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A New Method for Evaluating Experimental Cryptosporidial Parasite Loads Using Immunofluorescent Flow Cytometry
Michael J. Arrowood, Margaret R. Hurd and Jan R. Mead
The Journal of Parasitology
Vol. 81, No. 3 (Jun., 1995), pp. 404-409
Stable URL: http://www.jstor.org/stable/3283822
Page Count: 6
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A flow cytometric method for the quantification of Cryptosporidium parvum oocysts in stool specimens was developed to replace conventional microscopic 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 μl per pellet, and homogenized by vortexing. Purified oocysts were added to the samples (105, 104, 103 and 102/ml). Aliquots (200 μl) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradients. The oocyst-containing fractions were collected, washed, and incubated with an oocyst-specific monoclonal antibody (labeled with fluorescein isothiocyanate) for 30 min at 37 C. Sample volumes were adjusted to 600 μl with phosphate-buffered saline and assayed by using logical gating of forward/side scatter and fluorescence signal on a flow cytometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from chronically infected mice demonstrated that the flow cytometry method was approximately 10 times more sensitive than conventional immunofluorescent assays.
The Journal of Parasitology © 1995 The American Society of Parasitologists