Mj. Arrowood et al., A NEW METHOD FOR EVALUATING EXPERIMENTAL CRYPTOSPORIDIAL PARASITE LOADS USING IMMUNOFLUORESCENT FLOW-CYTOMETRY, The Journal of parasitology, 81(3), 1995, pp. 404-409
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.