Protozoan and Rickettsial Pathogens in Ticks Collected from Infested Cattle from Turkey

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05–100% nucleotide identity with those deposited in GenBank (query cover range: 89–100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey.


Introduction
Ticks and tick-borne diseases (TBDs) significantly affect livestock production in many countries of the world. They are considered second only to mosquitoes in importance as vectors of disease agents causing great impact on animal and human health [1]. The global distribution of ticks and TBDs have led to considerable economic losses, such as loss of productivity, decreased meat and milk production, and death of livestock [2][3][4][5]. To date, Pathogens 2022, 11, 500 2 of 13 731 species of Ixodidae (hard ticks), 216 species of Argasidae (soft ticks), and 1 species of Nuttalliellidae (Nuttalliella namaqua) have been described worldwide [6]. Due to suitable climatic conditions and the large amounts of wild and domestic animals, a total number of 51 tick species have been identified in Turkey (43 from the family Ixodidae and 8 from the family Argasidae). Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus ticks are widely present throughout Turkey [7][8][9][10].
In Turkey, 27% of farm animals are cattle, with approximately 90% of milk and meat produced from bovines [11]. However, TBDs have a significant impact on the economy and animal health. The epidemiology of TBDs in Turkey had been reported previously [12][13][14], but there is still limited data about pathogens carried or transmitted by ticks. Therefore, the aims of this study were to investigate the presence of and genetically characterize the pathogens harbored by ticks collected from cattle to improve understanding of their distribution towards effective control in Turkey.
such as loss of productivity, decreased meat and milk production, and death of livesto [2][3][4][5]. To date, 731 species of Ixodidae (hard ticks), 216 species of Argasidae (soft tick and 1 species of Nuttalliellidae (Nuttalliella namaqua) have been described worldwide Due to suitable climatic conditions and the large amounts of wild and domestic anim a total number of 51 tick species have been identified in Turkey (43 from the family Ixo dae and 8 from the family Argasidae). Dermacentor, Haemaphysalis, Hyalomma, Ixodes, a Rhipicephalus ticks are widely present throughout Turkey [7][8][9][10].
In Turkey, 27% of farm animals are cattle, with approximately 90% of milk and m produced from bovines [11]. However, TBDs have a significant impact on the econo and animal health. The epidemiology of TBDs in Turkey had been reported previou [12][13][14], but there is still limited data about pathogens carried or transmitted by tic Therefore, the aims of this study were to investigate the presence of and genetically ch acterize the pathogens harbored by ticks collected from cattle to improve understand of their distribution towards effective control in Turkey.

Analyses of Tick-Borne Pathogen DNA Sequences
Positive samples of each pathogen were randomly selected as representative for sequencing and analysis. The NCBI BLASTn analysis of A. marginale (OL408894; H. excavatum),  (Table 2). Furthermore, T. annulata (OL408892) shared the highest percent identity (99.07%) with a dog isolate from Tunisia (KX130956) ( Table 2).

Phylogenetic Analyses of Rickettsial Agents
The A. marginale msp4 (OL408894) sequence from this study was found in the same clade with cattle isolates from Turkey (MF377455), Italy (KF739428), Algeria (KX179906), and Tunisia (KJ512170) (Figure 2). Meanwhile, the Ehrlichia sp. pCS20 sequence obtained in this study clustered with KT362172, an isolate from Rh. (Boophilus) microplus in China ( Figure 3). Phylogenetic trees inferred from 16S rRNA, gltA, and ompA genes of Rickettsia sp. are shown in Figure 4. The Rickettsia 16S rRNA sequences were grouped in two clades ( Figure 4A). Two sequences (OM541406 and OL377895) created a polyphyletic group with other R. aeschlimannii and R. massiliae sequences, while the remaining sequences (OM541405, OM541407, and OL377896) were grouped into R. conorii complex clade. Interestingly, OM541405 formed a well-supported branch with Rickettsia sp. isolated from a human with clinical Astrakhan fever in Chad. Phylogenetic analysis of gltA ( Figure 4B) and ompA ( Figure 4C) sequences further confirmed that OM717962 and OM717959 were most closely related to Rickettsia sp. Chad. Meanwhile, OM717963 and OM717960 were in the same group of R. aeschlimannii, whereas OM717964 and OM717961 were in the same group of Candidatus Rickettsia barbariae, respectively ( Figure 4).

