Molecular identification of Theileria species in naturally infected sheep using nested PCR–RFLP

Ovine theileriosis is an important tick-borne haemoprotozoan disease of sheep in tropical and subtropical regions, causing severe productivity and economic loss. There is a paucity of information related to molecular studies of ovine theileriosis from India. The present study identified different Theileria spp. in naturally infected sheep using nested PCR-restriction fragment length polymorphism (nPCR-RFLP). Blood samples and ticks were collected from 204 sheep in different agro-climatic zones of Haryana state, India, during the tick active season. Microscopic examination of thin blood smears revealed 33.3% (68/204) infections with Theileria spp., while 44.6% (91/204) of blood samples were positive by nPCR assay. Different Theileria spp. were identified based upon RFLP patterns using four restriction enzymes: Hpa II, Bsh 1285I, Hae II and Rsa I. Out of 91 positive samples, 50.5% (46/91), 23.08% (21/91), 11% (10/91) and 2.2% (2/91) were positive for T. ovis, T. lestoquardi, T. luwenshuni (Theileria sp. China 1/Theileria sp. China) and T. annulata, respectively. Mixed infection was detected in 13.2% (12/91) of cases. Based upon HpaII enzymatic digestion pattern, two samples with T. lestoquardi and T. annulata, nine samples with T. lestoquardi and T. ovis and one sample with T. ovis and T. annulata were detected. The presence of these Theileria spp. was further confirmed by sequence analysis. The majority of ticks collected from sheep were identified as Rhipicephalus spp. followed by Hyalomma anatolicum and Hemaphysalis spp. The present investigation depicts the first comprehensive molecular report of naturally infected sheep with T. ovis, T. lestoquardi, T. annulata and T. luwenshuni from northern India.