Tick Fauna and Associated Rickettsia, Theileria, and Babesia spp. in Domestic Animals in Sudan (North Kordofan and Kassala States)

Ticks and tick-borne diseases (TBDs) have a major economic impact on animal production worldwide. In the present study, 2410 ticks were collected from January to August 2017 from livestock and other domestic animals in North Kordofan and Kassala, Sudan, for species identification and investigation of Rickettsia spp. and piroplasms, either individually or as pools containing up to 10 ticks by molecular methods. In total, 13 tick species were identified by morphology and 16S rDNA sequencing. The most frequent tick species were Hyalomma impeltatum (24.90%), Rhipicephalus evertsi evertsi (18.84%), Amblyomma lepidum (16.06%), and Rhipicephalus camicasi (12.49%). A pan-Rickettsia real-time PCR revealed an overall minimum infection rate (MIR) with Rickettsia spp. of 5.64% (136 positive tick pools/2410 total ticks). Rickettsia africae and Rickettsia aeschlimannii were the most frequently identified species by sequencing. Furthermore, the following highly pathogenic livestock parasites were detected: Theileria annulata, Theileria lestoquardi, Theileria equi, and Babesia caballi. The present study documented Rhipicephalus afranicus as well as Rickettsia conorii israelensis, Rickettsia massiliae, and Babesia pecorum for the first time in Sudan. These findings are significant for the animal production sector as well as in terms of One Health, as the detected Rickettsia spp. can cause serious illness in humans.


Tick Collection and Identification
In this cross-sectional study, tick specimens were collected from livestock and other domestic animals in the period from January to August 2017, in North Kordofan and Kassala states, Sudan [14]. Samples were collected from three localities in the state of North Kordofan, including Sheikan, Al-Rahad, and Um-Ruwabah, and from five localities in the state of Kassala, namely Aroomah, Wagar, Kassala, West Kassala, and Khashm el Griba (Figure 1). These localities were selected randomly and/or conveniently. In both areas, mostly sheep and goats were examined, followed by cattle, camels and dogs. Sampled animals were thoroughly examined for attached ticks by searching the head and ears, the neck (dewlap), the thoracic area and the abdomen, the udder or scrotum, the fore-and hindlimbs, perineum, and the tail. Attached ticks were either collected by hand-or forcepspicking and stored in 70% ethanol. All ticks collected from the same animal host were put into one tube. Tubes were labelled (location, animal species and date of collection) and sent to the Bundeswehr Institute of Microbiology, Munich, Germany, where ticks were identified to species level using morphological characteristics described by Apanaskevich and Horak [22], Apanaskevich and Horak [23], Apanaskevich and Horak [24], Apanaskevich, et al. [25], Voltzit and Keirans [26] and Walker, et al. [10,27].  (4), Kashm el Griba (5), Kassala (6), Aroomah (7) and Wagar (8). The map was created using ArcGIS v. 10 (esri Inc., Redlands, CA, USA).

Tick Collection and Identification
In this cross-sectional study, tick specimens were collected from livestock and other domestic animals in the period from January to August 2017, in North Kordofan and Kassala states, Sudan [14]. Samples were collected from three localities in the state of North Kordofan, including Sheikan, Al-Rahad, and Um-Ruwabah, and from five localities in the state of Kassala, namely Aroomah, Wagar, Kassala, West Kassala, and Khashm el Griba (Figure 1). These localities were selected randomly and/or conveniently. In both areas, mostly sheep and goats were examined, followed by cattle, camels and dogs. Sampled animals were thoroughly examined for attached ticks by searching the head and ears, the neck (dewlap), the thoracic area and the abdomen, the udder or scrotum, the fore-and hindlimbs, perineum, and the tail. Attached ticks were either collected by hand-or forceps-picking and stored in 70% ethanol. All ticks collected from the same animal host were put into one tube. Tubes were labelled (location, animal species and date of collection) and sent to the Bundeswehr Institute of Microbiology, Munich, Germany, where ticks were identified to species level using morphological characteristics Microorganisms 2020, 8, 1969 4 of 16 described by Apanaskevich and Horak [22], Apanaskevich and Horak [23], Apanaskevich and Horak [24], Apanaskevich, et al. [25], Voltzit and Keirans [26] and Walker, et al. [10,27].

