FIG 12.

Detection of neutral lipid stores in differentiated bradyzoites in culture. (A) EM of encysted bradyzoites (from the ME49 and Prugniaud [Pru] strains) differentiated for 4 or 7 days showing intracytoplasmic LD surrounded by ER elements. a, amylopectin granule; ap, apicoplast; CW, cyst wall; DG, dense granule; m, mitochondrion; n, nucleus; r, rhoptry; V, Vac compartment. Bars, 600 nm. (B) Quantification of cholesterol, CE, and TAG in encysted ME49 bradyzoites differentiated for 3 days after parasite release from host cells and purification as described previously (7). After washing, parasite lipid concentrations were measured by enzymatic and colorimetric assays. Data are means ± SD from 3 or 4 independent assays. (C) Fluorescence microscopy of encysted ME49 bradyzoites differentiated for 4 days and stained with BODIPY 493/503, TRITC-lectin for the cyst wall, and DAPI. OA at 0.2 or 0.4 mM was added to the medium at day 2 postdifferentiation, showing that the LD number is proportional to the OA concentrations in the medium. Parasite LD are intracytoplasmic, as evidenced by their corresponding images as dark inclusions seen on phase-contrast images (white squares) and by the boxed regions magnified in the orthogonal views (arrows). XY, optical slices taken in the center of the cyst; EF, extended-focus images combining multiple z-slice images.