Quantification of Trypanosoma evansi parasitic load in experimentally infected mice using real time PCR assay and its comparative evaluation with conventional parasitological techniques

Abstract

Trypanosoma evansi causes an economically important disease called surra, which is responsible for significant losses to livestock productivity in Asia, Africa, Central and South America. In the present study, a SYBR Green based quantitative PCR (qPCR) assay was optimized using primers targeting the internal transcribed spacer 1 (ITS–1) region of rRNA gene for quantitative estimation of T. evansi infection in experimentally infected mice. The sensitivity of qPCR assay was found approximate to the gold standard TBR1/2 primers based PCR assay and two times more than the conventional parasitological techniques, viz. wet blood film (WBF) stained thin blood smear (TBS) and microhaematocrit centrifugation test (MHCT). The detection limit of the assay was 1.5 parasite equivalence or 0.15 pg T. evansi DNA. On fourth day post infection, 100% (6/6) mice were detected positive by all parasitological techniques with parasitaemia ranging from 1.6×105 to 2.1×108 parasites. The study indicated that this assay can be applied for quantitative estimation of parasitaemia in animals suffering from surra and may be helpful for understanding disease status, risk analysis and efficacy of drug used in treatment of disease.

Keywords

• ITS-1
• Molecular diagnosis
• PCR
• qPCR
• Surra
• SYBER