Development and application of a quantitative real-time PCR for the diagnosis of Surra in water buffaloes.
Trypanosoma evansi (T. evansi) causes the disease called Surra in domestic animals, which is of great economic importance in South Asian countries. In order to improve the diagnosis of Surra, we endeavored to develop a real-time PCR assay for the detection and quantification of parasites in water buffaloes using specific primers for the T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 Variable Surface Glycoprotein (VSG) gene, which is a known diverse DNA region in trypanosomes. The quantitative detection limit of the assay was 10(2) trypanosomes per mL of blood, and the identity of the amplicon was confirmed in all assays by melting curve analysis. To evaluate the clinical applicability of this procedure, detection and estimation of parasitemia in blood samples obtained from water buffaloes and horses were conducted. T. evansi was detected in 17/607 (2.8%) blood samples, with parasitemia levels ranging from >10(1) to 10(7) parasites per mL of blood. Interestingly, out of the 17 PCR positive animals, 3 had previously received trypanocidal treatment and 1 had abortion history. These data indicate that real-time PCR for the estimation of putative parasitemia levels is a quantitatively and objectively applicable technique for clinical diagnosis of Surra, and could help to understand disease stage and risk of transmission of T. evansi.
Konnai
Satoru
S
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. konnai@vetmed.hokudai.ac.jp
Mekata
Hirohisa
H
Mingala
Claro N
CN
Abes
Nancy S
NS
Gutierrez
Charito A
CA
Herrera
Jesus Rommel V
JR
Dargantes
Alan P
AP
Witola
William H
WH
Cruz
Libertado C
LC
Inoue
Noboru
N
Onuma
Misao
M
Ohashi
Kazuhiko
K
D000818:Animals; D002020:Buffaloes / Q000469:parasitology*; D008562:Membrane Glycoproteins / Q000235:genetics; D018512:Parasitemia / Q000097:blood / Q000235:genetics / Q000662:veterinary; D010679:Philippines; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000662:veterinary*
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins