TgIMC3 as a marker to “birth date” bradyzoite replication within tissue cysts. TgIMC3 as a marker to “birth date” bradyzoite replication within tissue cysts. (A) Following an initial round of replication, emergent bradyzoites possess a strong TgIMC3 signal. In the absence of further replication, this signal dissipates with currently unknown kinetics (red boxed profile). In contrast, continued replication resets TgIMC3 intensity levels. (B) Tissue cysts harvested at weeks 3, 5, and 8 were stained at the same time with the same aliquots of TgIMC3 antibody. All images were acquired at random and at a fixed exposure. The mean TgIMC3 pixel fluorescence intensity was normalized to the imaged area of the cyst established. The intensity of TgIMC3 protein expression from 157 cysts (week 3 [n = 60], week 5 [n = 60], week 8 [n = 37]) was analyzed for cysts from three different weeks postinfection. One-way ANOVA demonstrates significant differences for TgIMC3 expression between weeks 3, 5, and 8 (F2,154 = 214.9; P < 0.0001 [indicated by four asterisks]). Tukey’s pairwise multiple-comparison test (α = 0.05) indicated that the mean intensity of TgIMC3 labeling of cysts harvested at each time point differed significantly from one another. The data indicate that tissue cysts harvested at week 3 are brightest, consistent with relatively recent or concurrent replication based on the level of TgIMC3 labeling. Tissue cysts at week 5 exhibit low overall levels of TgIMC3 intensity, while those harvested at week 8 have an intermediate level consistent with significant replication having occurred between weeks 5 and 8. (C) Representative images of tissue cysts defining the high, mean, and low TgIMC3 intensity levels for each population of cysts reveal not only different overall levels of TgIMC3 but also distinct patterns of labeling. (D) Potential models explaining the patterns of TgIMC3 between weeks 3 and 8. Rapid replication during the tachyzoite phase accounts for elevated TgIMC3 labeling that accounts for week 3 labeling exhibiting the brightest cysts with the greatest diversity. A reduction in growth rate, manifesting as greater intervals between replicative events, accounts for the decreased mean TgIMC3 intensity at week 5. The intermediate levels for TgIMC3 at week 8 can be accounted for by two distinct, although nonexclusive, models. In the first model, gradual, likely asynchronous growth results in a net increase in TgIMC3 intensity. Alternatively, a burst of replication within the population (depicted here between weeks 5 and 6), followed by an absence of replication and a time-dependent loss of TgIMC3 intensity, results in intermediate levels within cysts captured at week 8 postinfection. Elizabeth Watts et al. mBio 2015; doi:10.1128/mBio.01155-15