Electrophoretic and immunoblot analysis of Cryptosporidium oocysts

Abstract

Summary Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat-derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS-PAGE. Fifty-one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14 000-200 000 molecular weight (MW), two bands were less than 14 000 MW and one band was above 200 000 MW. Twenty-one bands were detected in the oocyst wall preparation, all within the range 14 000-200 000 MW. Immunoblot analysis oi Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two semm groups. Nine ofthe 10 human sera and all ofthe goat kid sera reacted with a 23 000 MW and 32 000 MW antigen. A 15 500 MW antigen was also detected by all the goat and four ofthe 10 human sera. Both serum groups reacted with various antigens above 40 000 MW. Surface labelling of three human isolatesofC/^p/o^^^onW/um oocysts with '25i was performed using the Boltonand Hunter reagent. The solubilized preparations were separated by SDS-PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15 500, 32 000. 47 500, 79 000 and 96 000 MW. Some variation in the molecular weight of polypeptides labelled with ^'^H was observed among the three isolates. These studies indicate that the 32 000 MW antigen appears to be a suitable candidate for the preparation of an antibody probe because it is common to many isolates and is located on the exterior of the oocyst wall.

Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

“…Proteins present in Cryptosporidium isolates have been characterized previously (28); however, amylopectin granules and residual bodies were not removed from oocyst walls prior to SDS-PAGE analysis. Because the residual bodies have not been fully characterized, SDS-PAGE analysis of excysted oocysts containing residual bodies may produce bands that are not truly associated with the oocyst wall.…”

Cryptosporidiosis

“…Results of recent studies (314)(315)(316) compared with others (191,192,215,315,334) suggest that at least two C. parvum sporozoite surface proteins are being confused. A 23-kDa molecule is weakly labeled by iodination (315), is highly immunogenic (191,192,215,334), and may have several epitopes that cross-react with some higher-molecular-size species (192,215). An 18-to 20-kDa protein, often referred to as P20, is intensely labeled by iodination (315) and appears to be less immunogenic than P23.…”

Localization of a 23 000 MW antigen of Cryptosporidium by immunoelectron microscopy

“…The oocysts were concentrated, purified, solubilized, electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to nitrocellulose filter paper as described previously (2). The strip of filter paper containing the 23 000 MW antigen was excised and then dissolved in a small volume of dimethyl sulfoxide and diluted in an equal volume of pyrogen-free saline.…”

“…Because immunocompetent hosts usually recover spontaneously and prove refractive to reinfection, various immunological studies have been performed to identify parasite antigens using immune sera from naturally infected hosts. Immunoblot studies have resulted in the detection of a small molecular weight (MW) antigen (20 000-23 000 MW) in either intact oocyst or sporozoite life cycle stages which is frequently recognized by acute and convalescent sera from humans, goats, cattle and horses (1)(2)(3). However, radiolabelling of intact oocysts with '2^1 failed to locate this antigen on the exterior oocyst wall (2), whereas fluorescent-antibody studies indicated it may be located on the sporozoite and merozoite membranes (3).…”