Molecular Characterization of Some Equine Vector-Borne Pathogens and Identification of Their Vectors in Egypt

Background Equine vector borne diseases (EVBD) have been considered emerging and reemerging diseases transmitted by arthropods and most of these diseases have zoonotic concern. This study was designed to screen EVBD in equines and their vectors using molecular analyses and identify vectors by MALDI-TOF and molecular techniques. Methods A total of 335 blood samples were collected from apparently healthy equines (320 from horses and 15 from donkeys) from Cairo and Beni-Suef provinces in Egypt. A total of 166 arthropods (105 sucking flies and 61 ticks) were collected from the same animals. MALDI-TOF and molecular techniques were used to confirm the findings of morphological identification of vector. Quantitative PCR and Standard PCR coupled with sequencing were performed in equines and vectors DNA for screening multiple pathogens. Results MALDI-TOF and molecular techniques confirmed that Hippoposca equina (louse fly), Rhipicephalus annulatus ( Rh. annulatus ) and Rh. microplus ixodid ticks were found. In vectors, we identified Anaplasma marginale ( A. marginale ; 1.6%), A. platys -like (1.6%) and a new Ehrlichia sp. (4.9%) in Rh. microplus , while Ehrlichia rustica ( E. rustica ) was found in Rh. microplus and Rh. annulatus . Likewise, Borrelia theileri was identified in Rh. microplus (3.3%). For H. equina , Anaplasma and Borrelia sp. DNA were detected by qPCR only. In equines, A. marginale (0.6%), A. ovis (0.6%) and Theileria ovis ( T. ovis ; 0.6%) were found in donkeys. In horses, T. equi (1.2%) and a new Theileria sp. Africa (2.7%) were identified.


Abstract
Background Equine vector borne diseases (EVBD) have been considered emerging and reemerging diseases transmitted by arthropods and most of these diseases have zoonotic concern. This study was designed to screen EVBD in equines and their vectors using molecular analyses and identify vectors by MALDI-TOF and molecular techniques.
Methods A total of 335 blood samples were collected from apparently healthy equines (320 from horses and 15 from donkeys) from Cairo and Beni-Suef provinces in Egypt. A total of 166 arthropods (105 sucking flies and 61 ticks) were collected from the same animals. MALDI-TOF and molecular techniques were used to confirm the findings of morphological identification of vector. Quantitative PCR and Standard PCR coupled with sequencing were performed in equines and vectors DNA for screening multiple pathogens.
Conclusions For the first time, we reported here the presence of Rh. microplus as a competent tick for Rh. annulatus in Egypt using MALDI-TOF and molecular identification. To the best of our knowledge, we provided the first detection of different pathogens as A. marginale, A. platys-like, E. rustica, new Ehrlichia sp., B. theileri in Rh. microplus, A. marginale, A. ovis and T. ovis in donkeys and a new Theileria sp. Africa in horses in Egypt.

