MOLECULAR STUDY AND PHYLOGENY OF Babesia spp. IN NATIVE SHEEP FROM SULAIMANI GOVENORATE/ NORTHERN IRAQ

This study was conducted to investigate Babesia parasites infecting sheep in eight districts of Sulaimani governorate/north Iraq from April to October 2017. Forty flocks of small ruminants were selected to collect blood samples randomly from 450 sheep. The samples were examined for babesiosis by microscopic examination and PCR. Primers based on the 18S rRNA were used for Babesia diagnosis, followed by sequencing of the amplicons for confirmation of the PCR product identities. Seventy-four samples (16.44%) showed the presence of Babesia piroplasms microscopically, while 116 (25.78%) samples were positive using PCR. Results showed that B. ovis was reported in 15.78% (n = 71), and B. motasi in 10.0% (n = 45) of the samples. Also, BLAST analysis of the obtained partial sequences of the 18S rRNA gene from current study isolates revealed the existence of both B. ovis and B. motasi, with a high homology degree of nucleotide identity with other nucleotide sequences of Babesia spp. in GenBank database. Distribution of babesiosis, according to the sampling time, revealed that high-frequency rates occur during July and August. Based on the result data, babesiosis was mainly caused by B. ovis and B. motasi.


INTRODUCTION
Protozoan parasites of the genus Babesia are known to cause babesiosis (13). The genus includes a large number of classified and unclassified species that infect different wild and domestic animals, humans, and birds. Babesia parasites are different in size, shape, and capability to produce disease (14). Babesia species are widespread in tropical and subtropical areas of the world. In sheep, babesiosis is a crucial hemoparasitic disease caused by Babesia ovis, B. motasi, B. crassa, and Babesia sp. BQ1 (Lintan) (20). Other species, including B. foliate and B. taylori, were described microscopically, while there was no molecular data in GenBank supporting the availability of these two Babesia species (21). The clinical signs of babesiosis vary, ranging from subclinical infection to lifethreatening diseases that may result in severe economic losses among livestock, particularly cattle (7). The clinical signs are not very prominent in small ruminants, although typical signs of pyrexia, hemoglobinuria, and anemia occur in the severe form of the disease (15). The main tick vectors responsible for the transmission of B. motasi and B. ovis are Haemaphysalis sp. and Rhipicephalus bursa, respectively (14), although B. ovis infection has been confirmed in Hyalomma anatolicum anatolicum and Hyalomma marginatum too (9). Also, the transmission of B. motasi by Dermacentor silvarum has been reported (1). Blood smears and clinical symptoms are useful in the diagnosis of acute piroplasmosis, but they are insufficient in subclinical cases. On the other hand, accuracy in diagnosis of specific-species existence in certain regions or countries required sensitive diagnostic method such as sequencing and phylogenetic analysis (3), so molecular methods could allow direct, specific, and sensitive detection of parasites. PCR is the most commonly used molecular assay, and the 18S rRNA genes have been successfully applied to identify and classify several unknown Babesia parasites (29). Moreover, detection of Babesia infection in carrier animals by DNA amplification is considered a powerful tool for epidemiological investigations, since it is more sensitive and specific than Giemsa-stained blood smears (2). Babesiosis has been documented in Sulaimani province/north Iraq using microscopic examinations. However, a comprehensive molecular study has not been previously conducted to determine the prevalent Babesia species in the area, especially in clinically healthy animals. This study was designed to investigate the occurrence of Babesia in naturally infected sheep from Sulaimani governorate in the north region of Iraq and to conduct molecular characterization and phylogenetic analysis of the isolates with reference isolates of Babesia spp., deposited in the GenBank database.

