In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of ~67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

The cyst wall of Microphallus madrasensis resisted 6 N and 11·3 N hydrochloric acids (HCL) 36 N concentrated sulphuric acid (H2S04) and 0·35 M sodium hypochlorite. It was partially dissolved in 0·6 M sodium azide and completely dissolved in 16 N nitric acid (HNO3). An HCl hydrolysate of the cyst wall contained 10 amino acids viz. alanine, leucine, aspartic acid, glutamic acid, lysine, histidine. phenylalanine. tyrosine. cystine and proline. Polyacrylamide gel electrophoresis revealed the presence of a fast moving fraction which stained with periodic acid-Schiffs (PAS) reagent and toluidine blue was noticed in tris-glycine buffer soluble and methanol water soluble substances present in the cyst wall. Triton X-100 soluble substances present in the cyst wall revealed the presence of two slow-moving fractions stained only with PAS, and also a fast-moving fraction stained only with toluidine blue. Sodium dodecyl sulphate (SDS) soluble substances in the cyst wall revealed the presence of two fast-moving fractions which stained with PAS and toluidine blue. The slow-moving fractions are protein-bound carbohydrates. The fast-moving fractions obtained after fractionation with SDS are acid mucopolysaccharides or nucleoproteins whereas the single fraction (Triton X-100 soluble substances) which stained with toluidine blue appears to be a carbohydrate-free protein. The cyst wall of M. madrasensis also consisted of phospholipids and neutral lipids.