MOLECULAR DETECTION OF THEILERIA SPP. AND BABESIA SPP. IN SHEEP AND IXODID TICKS FROM THE NORTHEAST OF IRAN Gholamreza Razmi, Moslem Pourhosseini, Saeed Yaghfouri, Ahmad Rashidi, and Mohsen Seidabadi Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, P.O. Box 91775-1793, Mashhad, Iran. e-mail: razmi@fum.ac ABSTRACT: Theilerioses and babesioses are important diseases in Iranian sheep. The present study was undertaken to identify and classify/specify Theileria spp. and Babesia spp. in sheep and vector ticks. Investigation was carried out from 2009 to 2011 in the Khorasan Razavi Province, Iran. In total, 302 sheep originating from 60 different flocks were clinically examined and their blood collected. In addition, from the same flocks, ixodid ticks were sampled. Stained blood smears were microscopically examined for the presence of Theileria and Babesia organisms, and a semi-nested PCR was used for subsequent molecular specification. From the ticks, salivary glands and uterus were isolated and subsequently analyzed by semi-nested PCR. Piroplasm organisms were observed in 29% of the blood smears with low parasitemia, whereas 65% of the blood samples yielded positive PCR findings. The presence of Theileria ovis (55.6%), Theileria lestoquardi, and mixed infection with Theileria spp. and Babesia ovis were detected by semi-nested PCR in 0.3%, 5.6%, and 0.99%, respectively. In total, 429 ixodid ticks were collected from different areas of the province. The most prevalent ticks were Rhipicephalus turanicus (n ¼ 376; 87.6% of the total), followed by Hyalomma marginatum turanicum (n ¼ 30; 7.0%), Dermacentor raskemensis (n ¼ 12; 2.8%), Hyalomma anatolicum anatolicum (n ¼ 7; 1.6%), Dermacentor marginatus (n ¼ 2; 0.5%), Rhipicephalus bursa (n ¼ 1; 0.2%), and Haemaphysalis sp. (n ¼ 1; 0.2%). Of the positive R. turanicus samples, 5 (5.7%) were infected with T. ovis and 2 (2.9%) with T. lestoquardi. Neither Babesia ovis nor Babesia motasi infection was detected in salivary glands or uterine samples of the ticks. The results also suggest that R. turanicus could be the vector responsible for transmission of the 2 Theileria species. Theileriosis and babesiosis are both important diseases in sheep. Theileria spp. infections in small ruminants are caused by at least 6 species, with Theileria lestoquardi, Theileria luwenshuni, and Theileria uilenbergi considered as highly pathogenic in sheep and goats. The other 3 species, Theileria separata, Theileria ovis, and Theileria recondita, are generally considered as non-patho- genic, or mildly pathogenic, in small ruminants (Uilenberg, 1995, 1997; Schnittger et al., 2000; Perston, 2001). Babesia species of small ruminants are commonly grouped together, but this may be an oversimplification, as the suscepti- bility of sheep and goats is highly variable (Uilenberg, 2006). Babesia ovis and Babesia motasi are generally regarded as valid taxa. Two other parasites have also been described, i.e., Babesia taylori and Babesia foliata, but their validity is doubtful (Uilenberg, 2001, 2006). A third distinct species is Babesia crassa, isolated in Iran; it is a large species and multiplies by quadruple division as well as binary fission. Many red cells contain 4 parasites, which can be the result of quadruple division, but also of 2 successive binary divisions (Hashemi-Fesharki and Uilen- berg, 1981). Theileria lestoquardi, T. ovis, B. ovis, B. motasi, and B. crassa have all been reported in sheep and goats in Iran (Hashemi- Fesharki, 1997). The prevalence of Theileria spp. infection has been reported to range from 10% to 36% from different areas of the country (Navidpour, 1996; Maleki, 2002; Hajikolaei et al., 2003; Razmi, Hossieni, and Aslani, 2003; Razmi et al., 2006). The seroprevalence of Babesia spp. infection is also variable, from 12% to 58% in different geographic areas (Tavassoli and Rahbari, 1998; Hashemzadeh et al., 2006). Hyalomma anatolicum anatolicum appears to be the only proven vector of T. lestoquardi in Iran (Hooshmand-Rad and Hawa, 1973; Hashemi-Fesharki, 1997). However, molecular studies have demonstrated that Rhipicephalus sanguineus can serve as a vector for T. ovis (Telmadarraiy et al., 2010), and Rhipicephalus bursa, Rhipiceph- alus turanicus, and R. sanguineus can serve as a vector for B. ovis (Shayan et al., 2007). The Khorasan Razavi Province is located in northeastern Iran and is one of the larger centers of sheep breeding in the country. The northern part of the province has a mountainous climate, while the central and southern areas are desert and semi-desert. Theileria spp. infection is more common to southern areas of Khorasan, while babesiosis is more prevalent in the north (Razmi et al., 2002, 2006). The aim of present study was to determine the extent of Theileria spp. and Babesia spp. in sheep and tick vectors of the Khorasan Razavi Province. MATERIAL AND METHODS Field study The Khorasan Razavi Province is located in northeastern Iran between 33830 0 –37841 0 N latitude and 56819 0 –61818 0 E longitude, with an area of more than 127,000 km 2 . It is adjacent to the northeastern and eastern borders of Iran, to the south of Turkmenistan and west of Afghanistan (Fig. 1). The climate is semi-arid, with cold winters and moderate summers. The mean annual temperature in the province increases from north to south, ranging between 13.6 and 17 C. In terms of habitat, the Khorasan Province is divided into northern and southern regions. While the northern part is mountainous, its lower areas possess valleys that provide suitable conditions for agricultural activity and animal husband- ry. The southern part is mostly semi-desert and desert, with poor vegetation cover. The number of sheep in this province has been estimated at approximately 5 million animals for 2008 (Khorasan Razavi Provincial Veterinary Service, pers. obs.). Blood samples We selected flocks with clinical histories of theileriosis or babesiosis in 10 different areas of the Khorasan Razavi Province during 2009 and 2011. Five sheep from each of these flocks were randomly selected and clinically examined. Blood was drawn from the jugular vein and collected in EDTA tubes; blood smears were also directly prepared from each sample. Simultaneously, the body of each animal was inspected, and, if ticks were found, these were placed into appropriately labeled vials. The blood and tick specimens were kept cool in transit to the laboratory. Microscopy of blood smears The blood smears were air-dried, fixed in methanol, and subsequently stained with 10% Giemsa solution in phosphate-buffered saline (PBS), pH 7.2. The slides were microscopically examined using oil immersion lens at a total magnification of 1,000 3. Received 15 May 2012; revised 5 August 2012; accepted 17 August 2012. DOI: 10.1645/GE-3202.1 77 J. Parasitol., 99(1), 2013, pp. 77–81 Ó American Society of Parasitologists 2013