Phylogenetic Analyses of Rickettsial Agents
The A. marginale msp4 (OL408894) sequence from this study was found in the same clade with cattle isolates from Turkey (MF377455), Italy (KF739428), Algeria (KX179906), and Tunisia (KJ512170) (Figure 2). Meanwhile, the Ehrlichia sp. pCS20 sequence obtained in this study clustered with KT362172, an isolate from Rh. (Boophilus) microplus in China ( Figure 3). Phylogenetic trees inferred from 16S rRNA, gltA, and ompA genes of Rickettsia sp. are shown in Figure 4. The Rickettsia 16S rRNA sequences were grouped in two clades ( Figure 4A). Two sequences (OM541406 and OL377895) created a polyphyletic group with other R. aeschlimannii and R. massiliae sequences, while the remaining sequences (OM541405, OM541407, and OL377896) were grouped into R. conorii complex clade. Interestingly, OM541405 formed a well-supported branch with Rickettsia sp. isolated from a human with clinical Astrakhan fever in Chad. Phylogenetic analysis of gltA ( Figure 4B) and ompA ( Figure 4C) sequences further confirmed that OM717962 and OM717959 were most closely related to Rickettsia sp. Chad. Meanwhile, OM717963 and OM717960 were in the same group of R. aeschlimannii, whereas OM717964 and OM717961 were in the same group of Candidatus Rickettsia barbariae, respectively ( Figure 4).

