Time-dose-response of Trypanosoma brucei brucei to diminazene aceturate (Berenil) and in vitro simulation of drug-concentration-time profiles in cattle plasma.

Bloodstream form Trypanosoma brucei brucei of axenically growing populations were incubated in the presence of 10.0, 1.0 or 0.1 micrograms/ml diminazene aceturate (Berenil) at 37 degrees C for various periods and, subsequently, either inoculated into mice or further propagated in vitro in drug-free medium. Growth was monitored for 10 days. The ability of trypanosomes of drug-sensitive CP 2137 (clone 1) to grow in vitro was irreversibly damaged after short incubation (< 1 min) with 10.0 micrograms/ml or after 15 min with 1.0 micrograms/ml diminazene aceturate. In contrast, drug-resistant CP 2469 (clone 1) trypanosomes tolerated incubation with 10 micrograms/ml of drug for up to 6 h and 1.0 micrograms/ml of drug for up to 24 h. Differences in drug susceptibility were also detected regarding infectivity to mice and changes in trypanosome cell volume. The results demonstrated that less than 1 min exposure to diminazene aceturate at concentrations as seen in bovine plasma at the initial peak after diminazene aceturate treatment is enough to irreversibly damage drug-sensitive trypanosomes. However, these concentrations were not sufficient to completely eliminate drug-resistant trypanosomes after exposure for 1-6 h; trypanosomes continued to grow for 48 h before the majority of them died and only a few organisms survived to revive the cultures. When drug-sensitive trypanosomes were exposed in vitro for 24 h to diminazene aceturate at the level of concentrations found in cattle after treatment with 3.5 mg/kg, most of the trypanosomes died and none of the surviving parasites could be propagated in vitro in the absence of drug for more than 2 days. However, a small population of drug-resistant trypanosomes was not irreversibly damaged and a few surviving trypanosomes were able to establish growing cultures. The addition of feeder layer cells did not change the outcome of these experiments.