Estimation of Generation Doubling Time of Trypanosoma evansi in Continuous Axenic Culture System

In vitro, axenic culture system is urgently needed to screen drugs and understand Trypanosoma evansi physiology and biochemistry. The present study was carried out to determine the adaptation period, growth kinetics, and generation doubling time of the trypomastigote forms of T. evansi in the in vitro system using an HMI-9 medium. The cryostabilate of T. evansi, isolated from an infected pony was propagated in mice and cultured in vitro in an HMI-9 medium. For the growth kinetics study, the population of trypanosomes in the HMI-9 medium was estimated every 24 h in three independent experiments from day 1 to day 90, and generation doubling time was estimated in a continuous axenic culture system. Data generated showed a gradual fall in T. evansi parasite numbers for the initial 7 days (1.75 x 10⁶ ± 0.2 x 10⁶ to 0.01 x 10⁶) followed by a steady rise of parasite population up to 32 days (3.25 x 10⁶ ± 0.2 x 10⁶). The generation doubling time of the healthy trypomastigote form of T. evansi was calculated as 22.3 ± 1.04 h in the exponential phase (day 60 onwards) of the culture. Based on the statistical analysis of data generated, the 90 days duration of culture was divided into lag phase (1-30 days), progressive phase (31-60 days), and logarithmic phase (61-90 days). To determine the infectivity, trypanosomes of 90th day culture were inoculated in mice. The parasitemia was observed on 6th day post-infection. In conclusion, the continuous in vitro culture of T. evansi can be established with no loss of morphology as well as infectivity of the parasite. The same strain could be used for in vitro drug screening, followed by ex vivo and/or in vivo testing of the candidate trypanocidal molecules.