Phylogenetic study of Theileria lestoquardi based on 18 SrRNA gene Isolated from sheep in the middle region of Iraq

Theileriosis is parasitic infection causes by obligate intracellular protozoa of the genus Theileria. T. lestoquardi is the most virulent species in sheep and goats which causes a severe disease with a high morbidity and mortality rate. In this study the phylogenetic relationships between two local isolate of T. lestoquardi and nine T. lestoquardi global isolates as well as Babesia ovis out-group isolate were analyzed using the 18S rRNA gene sequence. The multiple sequence alignment analysis and neighbor joining phylogenetic tree analysis were performed by using ClustalW multiple sequence alignment online based analysis of 1098bp 18S rRNA gene was amplified by polymerase chain reaction. Phylogenetic analysis results of these gene sequences revealed that T. lestoquardi local isolates were closely related to T. lestoquardi Iran isolate (JQ917458.1) and two Iraq Kurdistan isolates (KC778786.1 and KC778785.1) more than other countries. This study represents the first report on the use of molecular phylogeny to classify T. lestoquardi obtained in Middle Region of Iraq.


Introduction
Tick-borne protozoan parasites of the genus Theileria infect wild and domestic ruminants in the tropical and subtropical regions of the world.Although Theileria infection in cattle has been extensively studied little is known about theileriosis in sheep (1).Recently, interest has arisen in sheep-infecting Theileria parasites.Among known Theileria parasites of sheep, T. lestoquardi and Theileria spp.from North China are considered highly pathogenic.The other species, Theileria ovis, Theileria separata and Theileria recondita cause subclinical infection in small ruminants (2).In order to prioritize future research on the development of improved control measures against tickborne diseases, it is essential to define the prevalence of tick borne pathogens in target populations (3).The precise identification of these organisms is essential to understand their epidemiology and classification.The methods traditionally used to detect and identify these hemoparasites consist of microscopic examinations of thin blood smears and serological tests.In contrast to these conventional methods, the application of molecular techniques would allow direct, specific and sensitive detection of parasites, and rapid, simultaneous detection and differentiation of different Theileria infecting a given animal (4).The tools of molecular biology are increasingly relevant to veterinary parasitology.The techniques used with eukaryotic cells are generally applicable to the study of parasites and their hosts.The sequencing of the complete genomes of helminthes and protozoa is allowing great advances in studying the biology, and improving diagnosis and control of parasites, especially sequence data analysis allowed the researchers to identify and characterize the hemoparasites species in particular Theileria group (5)(6).The comparison of srRNA has been introduced for deducing phylogenetic relationships of the Piroplasmidia and other Apicomplexa (7)(8).These studies on srRNA confirmed that the pathogenic and benign Theileria parasites existed in many areas of the world and defined their phylogenetic relationship.Nevertheless, the status of Theileria spp of the Iraq remained unclear.
Aim of study: In this study the small subunit ribosomal RNA genes were examined and analyzed for construction of phylogenetic trees of Iraqi Theileria parasite in comparison to those of other Theileria spp.

Samples collections
43 blood samples collected from sheep clinically infected by theileriosis in Wasit province and placed in anticoagulant tubes, then transported to laboratory and stored in refrigerator until genomic DNA extraction step.

Genomic DNA Extraction
Genomic DNA was extracted from frozen blood by using (Genomic DNA Mini Kit, Geneaid.USA).The extraction was done according to company instructions by using frozen blood extraction Protocol method with Proteinase K.After that, the extracted gDNA was checked by Nanodrop spectrophotometer, and then stored at -20C at refrigerator until used in PCR amplification.

Polymerase Chain Reaction (PCR)
PCR assay was carried out by using specific primer which was designed by (7) to amplify a 1098bp fragment of highly conserved regions of 18S ribosomal RNA gene in all Theileria spp.18SrRNA forward primer (AGTTTCTGACCTATCAG) and 18SrRNA Reverse primer (TTGCCTTAAACTTCCTTG) were provided by (Bioneer company.Korea).Then PCR master mix was prepared by using (AccuPower ® PCR PreMix kit.Bioneer.Korea).The PCR premix tube contains freeze-dried pellet of (Taq DNA polymerase 1U, dNTPs 250 µM, Tris-HCl (pH 9.0) 10 mM, KCl 30 mM, MgCl2 1.5 mM, stabilizer, and tracking dye) and the PCR master mix reaction was prepared according to kit instructions in 20 µl total volume by added 5 µl of purified genomic DNA and 1.5 µl of 10 pmole of forward primer and 1.5 µl of 10 pmole of reverse primer, then complete the PCR premix tube by deionized PCR water in to 20 µl and briefly mixed by Exispin vortex centrifuge (Bioneer.Korea).The reaction was performed in a thermocycler (Techne TC-3000.USA) by set up the following thermocycler conditions; initial denaturation temperature of 95°C for 5 min; followed by 30 cycles at denaturation 95°C for 30 s, annealing 55°C for 30 s, and extension 72°C for 30 s and then final extension at 72°C for 7 min.The PCR products were examined by electrophoresis in a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV illumination.

