These represent a major risk to veterinarians and livestock or dairy farmers [62]. This bacterium has been detected in more than 40 hard and at least 14 soft tick species, but they are considered to play only a secondary role in the transmission to humans [63]. In Algeria, C. burnetii has been detected in ticks (Rhipicephalus and Hyalomma) and in the blood of small ruminants [10,64] and camels [10], but only one human case was reported in 2012 in the northwest of the country [65]. Coxiella burnetii has also been found in camels and their ticks in several countries [7,66,67]. The long-term persistence of C. burnetii is possible in camels, and this may pose a threat to public health as well as breeding farms. However, the role of camels in the transmission of C. burnetii should be further evaluated [7]. In our survey, C. burnetii was amplified in H. impeltatum and H. dromedarii. However, a recent study on C. burnetii isolated it from H. impeltatum, H. dromedarii and H. excavatum in Algeria [10]. In Tunisia, C. burnetii was reported in H. impeltatum and H. dromedarii, and in Egypt, H. dromedarii was positive for C. burnetii [68,69].
In our study, we report the first detection in Algeria of an Anaplasma sp. in H. dromedarii, and the species is closely related to canine A. platys. A potential new Ehrlichia sp. was also detected in H. impeltatum. Bastos et al. [70] reported a novel species genetically close to A. platys in camels and named it "Candidatus Anaplasma camelii". Other studies have recorded Ca. A. camelii in dromedary camels from Saudi Arabia [70], Tunisia, Morocco [20], Nigeria [18], Kenya [71] and Iran [72]. Recently, the occurrence of "Candidatus Anaplasma camelii"was reported in cattle and deer in Malaysia [73] and camel keds (Hippobosca camelina) [71]. The prevalent variations are likely to be the result of differences in the distribution of vectors in camel-sampling localities, reservoir hosts or tick control programs [56]. In Xinjiang, China, Li et al. (2015) reported the presence of canine A. platys in Camelus bactrianus (two-humped camels) and their ticks [74]. However, this strain was genetically divergent from the Anaplasma strain infecting dromedaries.
In order to characterize the obtained Anaplasmataceae species, we amplified fragments of three genes, one of which (groEL) is specific to Ehrlichia. The phylogenetic tree analysis based on the 16S rRNA gene revealed that the Anaplasma strain from H. dromedarii in our study clustered with "Candidatus Anaplasma camelii" from the dromedary camel of Kenya and A. platys from dog blood. To see whether the obtained sequences were identical to "Candidatus Anaplasma camelii"or A. platys and to differentiate the two Anaplasma strains, we targeted the 23S rRNA gene because of its ability to identify a large panel of Anaplasmataceae. As reported, this marker is ideal for discriminating between two closely related Anaplasma species and potentially detecting new species [37]. The phylogenetic analysis revealed that the obtained sequences formed an independent subcluster belonging to A. platys. In addition, the partial 23S rRNA sequence of "Candidatus Anaplasma camelii"does not exist in GenBank. Indeed, the 23S rRNA gene was able to separate Anaplasma sequences isolated from H. dromedarii and A. platys sequences. Consequently, we followed the proposition of Bastos et al. on the status of "Candidatus Anaplasma camelii".
Furthermore, phylogenetic analysis based on 23S rRNA revealed that the sequence of Ehrlichia sp. detected in H. impeltatum formed an independent branch close to the subcluster of E. canis. The phylogenetical analysis based on groEL and a gene specific for Ehrlichia showed that the sequence of Ehrlichia formed an independent branch and was separate from other species of Ehrlichia. Previously, Bastos et al. confirmed the presence of a novel Ehrlichia species that is genetically close to E. canis, and another study revealed E. canis infections in camels in Saudi Arabia [16,70]. Just recently, a study reported the presence of an Ehrlichia sp. genetically related to E. canis, E. regneryi and E. ruminantium isolated from the blood of sick camels and ticks (Amblyomma spp. and Rhipicephalus spp.) in Kenya [75]. The occurrence of canine pathogens in camels may be due to the close association and cohabitation of camels and livestock in the desert area, which amplifies the chances of spreading vector-borne diseases [16,71]. This may also be due to the fact that some dog ticks and camel ticks have relatively low host specificity and/or are quite aggressive ticks. In addition, high temperatures make ticks particularly aggressive and further decrease host specificity. Nevertheless, further investigations are required to clarify the veterinary and medical importance of the novel species.

Conclusions
Our study showed that MALDI-TOF MS is a useful tool for the rapid identification of tick species stored in ethanol and that it could be used to overcome morphological limitations. We report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a new Ehrlichia sp. in H. impeltatum. Ehrlichia sp. and Anaplasma sp., genetically related to A. platys detected in camels, have not been characterized yet. However, further studies are necessary to clarify their potential zoonotic and veterinary risks. Additionally, we detected C. burnetii in camel ticks (H. dromedarii and H. impeltatum), as previously reported in Algerian camel ticks. In Algeria, camels and ticks could be a significant reservoir or source for the transmission of Q fever to animals and humans.