The prevalence of Theileria infection in tick vector Hyalomma anatolicum anatolicum collected from healthy animals of Ludhiana district, Punjab was recorded to assess the natural infection level of theilerial parasite in the field condition. A total of 60 semi-engorged H. a. anatolicum were collected from cattle and their salivary glands were dissected out. One half of the salivary gland was stained with methyl green pyronin (MGP) and the other half was utilized for DNA isolation for molecular detection of Theileria infection. A PCR and nested PCR assays were standardized for the detection of T. annulata infection in salivary gland of H. a. anatolicum. The prevalence of T. annulata infection was recorded as 8.3, 20.0 and 60.0 % by MGP staining, primary PCR and nested PCR, respectively. Further, the prevalence was higher in female ticks (8.8 %) than male ticks (6.6 %). The results demonstrated that both primary and nested PCR assays are a valuable technique for detection of T. annulata infection in vector tick under field conditions.

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