Development of a Species-Specific qPCR Assay for Theileria annulata Detection and Its Molecular Surveillance in Kazakhstan.
4 affiliations
- 1Scientific Research and Production Center "MVA GROUP" LLP, 1124, Accounting Quarter 096, Koksay Village, Karasay District, Almaty Region 040921, Kazakhstan.
- 2Research Institute for Biological Safety Problems, JSC "QazBioPharm" of the Ministry of Health of the Republic of Kazakhstan, Gvardeysky, Kordai District, Zhambyl Region 080409, Kazakhstan.
- 3Institute of Veterinary and Agrotechnology, Zhangir Khan West Kazakhstan Agrarian and Technical University, Uralsk 090009, Kazakhstan.
- 4Moscow State Academy of Veterinary Medicine and Biotechnology named after K. I. Skryabin, Moscow 109472, Russia.
Abstract
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.
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BibTeX
@article{myrzakhmet2026development,
title = {{Development of a Species-Specific qPCR Assay for Theileria annulata Detection and Its Molecular Surveillance in Kazakhstan.}},
author = {Asylzhan Myrzakhmet and Sabyrkhan Barmak and Kulyaisan Sultankulova and Aidar Namet and Aiganym Tussipova and Timur Davlyatshin and Dulat Inkarbekov and Oralkhan Tusipkanuly and Berik Khairullin and Bekzhassar Sidikhov and Fyodor Vasilevich and Mukhit Orynbayev},
journal = {Veterinary sciences},
year = {2026},
month = sep,
doi = {10.3390/vetsci13101028},
url = {https://europepmc.org/article/MED/42944124},
note = {CC BY},
}
RIS
TY - JOUR
TI - Development of a Species-Specific qPCR Assay for Theileria annulata Detection and Its Molecular Surveillance in Kazakhstan.
AU - Asylzhan Myrzakhmet
AU - Sabyrkhan Barmak
AU - Kulyaisan Sultankulova
AU - Aidar Namet
AU - Aiganym Tussipova
AU - Timur Davlyatshin
AU - Dulat Inkarbekov
AU - Oralkhan Tusipkanuly
AU - Berik Khairullin
AU - Bekzhassar Sidikhov
AU - Fyodor Vasilevich
AU - Mukhit Orynbayev
PY - 2026
DA - 2026/09/29
JO - Veterinary sciences
DO - 10.3390/vetsci13101028
UR - https://europepmc.org/article/MED/42944124
AB - 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.
SN - 2306-7381
ER -