Background Theileriosis is a parasitic disease caused by an obligate intracellular protozoan genus Theileria (Apicomplexa, Piroplasmida) and transmitted by ixodid ticks in the ruminants of the tropical and subtropical areas of the world and Iran (1). Theileria annulata is the cause of tropical theileriosis in Mediterranean regions, North Africa, South and SouthWest of Europe, Middle East, China, and Central Asia (2). Other species with moderate pathogenicity in the cattle are T. sergenti, T. buffeli, and T. orientalis from Asia and Europe, as well as T. mutans, T. thaortragi, and T. wulfartia from Africa (3). The causal agent of bovine theileriosis is T. annulata in Iran. Theileria orientalis also exists in the north part of the country (4). According to Guglielmone et al (5), 702 out of 896 species of ticks belong to ixodid ticks (Acarina, Metastigmata). Approximately 10% of ixodid ticks re fed on domestic animals, particularly cattle, buffaloes, sheep, and goats (6). The hard ticks fauna and the role of some of them in the transmission of T. annulata were first reported in 1949 and 1972 in Iran, respectively (7). In addition, the genus Hyalomma was reported as the vector of bovine theileriosis in different parts of Iran (6,8,9). The species of H. anatolicum anatolicum (Koch, 1844), H. anatolicum excavatum (Koch1844), H. asiaticum asiaticum (Schulze and Schlottke, 1930), H. dromedarii (Koch 1844), H. detritum (Schulz, 1919), and H. marginatum are common ixodid ticks in Iran (7). The accurate diagnosis of theileriosis is essential in epidemiological studies (10). Several laboratory methods (i.e., histopathology, immunology bioassay, and molecular tools) are now available for the detection of T. annulata infection in cattle. Of those, thin blood smear and lymph node examination were the most frequent methods for detecting acute theileriosis in Iranian cattle for many decades (4). However, these methods had low sensitivity due to difficulties in Theileria species discrimination and asymptomatic carrier detection (11). Accordingly, DNA-based techniques such as polymerase chain reaction (PCR), Semi-nested PCR, Nested PCR, and PCR-restriction fragment length polymorphism were developed to detect T. annulata infections in cattle with low levels of parasitemia in the chronic stage and carriers (10,12).