Iranian J Parasitol: Vol. 6, No.1, 2011, pp.66-72 node) and was first lysed in 100 µl lysis buffer and the proteins were degraded with 10 μl proteinase K for 10 minutes at 55˚C. After addition of 270 µl Bindings buffer and incubation for 10 minutes at 70˚C, 320 µl ethanol (100%) was added to the solution and after vortexing, the complete volume was transferred to the MBST-column. MBST column was first centrifuged and was then washed twice with 500 µl washing- buffer. Finally, DNA was eluted from the carrier with Elution buffer. PCR amplification Two pairs of primers were designed based on the nucleotide sequences of 18S rRNA gene of T. annulata/lestoquardi primer sense (5' CAC AGG GAG GTA GTG ACA AG 3' ) was derived from nucleotide 431 to 450 of complete sequence of 18srRNA gene of T. lestoquardi (Accession number AF081135). This area was common from nucleotide 274 to 293 in T. ovis (Accession number GU726904 ), from nucleotide 419 to 438 in B. ovis (Accession number AY260178). Primer antisense (5' CTA AGA ATT TCA CCT CTG ACA3') was derived from nucleotide 838 to 858 of complete se- quence of 18srRNA gene of T. lestoquardi (Accession.number AF081135). This area was common from nucleotide 685 to 705 in T. ovis (Accession number GU726904), from nucleotide 789 to 809 in B. ovis (Accession number AY260178). The PCR was performed in a total reaction volume of 100 µl containing 10 µl of 10X PCR buffer, 3 µl MgCl2 (50 mM), 2 µl of dNTP (10mM each), 0.5 µl Taq DNA poly- merase (5 U, Fermentas), 2 µl of each primer (20 µM), 79/5 µl dH2O and 1µl of template DNA (approximately 100 ng). The reaction was repeated for 35 cycles under the follow- ing conditions: 5 min at 94°C, 45'' at 94°C, 45'' at 56°C, 45'' at 72°C and finally, PCR was completed with the additional extension step for 10 min. Distilled water was used as 68 negative control in each PCR reaction and the DNA from T. annulata was used as posi- tive control. PCR products were separated on 1.8% agarose gel in 0.5× Tris–borate– ethylenediamine tetraacetic acid (EDTA) buffer and visualized using ethidium bro- mide and an UV illuminator. PCR product extraction from Agarose gel The PCR product isolated from the agarose gel using the gel extraction Kit (MBST, Iran) according to the manufacturer’s instructions. Briefly, the DNA bands were cut from the gel under UV illumination and dissolved in the binding buffer at 60°C. The dissolved agarose was transferred into the MBST col- umn. After washing twice with wash buffer, the bounded DNA to the carrier was eluted using 20 µl Tris–EDTA buffer. The ex- tracted PCR product from the agarose gel was sequenced by Kawsar Company in sanger method. Results During a lethal disease in a sheep herd consisting of 53 sheep at Rumais (Barka) region in the Sultanate of Oman, 11 sheep were died. One of these sheep died suddenly without any clinical signs, whereas the other ten showed anorexia and anemia before they were died. According to the frame work of “integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, blood smears, the samples from, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases (IRCTTD). In blood smears no schizonts or piroplasm form was detectable, whereas schizonts like structures was detected in the impression smears of lymph node, lung, liver and spleen (fig. 1A, B and C).