Abstract
Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.
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Data availability
Data are available from the authors upon reasonable request; sequences are available via GenBank.
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Acknowledgements
The authors are thankful to Department of Veterinary Medicine, LLR University of Veterinary and Animal Sciences, Hisar, for providing financial support, and to the Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, for providing the infrastructural facility.
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Sumnil Marwaha performed the experiments, analyzed the data, prepared the figures and tables, and authored and reviewed the drafts of the paper. Basanti Brar assisted in conceptual discussions and revision of the manuscript. Vinod Kumar Jain and Minakshi Prasad conceived and designed the experiments, and reviewed and edited the manuscript. Rachna Poonia assisted in sampling and revision of manuscript. All authors read and approved the final manuscript.
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In this study, blood was collected from the animals by the veterinarians as per the procedure in compliance with the ethical standards and guidelines of the Institutional Animal Ethics Committee (IAEC), LUVAS, Hisar, India, and permission was obtained via. Order no. VCC/IEAC/2625–2647/21–12-19.
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Marwaha, S., Brar, B., Jain, V.K. et al. Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle. Parasitol Res 122, 1189–1197 (2023). https://doi.org/10.1007/s00436-023-07819-1
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DOI: https://doi.org/10.1007/s00436-023-07819-1