Background
Equine vector borne diseases (EVBD) have been considered emerging and reemerging diseases and most of these diseases have zoonotic concern [1]. Among haematophagic arthropods affecting Equidae, two major groups are to be mentioned as prominent infectious diseases vectors: Ixodid ticks (Acari) and hexapod Diptera (true flies) [2]. As a result of the vectors' parasitism, Equidae suffer from allergy, paralysis, myiasis and risk of transmission of various viral, bacterial, spirochetal and rickettsial diseases [1]. Since there is a distinct correspondence between the epidemiology of EVBD and the distribution of vectors [3], equines and their vectors play an important role in the maintenance and circulation of EVBD throughout the world [1,4].
The Anaplasmataceae family includes intracellular bacteria such as Anaplasma and Ehrlichia, with a significant medical and veterinary importance [5,6]. Anaplasma spp. are distributed worldwide especially in tropical and subtropical Africa, and are responsible for granulocytic anaplasmoses and ehrlichioses in equine and canine populations and ruminants [7,8]. These diseases are tick-borne and occur during spring and autumn seasons regarding tick activity [9]. Equine anaplasmosis (EA) has been reported in most of European countries, but the prevalence and incidence of EA in African countries, including Egypt, is limited [10].
The genus Borrelia includes pathogenic spirochaetes, which cause relapsing fevers and Lyme borreliosis [11]. Lyme disease is transmitted by hard ticks, while the relapsing fever borreliosises are transmitted by soft ticks [12,13]. Equine borrelioses (EB) are characterized by shifting leg lameness, generalized stiffness, muscular weakness, lethargy and behavioral abnormalities [14]. Due to the neuromuscular and musculoskeletal effects of EB, it has been included in differential diagnosis in cases of lameness and poor performance in sport horses [15]. In Egypt, data regarding the incidence and prevalence of EB and their vectors are absent. Borrelia burgdorferi was detected in ticks [16,17] and Borrelia theileri was detected in Rhipicephalus annulatus [17]. The Borrelia flaB and 16S rRNA genes were used for Borrelia detection [18].
Equine piroplasmosis (EP) is a tick-borne disease which is endemic in Europe, Asia, Russia, Africa and USA [19]. EP is caused by one of the hemoprotozoan parasites; Theileria equi and Babesia caballi [3,20]. EP is characterized by fever, hemoglobinuria, jaundice, ventral edema, pale mucous membranes, anemia, weakness, lethargy, mild colic, abortion in mares, and death can occur in the acute phase of the infection [21,22]. The mortality rate for B. caballi is 10%, while it reaches 50% for T. equi [3,23]. The recovery from infection is possible, but recovered horses may become asymptomatic carriers in case of T. equi, while B. caballi is generally self-limited up to 4 years [24]. For these reasons, the equid movement across borders may be restricted [25]. Subsequently, EP is considered one of the biggest problems in international equid trade [19,26].
In Egypt, the most prevalent Ixodid tick genera infesting equines are Hyalomma and Rhipicephalus. The genus Hyalomma includes H. excavatum, H. dromedarii and H. marginatum that are commonest species that infest equines [27,28]. Besides, the genus Rhipicephalus (formerly Boophilus) includes Rh. Annulatus, which mainly infests cattle and is rarely found on equines [29]. The common dipteran infesting equines is the louse fly Hippobosca equina [1]. Recently, changes in tick distribution and introduction of exotic ticks with infectious agents into previously unaffected areas have been recorded [30][31][32]. Therefore, vector identification is essentially important for the epidemiological mapping of vectors and vector-borne diseases [33,34]. Recently, the proteomic approach was applied for vector identification, which is called matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) [35,36]. MALDI-TOF successfully identified vectors through the spectra obtained from fresh, frozen or even alcohol preserved vector leg specimens, allowing to avoid difficulties of morphological taxonomy for identification due to damaged samples or immature stages [36][37][38][39]. Furthermore, this approach has been characterized by time saving, low cost and applicability on large scale studies when compared to molecular identification [40,41].
The availability and significant increase in DNA sequence data due to the high sensitivity and accuracy of molecular techniques has attracted the interest of evolutionary researchers to more accurately identify and characterize previously detected and/or novel species and/or genotypes. Moreover, studying phylogenetic inferences and epidemiology of vector-borne pathogens contributes to the knowledge needed for disease control and prevention. In Egypt, reports for epidemiology of EVBD have been limited, except few reports regarding EP [20]. Therefore, the aim of this study was to screen Egyptian Equidae and their vectors for EVBD such as Piroplasma, Anaplasma, Borrelia, Rickettsia, Coxiella burnetti and Bartonella using molecular analyses. In addition, equine vectors were identified using MALDI-TOF and molecular techniques.

