Development of a Species-Specific qPCR Assay for Theileria annulata Detection and Its Molecular Surveillance in Kazakhstan

Theileria annulata causes tropical theileriosis in cattle and is endemic in Kazakhstan. Although molecular diagnostic methods for this pathogen exist, data on its molecular surveillance and genetic diversity in Kazakhstan remain limited. This study aimed to develop and experimentally validate a species-specific real-time PCR (qPCR) assay for the detection of Theileria annulata and to conduct molecular surveillance and phylogenetic analysis of circulating isolates. A total of 709 cattle blood samples from 10 regions of Kazakhstan were analysed. Species-specific primers and a probe were designed targeting the 18S rRNA gene, and diagnostic performance was evaluated against sequencing as the reference method. Theileria annulata infection was confirmed in 110 of 709 samples (15.51%), with positive samples identified in the Turkistan, West Kazakhstan, Kyzylorda, and Zhambyl regions. The developed assay showed high diagnostic sensitivity (99.09%), specificity (99.67%), and diagnostic efficiency (99.58%), with no cross-reactivity with Theileria orientalis, Theileria parva, Babesia bovis, Babesia bigemina, or Anaplasma marginale. Phylogenetic analysis of partial 28S rRNA sequences confirmed the species identity of the detected isolates and demonstrated their close relationship with previously reported Kazakhstani and Chinese isolates. The developed qPCR assay is suitable for the molecular diagnosis and surveillance of bovine tropical theileriosis in Kazakhstan.