In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.
Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n = 10) were infected orally (study group, n = 50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16 weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1 week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.
Toxoplasma gondii infection causes toxoplasmosis, an infectious disease with worldwide prevalence. The limited efficiency of drugs against this infection, their side effects and the potential appearance of resistant strains make the search of novel drugs an essential need. We examined Eurycoma longifolia root extract and fractions as potential sources of new compounds with high activity and low toxicity. The main goal of this study was to investigate the anti-T. gondii activity of crude extract (TACME) and four fractions (TAF 273, TAF 355, TAF 191 and TAF 401) from E. longifolia, with clindamycin as the positive control.