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. [62–64]. Therefore, MALDI-TOF MS was applied to identify Rhipicephalus sp. and H. equina. MALDI-TOF MS confirmed that we have two different species of Rhipicephalus: Rh. microplus and Rh. annulatus. The molecular identification confirmed MALDI-TOF MS results. CO1 of H. equina and 16S rRNAs of Rh. annulatus and Rh. microplus were used for the identification and were deposed in GenBank (MK737646, MK737648 and MK737647; respectively). The percentages of coverage and identity between sequences obtained from the same species were 99%. After molecular confirmation, MS spectra of H. equina, Rh. annulatus and Rh. microplus were deposed in the database as reference MS spectra, and then, a blind test was applied on all vector samples. The majority of Rhipicephalus sp. were identified as Rh. microplus that were collected from horses and donkeys from Beni-Suef province. To the best of our knowledge, Rh. microplus has never been reported in Egypt or even in North Africa. Rh. microplus is a Southeast Asian tick, introduced into Southeast Africa by cattle from Madagascar [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]. In the present study, the overall prevalence of Anaplasmataceae infection in Rhipicephalus sp. collected from Equidae was 14.7%. This study reports for the first time a novel potential Anaplasma sp. in Rh. microplus in Egypt. This Anaplasma sp. was genetically close to canine A. platys with 99% homology, so that it was commonly named A. platys-like. The phylogenetic tree revealed that our A. platys-like was grouped in the same clade with canine A. platys (bootstrap value 89; Fig. 4). However, it forms a separate clade with A. platys-like detected in cattle and sheep from Beni Suef province where Rh. microplus was collected from donkeys (Abdullah et al., unpublished). As far as we know, the presence of A. platys-like has never been reported in Africa in Rh. microplus. Recently, A. platys-like was reported in Rh. microplus in China [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].