Survey of theileriosis in sheep and goats in east and southeast anatolia region by microscopical and molecular methods

Survey of Theileriosis in Sheep and Goats in East and Southeast AnatoliaRegion by Microscopical and Molecular MethodsKursat AltayThis study was carried out to determine the presence and distribution ofTheileria ovis and T. lestoquardi in sheep and goats in East and SoutheastAnatolian regions between June 2004 and September 2005. Firstly, a nestedpolymerase chain reaction (PCR) method was developed for the specific diagnosisof T. ovis, and then the prevalence of T. ovis in sheep and goats was investigatedby the method. In addition, the presence of T. lestoquardi in the same region wasalso investigated.Two oligonucleotide primers were designed from the 18S SSU rRNA genesequence (Genbank No: AY508453) of T. ovis isolated from sheep in Erzincan.The primer sequences were tested with BLAST program in computer. Then,DNAs from T. ovis, T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesiaovis were tested in the PCR in laboratory. In addition, detection limit of the PCRfor T. ovis was determined using dilution of T. ovis-infected erythrocytes with %10-2, % 10-3, % 10-4, % 10-5, % 10-6, % 10-7 and % 10-8 parasitemia.A total of 819 whole blood (677 sheep and 142 goats) and 795 bloodsmear (656 sheep and 139 goats) were collected from sheep and goats at differentlocations in the region. At the same time, 27 lymph smear samples were collectedin sheep in Mardin. In addition, a total of 531 tick in sheep and 89 ticks in goatswere collected in Mus and Malatya provinces. Ticks were examined under stereo-microscope for species identification. T. ovis and T. lestoquardi DNAs extractedfrom sheep and goats blood were amplified by PCR using species-specificprimers. Blood and lymph smears were examined for Theileria piroplasms andschizonts forms by microscopic examination (ME).A 398-bp DNA fragment was specifically amplified from blood samplesfrom sheep naturally infected with T. ovis. No PCR products resulted from T.lestoquardi, T. annulata, T. parva, T. buffeli and B. ovis DNA using these specificprimers. The sensitivity of the nested PCR for T. ovis which was assessed showedthat one infected cell in 107 sheep erythrocytes, equivalent to a blood parasitemiaof 10-5 %, could be detected.In the microscopic examination of blood smears, Theileria spp. piroplasmswere observed in 120 out of 656 sheep (18.29 %) and 4 out of 139 goats (2.87 %),respectively. Schizont forms of the parasite were not seen in lymph node smears.Of the 819 field samples obtained from 677 sheep and 142 goats tested, 308(45.49 %) and 9 (6.34 %) were positive for the presence of T. ovis by the firstround PCR, respectively compared to 398 (58.78 %) and 16 (11.26 %) positive forT. ovis by nested PCR. T. lestoquardi was not amplified in the same animals byPCR.Ticks collected from sheep and goats were identified to be Rhipicephalusbursa and R. sanguineus on the basis of morphological features.The differences between ME and PCR results and between the first roundand nested PCR results were statistically significant (P<0.01). The frequency of T.ovis infection was higher in sheep than in goats and difference between theinfection rates of sheep and goats were found to be statistically significant(P<0.01).Keywords: Theileriosis, microscopic examination, PCR, sheep, goat.