Animals and blood sampling
This study is a cross-sectional study and included a total of 335 apparently healthy Equidae (320 horses and 15 donkeys) using a convenience sampling strategy. Animals were examined for the presence of vectors on different parts of their body. Blood samples were collected from jugular vein in a sterile EDTA tube from different localities of Capital Cairo and Beni-Suef province in Egypt, between 2016 and 2017. All blood samples were stored at -20 °C until DNA extraction for molecular investigations. In addition, data for each animal were recorded by sex, breed, age, health status and vector infestation.

Vectors collection
A total 166 arthropods (105 sucking flies and 61 ticks) were collected from equines from different localities, as mentioned above. All vectors (flies and ticks) have been carefully collected to avoid physical damage. The vectors collected from the same animal were counted and pooled in one tube containing 70% ethanol and transfer to the lab for morphological, molecular and MALDI-TOF MS analyses.

Sample preparation:
Ticks and sucking flies which reserved in 70% ethanol were rinsed twice in distilled water for 1 min then dried by sterile filter paper [36]. The legs of arthropod were cut by sterile scalpel, put in a sterile 1.5 ml Eppendorf and were then incubated at 37 °C overnight to evaporate any alcohol residue [38,39]. The rest of sample was cut longitudinally in two parts; one half was dissected into small species for molecular purpose and the remaining half was stored at − 20 °C as backup for any additional investigation.

Sample Homogenization and loading on MALDI-TOF target plates:
On the cut-off legs, a nip of glass powder (Sigma, Lyon, France) was added in addition 30 µl of a mix 70% (v/v) formic acid and 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland) [36]. The legs were crushed and homogenized by a TissueLyserII device (Qiagen, Hilden, Germany) with 30 movements per second for 1 min and repeated three times [45]. After centrifugation of homogenized arthropod legs (at 2000 g for 30 sec), 1 µl of the supernatant of extracted protein was dropped onto a spot of MALDI-TOF polished steel plate (Bruker Daltonic, Wissembourg, France) in quadruplicate [45]. At room temperature, the plate was left to dry and then each spot covered with 1 µl of CHCA matrix solution that composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water [41]. After drying, the plate was loaded into the Microflex LT MALDI-TOF MS apparatus (Bruker Daltonics, Germany) for analysis. Matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I) to control loading on the MS target plate, matrix quality and MALDI-TOF apparatus performance [45].

MALDI-TOF MS parameters:
The protein mass profiles of each sample were obtained using a Microflex LT spectrometer with the Flex Control software (Bruker Daltonics) with special parameters recommended by [36]. The spectra obtained were visualized by the Flex Analysis 3.3 software and transferred to ClinPro Tools version v.2.2 and MALDI-Biotyper v.3.0 (Bruker Daltonic, Germany) for analysis [37].

Spectra analysis and Reference database creation:
For spectra analysis, ClinPro Tools v2.2 and FlexAnalysis v3.3 software programs were used to evaluate the reproducibility of the MS spectra obtained from the same arthropods species and to assess intra-species specificity [47]. After morphological and molecular confirmation of arthropod identification, two to five high quality reproducible profiles with a high peak intensity of each species conserved in 70% ethanol were selected to serve as reference spectra [36,48]. As for upgrade arthropod MALDI-TOF database, reference spectra were established by spectra at least 2 specimens per species of both genders using the algorithm MALDI-Biotyper software v3.0 (Bruker Daltonics) [39,48].

Blind test:
All specimens were subjected to a blind test, except those used as reference spectra. The log score values (LSVs) were calculated by the MALDI-Biotyper software v.3.0 (Bruker Daltonics) to estimate the reliability of species identification. The LSVs ranged from 0 to 3; these correspond to the degree of similarity between the spectra submitted by blind test and the MS reference spectra in the database. The identification was considered reliable with a LSV of at least 1.8 [36,38]. The identification of blind tested samples was assessed by taking the highest LSVs associated with a spectrum quality.