Nucleic Acid Extraction
Total nucleic acid was extracted using the MagNA Pure LC RNA/DNA Kit (Roche, Mannheim, Germany) in a MagNA Pure LC instrument (Roche) according to the manufacturer's instructions. Total nucleic acid was extracted from individual ticks or pools containing 2-10 specimens per pool, if the ticks shared the same developmental stage and species and were collected from the same animal. The extracted total nucleic acid was stored at −80 • C until use.

Molecular Tick Species Identification
Identification of ticks that were either damaged or fully engorged, and thus not reliably identifiable based on morphological criteria (n = 23), as well as confirmation of primary morphological determinations (n = 13), was achieved by 16S rDNA sequencing (250 bp fragment) and phylogenetic analysis. The gene was amplified using polymerase chain reaction (PCR) protocols and sequenced in both directions as previously described by Mangold, et al. [28]. Tick sequences generated in this study are available in GenBank (MT535883-MT535906). Additional sequences from GenBank were chosen to cover the range of Rhipicephalus and Hyalomma species that occur in Sudan and closely related species. As the prevalence of misidentified tick species among sequence data in GenBank is a growing problem, the selected sequences were derived from recent studies that included large-scale taxonomic investigations to verify identification by phylogenetic analysis and correlated morphology [29][30][31][32][33][34]. Sequence data were aligned using MAFFT (Q-INS-I, 200PAM/k = 2, Gap opening penalty 1.53) [35], and the final alignment comprised 265 nucleotide characters. The alignment was inspected manually to ensure sequences were in reading frame. Phylogenetic analysis was based on maximum likelihood with 1000 bootstrap replicates in MEGA v7.0.14 [36] using a TPM2u + F + G4 model determined by Bayesian Information Criterion calculations in W-IQ-TREE [37].

PCR for Rickettsia spp. and Piroplasms
For detection of Rickettsia spp., a pan-Rickettsia real-time PCR was used [38,39]. Positive samples were further subjected to Rickettsia species identification by amplification, sequencing in both directions and phylogenetic analysis of the 23S-5S intergenic spacer region (330 bp fragment) as described by Chitimia-Dobler, et al. [40]. Additional sequences from GenBank were chosen to cover the range of Rickettsia species that occur in Africa and Eurasia [41,42]. Sequence data were aligned using MAFFT (Q-INS-I, 200PAM/k = 2, Gap opening penalty 1.53) [35], and the final alignment comprised 403 nucleotide characters. The alignment was inspected manually to ensure sequences were in reading frame. Phylogenetic analysis was based on maximum likelihood with 1000 bootstrap replicates in MEGA v7.0.14 [36] using an HKY + F + G4 model determined by Bayesian Information Criterion calculations in W-IQ-TREE [37].
To identify whether the collected ticks were infected with piroplasms, the pools were tested for Theileria spp. and Babesia spp. DNA by amplifying a part of the 18S rDNA in a conventional PCR, using the primers BJ1 and BN2 [43], as described by Springer, et al. [44]. Obtained 18S rDNA amplicons were custom Sanger-sequenced (Microsynth Seqlab Sequencing Laboratories, Göttingen, Germany), or-in case of weak bands-ligated into the pCR™4-TOPO ® TA vector and cloned into One Shot Top10 chemically competent Escherichia coli (TOPO ® TA Cloning kit, Thermo Fisher Scientific GmbH, Dreieich, Germany). After plasmid extraction and purification (NucleoSpin Plasmid kit, Macherey-Nagel GmbH & Co. KG, Düren, Germany), the insert was custom Sanger-sequenced, as indicated above. Rickettsia spp. and piroplasms' sequences generated in this study are available in GenBank under the accession numbers MW152276-MW152327 for Rickettsia spp., and MW131349-MW131365 for piroplasms. Minimum infection rates (MIRs) were calculated under the assumption of only one positive tick per pool (MIR = number of positive pools/total number of ticks).

