Application of multiplex PCR for the simultaneous detection of natural infection of theileriosis, babesiosis and trypanosomosis in cattle

Multiplex-PCR, consisting of multiple primer sets within a single reaction mixture to produce amplicons of varying sizes that are specific to haemoprotozoans of bovines, has been standardized and employed for the simultaneous detection of infections. Blood samples were collected from cattle suspected for haemoparasitic infections viz. Theileria spp, Babesia bigemina and Trypanosoma evansi and tested by multiplex PCR to visualize desired amplicons 1098 bp, 689 bp and 275 bp, respectively. Results of study revealed that 20% samples were positive for multiple (17.7%) and single (2.2%) haemoparasitic infections as compare to 8.8% (single-6.6% and multiple-2.2%) positive by conventional microscopy. Multiplex PCR was able to detecteach of the three haemoparasites with same sensitivity irrespective of amplification in isolation or together with the DNA of other haemoparasites. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used and multiplex PCR was found to be 100% sensitive and 81.81% specific with respect to blood films examination for the detection of mixed infections.