1-DNA extraction:
Each dissected half of arthropod was put in a sterile 1.5 ml tube containing 200 µl of G2 lysis buffer and 25 µl of proteinase K (Qiagen, Hilden, Germany), cut in pieces and incubated at 56 °C overnight. 200 µl of supernatant was transferred into a new sterile tube after centrifugation of the mixture. DNA was extracted from 200 µl of each blood samples and arthropods using EZ1 DNA Tissue Kit (Qiagen) according to the manufacturer's instructions. The extracted DNA was stored at -20 °C until use in molecular methods.

2-Molecular Identification of vectors:
Standard PCR was applied to confirm MALDI-TOF identification. Mitochondrial genes (CO1 and 16S rRNA gene) sequencing was used for the identification of flies and tick species through DNA automated thermal cycler (Applied Biosystem, Paris, France) under the same condition as previously described (Table 1) [48,49]. PCR products were purified and sequenced as mentioned earlier [48]. The obtained sequences were assembled and corrected by ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia), and blasted against the reference sequences available in GenBank (http://blast.ncbi.nlm.nih.gov). The obtained sequences of Egyptian vectors (ticks and flies) were submitted in GenBank. Table 1 Primers and probes used for qPCR, Standard PCR and sequencing in this study.

3-Molecular detection of pathogen DNA in equines and their vectors:
Screening of pathogen DNA by qPCR: Quantitative PCR (qPCR) was performed in all extracted DNA samples (equines and their vectors) for multipathogen DNA screening using genus-specific primers and probes targeting the 5.8S rRNA gene of Piroplasmida [50], the 23S rRNA gene of Anaplasmataceae [51], the gltA gene Rickettsia sp. [52], the 16S rRNA gene Borrelia sp. [53], the IS1111 intergenic spacer for C. burnettii [54] and 16S-23S intergenic spacer for Bartonella sp. [55] ( Table 1). The qPCR was applied using the CFX96 Real Time System (Bio-Rad, Marnes-La-Coquette, France). The mixture of qPCR contained 10 µl of Eurogentec Probe PCR Master Mix (Erogentec, Liège, Belgium), 0.5 µM of primers and FAM-labeled probe, 5 µl of DNA template 3.5 µl sterile distilled water to complete the reaction volume to 20 µl. The negative controls (without any DNA) and positive controls (corresponding pathogen DNA) were added to each reaction to evaluate the reaction. The samples were measured positive with the cycle threshold (Ct) lower than 35 Ct [56].

Standard PCR and Sequencing
The positive qPCR samples were subjected to standard PCR and sequencing. For identification of Piroplasmida (Thierleria spp. and Babesia spp.), Anaplasmataceae (Anaplasma spp. and Ehrlichia spp.) and Borrelia spp. 1100 bp of the 18S rRNA gene, a 520 bp fragment of the 23S rRNA gene and a 1200 bp of 16S rRNA gene were used; respectively [50,51,53]. The PCR reactions were performed on a Thermocycler (Applied Biosystem, Paris, France) using the AmpliTaq Gold® 360 Master Mix (ThermoFisher Scientific, USA) according to the manufacturer's recommendations. Negative and positive controls were included in each reaction. The PCR products were visualized by electrophoresis on 1.5% agarose gel stained with SYBR® Safe (Invitrogen, USA) and examined and analyzed by Lab Image software (BioRad, Marnes-La-Coquuette, France).
The purification of PCR products was applied using NucleoFast 96 PCR plates (Macherey-Nagel, Düren, Germany), in accordance with the manufacturer's recommendations. The purified PCR products were sequenced using the BigDye Terminator Cycle Sequencing Kit (3130 × 1 Genetic Analyzer, ABI-PRISM). The sequences obtained were assembled and edited by ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia) and the corrected sequences were compared with the reference sequences available in GenBank by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences obtained of Egyptian EVB genotypes were recorded in GenBank.

Phylogenetic analyses
Multiple sequence alignments were performed between the obtained sequences and other reference sequences in GenBank using CLASTAL W in MEGA software version X [57]. The phylogenetic trees were inferred using the Maximum-Likelihood method and Tamura-Nei model with 500 bootstrap replicates in MEGA X software [57,58].

