**2.3 Isolation of** *T. gondii* **bradyzoites and interaction with FIEC**

*T. gondii* cysts from the ME-49 strain (Type II) previously isolated from infected mice were inoculated intraperitoneally into female C57BL/6 mice (15–18 g) with 50 cysts/animal. Four and 12 weeks after infection, the mice were sacrificed and the brain cysts isolated, as described by Freyre [28] and modified by Guimarães et al. [29]. In addition, bradyzoites were obtained from isolated tissue cysts according to Popiel et al. [30].

Confluent FIEC cultures were infected with *T. gondii* bradyzoites. The assays were performed at 1:5, 1:10, and 1:20 (parasite: host cell) ratios for periods ranging from 1 to 9 days postinfection to analyze the infection course and to evaluate the parasite's intracellular fate. After different periods of interaction, the cells were washed in PBS and processed according to the experiments to be performed.

The ability of the *T. gondii* bradyzoites to infect FIECs *in vitro* was examined by fixation of the cells in Bouin's solution and Giemsa staining. The infection analysis was carried out with 200 cells per coverslip in three independent experiments at interaction times of 24, 48, 72, and 96 hours, in duplicate. Data were analyzed in GraphPad Prism 7.0 using Two-Way ANOVA with Tukey's multiple comparisons test, and results were expressed as mean with SEM (graph) or SD (table). Differences were considered statistically significant when the *p*-value was <0.05.