Discussion
Tick-borne protozoan and rickettsial diseases are major health problems for cattle and cause economic losses worldwide [4,14,15]. In recent years, studies related to ticks and TBDs have increased in Turkey. Understanding the complex relationships between ticks and pathogens is important to public health in mitigating tick-borne pathogen (TBP) transmission. In this study, we performed a molecular survey of tick-transmitted pathogens in ticks obtained from cattle in Diyarbakır, Gaziantep, Kahramanmaraş, Karaman, anḑ Sanlıurfa provinces located in the south of Turkey. Overall, seven tick-associated microorganisms, including A. marginale, B. bovis, B. occultans, Coxiella sp., Ehrlichia sp., Rickettsia sp. (Candidatus R. barbariae, R. aeschlimannii, and Rickettsia sp. Chad), and T. annulata were found in four tick species identified, namely H. excavatum, H. anatolicum, Rh. turanicus, and Rh. bursa.
Six Anaplasma sp., including A. bovis, A. centrale, A. marginale, A. ovis, A. platys, and A. phagocytophilum, cause a range of diseases in humans and other vertebrates [16]. The most common agent for bovine anaplasmosis is A. marginale, which is highly pathogenic and can be fatal in susceptible cattle [17]. Major surface protein 4 (MSP4) is an important protein, which is considered as a stable marker for genetic characterization of A. marginale strains [18,19]. In this study, a molecular survey based on the PCR amplification of the target gene msp4 was performed and A. marginale was detected in H. excavatum and Rh. turanicus ticks with a minimum detection rate of 2.5%. Previously, A. marginale has been reported in many regions of Turkey, which has been confirmed as the most prevalent tick-borne pathogen in cattle [10,15,[20][21][22][23][24]. However, A. marginale infection in ticks was rarely reported in Turkey. The phylogenetic analysis of A. marginale revealed that the msp4 sequence obtained from this study was highly similar to isolates from other Mediterranean countries. This result demonstrates the utility of A. marginale msp4 as a marker for phylogeographic characterization of A. marginale isolates from the field [18].
Bovine babesiosis is one of the most important TBDs of cattle in Turkey and has been reported in all Turkish provinces [11]. The most important causative agents of bovine babesiosis are B. bovis and B. bigemina, which are transmitted by Rhipicephalus ticks. The minimum detection rate of B. bovis in ticks was 7.9% and was only detected in ticks from Gaziantep and Kahramanmaraş. Interestingly, we did not identify ticks harboring B. bigemina. In Turkey, B. bigemina is the most frequent Babesia species and is transmitted by Rh. annulatus ticks [10]. Such results may be explained by the limited number of collected tick species and the number of samples in this study. Meanwhile, B. occultans was first described in South Africa and Nigeria and has been identified in Hyalomma ticks [25,26]. Of the 90 pools, 2 (minimum detection rate 0.7%) pools fromŞanlıurfa and Karaman were found positive for B. occultans, which was carried by H. excavatum in this study. Previous reports have proved the presence of B. occultans in Turkey [27] and found that it was not only carried by Hyalomma ticks, but was also observed in Rh. turanicus ticks [22].
Coxiella burnetii causes Q fever, a zoonotic disease which threatens human health and commonly causes abortions in cattle and sheep. In the present study, the Coxiella sp. positive tick species were H. excavatum, Rh. turanicus, and Rh. bursa. The presence of Coxiella sp. in ticks has been reported previously in Turkey wherein it was detected in H. marginatum, H. anatolicum excavatum, H. detritum, and Rh. annulatus ticks [28]. In another study, Coxiella sp. DNA was detected in H. excavatum, Rh. turanicus, and Rh. bursa ticks [29]. This suggests that Coxiella sp. is widespread in a variety of Hyalomma and Rhipicephalus species in Turkey. Additional studies are warranted to determine the presence of C. burnetii in the tick samples from this study.
Ehrlichia ruminantium causes heartwater disease in cattle and the main vector is the Amblyomma tick, particularly A. hebraeum and A. variegatum [30]. The partial sequences of E. ruminantium were reported previously in Rh. evertsi evertsi, H. truncatum, and H. marginatum ticks [31]. In this study, we detected Ehrlichia sp. in different tick species, including H. anatolicum, H. excavatum, Rh. turanicus, and Rh. bursa, suggesting that Ehrlichia sp. may be carried by Hyalomma and Rhipicephalus ticks. So far, only a few studies have documented the detection of Ehrlichia in ticks. Although Ehrlichia sp. was detected in H. excavatum and Rh. bursa in studies conducted in Turkey, that Ehrlichia sp. sequence found was similar to E. canis and Ehrlichia sp. Omatjenne strain [21,22]. The present study used an E. ruminantium-specific PCR assay, but the obtained Ehrlichia sp. pCS20 gene sequence has a 99.28% identity with the Ehrlichia sp. (KT362172) sequence, previously reported in Hunan province, China [32]. Further studies are needed to confirm the species of the detected Ehrlichia, to investigate its presence in the blood samples of bovines where the positive ticks were collected from, and assess its significance for animal health.
Three species of Rickettsia were confirmed by phylogenetic analysis based on the 16S rRNA, gltA, and ompA genes in this study, including Candidatus Rickettsia barbariae (OM541407, OM717964, and OM717961 from one sample), R. aeschlimannii (OM541406, OM717963 and OM717960 from one sample), and Rickettsia sp. Chad (OM541405, OM717962, and OM717959 from one sample). Our findings confirmed the presence of Candidatus Rickettsia barbariae in Turkey, which is in concordance with a previous report [33]. This rickettsial agent's DNA was firstly detected in Rh. turanicus ticks collected from European hare (Lepus europaeus) in 2020 [34]. The present study revealed the presence of Candidatus Rickettsia barbariae in ticks and reported the presence of this agent in Turkey for the second time. Moreover, the Rickettsia sp. Chad isolated from a patient was reported previously in a clinical Astrakhan fever case [35]. There is no information about the vector of Rickettsia sp. Chad. We have detected Rickettsia sp. Chad in Rh. turanicus in this study. Although there are only a few reports of Rickettsia sp. Chad infections in humans, our result suggests that Rickettsia sp. Chad infection may be a potential threat for humans, which manifests as fever and maculopapulous rash after infection [35]. As it was detected in ticks of cattle, further studies regarding its infectivity on cattle should be conducted.
Theileriosis is another tick-borne protozoan disease of cattle. The causative agents are transmitted from an infected animal to others by transstadial transmission via ticks. Theileria annulata transmitted by Hyalomma is the main TBP affecting cattle in Turkey [10]. The current study detected T. annulata infection in H. excavatum, H. anatolicum, and Rh. turanicus with a minimum detection rate of 5.8%. The main vectors of T. annulata are some species in the genus Hyalomma, and there are studies conducted in Turkey reporting that T. annulata was detected in H. excavatum [22,33]. Rhipicephalus turanicus harboring T. annulata was also reported in a previous study [36]. Therefore, it is necessary to evaluate whether Rh. turanicus will be a risk for T. annulata transmission in Turkey.
The detection of Coxiella sp. from ticks in this study despite using specific PCR primers, coupled with the results in BLASTn analysis, indicates that this species is an endosymbiont. Endosymbionts are non-pathogenic microorganisms that have evolved in the carriers or hosts either as obligate or facultative symbionts [37]. Ticks were reported previously to be highly infected with endosymbionts [38,39]. In this study, an isolate highly similar to Candidatus Coxiella mudrowiae, a known Coxiella endosymbiont, was detected in Rh. turanicus ticks, as it was similarly described in a previous study [40]. The extent to which the endosymbionts of ticks in Turkey affect transmission of other TBPs needs to be studied in the future.
In the study, a large number of single or multiple pathogenic microorganisms' DNA was detected in cattle tick pools. It is difficult to determine whether these microorganisms originated from ticks or from hosts blood since all ticks were collected while sucking blood from cattle. Karim et al. [38] reported that the characterization of multiple infections in ticks poses a major scientific challenge for understanding the epidemiology of tick-borne infectious diseases. However, since the tick DNA samples in this study were not extracted from each individual ticks but from 1-10 ticks collected in pools, this limits the discussion of single and coinfection status. Additionally, our study does not discuss the co-transmission of many microorganisms from ticks to cattle, and further studies are needed to obtain information about potential tick-borne pathogens and the dynamics of tick-borne infections in a region [38,39]. The limitation of this study was that it was carried out on a restricted number of cattle. The collection of ticks at a certain time of the year and the lack of periodic tick collection according to the seasons are considered as other limiting factors of this study. The study is also limited in choosing sampling locations and, thus, could not cover the whole of Turkey to provide a better representation of tick and tick-borne pathogens. Another limiting factor is the inability to obtain blood from infested cattle. Although we were aware of the importance of this, it was outside the scope of the present study.
In conclusion, the results obtained from the present study will contribute to the present knowledge on the distribution of ticks and the possible TBPs they may carry. Further investigation for the vector competence of tick species in transmitting these pathogens and more research with a greater number of ticks, blood samples, and sampling locations should be conducted in order to fully assess the risk factors for tick-transmitted livestock diseases in Turkey.