Morphological identification of vectors:
A total of 105 louse flies were collected from 311 horses from Cairo and morphologically identified as H. equina (70 males and 35 females; Fig. 1). We examined 15 donkeys and 9 horses from Upper Egypt province (Beni-Suef) and found 7 Rh. annulatus on 3 horses and 4 donkeys. Moreover, we found 54 Rhipicephalus sp. on 15 donkeys and 8 horses, whose morphological identification could be confirmed using molecular biology and MALDI-TOF techniques. The details of collected vectors are listed in Table 2.

MALDI-TOF MS analyses and Validation of vectors identification by blind tests:
In-lab MS reference spectra database was essential for accurate identification of vectors specimens [39,48]. After morphological and molecular confirmation, H. equina (Fig. 2), Rh. annulatus and Rh. microplus (Fig. 3)

Pathogens detection in vectors
All arthropods DNA samples were screened for the detection of pathogen DNA of Piroplasma sp., Anaplasma sp., Rickettsia sp., Borrelia sp., C. burnetii and Bartonella sp. The result revealed that DNA of Anaplasma and Borrelia sp. were detected in Rhipicephalus sp. collected from donkeys and H. equina from horses. While, Rhipicephalus sp. collected from horses were free from any pathogen DNA (Table 3). All vectors were found to be free from Piroplasma, Rickettsia, C. burnetii and Bartonella infections.   (Fig. 4). The phylogenetic position of these genotypes was illustrated in Fig. 4 Fig. 5.

Pathogens detection in Equidae:
All blood samples were screened by qPCR to screen the presence of pathogen DNA of Piroplasmids, Anaplasmataceae, Rickettsia spp., Borrelia spp., Coxiella burnetii and Bartonella spp. DNA of Piroplasmida and Anaplasma spp. were detected in horses and donkeys, while blood samples were free from the other DNA pathogens (Table-3 , that clustered in a separate clade with a good bootstrap support with the other Theileria sp. "Africa" previously detected in African horses (Fig. 6). By BLAST analyses, one genotype from horse was considered to be a new genotype with 99% (917/918) identity to T. equi detected in horse blood from Brazil (GenBank:MG052913). Three other genotypes were identified as T. equi with 100% (923/923) identity to those detected in horse blood from Turkey and Sudan (GenBank: MG569896, MG569893 and AB515309; respectively). In donkeys, T. ovis shared 100% (919/919) identity with sheep and buffalo previously detected in the same province in Egypt (GenBank: MN625886 & MN625887).

