Cryptosporidium merozoite isolation and purification using differential centrifugation techniques.

Abstract

Simple modifications to a recently published merozoite purification procedure (Bjorneby et al., J. Immunol. 145:298, 1990) increased yields 3- to 5-fold. Calves were infected with 2.5 x 10(8) Cryptosporidium parvum oocysts and sacrificed 65 h post-infection. The ilium and caecum were removed. The tissue was sieved through a large strainer (2 mm2) to produce a homogeneous suspension. Red blood cells were removed by differential centrifugation (600 g); merozoites remained in the supernatant. The merozoites were pelleted (2,100 g) and washed in modified Hank's balanced salt solution deficient in Mg+2 and Ca+2. Percoll purification (density 1.070 g/ml and centrifugation speed of 22,000 g for 30 min) yielded 8 x 10(8) merozoites. Nineteen monoclonal antibodies (MAb) detected by either an enzyme-linked immunosorbent assay or an immunofluorescence assay, have been generated against the merozoite stage. Gels of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-stained showed that sporozoites and merozoites have many common lower molecular weight proteins. Western blots of sporozoite and merozoite antigens reacted with anti-sporozoite MAb showed several cross-reacting antigens shared by these life-cycle stages.