Ethical Statements
The owners of the cattle were informed about the study, and their approvals were obtained for tick collection. Tick sampling, as well as sample processing, were carried out according to the ethical guidelines permitted by Obihiro University of Agriculture and Veterinary Medicine (Permit for DNA experiment: 1723-4 and 1724-4; Pathogen: 201712-5).

Tick Samples and DNA Extraction
Tick samples were collected from March to June 2013. Seventy apparently healthy cattle were examined. Tick infestations (1-45 ticks per animal) were determined in 22 (31.43%) of these cattle. The study consisted of 277 adult ticks, which were collected in 5 provinces (Diyarbakır, Gaziantep, Karaman, Kahramanmaraş,Şanlıurfa) of Turkey. All ticks were removed from cattle skin with the consent of the livestock owner, and care was taken to minimize animal discomfort. The tick samples were kept in 70% ethanol prior to identification using a binocular microscope (Olympus SZX16, Tokyo, Japan) based on standard taxonomic keys [41,42]. Tick identification was done at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan. After microscopic identification, the tick samples were pooled according to species, gender, and sampling site. Ninety pools were generated based on sample size (1 to 10 ticks per pool). Tick DNA extraction was done as previously described [43] by using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was eluted with 50 µL of double-distilled water and stored at −30 • C until use.