MATERIAIS AND METHODS Blood sampling and study area
This study was carried through in eight districts of Sulaimani, north of Iraq, from April to October 2017. Forty small ruminants' flocks were selected in seven districts of the province, namely Arbat, Bakrajo, Bazian, Mawat, Nal Parez, Sayid Sadiq, Sharazoor, and Sitak. A total of 450 sheep blood samples were collected randomly. About 5 mL of blood was withdrawn from the jugular vein and collected in EDTA-coated vacutainer tubes. Samples were collected indiscriminately from male and female one-to-six-year-old sheep. Thin blood smears were fixed on glass slides, and the remainder of each blood sample was stored at -80°C till DNA extraction. The blood smears were fixated in methanol and stained with Giemsa stain using standard procedures. The slides were examined microscopically under 1000× magnification for the presence of Babesia organisms. DNA extraction DNA was extracted from aliquots of 200 μL of whole blood samples using a DNA extraction kit for blood (GeNet Bio, South Korea) according to the manufacturer's instructions. The DNAs were eluted into 200 μL buffer, at room temperature, and extracted DNA yields stored at -80°C and used as templates when PCRs performed.

Molecular detection
All DNA samples were screened using PCR with primers designed based on the 18S rRNA. The reactions were achieved in a total volume of 20 μL using 2× Prime Tag Premix (GeNet Bio), in a programmable thermal cycler (Prime, UK) in two steps. The first step included amplifying about 1700 bp of Piroplasma 18S rRNA with the primer sets BaTh F 5'-CCTGGTTGATCCTGCCA GTAGTC-3 ' and BaTh R5'-CCTTCTGCAGGTTCACCTA CGG -3'. Five microliters of the DNA template were used, and the PCR protocol started with an initial denaturation 95°C/5 min, with 38 cycles of 95°C/1 min, 60°C/30 sec, 72°C/2 min, and a final extension of 72°C/5 min. The second step included PCR amplification for DNA samples being positive in the first PCR step by using the previously described primer sets Bbo-F5'-TGGGCAGGACCTTGG TTCTTCT-3' and Bbo-R 5'-CCGCGTAGCGCCGGCTAAATA-3' to amplify a 549 bp fragment of the 18S rRNA gene of B. ovis (6). For B. motasi, primer sets described by Shayan et al (28) were applied by semi-nested PCR. In the first reaction, 5.0 μL of DNA with primer sets P1 5'-CACAGGGAGGTAGTGACAAG-3' and P2 5'-AAGAATTTCACCTATGAC AG -3' were used to amplify 389-402 bp of DNA fragment. The second reaction was run with 1.0 μL of the PCR product as a template with primer sets P3 5'-AAGAATTTCACC TATGACAG-3' and P4 5'-CGCGATTCCG TTATTGGAG-3' for amplifying a 205 bp fragment. PCR protocols for Babesia species differentiation were executed following previously described procedures (6 and 28) with some modification. Finally, PCR products were separated by loading 10 μl on 1% agarose gels in 1 × Tris/Borate/EDTA buffer for electrophoresis and visualized using ethidium bromide for checking the amplicon size, and by comparing them to a 100 bp DNA ladder. Sequencing and phylogenetic analysis PCR amplicons from eight Babesia-positive samples five from B. ovis and three from B. motasi were selected randomly for nucleotide sequencing by Sanger DNA sequencing system in South Korea, using the primer sets for amplifying 1700 bp of Piroplasma 18S rRNA. The obtained partial sequences identified in the study were deposited in the GenBank database, and the accession numbers MN545599, MN545600, MN545603, MN551067, MN560046 were obtained for B. ovis isolates. Moreover, the B. motasi isolates received the accession numbers MN548430, MN551068, and MN548425.The nucleotide sequence identities and similarities were studied by nucleotide sequence homology using BLASTn at the network server of NCBI (National Center for Biotechnology Information). Sequence alignment was performed using CLUSTAL Omega multiple sequence alignment online tool, and phylogenetic analysis was done using MEGA X software (18). The maximum parsimony method was used to construct phylogenetic trees for Babesia spp., and Bootstrap with 1000 replications was used to estimate the confidence of the nodes, and branches of the trees.

