A NEW METHOD FOR EVALUATING EXPERIMENTAL CRYPTOSPORIDIAL PARASITE LOADS USING IMMUNOFLUORESCENT FLOW-CYTOMETRY

A flow cytometric method for the quantification of Cryptosporidium par vum oocysts in stool specimens was developed to replace conventional m icroscopic immunofluorescent assays. Fecal pellets were collected from control (uninfected) severe combined immune-deficient mice, suspended in 2.5% potassium dichromate at a ratio of 400 mu l per pellet, and h omogenized by vortexing. Purified oocysts were added to the samples (1 0(5), 10(4), 10(3), and 10(2)/ml). Aliquots (200 mu l) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradie nts. The oocyst-containing fractions were collected, washed, and incub ated with an oocyst-specific monoclonal antibody (labeled with fluores cein isothiocyanate) for 30 min at 37 C. Sample volumes were adjusted to 600 mu l with phosphate-buffered saline and assayed by using logica l gating of forward/side scatter and fluorescence signal on a flow cyt ometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from c hronically infected mice demonstrated that the flow cytometry method w as approximately 10 times more sensitive than conventional immunofluor escent assays.