Development of a duplex PCR assay for simultaneous detection of Babesia bigemina and Theileria annulata infections in cattle

BISWA RANJAN MAHARANA; ANITA GANGULY; RANBIR SINGH BISLA; ANKIT KUMAR; SANDEEP POTLIYA; DEVAN ARORA

doi:10.56093/ijans.v88i4.78766

Authors

BISWA RANJAN MAHARANA Scientist, Department of Veterinary Parasitology, LUVAS, Karnal, Haryana 132 001 India
ANITA GANGULY Scientist, Department of Veterinary Biochemistry, LUVAS, Karnal, Haryana 132 001 India
RANBIR SINGH BISLA Regional Director cum Principal Scientist, Department of Veterinary Surgery and Radiology, LUVAS, Karnal, Haryana 132 001 India
ANKIT KUMAR Scientist, Department of Veterinary Medicine, LUVAS, Karnal, Haryana 132 001 India
SANDEEP POTLIYA Scientist, Department of Veterinary Surgery and Radiology, LUVAS, Karnal, Haryana 132 001 India
DEVAN ARORA Scientist, Department of Veterinary Epidemiology and Public Health, Referral Veterinary Diagnostic and Extension Centre

https://doi.org/10.56093/ijans.v88i4.78766

Keywords:

Babesia bigemina, Cattle, Duplex PCR, Microscopy, Theileria annulata

Abstract

Bovine babesiosis and theileriosis are fatal tick borne haemoparasites of vertebrates imposing serious constraints on health and productivity of livestock. Additionally, the recovered animals become persistent carriers and play a significant role in disease epidemiology. The present investigation describes the development and evaluation of duplex PCR assay for simultaneous detection of Babesia bigemina (B. bigemina) and Theileria annulata (T. annulata) in cattle. Following in silico analysis for candidate target genes representing each of the haemoparasites, an optimised duplex PCR assay was established using two sets of primers, ssurRNA and cytob1 for genomic DNA amplification of B. bigemina and T. annulata encoding product size of 689 and 312 bp, respectively. The results were compared with conventional microscopy and monoplex PCR assay. The sensitivity of each primer pair was checked using serial dilutions of parasite DNA, while specificity was determined by testing for amplification from DNA of different stocks of each pathogen. The duplex PCR detected each parasite species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or with DNA mixture representing the other pathogens. Additionally, single and duplex PCRs could able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of both the pathogen, and nonspecific amplification from non target species was not observed. The developed assay represents an economical, simple, sensitive, specific and reproducible diagnostic tool for simultaneous detection of tropical theileriosis and bovine babesiosis and boosting targeted selective control strategy in endemic areas.

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