RESUITS AND DISCUSSION
Seventy-four (16.44%) out of the total 450 examined sheep included in this study were positive for ovine babesiosis by microscopic examination. Positive smears showed different intraerythrocytic forms of hemoprotozoa, which were morphologically compatible with Babesia piroplasms. In different to our finding (5) reported (26.9%) from Al-Najaf province. The incidence of babesiosis was also reported from other countries using microscopy, in Iran (46.0%, 24.67%) were reported by (16 and10), in Turkey 27.3% was reported (25), also in India, the prevalence rate of babesiosis was 26.8% (15). In difference to our finding low prevalence of (9.67%, 2.9%) were reported by (22 and 11) from Pakistan and Tunisia respectively. Following the finding of Shayan et al (28), polymorphic characters of small ruminant Babesia were prominent, including double pyriform with an acute or obtuse angle, single pyriform, and ring form. Different sizes of round forms were also evident. Previously, according to morphological data, the large Babesia species was routinely diagnosed as B. motasi and small-sized as B. ovis. Also, the presence of polymorphisms could be the main problem in Babesia species differentiation microscopically, which can be dissolved by PCR analysis (10). In this regard (28) described that the large-sized B. ovis resembled B. motasi in morphological and biometrical parameters. Detection of Babesia infection in carrier animals through DNA amplification is a powerful tool for epidemiological investigations, as it is more sensitive and specific than Giemsa-stained blood smears examination (2). The PCR results of 450 examined sheep revealed that 25.78% (n = 116) were infected with Babesia spp. Of these, B. ovis was reported in 15.78% (n = 71) of the studied sheep, while 10.0% (n = 45) of examined sheep were positive for B. motasi. Based on result data, B. ovis and B. motasi were reported as the main causative agents of small ruminant babesiosis, with various prevalence rates in different geographical areas. The prevalence rate of 45.0% was reported by Alkhaled and Abdul-Hassan (4) from Iraq, with infection rates of 15.0% and 5.0% for B. ovis and B. motasi respectively, also in Spain, Nagore et al. (19) reported prevalence rates of 2.5% for B. ovis and 2.0% for B. motasi by RLB. However, indifferent to the study data, higher infection rate of B. motasi than B. ovis were reported by Ros-García et al. (24) et al which were 12.3% and 6.3% for both species respectively. Other researchers reported higher incidences of B. ovis than the current study in Iran (12) reported an incidence of 18.5%, and (8) found that 17.5% of sheep were infected. In Turkey, a prevalence rate of 48.0% was reported by Sarayli et al. (25). In Pakistan, Iqbal et al. (17) reported prevalence rate of 50.0% by PCR. Also, in Tunisia, an incidence of 17.4% was reported by Rjeibi et al. (23). Different factors might cause variations in the frequency of babesiosis, such as the animals breed, the immunological status of the host, geographical diversity, and parasite strains (26). This study result constituent with the finding of Faraj and Al-Amery (13) that PCR technique was with sensitivity even in low level of parasitemia in compare to Giemsa stained blood smear for detecting Babesia organisms. The existence of subclinical babesiosis in an endemic area might be related to enzootic stability phenomenon, due to the presence of a relatively high number of infected ticks (11). Due to the existence of many infected ticks, host immunity is being maintained at a high level through repeated challenges. So, with the traditional management system, where tick control is loose, the disease represented to be less serious. Month's wise prevalence of babesiosis revealed high-frequency rates of Babesia spp. during July and August. In contrast, the lowest frequency rate was found in October (Table 1), which might be related to the favorable conditions for tick vectors activation and disease transmission during this period. Ovine babesiosis as a significant tickborn disease result in high economic losses globally due to high morbidity and mortality (27). ovis sequence isolates represented the presence of diversity. The isolates were clustered in different subgroups of phylogenic branches (Fig. 1), whereas B. motasi isolates with accession no. MN548425 and MN548430 were clustered separately, and The MN551068 isolate was clustered in another subgroup of the phylogenic tree (Fig. 2).

Figure 2. Phylogenic tree based on the 18S
rRNA gene partial sequences of Babesia motasi isolates with previously registered sequences in the GenBank database. The relationship of the genotypes was determined by maximum parsimony with bootstrap test of (1000 replicates) in MEGA X ACKNOWLEDGEMENT The University of Sulaimani financially supported this study. The authors acknowledge the assistance of all veterinary staff in the districts where samples were collected.