Axenic Culture of Trypanosoma evansi : An Application to the Simple Detection of Sensitivity of Bloodstream Trypomastigotes to Trypanocidal Drugs

Abstract

An attempt to the development of a simple in vitro method to detect the drug sensitivity of Trypanosoma evansi was made. Bloodstream forms (BSFs) of kinetoplastic T. evensi, T. evansi IL3354 and T. evensi IL3960 populations, and akinetoplastic T. evensi AK, T. evensi IL1934 and T. evensi IL3960-AK populations were propagated in vitro in an axenic culture system, in which bloodstream forms of T. evansi were cultured in the absence of feeder layer cells using HMI-9 culture medium following the procedure described earlier (Hirumi and Hirumi 1994).
With a minor modification of the original drug sensitivity test (Hirumi et al. 1993), the minimum effective concentration of diminazene aceturate (DA) (Berenil ® and TRYPAN®) was tested for theT. evensi populations. Five hundred μl aliquots of trypanosome suspension in the medium, containing 2 × 10⁴ BSFs, were placed in each well, added an equal volume of drug solution containing various concentrations of DA in culture medium and maintained at 37℃ with 5% CO₂ in air for 10 days without medium change. Effects of the drug were examined by phase-contrast microscopy every 24 hrs. In the control group with no drug, trypanosomes increased in number during the initial 3 days and reached the maximum cell density (1-2 ×10⁶ BSFs/ml). Trypanosomes, thereafter, rapidly decreased during the following 2 days and died by day 7 due to overpopulation. In contrast, trypanosomes maintained in the medium contained ineffective doses of DA, rapidly decreased in number and died by days 3-5. While, in the medium contained lower doses, numbers of BSFs gradually decreased to the levels of 10-10³ BSFs/well during the initial 3-5 days. Thereafter, the survived parasites increased in number reaching the maximum density during the following 3-5 days and then died by days 7-10.
Although no significant difference was observed between the kinetoplastic and chemically induced akinetoplastic populations tested, the results demonstrated that the minimum effective concentration of the drug tested can be readily detected by means of phase-contrast microscopy without aids of sophisticated equipments, including a freeze-dryer which was used in the original method. The method may be simple enough to apply in ordinary research laboratories which are equipped with standard cell culture equipments including a CO₂ incubator and an inverted phase-contrast microscope. The results were highly reproducible.