Molecular Characterization of Ticks
To further confirm the tick species, molecular identification of the tick species was conducted through PCR assays. A primer set targeting the 16S rDNA of ticks was used, followed by cloning and sequencing of amplicons (Table S2). DNA amplification was done with a 10 µL PCR reaction volume containing 1 µL of 10 × Thermopol ® Buffer (New England Biolabs, Ipswich, MA, USA), 0.2 µL of 10 mM dNTP mix (New England Biolabs, Ipswich, MA, USA), 0.2 µL of 10 µM forward and reverse primers, 0.05 µL of Taq polymerase (New England Biolabs, Ipswich, MA, USA), 1 µL of DNA sample, and 7.35 µL of double distilled water. The thermocycling conditions consisted of an initial denaturation (95 • C for 2 min); 30 cycles of denaturation (95 • C for 30 s); annealing (52 • C for 30 s) and extension (68 • C for 1 min), then final extension (68 • C for 5 min) [44]. PCR products were checked by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized under UV light. All amplicons were extracted from gels using a Gel Extraction Kit (QIAGEN, Hilden, Germany) and sequenced.

Detection and Characterization of Tick-Borne Pathogens
A total of 90 tick DNA pools were generated in this study. All tick pool samples were primarily screened using primers targeting the genes 18S rRNA (V4 hypervariable region) for Babesia and Theileria [45], 16S rRNA for Anaplasma and Ehrlichia [46], and 16S rRNA for Rickettsia [47], as listed in Table S2. The positive samples based on these TBP assays were selected for species-specific detection. Partial sequences of B. bovis spherical body protein 4 (sbp4) [48], B. bigemina rhoptry-associated protein 1a (RAP1a) [48], T. orientalis major piroplasm surface protein (MPSP) [49], T. annulata merozoite surface antigen 1 (Tams-1) [50], Anaplasma marginale major surface protein 4 (msp4) [51], Ehrlichia pCS20 [52], C. burnetii 16S rRNA [53], and Rickettsia citrate synthase gltA and ompA [54,55] genes were amplified. The primers used are shown in Table S2. The DNA amplification and PCR cycling for all pathogens followed the same conditions as aforementioned, except for the annealing temperatures, wherein those documented in referenced publications were used. PCR products were checked and purified as described above. The samples were further confirmed by sequencing.

Cloning and Sequencing
The amplicons (tick 16S rDNA or pathogen DNA) were cloned in pGEM-T Easy Vector and sequenced as described [56] using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) and the ABI PRISM 3100 genetic analyzer (Applied Biosystems). The GenBank accession numbers are shown in Tables 2 and S1.

Sequence Alignment and Phylogenetic Analyses
The phylogenetic analyses of the sequences obtained from the PCRs specific for A. marginale, Ehrlichia sp., and Rickettsia sp. were performed subsequently, as rickettsial pathogens are relatively rarely studied in Turkey. Sequenced DNA samples were analyzed using the BLASTn tool of NCBI GenBank database and Clustal X program. In addition, phylogenetic analyses were inferred using MEGA version 7.0 software. The maximum likelihood method was employed as the method for tree construction because of its use of complex models to simulate biological reality and infer sequence evolution [57]. The sequences included in the phylogenetic analysis were chosen based on the BLASTn search results and geographical origin. Bootstrap analysis was performed with 1000 replicates to estimate the confidence of branching patterns of the tree.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens11050500/s1, Table S1. Tick species identification based on the mitochondrial 16S rDNA gene. Table S2. Primer sets used for the detection of tick-borne pathogens.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.