DEVELOPMENT OF SIMPLEX AND MULTIPLEX PCR ASSAY FOR RELIABLE IDENTIFICATION OF HAEMOPARASITIC DISEASES OF CATTLE, BANGLADESH

Abstract

Bangladesh is a tropical, agro-based developing country. Anaplasmosis, Babesiosis, and Theileriosis are the most common vector-borne haemoparasitic diseases in cattle. This study not only describes the development and evaluation of a multiplex PCR assay for simultaneous detection of haemoparasitic diseases (Theileria annulata, Babesia bigemina, and Anaplasma marginale), but also their prevalence ratio. In the multiplex PCR assay, three sets of pre-designed primers were used that targeted the genes Tams1 for T. annulata, 18S rRNA for B. bigemina, and 16S rRNA for A. marginale, with desired amplicons sizes of 751 bp, 504 bp, and 270 bp, respectively, with the use of 2% agarose gel for electrophoresis of amplified PCR products. A total of 350 blood specimens were collected that were tentatively diagnosed as haemoparastic diseases on the basis of clinical signs from three districts of Bangladesh: Chattogram, Rangpur, and Sylhet. Blood samples were stained and preserved at -20°C for further molecular study. In this study, 52% (out of 350) cases were tentatively diagnosed as positive by microscopic examination, among the isolates A. marginale (30.3%), Babesia sp. (12.9%), and Theileria sp. (9%). (Whereas, 21.7% in Chattogram, 17.1% in Sylhet, and 13.1% in Rangpur.) However, in the simplex PCR assay, 35.7% of the cases (14.9% in Chattogram, 12% in Sylhet, and 8.9% in Rangpur) showed a positive band in electrophoresis. Mixed haemoparasitic infection is very common in cattle. Overall, 13.1% of infections were detected positive by multiplex PCR; among them, 2% were diagnosed as positive for all three haemoparasites and 3.71% were diagnosed as anaplasmosis+Babesiosis and so. The partial gene sequencing and phylogenetic analysis of the nucleotide sequences expressed the fidelity of the primer pairs that were used in multiplex PCR, which was found to be 100% sensitive and 85% specific for the detection of infections. In multiplex PCR, amplification of multiple target sequences in one assay is helpful for diagnosis of multiple organisms at a time. This is also time-saving and costeffective compared to other two methods, but requires several trials for optimization of annealing temperature and shows false negative results in minor errors. Beyond all limitations, multiplex PCR assay is precise and could be used as a robust tool for easy, sensitive, specific, and simultaneous diagnosis of haemoparasitic diseases in cattle.