Toxoplasma Cystozoite Antigenic Change in Tissue Culture

Abstract

Antigenic differences between the endozoite and cystozoite of Toxoplasma gondii were revealed by fluorescent antibody staining (Lunde and Jacobs, 1983, Journal of Parasitology 69: 806-808). Antisera against the cystozoite reacted only against the cystozoite while anti-endozoite sera reacted partially against the cystozoite as well as against the endozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. It was of interest to determine whether or not cystozoite antigens persisted on the surface of organisms derived from tissue cultures infected with cystozoites. Bovine embryo skeletal muscle cell cultures were infected with cystozoites derived from Toxoplasma strain ME 49 cysts separated from triturated mouse brain by centrifugation at 5,000 g for 30 min in 17% Ficoll-PBS (pH 7.2). The cystozoites were obtained by digestion with pepsin-HCl (Jacobs et al., 1960, Journal of Parasitology 46: 11-21) for 10 min and were removed from the digestion medium by 2 washings and centrifugations and resuspended in Eagle's complete medium with glutamine and 10% fetal calf serum. Approximately 100 x 103 cystozoites were added to each of 2 tissue cultures in 35 x 100 mm disposable culture dishes and incubated at 37 C with 5% CO2. The tissue cultures were confluent when inoculated. Gentamicin, 50 ,g/ml, was added to the culture medium to inhibit possible bacteria contaminants. On day 3 the cultures showed cytopathic effect, cell lysis and rounding, and were examined. Viable zoites were observed. Cells were mechanically removed from the culture dishes, centrifuged at 1,000 g and washed once with PBS pH 7.2. Portions of the sedimented zoites were then placed within circumscribed areas on microslides. Toxoplasma endozoites obtained from bovine embryo skeletal muscle cells infected with endozoites, RH strain, were also added to similar slides. The zoites were air dried and fixed briefly in absolute methanol. Fluorescein-labeled rabbit antiserum against either endozoites or cystozoites (Lunde and Jacobs, 1983, loc. cit.) was then added to the slides. After 60 min at 37 C the slides were washed and examined for immunofluorescence. Toxoplasma derived from tissue cultures infected with cystozoites or with endozoites reacted identically with the fluorescein-tagged anti-sera. The organisms all reacted positively with anti-endozoite antibody and showed no reaction with anti-cystozoite antibody. It appears, therefore, that only a few generations [we estimate about seven (Kaufman et al., 1958, American Journal of Ophthalmology 46: 255-260)] of the parasites within cultured cells results in the loss of the specific surface determinants of the cystozoites. The fact that this transformation occurs suggests that it probably takes place at the first division of the cystozoites within the cultured cells, because new membranes are formed in the division process. It was difficult to obtain enough cystozoites to infect the cultures, and recover and identify adequate numbers of individual parasites to determine their surface antigens. Because of this, a series of experiments to determine precisely when this change occurred was not done.