Efforts to develop therapies against Toxoplasma reactivation have been hampered by the lack of an in vitro model of bradyzoite recrudescence. To overcome this issue, we established a new ex vivo model of bradyzoite recrudescence using bradyzoites from an unadapted Type II ME49 strain isolated from murine brain tissue to infect human foreskin fibroblasts and neonatal murine astrocytes. Bradyzoite infection of both host cell types produced two sequential tachyzoite growth stages that appeared similar to previous sporozoite in vitro infections; a fast-growing stage was followed by spontaneous formation of a slowergrowing stage. In astrocytes, but not in fibroblasts, there was evidence of second bradyzoite to bradyzoite recrudescent pathway that occurred simultaneously with the bradyzoite to tachyzoite pathway.Thus, the host cell environment strongly influenced the pathway of bradyzoite recrudescence. Infections of mice with either the fast-growing or slow-growing recrudescent tachyzoites showed progressive decreases in brain cyst formation that could be fully recovered using serial tissue cyst passage demonstrating the loss of developmental capacity was reversible. By contrast, poor tissue cyst formation of laboratory-adapted tachyzoites was not reversible by these approaches indicating developmental incompetence was permanent in these strains. In order to develop methods to distinguish strain developmental competency, we identified Toxoplasma genes highly expressed in ME49EW in vivo tissue cysts and developed a qPCR approach that differentiates immature from mature bradyzoites. In summary, the results presented describe a new ex vivo bradyzoite recrudescence model that fully captures the reported growth and developmental processes observed during toxoplasmosis reactivation in vivo opening the door to the further study of these important features of the Toxoplasma intermediate life cycle.

Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central feature of reactivation. We have developed an innovative ex vivo model of bradyzoite recrudescence and applied this method to successfully modify Toxoplasma genes while avoiding the problems caused by continuous in vitro cell culture. We present four protocols required to engineer in vivo transgenic tissue cysts: 1) the reliable production of in vivo tissue cysts and excysted bradyzoites, 2) the use of fast-growing parasites from ex vivo bradyzoite infections to successfully generate transgenic tissue cysts in mice, 3) the cloning of transgenic bradyzoites via single cyst infections, and finally, 4) the long term cold storage and recovery of transgenic tissue cysts in brain tissue homogenates. We demonstrated these protocols by knocking out the Toxoplasma TgHXGPRT gene and the gene encoding the ApiAP2 transcription factor, AP2IX-9 in tissue cysts from mice. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite; host-dependent bradyzoite-to-bradyzoite replication.