1993 Volume 3 Issue 2 Pages 52-63
An axenic culture system for Trypanosoma congolense bloodstream trypomastigotes was used for in vitro detection of their sensitivity to diminazene aceturate (DA), homidium chloride (HC), isometamidium chloride (IC) and quinapyramine sulphate (QS). Bloodstream trypomastigotes of 4 stocks (IL2079, IL2466, IL3266 and IL3338), 4 clones (IL1180, IL2642, IL3000 and IL3035) and 16 clones obtained in vitro from IL2079, IL2466, IL3000, IL3266, and IL3338, were propagated in vitro using HMI-93 medium (Hirumi and Hirumi 1991). Among these populations IL1180, IL2466, IL2642 are known to be sensitive to DA and/or IC as tested in mice and/or cattle, while IL3035 and IL3338 are highly resistant to DA, IC and/or HC when examined in cattle. Each well of a 24-well culture plate received 100 μl of distilled water containing various amounts of the drugs. Test plates were then freeze-dried and stored at room temperature. Levels of the resistance for each drug were then expressed in 10 steps from 10 to 1, denoting the following concentrations: DA at 600, 500, 400, 300, 200, 100, 80, 60, 40 and 20 ng/ml; HC, IC and QS at 10-fold serial dilutions from 10 μg to 10 fg/ml. Five hundred μl aliquots of trypanosome suspension in the medium, containing 4x105 trypanosomes/ml, were then placed in each well and maintained at 34℃ in a CO2 incubator for 5 days without medium change. Effects of the drugs were examined by phase-contrast microscopy every 24h. Growth inhibition of trypanosomes could be detected by day 3 and affected trypanosomes died during the next 24-48h. In contrast, trypanosomes which were resistant to the given concentrations continued to grow reaching the maximum population density by day 3-5, and died during the next 24h due to overgrowth. The pH indicator, phenol red, in medium in wells which contained affected populations indicated pH 8.0-8.5, while that in wells in which trypanosomes reached the maximum population density indicated pH6.5 by day 3-5. Colorimetry of the media on day 5 was thus also used to distinguish the drug sensitivity of trypanosome populations. Resistance levels of the 4 stocks and the 4 clones against DA, HC, IC and QS were IL1180: 4, 7, 6 ＆ 6, IL2079: 5, 6, 6 ＆ 6, IL2466: 4, 6, 6 & 8, IL2642: 2, 4, 3 & 5, IL3000: 5, 7, 7 ＆ 6, IL3035: 8, 7, 6 ＆ 8, IL3266: 4, 6, 5 ＆ 5 and IL3338: 8, 7, 7 ＆ 6, respectively. The levels of resistance of the in vitro cloned populations were similar to those of their parental populations. The information obtained in this system using 3 plates per drug per trypanosome population for 5 days provided an equivalent amount of information about the drug sensitivity of a trypanosome population as an in vivo test which uses 36 mice and lasts 2 months.
