María Elena García

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

Abstract 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.

Trypanosomiasis in Venezuelan water buffaloes: association of packed-cell volumes with seroprevalence and current trypanosome infection

Abstract The seroprevalence of trypanosomiasis and the prevalence of current trypanosome infection in water buffaloes from the most important livestock areas of Venezuela were evaluated by IFAT and the microhaematocrit centrifugation technique, respectively. The usefulness of a PCR-based assay for identifying the trypanosome species in the buffaloes was also evaluated. Of the 644 animals investigated, 40 (6.2%) were found infected with trypanosomes by blood centrifugation, and 196 (30.4%) were found positive for anti-trypanosome antibodies, by IFAT. The results of the PCR-based assay indicated that 92.5% of the animals with current infections were infected with Trypanosoma vivax and the rest with T. theileri (the first molecular confirmation of T. theileri in Venezuelan water buffaloes). The national programme to treat and prevent trypanosome infections in the buffaloes does not appear to be meeting with great success, even though it is focused on T. vivax. Although the level of parasitaemia was categorized as low for 28 (70%) of the infections detected (and packed-cell volumes appeared to be unassociated with IFAT result, and uncorrelated, in the infected animals, with level of parasitaemia), the 40 infected buffaloes had a significantly lower mean packed-cell volume than the uninfected animals (P<0.05). Farmers should therefore be made aware of the probability of trypanosome-attributable losses in buffalo productivity.