Toxoplasma gondii: Prolonged in-vitro maintenance of virulent tachyzoites in fluid media at low temperatures

Abstract

Background: Prolonged maintenance of infective Toxoplasma gondii tachyzoites (T.g.T.) is an important subject for research purposes. This study aimed to evaluate four serum free fluid media for prolonged in vitro maintenance of T.g.T. Methods: The four fluid media Phosphate buffered saline (PBS) pH 7.2 and Roswell Park Memorial Institute (RPMI-1640) with or without 3% fetal bovine serum (FBS) were evaluated for maintenance of virulent T.g. T. The four media were tested after incubation at three different temperature degrees in the darkness. Results: Prolonged maintenance period for infective T.g.T. was recorded especially in the absence of FBS supplement. RPMI without FBS was able to maintain infective T.g.T. for 16 days post incubation (dpi) at refrigerator temperature. This period decreased to 10 dpi and 6 dpi after incubation in the same media at 18-22°C and 37°C, respectively. Cultivation of T.g.T. in RPMI supplemented with 3% FBS and in PBS proved to maintain infective T.g.T. for 14 dpi at refrigerator temperature, and for 9 and 5 dpi when the two media were incubated at 18-22°C and 37°C, respectively. Shorter periods for keeping the T.g.T. infectivity were recorded using PBS supplemented with 3% FBS under all tested temperature conditions. Conclusion: This method allows economic long-lasting maintenance of tachyzoites for 16th dpi in RBMI that can be reactivated by reinoculation in mice.

Comparative evaluation of amniotic fluid as an alternative to fetal bovine serum in the maintenance of Leishmania major and Toxoplasma gondii

“…The authors in the recent study concluded that the best condition for T. gondii tachyzoite maintenance was PBS at 4°C, in which the parasite survived for 12 days (Kalani et al 2016a). However, this finding needs further evaluation since other researchers have showed that RPMI-1640 is more successful than PBS in the maintenance of T. gondii tachyzoites (El-Bahy et al 2018). In the present study, we observed that the FBS-supplemented medium is more appropriate than AF for T. gondii tachyzoite maintenance.…”

“…However, this finding needs further evaluation since other researchers have showed that RPMI-1640 is more successful than PBS in the maintenance of T . gondii tachyzoites (El-Bahy et al 2018 ). In the present study, we observed that the FBS-supplemented medium is more appropriate than AF for T. gondii tachyzoite maintenance.…”

Viability of Toxoplasma gondii tachyzoites in different conditions for parasite transportation

“…The results were comparable to other studies in which nutrient-free media, such as NS and PBS, were more suitable than minimum essential media for maintaining T. gondii tachyzoites. Indeed, the media without nutrients, vitamins, carbohydrates, and fats could prolong the viability of free T. gondii tachyzoites better than those containing these kinds of supplements [12,13]. The osmotic coefficient of NS is about 0.93 [21] and is similar to the osmolarity of blood in which circulating tachyzoites have been found in a range of host species [22][23][24][25][26].…”

“…It was likely that these biological activities produce harmful materials and cause changes in solution pH, threatening the parasite's survival. In addition, it showed that tachyzoites could survive longer at 4°C than at 18-22°C and 37°C because they retain a minimum metabolic rate [12,13]. Therefore, enrichment of the nutrient solution, such as with the MEM or MEM-FBS media, and at RT might be more harmful to free-living tachyzoites than NS and PBS for the IC condition.…”

“…To maintain high viability and survival rates for the parasites, T. gondii tachyzoites should be used for inoculation in an animal model [9,10] and in vitro study immediately after purification. Available at www.veterinaryworld.org/Vol.15/January-2022/25.pdf Heider et al [9] and Räisänen [11] demonstrated that tachyzoites survive and remain infectious better in serum solutions than the free nutrient media, the viability of the parasites in phosphate-buffered saline (PBS) or normal saline (NS) was significantly high, compared to enriched media in other studies [12,13]. Besides, the sensitivity of T. gondii tachyzoites against low pH was found in an experiment in which simulated gastric fluid was used to examine the tachyzoites infectivity retention in different acidity [14].…”