Rickettsia Species
In total, 783 tick pools were tested for Rickettsia species by real-time PCR. Of these, 136 were Rickettsia-positive, resulting in an MIR of 5.64% (136/2410). Rickettsia DNA was detected in 11 out of 13 tick species (Table 1). Rickettsia species composition among the positive tick pools from North Kordofan and Kassala is shown in Figure 2B.
In Amblyomma spp., the MIR was 12.13% (54/445), and sequencing of the 23S-5S IGS region confirmed Rickettsia africae in 37 Amblyomma pools ( Figure 5). In the remaining 17 samples, the Rickettsia DNA content was too low for species identification. The MIR in Hyalomma spp. was 4.4% (44/998). Twelve of the 44 samples were successfully sequenced, leading to the identification of Rickettsia aeschlimannii. Among the observed Rhipicephalus spp., the MIR was 3.93% (38/967). Unfortunately, the majority of Rickettsia-positive Rhipicephalus samples did not contain enough Rickettsia DNA for 23S-5S sequencing. Regardless, Rickettsia conorii israelensis was detected in two Rh. camicasi pools and R. aeschlimannii in one Rh. evertsi evertsi pool. The single Rh. afranicus specimen was also Rickettsia-positive, and subsequent sequencing identified Rickettsia massiliae.       (44/998). Twelve of the 44 samples were successfully sequenced, leading to the identification of Rickettsia aeschlimannii. Among the observed Rhipicephalus spp., the MIR was 3.93% (38/967). Unfortunately, the majority of Rickettsia-positive Rhipicephalus samples did not contain enough Rickettsia DNA for 23S-5S sequencing. Regardless, Rickettsia conorii israelensis was detected in two Rh. camicasi pools and R. aeschlimannii in one Rh. evertsi evertsi pool. The single Rh. afranicus specimen was also Rickettsia-positive, and subsequent sequencing identified Rickettsia massiliae.

