The detection and PCR-based characterization of the parasites causing trypanosomiasis in water-buffalo herds in...

The usefulness of PCR-based assays for detecting trypanosomiasis in water buffaloes and other livestock was explored, under field conditions, in Venezuela. The sensitivity and specificity of the assays, which were based on established primer pairs (21-mer/22-mer and ILO1264/ILO1265), were evaluated, partly by comparison with the results of parasitological tests (stained bloodsmears and microhaematocrit centrifugation) and immunological assays (IFAT) run in parallel. The optimised PCR-based assays showed a sensitivity of 10 pg DNA. The use of the 21-mer/22-mer primer pair gave a test that was specific for species in the subgenus Trypanozoon (including Trypanosoma evansi), whereas use of ILO1264/ILO1265 produced a test that was specific for T. vivax. The results of a hybridization assay using T. evansi-DNA and T. vivax-DNA probes indicated no cross-hybridization between the T. evansi and T. vivax PCR products.The results of the bloodsmear examinations, microhaematocrit centrifugations (MHC) and IFAT indicated that 23 (6.7%), 39 (11.4%) and 135 (39.5%) of the 342 blood samples investigated (including 316 from water buffaloes) contained trypanosomes, respectively. The results of the PCR-based assays indicated that 68 (19.9%) of the same blood samples contained T. vivax (or at least T. vivax DNA), and that none contained T. evansi or any other member of the subgenus Trypanozoon. For the detection of trypanosomes, the assay therefore appeared almost twice as sensitive as the MHC. These results are the first on the molecular characterization of the trypanosomes infecting water buffaloes in Venezuela. When the results of the MHC (which is the most practical, and frequently used, alternative detection method) were used as the gold standard, the PCR-based assay for T. vivax was found to have 100% sensitivity, 90.4% specificity, a positive predictive value of 0.57, a positive likelihood ratio of 10.45, and a negative likelihood ratio of 0.00. The assay therefore appears a reasonable choice for detecting T. vivax in the mammalian livestock of Venezuela and elsewhere.