Materials and Methods:
A cross-sectional study was conducted in the Jabalpur region (23.1815° N, 79.9864° E), Madhya Pradesh between March, 2018- June, 2019 to investigate the prevalence of CCHF and theileriosis in livestock’s and its associated risk factors (Fig.1). To cover urban and rural/tribal areas, five villages were selected purposely from peripheral villages of study region.. Simple random sampling method was applied to select the animals from the herd. The sample size was estimated based on the assumption of 10% sero-positivity rate, the level of confidence as 95 percent and a absolute error of 5 %. A minimum desired sample size was 140, so the desired sample bovine and goat/sheep was 140 each. Overall, a total of 155 bovine and 238 goats’ blood samples were collected from the jugular veins following biosafety measures. The pre-designed structured interview schedule was used to collect information on animal husbandry activities and risk factors. Sera were separated and subjected to two commercial ELISA kits (1 for bovine -Catalogue No.CCHFCTIGG-48, and 1 for goats -Catalogue No. CCHFSGIGG-48) (Zydus Diagnostics, Ahmedabad, India). The manufacturer’s instructions were followed for testing samples and based on the ratio >1.5, the positivity was determined for both the kits. The DNA was isolated from the CCHF IgG negative bovine blood samples using DNA Sure Mini Kit (Nucleo-pore, GENETIX, India) as per the manufacturer’s protocol. The isolated DNA samples were subjected to PCR amplification targeting the cytochrome b (312 bp segment) gene of the Theileria annulata (Bilgic et al., 2010). The PCR products were sequenced analyzed and submitted to GenBank. The necessary ethical permission for the conduct of this study was obtained from the Institutional Animal Ethics Committee (IAEC) and Institutional Ethics Committee (NIRTH/IEC/3756/2018).