Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone

• Published in: Veterinary parasitology 2024-06, Vol.328, p.110183-110183, Article 110183
• Main Authors: Su, Shuxiao, Zhao, Shuaiyang, Liu, Junlong, Zhang, Chuhan, Zhu, Haohan, Guan, Guiquan, Yin, Hong, Luo, Jianxun
• Format: Article
• Language: English
• Subjects: Animals; Antiprotozoal Agents - pharmacology; Antiprotozoal Agents - therapeutic use; Buparvaquone; Cattle; China - epidemiology; Drug Resistance - genetics; Mutation; Naphthoquinones - pharmacology; Protozoan Proteins - genetics; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - veterinary; Reproducibility of Results; Resistant; Sensitivity and Specificity; T. annulata; TaPIN1; TaqMan Real-time PCR; Theileria annulata - drug effects; Theileria annulata - genetics; Theileria annulata - isolation & purification; Theileriasis - diagnosis; Theileriasis - drug therapy; Theileriasis - parasitology
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/j.vetpar.2024.110183

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application. •T. annulata resistant to buparvaquone was found in China where buparvaquone was forbidden to be used to treat cattle.•The mutation of TaPIN1 where most likely lead to buparvaquone-resist for Theileria annulata was detected.•Established a TaqMan real-time PCR method to detect the mutation of TaPIN1.•The distribution of Theileria annulata resistant to buparvaquone was investigated in Xinjiang, China.