Molecular Detection and Differentiation of Different Theileria Species in Naturally Infected Goats Using Nested PCR-RFLP: A First Report from Northern India.

Purpose: Theileriosis is an economically important tick-borne pathogen with a serious impact on livestock health and productivity. Despite the fact that bovine theileriosis has been widely investigated, there exists a paucity of information on these infections in small ruminants, especially in India. The present study was carried out to detect and differentiate different Theileria spp. in goats using nested PCR RFLP.

Methods: Blood samples and ticks were collected from 405 goats in various agro-climatic zones of Haryana state, India. The blood samples were screened by microscopy, nested PCR-RFLP, and sequence analysis. The nested PCR-RFLP was performed with four restriction enzymes viz., Hpa II, Bsh 1285I, Hae II and Rsa I. Six nested PCR amplicons with different RFLP patterns were sequenced and submitted to NCBI (OM666861, MZ220430, OM666628, MZ220437, OM666637, OM721806).

Results: Microscopy revealed 18.27% (n = 74) infection with Theileria spp., while 33.58% (n = 136) of blood samples were confirmed positive by nested PCR. Out of 136 positive samples, 43.38% (n = 59), 11.02% (n = 15) and 20.58% (n = 28), were positive for T. ovis, T. lestoquardi and T. luwenshuni (Theileria sp. China 1), respectively. Mixed infection was detected in 25% (n = 34) cases. Based upon Hpa II digestion pattern, 13 samples with T. lestoquardi and T. ovis, and 21 samples with T. ovis and T. luwenshuni were detected. Sequence study further confirmed their identity. The majority of ticks collected from goats were identified as Rhipicephalus spp., Hyalomma anatolicum and Hemaphysalis spp.

Conclusion: This study represents the first confirmed molecular report of goats infected with T. ovis, T. lestoquardi, and T. luwenshuni from northern India.