DNA sequencing method
DNA sequencing method was performed for confirmative detection and Phylogenetic relationship of T. lestoquardi based on 18SrRNA gene by Phylogenetic tree analysis using ClustalW multiple sequence alignment program.1098bp PCR product was purified from agarose gel by using (EZ EZ-10 Spin Column DNA Gel Extraction Kit, Biobasic.Canada).The purified 18S rRNA gene PCR product samples were sent to Bioneer Company in Korea for performed the DNA sequencing using 18SrRNA forward primer by (AB DNA sequencing system).

Results
Out of 43 blood samples of clinical suspected theileriosis were tested by convential PCR assay, only 10 samples which appeared positive for Theileria spp at 1098bp PCR product of 18S rRNA gene on agarose gel electrophosis (Fiq.1).Sequence analysis of two samples positive for T. lestoquardi was performed to confirm the PCR results.These sequences for the 18S ribosomal RNA genes of the pathogens can be found under the accession numbers KJ024366 and KJ024367 at NCBI -GenBank submissions.The DNA sequencing analysis of 18S rRNA gene 1098bp PCR product by multiple sequence alignment (ClustalW2) showed specific detection of T. lestoquardi Where, the results of Phylogenetic sequence alignment of (Theileria lestoquardi local isolate) was 99% identity to Theileria lestoquardi strain (KF429800.1)more than other Theileria spp. as following(Fig.2).The phylogentic tree was constructed based on the two T. lestoquardi Iraq isolate, seven T. spp and out group sequences including Babesia ovis (Fig. 3) The result of current study was refer to that the Iraq isolate more closely related to Iran_ 1 (JQ917458.1)more than other of countries included in phylogenetic tree (Fig. 4).   Figure 4: The phylogenetic tree of selected seven T. lestoquardi strain and isolate from some countries, two Iraq Kurdistan isolate with two Iraq isolate based upon 18S ribosomal RNA genes using neighbor joining phylogenetic tree analysis by CLUSTAL 2.1 multiple sequence alignment.
The identity percent of two T. lestoquardi Iraq isolate and other T. lestoquardi of selected countries were 98% to 99%, furthermore the score were high in china and iran3, moderate in iran2, iran1, Iraq K1and Iraq K2 comparable to low score in Tanzania1, tanzania2 and Sudan (Table 1).

Discussion
Ribosomal RNA is the most abundant constituent of nucleic acids in any non-viral organism with the eukaryotic RNA transcription unit.The rRNA gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (9-10).Moreover, the 18S rRNA gene is valuable for phylogenetic analysis due to its high levels of conservation (11).However, the molecule also possesses phylogenetically informative variable regions that are useful for determining relationships among species (12).Results from the present study illustrate relationships among Theileria parasites where their previous taxonomic classification was not clear.Molecular techniques such as sequencing of S r RNA genes described in this study are promising tools for classification of these parasites.However, basic information based on life cycle differences, vectors, modes of transmission, virulence and genetic compatibility is essential for clearer taxonomic definition of the Theileria parasites.A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iraq isolates, and other species of Theileria available in GenBank.In the constructed tree, Theileria lestoquardi (Iraq isolates) was closely related to T. lestoquardi (AF081135.1).Based on phylogenetic analysis study of T. lestoquardi Iraq isolates which isolated from clinically infected sheep in middle of Iraq, by using 18S rRNA gene sequence.In addition, Theileria species are host and vector specific (13)(14) but in Iraq more than one species can infect sheep (15) which causes a problem in diagnosis and epidemiology.In this study, the fragment of 18S rRNA gene sequences of T. spp1098bp were amplified for examined samples.Nucleotide sequence identity data demonstrated that the T. lestoquardi Iraq isolate has nucleotide identity percent of 99% with T. lestoquardi (AF081135.1)and with T. annnulata(KF429800.1)were 99%.The results of the current study was nearly identical with (16) when he show the similarity identity percent between T. annulata Iran strain and T. lestoquardi was 99.5%.In spite of The identity percent of two T. lestoquardi Iraq isolate and other T. lestoquardi of selected countries were 98% to 99% but the score of identity was higher in china and iran3, moderate in iran2, iran1, Iraq K1and Iraq K2 From this perspective, the Iraq isolate has a great homology with other T. lestoquardi isolates from above countries which most of them in neighborhood.

Figure 1 :
Figure 1: Agarose gel electrophosis image, which show the PCR product results for Theileria spp of 18S rRNA gene at 1098bp PCR product size, where M: Marker 100bp, Lane (1-10) are positive samples.

Table 1 :
Homology sequence identity for T. lestoquardi Iraq isolate