Discussion
Equidae are used in many beneficial activities for human such as police services, agriculture and pharmaceutical purposes, in addition to competitive and non-competitive leisure pursuits [59]. Generally, Equidae, especially donkeys, play a significant role in the transmission of vector borne diseases by acting as a domestic reservoir and carrying vectors to a broad host range or even to human [1]. Recently, the spectrum of EVBD has increased and drawn the attention of veterinarians and clinicians to diseases such as, piroplasmoses, anaplasmoses, borrelioses, rickettsioses, bartonelloses and Q fever [33]. In addition, advances in molecular biology tools and the availability of DNA sequence data facilitate the detection of new pathogen species and even genotypes [60]. The present study summarized epidemiological and entomological data on the prevalence of EVBDs infecting Equidae and their vector in two regions of Egypt (Capital Cairo and Beni-Suef province). Besides, equine arthropod parasites were identified by MALDI-TOF and molecular techniques.
Hyalomma and Rhipicephalus Ixodid ticks and the dipteran H. equina are the most common vectors infesting equines [1,27,28]. In this study, the morphological identification of vectors revealed the presence of Rhipicephalus sp. as ixodid ticks collected from horses and donkeys of Beni Suef province. Also H. equina was morphologically identified from horses in Cairo. In support of these morphological identification results, previous studies have reported the presence of Rhipicephalus sp. (especially Rh. annulatus) in Egypt as the main ixodid ticks infesting cattle and that may infest equines [29] and H. equina was louse fly of horses [61]. The morphological similarities at both intra-and inter-species level limit the worth of morphological taxonomic key such as in Rhipicephalus sp. [62][63][64].  [42]. In 2007, it was reported for the first time as an invasive tick in West Africa [65]. Then, it spread and was reported in other West African countries as Togo and Burkina Faso [66], Benin [67], Mali [39,66] and Côte d'Ivoire [60,68]. That indicates the rapid spread of Rh. microplus through the African countries and the risk of its invasion to North Africa. The change in tick distribution and introduction of exotic ticks might be attributed to climatic change, host availability and animal movements [30,31].
Family Anaplasmataceae includes two significant genera, Anaplasma and Ehrlichia, which can cause significant infections in a wide range of animal hosts and humans. These infections are mainly transmitted by ticks [69].  [70] and Pakistan [71]. Later, A. platys-like was also identified in different animal hosts other than dogs as cattle in Italy [72], Algeria [73] and Tunisia [74], and sheep and goat in South Africa [75] and Senegal [76]. Similarly, our study is the first to report the presence of A. marginale in Rh. microplus in Egypt. A. marginale has previously been reported in Rh. microplus in Côte d'Ivoire [60], Ecuador [77], India [78] and Pakistan [79]. As for genus Ehrlichia, we recorded for the first time two different genotypes of "E. rustica" in both Rh. microplus and Rh. annulatus in Egypt. One genotype of "E. rustica" was identified in Rh. microplus and Rh. annulatus with 100% homology to those of E. rustica found in Amblyomma vargiegatum from Côte d'Ivoire, and another genotype was identified in Rh. microplus only with 99% homology to the same reference [60]. Moreover, we have also identified a new potential Ehrlichia sp. in three Rh. microplus with 98% similarity to those of Candidatus E. urmitei detected in Rh. bursa from France (Fig. 4). The sequence of this potential Ehrlichia sp. clustered in a separated clade with E. ruminantium (bootstrap value 55; Fig. 4). As a result, we had a new potential Ehrlichia sp. in Rh. microplus and E. rustica in Rh. annulatus that had never been reported before in Egypt. Interestingly, these potential new species were identified in three different regions in the world (France, Côte d'Ivoire and Egypt) and from different tick species (Rhipicephalus, Amblyomma, and Hyalomma sp.) [60]. Thus, Rh. microplus could be an alternative vector for Anaplasmataceae alongside Rh. annulatus in Egypt, and there is a risk of transmission of other potential new vector-borne diseases and this should be evaluated in future studies.
In Africa, most of the Borrelia species were detected in soft ticks, such as Ornithodoros sp. which is the main vector [80]. To date in Africa, Borrelia sp. was identified in hard ticks (Amblyomma and Rhipicephalus sp.) in Ethiopia [81,82], Mali [18], Côte d'Ivoire [60], Egypt [17], Madagascar [83] and Ecuador [77]. In the present study, a new potential B. theileri was identified in two Rh. microplus with 3.3% infection rate. The obtained sequence was 99% identical to B. theileri found in Rh. geigyi in Mali [18]. Likewise, the phylogenetic analysis revealed that a new genotype of B. theileri was clustered in the same clade with B. theileri detected in sheep and cattle from the same locality of Beni-Suef province (GenBank: MN621893 & MN621894; Abdullah et al., unpublished). B. theileri in Rh. microplus has been reported in Madagascar [83], Ecuador [77], Brazil [84] and Argentina [85]. Thus, this is the first time that B. theileri has been detected in Rh. microplus in Egypt. It had previously been identified in Rh. annulatus [17].