Discussion
Ticks and TBDs constitute a global economic and health burden. In countries with a socioeconomic status similar to that of Sudan, a substantial proportion of livestock are owned by subsistence farmers, who are especially vulnerable to the impact of ticks and TBDs [45]. Hassan and Salih [17] stated that the natural population of ticks infesting livestock is changing in Sudan. Therefore, monitoring of the local tick fauna is necessary. In this study, we classified 2410 ticks collected from livestock and other domestic animals in two regions in Sudan into 13 different tick species belonging to the genera Hyalomma, Amblyomma, and Rhipicephalus. Tick screening for Rickettsia spp. and piroplasms revealed Rickettsia spp., like R. africae and R. aeschlimannii, as well as several piroplasms of veterinary relevance. Interestingly, we report Rhipicephalus afranicus, Rickettsia conorii israelensis, Rickettsia massiliae, and Babesia pecorum for the first time in Sudan. These findings are of high significance for the animal and public health sectors, particularly from a One Health point of view, as rickettsiosis is an important zoonotic disease.
With the exception of Rh. afranicus, which can experimentally transmit Babesia trautmanni to pigs [29], all of the other detected tick species have formerly been reported in Sudan [15,[46][47][48]. Both sampling areas are characterized primarily by Sahelian dry savannah ecosystems; nevertheless, regional differences were noted. Although there was variation in the species composition of the examined host populations, limiting comparability between both regions, the majority of examined animals in both regions were sheep, goats and cattle. Therefore, it was remarkable that the tick fauna in North Kordofan was dominated by Hyalomma spp., while Rhipicephalus spp. were the most frequent ticks in Kassala. H. anatolicum, the main vector of T. annulata, which causes bovine tropical theileriosis, has undergone a south-and west-ward spread in Sudan since the 1980s, probably due to animal movement and environmental change [17]. Recently, H. anatolicum represented more than 50% of collected ticks in West Darfur, Al-Jazeera, and River Nile states [15]. This indicates that the distribution of H. anatolicum has reached the western border of Sudan. In the present study, it was the most frequently observed Hyalomma spp. in Kassala, but was also collected in North Kordofan, coinciding with the detection of bovine tropical theileriosis in North Kordofan [49].
In Kassala, Rh. evertsi evertsi and Rh. camicasi together accounted for more than 50% of collected ticks. Similarly, Rh. evertsi evertsi was frequently encountered on cattle in Gezira, central Sudan, and on different domestic animals in West Darfur and River Nile [15]. Both species were also reported by Jongejan, et al. [46] in the Blue and White Nile ecosystems. However, Rh. camicasi can be difficult to distinguish morphologically from other ticks of the Rh. sanguineus group, which may explain why this species has been less frequently reported in other studies [48,50].
Contrary to the findings of Shuaib, et al. [15], Elghali and Hassan [51] and Ahmed, et al. [50], A. lepidum was found in both states, North Kordofan and Kassala, and accounted for approximately 16% of all identified ticks. This tick has historically been abundant in the eastern part of the country, such as Kassala [17]. In recent decades, a westward (towards Kordofan and Darfur regions) spread of A. lepidum has been observed [17]. Indeed, the importance of A. lepidum lies in the fact that it is the main vector of Ehrlichia ruminantium, the causative agent of heartwater, which results in significant morbidities and mortalities in domestic ruminants [52].
One Rh. afranicus specimen, collected from a sheep, was identified by sequencing of the 16S rRNA gene. This taxon was historically confounded with Rhipicephalus turanicus, however, it was recently described as a distinct species [29]. It has further been confirmed in South Africa [29] and Uganda [53] to date. These Rh. afranicus populations may represent two distinct lineages within the species given molecular distances between southern (i.e., South Africa) and northern (i.e., Uganda, Sudan) regions [53]. Interestingly, this specimen carried R. massiliae DNA.
The most relevant tick-borne rickettsiae in Africa are R. africae, primarily transmitted by Amblyomma spp., R. aeschlimannii, mainly transmitted by Hyalomma spp., and R. conorii conorii, which is transmitted by Rhipicephalus ticks [54]. In this study, the detected MIR (12.13%) of Amblyomma ticks with Rickettsia spp. and the confirmation of R. africae in the majority of samples denote to a considerable risk of infection of humans with R. africae, the causative agent of African tick-bite fever. Similar infection rates of ticks with R. africae have been noted in Sudan before [15]. However, high rickettsial infection rates of up to 100% have been described in Amblyomma spp. in other regions of Africa, probably due to effective transovarial transmission [54]. Furthermore, MIRS of 4.4% and 3.9% were detected in