Comparative evaluation of diagnostic methods for detection of Theileria spp. in cows

Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man® probe for detection and quantification of Theileria orientalis and Theileria annulata simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically Theileria–suspected cows of three Theileria–endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed T. annulata and T. orientalis in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the Theileria spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of T. annulata and T. orientalis simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of Thelieria spp.