Regarding H. equina, Anaplasma and Borrelia sp., DNAs were detected by qPCR. However, we were unable to amplify and sequence these samples, which could be attributed to the high sensitivity of qPCR compared to standard PCR, or to the low concentration of pathogenic DNA in fly tissues. Kowal and his colleagues [86] reported the role of Hippoboscids in the transmission of bacterial pathogens such as Anaplasma and Bartonella. Moreover, Boucheikhchoukh and his colleagues [87] detected Bartonella and Wolbachia sp. in H. equina.
In Equidae blood, we reported Anaplasmataceae DNA in donkeys and Piroplasimda DNA in horses. For Anaplasmataceae, the overall prevalence of anaplasmoses in donkeys was 26.6%, while horses were found free from Anaplasma sp. This result was in accordance with [60], who did not find any Anaplasma in horses. A. ovis and A. marginale were the common Anaplasma pathogens of sheep and cattle; respectively [76]. However, in our study, we found A. ovis and A. marginale in donkeys. A. ovis shared 100% identity to those of A. ovis in sheep and cattle blood from the same locality of Beni-Suef province (GenBank: MN626392 & MN625933), as well as in the blood of sheep from Niger (GenBank: KY644694). Another Anaplasma sp. was A. marginale that shared 100% similarity with those of A. marginale detected in blood of cattle collected from the same locality of Beni-Suef province (GenBank: MN625935; Abdullah et al., unpublished). To the best of our knowledge, A. ovis and A. marginale have never been reported yet in donkeys in Egypt and even Africa. Also, A. marginale has been reported in donkeys in Pakistan [88]. Therefore, donkeys should be involved in the epidemiology of tick-borne pathogens and other associated agents such as Anaplasmoses of health importance.
EP is a protozoan disease caused by T. equi and B. caballi [3,20]. Our study reported an overall prevalence of EP at 4.5% (1.2% for T. equi, 2.7% for Theileria sp. "Africa" and 0.6% for T. ovis), but we did not detect B. caballi. This might be attributed to self-limiting of B. caballi infection and the lifetime persistence of T. equi [89]. In this study, two genotypes of T. equi were pooled in a separate clade with T. equi that has already been reported in horses in America [90] and Israel [91]. Yet, a new potential Theileria sp. "Africa" genotypes were clustered in a separate clade of a good bootstrap support with the other Theileria sp. "Africa" previously detected in African horses in Senegal and Chad (Fig. 6) [50]. As far as we know, Theileria sp. "Africa" has never been reported yet in Egypt. T. ovis was detected in donkeys with a prevalence rate of 0.6%, representing its first detection in donkeys in Africa. Recently, T. ovis was reported in horses and donkeys in Turkey [92]. In the last decade, several studies have reported the existence of other piroplasmid species in horses and donkeys and have reduced the host specificity of piroplasmids [93,94].

Conclusion
In conclusion, the present study summarized the epidemiological and entomological data of the prevalence of EVBD infecting equines and their vector in two regions of Egypt (Cairo and Beni-Suef province). We reported, for the first time, the presence of Rh. microplus as a competent tick for Rh. annulatus in Egypt using MALDI-TOF and molecular identification, which increases the risk of transmission of other potential new vector-borne diseases, and this should be assessed in future studies. Also, we reported the first detection of A. marginale, A. platys-like, "E. rustica", new Ehrlichia sp., B. theileri in Rh. microplus, A. marginale, A. ovis and T. ovis in donkeys and a new Theileria sp. "Africa" in horses in Egypt. Therefore, equines, especially donkeys, should be involved in the epidemiology of tick-borne diseases as they serve as reservoirs for these emerging and remerging pathogens to other animals.

Ethical approval and Consent to participate
This study was approved by the Medical Research Ethics Committee at the National Research Centre, Egypt under the number 19059.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. In addition, the obtained sequences in this study were submitted to the GenBank database under their accession numbers. Figure 1 The arthropod vectors were collected from horses and donkeys, the louse fly Hippobosca equina (a-c) and ixodid tick Rhipicephalus (formerly Boophilus) annulatus (d-g