Tick Distribution and Detection of Babesia and Theileria Species in the East and South regions of Kazakhstan


 Background: Piroplasmosis, caused by Theileria and Babesia species, is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks from different host species in Kazakhstan. Method: We collected adult ticks at 26 sampling sites in 16 districts in the East and South regions of Kazakhstan during 2016 - 2019. Tick species were identified according to morphological and molecular characteristics. Two fragments of the 18S ribosomal RNA (18S rRNA) were used to determine the species of piroplasms. The genotype characteristics of Babesia caballi and Theileria equi were analyzed further. Results: A total of 6107 adult ticks were collected, including 2920 Dermacentor marginatus, 1442 Argas persicus, 903 Hyalomma asiaticum, 464 Hyalomma scupense, 225 Hyalomma anatolicum, 122 Rhipicephalus turanicus, 16 Haemaphysalis erinacei and 15 Dermacentor reticulatus. In these ticks five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected. Theileria equi and Babesia caballi belonged to their E and A genotype, respectively.Conclusions: Apart from Dermacentor marginatus, three Hyalomma species predominate among ticks infesting herbivorous livestock in East and South regions of Kazakhstan. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by D. marginatus and Ha. erinacei, were clustered together with conspecific ticks reported from China. Babesia occultans was found for the first time in D. marginatus in Kazakhstan. Altogether five piroplasm species were detected, indicating that enhanced control measures are necessary to prevent piroplasm infection of domestic animals in this region.

Historically, tick-borne diseases have always represented important public health issues in Central Asia.
Among the others, tick-borne encephalitis, rickettsial diseases, Q fever and Crimean Congo hemorrhagic fever are known to occur in Kazakhstan [3][4][5][6]. Piroplasms (Apicomplexa: Piroplasmida) are tick-borne parasitic protozoa, most of which belong to the genera Theileria and Babesia [7]. A number of these parasites are highly pathogenic for cattle, horses, dogs, sheep, camels and in some cases, even to humans, as they may cause high fever, lethargy, anorexia, peripheral edema, petechiation of the mucous membranes, hemolysis, tachycardia and pigmenturia [8,9]. Bovine theileriosis caused by Theileria annulata and equine piroplasmosis caused by Theileria equi were reported in Kazakhstan [10][11][12]. However, the detection of Babesia and Theileria species in ticks from other livestock and wildlife species is still poorly known in this country, especially in East and South regions.
In the present study, the distribution and abundance of tick species were investigated in the East and South regions of Kazakhstan. These ticks were also screened for the presence of Babesia and Theileria DNA. In addition, genotype characteristics of T. equi and Babesia caballi in ticks of horses were evaluated.

Methods
Tick sampling and morphological identi cation From March to May during 2016 -2019, contemporaneously with the peak activities of adult ticks in Kazakhstan, ticks were collected from their hosts and in the environment at 26 sampling sites in East Kazakhstan (8), Almaty (9), Jambyl (2), Southern Kazakhstan (6) and Kyzylorda (1). Off-host ticks were collected by the dragging-agging and arti cial trapping methods [13,14]. Engorged ticks were sampled over the entire body of each animal including cattle, horses, sheep, camels, shepherd dogs, hedgehogs and chickens [15]. Data of all specimens were recorded, including date and location of collection, host species (if relevant) and individual number. All ticks were identi ed morphologically according to previous reports [16,17].

DNA extraction and molecular identi cation of ticks
After detailed morphological analysis, the genomic DNA was extracted from 209 ticks individually (2 to 40 individuals per tick species representing each sampling site) with the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). These DNA extracts were used for molecular taxonomic analysis of the 16S rDNA gene [18].

Molecular and phylogenetic analyses of Babesia and Theileria species
The presence of Babesia and Theileria species was screened with a PCR amplifying two fragments (487bp and 438bp) of the 18S ribosomal RNA (18S rRNA) gene, using the primers BJ1/BN2 [19] and PIRO-A/B [20]. Sequence-con rmed Babesia occultans and Theileria ovis DNA ampli ed in our laboratory and double distilled water were used as positive and negative controls, respectively [21]. In addition, samples positive for B. caballi and T. equi were evaluated further by amplifying longer the 18S rRNA gene fragments ( 1, 380 bp for B. caballi and 1470 bp for T. equi). Nucleotide sequences of the primers used for the identi cation of piroplasm and tick species were shown in Appendix Table S1. PCR products were puri ed using the TIANgel Midi Puri cation Kit (TIANGEN, Beijing, China) and cloned into the pGEM-T Easy vector and subjected to sequencing. The sequences obtained here were compared to others deposited in GenBank with the BLASTn programme (http://www.ncbi.nlm.nih.gov/BLAST/). Phylogenic trees were constructed using the Maximum Likelihood method with MEGA 7.0 software [22]. Piroplasms were molecularly detected in 698 out of 6107 sampled ticks by amplifying 487 bp and 438 bp 18S rRNA fragments. Among these, 1.43% (10/698) and 3.44% (24/698) of samples were positive for Babesia and Theileria species, respectively. Based on sequence comparisons, two Babesia species (B. occultans and B. caballi) and three Theileria species (T. ovis, T. annulata and T. equi) were identi ed ( Table 1, Fig. 2 and Appendix Table S3). Phylogenetic analyses using 18S rRNA fragments revealed that T. equi belonged to genotype E and B. caballi to genotype A ( Fig. 3A and 3B).  [24]. Furthermore, the phylogenetic tree based on 16S rDNA sequences showed that D. marginatus ticks in Almaty, Jambyl and South Kazakhstan clustered together with those from XUAR (GenBank: MF002564). In addition, Hy. asiaticum (MN907445) in Almaty represented the same phylogenetic group as ticks from Turkmenistan (KU13042), Turkey (MG418675) as well as XUAR of China (MF973043) (Fig. 4). Hy. scupense, however, showed different abundances on domestic animals in Southeastern Kazakhstan compared to Northwestern China. According to the present results, this species is widely distributed in East Kazakhstan and Almaty on cattle and goats, while it is rare in XUAR [24].

Results
1. B. occultans was rstly described from Hyalomma ru pes in 1981 in South Africa [25]. Subsequently, it was found in engorged ru pes, Hyalomma truncatum, Hyalomma impressum, Hyalomma marginatum and Hyalomma impeltatum ticks in 1984 in Nigeria [26]. For a long time, the distribution of B. occultans was believed to be con ned to sub-Saharan Africa. However, in 2017 it was shown to be present in Dermacentornuttalli in XUAR [21]. Here B. occultans detected for the rst time in D. marginatus ticks in Kazakhstan clustered phylogenetically with B. occultans (MF120939) reported from Jimunai County of XUAR (Fig. 2). Taken together, in this study ve species of piroplasms, i.e. B. occultans, B. caballi, T. ovis, T. annulata and T. equi were detected in South Kazakhstan, suggesting that enhanced control measures are necessary to prevent piroplasm infection of domestic animals in this region.
The 18S rRNA gene is widely used as a genetic marker for phylogenetic analyses of piroplasms [27]. Previous studies reported that T. equi genotype E was associated with fatal cases of equine piroplasmosis in Greece and Spain [28,29]. Although increasing amounts of data on equine piroplasmosis had become available from Spain, Mongolia, Egypt and China [28,[30][31][32]    caballi obtained in this study are indicated by solid diamond ( ) and solid triangle ( ), respectively.