SNP-based molecular diagnostic platform: rapid single-step identification of Theileria annulata and its...

Theileria annulata, a tick-borne protozoan that causes tropical theileriosis, poses a serious threat to livestock production in endemic regions. The emergence of resistance to buparvaquone, the primary chemotherapeutic treatment, has been attributed to acquired mutations in the cytochrome b (Cytb) gene, with identical resistance-associated polymorphisms observed in both laboratory-adapted strains and field isolates from China.

A dual probe-specific real-time polymerase chain reaction (PCR) assay was developed to detect point mutations in the Cytb gene. The specificity, sensitivity, and reproducibility of the assay were validated, and its field applicability was evaluated via cattle blood samples (n = 531) collected from five endemic Chinese provinces.

Six point mutations were identified in the Cytb gene, and the developed dual probe-specific real-time PCR assay simultaneously detected T. annulata infection and distinguished between the buparvaquone-sensitive and buparvaquone-resistant genotypes. The assay demonstrated a detection limit of 1 × 10¹ copies/μl, high specificity, and satisfactory repeatability, with results consistent with those of Sanger sequencing. Field screening revealed a 21.7% (115/531) prevalence of T. annulata and a 4.3% (23/531) occurrence of resistant genotypes. Moreover, two-dimensional scatterplot visualization enabled clear genotype discrimination without post-PCR processing.

The developed dual probe-specific real-time PCR assay enables efficient detection of buparvaquone-resistant genotypes, providing important implications for guiding the treatment of tropical theileriosis and enhancing epidemiological surveillance of emerging resistance in endemic regions.