AXENIC CULTURE OF TRYPANOSOMA-CONGOLENSE - APPLICATION TO THE DETECTION OF SENSITIVITY LEVELS OF BLOOD-STREAM TRYPOMASTIGOTES TO DIMINAZENEACETURATE, HOMIDIUM CHLORIDE, ISOMETAMIDIUM CHLORIDE AND QUINAPYRAMINE SULFATE

An axenic culture system for Trypanosoma congolense bloodstream trypom astigotes was used for in vitro detection of their sensitivity to dimi nazene 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, IL246 6, IL3000, IL3266 and IL3338, were propagated in vitro using HMI-93 me dium (Hirumi and Hirumi 1991). Among these populations IL1180, IL2466, IL2642 are known to be sensitive to DA and/or IC as tested in mice an d/or cattle, while IL3035 and IL3338 are highly resistant to DA, IC an d/or HC when examined in cattle. Each well of a 24-well culture plate received 100mul of distilled water containing various amounts of the d rugs. Test plates were then freeze-dried and stored at room temperatur e. Levels of the resistance for each drug were then expressed in 10 st eps from 10 to 1, denoting the following concentrations: DA at 600, 50 0, 400, 300, 200, 100, 80, 60, 40 and 20 ng/ml; HC, IC and QS at 10-fo ld serial dilutions from 10mug to 10 fg/ml. Five hundred mul aliquots of trypanosome suspension in the medium, containing 4x10(5) trypanosom es/ml, were then placed in each well and maintained at 34-degrees-C in a CO2 incubator for 5 days without medium change. Effects of the drug s were examined by phase-contrast microscopy every 24h. Growth inhibit ion of trypanosomes could be detected by day 3 and affected trypanosom es died during the next 24-48h. In contrast, trypanosomes which were r esistant to the given concentrations continued to grow reaching the ma ximum 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 wel ls in which trypanosomes reached the maximum population density indica ted 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. R esistance levels of the 4 stocks and the 4 clones against DA, HC, IC a nd QS were IL1180: 4,7,6 & 6, IL2079: 5,6,6 & 6, IL2466: 4,6,6 & 8, IL 2642: 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 th e in vitro cloned populations were similar to those of their parental populations. The information obtained in this system using 3 plates pe r drug per trypanosome population for 5 days provided an equivalent am ount of information about the drug sensitivity of a trypanosome popula tion as an in vivo test which uses 36 mice and lasts 2 months.