Trypanosoma evansi: measurement of pyruvate production as an indicator of the drug sensitivity of isolates in vitro.

In a previous study, three in vitro methods for the assessment of drug sensitivity among Trypanosoma evansi isolates were compared--a direct counting method, pyruvate production method and uptake of radiolabelled hypoxanthine. The pyruvate assay system, which measures the amount of pyruvate in the supernatant of growing populations of trypanosomes by a spectrophotometric method, was selected for further investigation with regard to its suitability for field studies. The effect of initial seeding density and incubation time on the growth of three stocks of T. evansi--TREU 1840 and TREU 1981 (suramin sensitive) and TREU 2136 (suramin resistant)--and drug sensitivities revealed by the pyruvate assay and direct counting were examined to optimise assay conditions. Maximum densities and pyruvate production achieved were not affected by varying the initial seeding densities in the range of 5 x 10(4)-5 x 10(5)/ml and had been reached after 48 hours incubation with one exception: Pyruvate levels continued to increase up to 72 hours in the suramin resistant stock. However, inhibition curves were affected by initial seeding density and incubation period. Results suggested that an initial seeding density of 1 x 10(5)/ml and an incubation time of 48 hours are optimal for the assay. Using these assay conditions, the isolates were screened against suramin, quinapyramine sulphate and Cymelarsan, the trypanocides used most commonly against T. evansi. This assay proved to be a relatively simple and cheap technique applicable to screening large numbers of isolates of differing sensitivities to trypanocidal drugs.

A comparison of in vitro assay systems for the measurement of drug sensitivity of Trypanosoma evansi.

Two in vitro assay systems were investigated for their effectiveness in detecting the sensitivity of Trypanosoma evansi stocks to the trypanocide suramin. These assay systems measured 1) incorporation of radiolabelled nucleic acid precursor, hypoxanthine; and 2) pyruvate production. They were compared with the direct counting method in which numbers of motile trypanosomes were estimated using a Neubauer haemocytometer chamber. Three stocks of T. evansi were tested-2 suramin sensitive stocks from Indonesia, TREU 1840 and TREU 1981, and a suramin resistant stock from the Sudan, TREU 2136. Each assay system distinguished between the suramin sensitive and resistant stocks. However, inhibition compared to untreated control cultures was less when assessed from pyruvate concentration in culture supernatants than by direct counting. The length of incubation with drug before addition of radio-label was the most important variable in the hypoxanthine incorporation assay. A pre-incubation time of 16 hours with the drug before adding the label for the further 8 hours of the assay was found to be the most sensitive. Under these conditions, the IC50 values (drug concentrations causing 50% inhibition) were similar to those obtained from direct counts. Pre-incubation of parasites with drug before adding the label resulted in a decrease of the IC50. These results suggest that the discrepancy between the levels of pyruvate production and relative growth at inhibitory concentrations of the drug are due to metabolism by the parasites during the initial stages of the assay, before the drug has began to inhibit cell growth.

Trypanosoma congolense: re-expression of a deleted metacyclic variable antigen type in vivo and in vitro.

The expression of variable antigen types (VATs) was determined among dividing populations of T. congolense growing in vivo in rabbit chancres and in vitro on bovine aorta endothelial cell monolayers. Experiments were performed in which a single metacyclic VAT (M-VAT) was deleted from a cultured metacyclic population by neutralisation with a monoclonal antibody and complement. Subsequent expression of the deleted M-VAT and two unrelated M-VATs was determined by an indirect immunofluorescent antibody test. The deleted M-VAT was re-expressed both in vivo and in vitro and the proportions of unrelated M-VATs were not markedly affected by the neutralisation of this single M-VAT. In addition, an overall similarity was observed between M-VAT expression and re-expression in vivo and in vitro.