Detection of the initial site of Toxoplasma gondii reactivation in brain tissue

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.