Specific detection and identification of African trypanosomes in bovine peripheral blood by means of a PCR-ELISA assay.
The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n=18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n=42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.
Cabrera
Leyda
L
Alexander von Humboldt Institute of Tropical Medicine (IMTAvH), Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, San Martín de Porras, Lima 31, Peru.
De Witte
Jacob
J
Victor
Björn
B
Vermeiren
Lieve
L
Zimic
Mirko
M
Brandt
Jef
J
Geysen
Dirk
D
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014345:Trypanosoma / Q000302:isolation & purification*; D014353:Trypanosomiasis, African / Q000097:blood / Q000662:veterinary*