Hyalomma and Rhipicephalus ticks, respectively. In previous studies from eastern Africa, these tick genera mostly showed a lower Rickettsia prevalence than Amblyomma spp., ranging from approximately 10 to 46% in Hyalomma spp. [55][56][57] and 0 to 1.1% in Rhipicephalus spp. [55,57]. Species identification was only possible in approximately one third of the positive Hyalomma pools and the pathogen was confirmed as R. aeschlimannii. In the same way, the low rickettsial DNA content did not allow for species identification in most of the Rhipicephalus samples. Probably, low rickettsial DNA content is indicative of the fact that the last blood meal of the tick contained rickettsiae, rather than indicating true infection of the tick. Nevertheless, R. conorii israelensis was identified in two Rh. camicasi pools and R. aeschlimannii in one Rh. evertsi evertsi pool. Rickettsia conorii israelensis is the causative agent of Israeli spotted fever and occurs mainly in the Mediterranean countries [54]. Nevertheless, it has been occasionally detected in Africa, e.g., in Tunisia [58], Nigeria [59] and Kenya [60]. Studies proved that Rh. sanguineus s.l. acts as a vector of R. conorii israelensis, while the competency of Rh. camicasi as a vector is yet to be confirmed [61].
Of note, most of the ticks collected in the present study infest humans only occasionally [62,63]. Nonetheless, this does not rule out the risk of Rickettsia spp. transmission to humans. Currently, there are no published data on human Rickettsia exposure in Sudan or on the incidence of African tick-bite fever or other rickettsioses. Regarding livestock, high seroprevalences have been observed in sheep (59.3%) and cattle (64.4%) [64]. Considering these high seroprevalence rates and the reported MIRs in this study, investigations into the epidemiology of rickettsiosis in humans are required, concentrating on at-risk populations, especially rural communities with frequent contact with livestock.
Furthermore, relevant tick-borne pathogens for domestic animal health were detected in this study. MIRs were 0.58% for Theileria spp. and 0.12% for Babesia species. Serological and molecular evidence for circulation of these piroplasms among livestock has been reported in Sudan before [13,65]. Molecular characterization by sequencing of the 18S rDNA revealed that the investigated ticks carried T. annulata. In Sudan, bovine tropical theileriosis has been recognized as one of the main limitations that slow the development of the dairy industry [66]. A westward spread of T. annulata with its main vector, H. anatolicum, has occurred in Sudan, and the pathogen is now also present in North Kordofan, where it was believed to be absent until 2015 [49]. In the present study, we detected H. anatolicum in North Kordofan, but all T. annulata-positive ticks (one H. anatolicum and one Rh. evertsi evertsi pool) were from Kassala. Therefore, further studies are needed to assess the risk of T. annulata transmission to cattle in North Kordofan.
Overall, the high diversity of pathogenic piroplasms detected in the present study indicates that tick control is relevant for all livestock species in Sudan. Besides T. annulata, T. lestoquardi that leads to malignant ovine theileriosis, as well as T. equi and B. caballi, the etiological agents of equine piroplasmosis were also detected, in addition to the apathogenic species T. ovis and T. velifera [67]. Regarding equine piroplasmosis, T. equi was detected in H. anatolicum and B. caballi in A. variegatum. While H. anatolicum is a relevant vector for T. equi, the detection of B. caballi in A. variegatum may indicate that this tick had simply ingested infected blood, as Amblyomma ticks are not known to act as vectors of Babesia spp. [68].
In addition, B. pecorum was detected in two H. impeltatum pools. This finding suggests that this parasite is globally widespread, since it has been reported in wild animals in South Africa and Spain and in sheep in China [69][70][71]. For transmission of this parasite, H. anatolicum showed vector competency in China, whereas in Spain, H. lusitanicum was suggested to be the vector of B. pecorum [69,71]. It is unlikely that B. pecorum is of any relevance for domestic animal health, as experimentally infected non-immunosuppressed sheep and calves did not show any clinical signs [69,71].

Conclusions
The present study demonstrated a diverse tick fauna on livestock and other domestic animals in Sudan, with Hyalomma spp. predominating in North Kordofan and Rhipicephalus spp. in Kassala. In addition, the newly described species Rh. afranicus was detected. The high Rickettsia infection rates indicate a non-negligible risk for humans, especially in pastoral communities and rural areas. The presence of R. conorii israelensis in Sudan was documented for the first time. The detection of the highly pathogenic livestock piroplasms (T. annulata, T. lestoquardi, T. equi and B. caballi) is an indicator of the need for control programs to reduce the potential economic losses due to ticks and TBDs, as well as for further studies to provide a full picture of their epidemiology.