A characteristic structural feature of Toxoplasma gondii and Neospora caninum is the presence of a triple-membrane pellicle, on the zoite stages of their complex life-cycle. Here we report the results of electron microscopic studies which show that the pellicle is made of a typical plasmalemma covered on its cytoplasmic side by a system of flattened vesicles named the inner membrane complex. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, we have evaluated the same methodology for the preparation of pellicles and plasmalemma from N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and the proteins and antigens present in them were subsequently studied by SDS-PAGE and Western blotting. Electron microscopy of the pellicle fractions of N. caninum demonstrated preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained 2 abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report, for the first time, a detailed molecular characterization of the pellicle and plasmalemma of N. caninum.

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0·7M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS–PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20–100 nm) which possess a low density (1·085–1·090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1·12–1·16 g/cm3).