Molecular diagnosis of tropical theileriosis: Development and validation of species-specific qPCR assays in Pakistan.

BACKGROUND

Theileria spp. are economically important tick-borne hemoprotozoan parasites that threaten livestock health and productivity, particularly in tropical and subtropical regions. In this study, we developed and validated a qPCR-based diagnostic panel for the sensitive and specific detection of Theileria infections in ruminants from Khyber Pakhtunkhwa (KP), Pakistan. The panel included a broad-range Pan-Theileria assay as well as four species-specific assays for Theileria annulata, T. parva, T. lestoquardi, and T. ovis, the main species infecting domestic ruminants in the region.

METHODS

A total of 1026 tick-infested animals were examined, including sheep (n = 514), goats (n = 462), and cattle (n = 50). Blood was collected from symptomatic animals, and 51 microscopically confirmed Theileria-positive samples were selected for further analysis. DNA was extracted using the phenol-chloroform method for clinical validation. Primers and hydrolysis probes were designed to target the hypervariable V4 region of the 18S rRNA gene, allowing high-resolution species identification.

RESULTS

Analytical validation using synthetic gBlock™ gene fragments showed strong assay performance, with excellent linearity (R2 = 0.982-0.9965), high PCR efficiency (86.2 %-105.2 %), and detection limits of 10-100 copies per reaction. Reproducibility was confirmed with coefficients of variation ≤5 %. The Pan-Theileria assay detected 47 positives. Sequencing of the 18S rRNA gene confirmed these results and additionally identified three cases of T. orientalis, two of which were also detected by qPCR. One sample tested positive for T. ovis. Overall, the assay achieved 100 % clinical sensitivity and specificity within this validation set, though larger multi-site field evaluations are needed to confirm these findings.

CONCLUSION

This is the first comprehensive qPCR-based diagnostic platform for simultaneous detection and speciation of Theileria spp. in Pakistan. The tool provides a powerful approach for large-scale surveillance, timely diagnosis, and improved control of tropical theileriosis in endemic areas.