Quantitative detection of fecal oocyst load

Oocysts were extracted via a modified discontinuous sucrose gradient technique [50]. Briefly, Sheather’s solution was freshly prepared by dissolving 156.25 g sucrose and 2.5 mL phenol in 100 mL water. Fecal pellets were removed from storage then homogenized by vortexing and pipetting. In microcentrifuge tubes, 0.2 mL fecal homogenate was overlaid on 0.75 mL of solution with a specific gravity of 1.064 (20% Sheather’s and 0.1% Tween 80 in PBS), overlaid on 0.75 mL of solution with a specific gravity of 1.103 (33% Sheather’s and 0.1% Tween 80 in PBS). Oocysts were floated from feces by centrifugation at 1,000×g for 20 min and collected with a pipet tip from the 1.064/1.103 specific gravity interface. Oocysts were rinsed once in cold PBS, pelleted by centrifugation at 15,000×g for 10 min, and resuspended in PBS. 50 μL aliquots were incubated for 30 min at room temperature with 0.25 μg fluorescein isothiocyanate-conjugated mouse anti-Cryptosporidium antibody (OW50-FITC, BioRad 2402–3007), then diluted to 200 μL with PBS. Samples were analyzed using a Guava EasyCyte flow cytometer and CytoSoft Data Acquisition and Analysis software (v5.3; Guava Technologies, Inc.), using a 100 s sampling interval, 0.59 μL/s flow rate, and logical gating of forward and side light scatter and OW50-FITC fluorescence signals. Each experimental run included positive and negative controls to calibrate region settings discriminating the oocyst-FITC population from background signals. Oocyst counts/mL sample values were exported to Excel (Microsoft Corp.) for normalization to counts/mg feces. Final graphing and statistical analyses of data were done using GraphPad Prism software (v6, GraphPad Software, Inc.). Two-way ANOVA with Dunnett’s correction (Fig 4A) and unpaired multiple t-tests with False Discovery Rate approach (Fig 4B) were performed on % Vehicle data to determine significance between vehicle-treated and compound-treated mice.