Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 9 1 2014 03 15 99 106 EN Seyedeh Missagh Jalali Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran AND Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Zohreh Khaki Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Bahram Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Sadegh Rahbari Parasitology Department, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Parviz Shayan Parasitology Department, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Mojgan Bandehpour Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Seyedeh Parastoo Yasini Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 2015 10 14   Background: The present study was carried out to investigate the accurate status of ovine Theileria infection in sheep from Ahvaz and surrounding region, a tropical area southwest Iran. Methods:A PCR-RFLP method based on 18S ribosomal RNA gene was designed which could detect and differentiate Theileria and Babesia spp. and also differentiate main Theileria species in sheep at the same time. 119 sheep blood samples were col-lected from Ahvaz and surroundings. Results: Microscopic examination of blood smears revealed 69.7% (83/119) infec-tion with Theileria spp. Of the total samples subjected to PCR, 89% (106/119) were found to be positive, all of which were identified as Theileria by RFLP analysis using enzyme Hind II. In enzymatic digestion of PCR products by Vsp I, 91.5% (97/106) of Theileria positive samples were identified as T. ovis while mixed Theileria infections were found in 9 samples. The samples with mixed infections were analyzed with an additional nested PCR-RFLP method, by HpaII enzyme digestion. 3 samples with T. lestoquardi infection, 1 sample with T. ovis and T. annulata, 1 sample with T. lestoquardi and T. annulata, and 4 samples with T. ovis, T. lestoquardi and T. annulata mixed infections were detected. Conclusion: Ovine theileriosis caused by T. ovis is highly prevalent in southwest Iran while T. lestoquardi and T. annulata infection can be detected in a lesser propor-tion of sheep in this region. The new PCR-RFLP method that was designed in this study, can serve as a beneficial diagnostic tool, especially in T. ovis prevalent re-gions. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/428 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/428/310