Application of Real-time polymerase chain reaction for diagnosis of Trypanosoma evansi infection in cattle and buffaloes

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Date
2011
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GADVASU, Ludhiana
Abstract
The present study was conducted for detection of Trypanosoma evansi which causes the disease surra in domestic animals. Real time PCR was applied in field cases of T. evansi in cattle and buffaloes. It is more sensitive assay using TaqMan primer and probe for T. evansi and targets the internal transcribed spacer 1 (ITS-1) region of rRNA. Real time PCR was standardized by using purified as well as whole blood trypanosomes of infected mice blood. Purification and isolation of trypanosomes were carried out. DNA extraction was carried out as per the standard protocol of QIAGEN blood mini kit. The sensitivity of TaqMan assay was evaluated by using purified as well as whole blood trypanosomes DNA of infected mice and found that the minimum detection limit for purified trypanosomes was 0.01 ng (0.33 genomic DNA of trypanosoma ) whereas for whole blood the minimum detection limit was 0.1 ng (6.12 genomic DNA of trypanosoma). T. evansi infected mice blood samples were collected at different interval post infection and analyzed by CPT and real time PCR and observed that TaqMan assay is very sensitive than other CPT in case of in vivo infectivity in mice and gave positive signal at 36 h post infection. A total 109 (80 cattle and 29 buffaloes) blood samples of cattle and buffaloes were collected from in and around Ludhiana district and analyzed by conventional parasitological techniques and real time PCR. The results revealed that real time PCR was more sensitive than other conventional method like wet blood smear, thin blood smear, thick blood smear and haematocrit centrifugation technique. The overall prevalence of T. evansi in and around Ludhiana by conventional parasitological techniques was 2.75 per cent. The prevalence of T. evansi in case of cattle was 2.5 per cent and in case of buffaloes 3.45 per cent by conventional methods.The overall prevalence rate by real time PCR in cattle and buffaloes was 12.84 per cent however buffaloes (13.79%) showed the higher prevalence rate than cattle (12.50%) by both conventional and TaqMan assay.
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