Figure 2

Approaches to increase intracellular concentration of CPA. Increasing concentrations of bleach, trehalose and DMSO were applied to maximize intracellular permeation of CPA among C. parvum oocysts. (a) Viability of oocysts permeabilized with either 5% or 20% bleach, followed by incubation with a CPA cocktail containing varying concentrations of trehalose and DMSO over a duration of 5–15 min. Viability was measured by PI exclusion after removal of CPA (n = 3). Differences in viability in response to bleaching and trehalose/DMSO treatment were measured using two-way ANOVA (***p < 0.0001; **p = 0.002). (b) Infectivity was measured as percent of intracellular parasitic stages established in MDBK cells in comparison to control oocysts incubated with PBS in lieu of DMSO (n = 4). Differences in infectivity in response to DMSO concentration were measured for both permeabilization/ dehydration protocols using two-way ANOVA (***p < 0.0001). (c) Reduced CPA toxicity was accomplished through the introduction of a two-step CPA loading protocol. Toxicity due to CPA alone (pre-freeze) was measured in terms of viability and in vitro infectivity following treatment with 50% DMSO in either one- or two-step additions. Oocysts were bleached (20%), dehydrated (1.6 M trehalose, 10 min) and then incubated with 50% DMSO for 5 min or for 1 min following 5 min 30% DMSO incubation. Viability is reported as percent of PI⁻ oocysts and infectivity as percent of intracellular parasitic stages established in MDBK cells in comparison to control oocysts incubated with PBS in lieu of DMSO (n = 3). Differences between treatments were measured using Mann–Whitney test (*p = 0.05). All values indicate means and error bars indicate standard deviation.