Shayan et al. : Molecular Study of Sheep Malignant Theileriosis… Introduction Tconstitute a complex of several he tick-borne diseases of livestock diseases with the different etiological agents, such as protozoa, rickettsia, bacteria, and viruses. The only common feature between these diseases is that they can all be transmitted by ticks. Theileriosis and Babesiosis belong to this complex and cause diseases in the livestock with high morbidity and mortality and therefore they give rise to the high economical looses worldwide (1-5). For the long time, it was assumed that T. lestoquardi is the only pathogenic parasite in small ruminants (6). However, recently a previously identified parasite has been described as a T. china I and II spp., which are pathogenic for small ruminants as well, causing a fatal diseases of small ruminants distributed in North China (6-10). Interestingly, the purpose of reviews of tick- borne diseases has been increasingly recognized on the world as public health problems. The Geimsa stained of blood smear is the most used method for the identification and characterization of these piroplasms, which accompanied with some technical problems causing false morphological diagnosis and in some cases, needs special diagnostic knowledge. In cer- tain cases serological methods such as the immuno fluorescence antibody test (IFAT) or immunoperoxidase test, have also been applied (4,11-13). Shayan and Rahbari (2005) showed the DNA extracted from infected blood smear with Theileria spp. and Babesia spp. could be amplified with the common primer pair derived from hyper variable region V4 of 18S rRNA. They showed the theilerial hypervariable region V4 of 18S rRNA con- sists of more nucleotides than in Babesia spp. Therefore, the application of the reported primer pair could easily and simultaneously 67 differentiate between Theileria spp. and Babesia spp. (14). The PCR product of Theileria spp. and Babesia spp. will be then 426-430 bp and 389-402 bp respectively. The difference of ca. 30 bp in the length of PCR product will be easily determinable in 1.8% agarose gel. In the present study, we used the PCR tech- nique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples col- lected from Sultanate of Oman. Material and Methods Collection of samples After lethal diseases in a sheep herd (11dead from 53 sheep) at Rumais (Barka) region in the sultanate of Oman, samples were pre- pared from the alive and dead sheep. Small pieces of lung, spleen, liver, and lymph node were cut and transferred into the tubes con- taining 70% ethanol. Additionally, impres- sion smears from the above-mentioned or- gans were also prepared. The ethanol fixed samples were sent to the Iranian Research Center for Ticks and Tick-borne Diseases for molecular analysis and the impression smears were stained in 10% Geimsa solution in phosphate-buffered saline (PBS, pH of 7.2) at the Sultan Qaboos University, Col- lege of Agricultural and Marine Sciences, Department of Animal and Veterinary Sci- ences. Blood smears were also stained with Geimsa. DNA extraction DNA was extracted from lung, liver, spleen and lymph node using DNA extraction kit (MBST, Tehran, Iran) and extracted DNA was stored at -20 ºC until subsequent analy- sis. Briefly, we cut 3×3 mm from each in- fected organ (lung, liver, spleen and lymph