Improved methods for examination of Toxoplasma gondii cytoskeleton at ultrastructural level.

To evaluate an improved method for identifying the presence of the structural elements of the cytoskeleton of Toxoplasma gondii and their influence on invasion of the parasite in host cells, copper grids coated with plastic film were used for adhesion of whole parasites. Tachyzoites were incubated with 0.5% Triton X-100 in PHEM buffer containing protease inhibitors, post-fixed in 1% glutaraldehyde, stained with uranyl acetate and submitted to critical point drying. In order to analyze the presence of the structural elements of the cytoskeleton, immunolocalization was carried out using colloidal gold. Invasion of the parasite was examined on cell culture after treatment of tachyzoites with cytochalasin B (CB). In order to observe this effect, an immunocytochemical assay using alkaline phosphatase was carried out. A very well conserved extraction of the cytoskeleton elements of T. gondii, such as conoid and microtubules, as well as the rhoptries, was observed. By immunolocalization with colloidal gold, it was possible to detect the actin in its globular form. In the assay of invasion of the parasite on the host cell, after treatment of the T. gondii tachyzoites with CB, the invasion process was totally inhibited.