Studies on the in Vitro Cultivation of Trypanosomos and Effects of Some Drugs Showing Trypanocidal Activities.

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Otigbuo, N.I
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University of Lagos
A modified medium for the in vitro cultivations of trypanosomes has been developed. The medium ME-99 comprised of Eagle Minimum Essential Medium (MEM) base and various components of medium 199, not found in MEM. The growth, survival and infectivity of T. b. brucei, T. b. gambiense and T. vivax were compared in the modified medium (ME-99), RPMI 1640, MEM, BCM and 199. The effects of the various sera and the Nu-serum on the culture system Baby Hamster kidney cells (BHK) in MEM, on the growth of T. b. brucei were compared. Parasites grew up to 2 x 1010 in medium utilizing Nu-serum, they were infective to mammalian hosts and they could be subcultured. Nu-serum was found to be suitable replacement for the traditional animal sera used in culture. After the exposure of a monomorphic slender T. b. brucei to the Nu-serum culture, they were observed to have reverted to pleiomorphic forms. It was observed that Nu-serum was responsible for this phenomenon. An attempt at culturing trypanosomes axenically was made. This new system grew the parasites but not as well as the cell systems. Blood stream forms of T. b. brucei were grown over BHK cells in MEM with various concentrations of antiprotozol drugs, (metronidazole, chloroquine and mefloquine) to study the effects of the drugs on the parasites. Drugs inhibited the multiplication of the parasites in vitro. The least effective in vitro concentration for methronidazole and chloroquine were 0.003 mg/ml and 0.0024 mg/ml respectively and for mefloquine 0.002 mg/ml. Groups of female mice were treated once to four times daily with metronidazole and chloroquine individually 24, 48 and 72 hours after T. b. brucei infection. The drugs (chloroquine 0.08mg/kgb w, metronidazole 0.1mg/kgw, 0.03mg/kgbw reduced the number of parasites in the mice but did not effect a cure; they prolonged the survival period of the animals. When mefloquine was administered daily at 0.03 mg/kg body weight for 4 consecutive days, a permanent cure was recorded. The combination of metronidazole (0.1 mg/kgbw) and chloroquine (0.08 mg/kgbw) given daily for four consecutive days, also cleared the parasites from the blood stream.Mice inoculated with culture medium containing a combination of both chloroquine and metronidazole at 0.0024 mg/ml and 0.003 mg/ml respectively confer protection against infection by trypanosomes for 14 days. Mefloquine alone at 0.002 mg/ml conferred protection against subsequent infection for more than 4 months. Mice inoculated with washed trypanosomes from cultures containing a combination of metronidazole and chloroquine, were protected against infection for 3 weeks. Mice initially inoculated with pre-treated and washed trypanosomes from drug combined culture medium, and later given a combination of both drugs intraperitoneally, were protected against infection for a period of 5 weeks.Mice injected with 0.03 mg/g mefloquine for 3 days consecutively were protected against T. b. brucei infection for 90 days and over.
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Trypanosomos , Trypanocidal , vitro cultivations
Otigbuo, N.I, (1987), Studies on the in Vitro Cultivation of Trypanosomos and Effects of Some Drugs Showing Trypanocidal Activities, University of Lagos School of Postgraduate Studies Phd Thesis and Dissertation Abstracts, 189pp.