Comparative evaluation of real time PCR assay with conventional parasitological techniques for diagnosis of Trypanosoma evansi in cattle and buffaloes

Abstract

For comparative evaluation, a real time PCR assay was standardized by using TaqMan primer and probe targeting the internal transcribed spacer 1 (ITS-1) region of rRNA for Trypanosoma evansi and sensitivity was evaluated by using DNA, extracted from diethyleamino ethane cellulose purified trypanosomes and trypanosomes infected whole blood of mice. The minimum detection limit for purified trypanosomal DNA was 0.01ng (∼0.33 genomic DNA of T. evansi) whereas for whole blood the minimum detection limit was 0.1ng (∼6.12 genomic DNA). T. evansi infected mice blood samples were collected at different interval post infection and were analysed by conventional parasitological methods (CPT) viz. wet blood smear, thin blood smear, thick blood smear, quantitative buffy coat and real time PCR and found that TaqMan assay was two fold sensitive than CPT in case of in vivo infectivity in mice and gave positive signal at 36h post infection where as QBC and blood smear examination was able to detect at 60h and 72h post infection respectively. A total 109 (80 cattle and 29 buffaloes) blood samples were collected from in and around Ludhiana district and analysed by CPT and real time PCR. The overall prevalence of T. evansi by CPT in cattle and buffaloes was 2.75 per cent. The prevalence rate was 2.5 per cent in cattle and 3.45 per cent in buffaloes. By real time PCR overall prevalence was 12.84 per cent in cattle and buffaloes, with a prevalence rate of 12.50 per cent in cattle and 13.79 per cent in buffaloes.