Fig. 1 | Nature Communications

Fig. 1

From: Cryopreservation of infectious Cryptosporidium parvum oocysts

Fig. 1

C. parvum oocyst response to CPAs. a Oocysts were incubated with high concentrations of several common CPAs. As no significant increase in oocyst toxicity was observed over long incubation intervals (30–120 min), it was concluded that the oocysts are impermeable to CPAs. Values indicate means and error bars indicate standard deviation (n = 3). b Hypochlorite treatment was used as a method to increase oocyst permeability to CPAs. Bleached and unbleached oocysts were incubated with 40% DMSO and cytotoxicity was quantified by flow cytometry using PI exclusion. Both bleached and unbleached oocysts incubated with PBS served as a normalizing control. While unbleached oocysts exhibited no toxicity, bleached oocysts incubated with DMSO exhibited substantial toxicity over the observed period (30–120 min), suggesting CPA permeability was achieved. Values indicate means and error bars indicate standard deviation (n = 3). c Oocyst volume was measured by flow cytometry to characterize dehydration induced by hyperosmotic solutions of NaCl and trehalose (V, estimated oocyst volume; Vo, volume of control oocysts). While NaCl had insignificant effect on volume (one-way ANOVA; p = 0.90, f = 0.33, df = 20), significant dehydration was observed using trehalose (one-way ANOVA; p < 0.0001, f = 18.65, df = 20), a common extracellular CPA. Data met requirements of normality (Wilk Shapiro test; p > 0.29 and p > 0.85 for all NaCl and trehalose concentrations, respectively) and homoscedasticity (Brown–Forsythe test; p = 0.80 and p = 0.88 for NaCl and trehalose, respectively) for inclusion in ANOVA analysis. Values indicate means and error bars indicate standard deviation. Panels show a significant decrease in cell volume in response to increasing concentrations of trehalose compared to the untreated PBS control (n = 3). Micrographs show oocysts exposed to trehalose or PBS as indicated. Scale indicates 5 µm. d The kinetics of CPA toxicity were measured by incubating bleached (5% bleach, 1 min) oocysts in DMSO, with our without dehydration in trehalose, for 5–60 min. Oocysts were dehydrated with 1 M trehalose for 10 min and then treated with a solution of DMSO to achieve a final concentration of 0.5 M trehalose/30–40% DMSO, or 30–40% DMSO only. Cytotoxicity was measured by PI inclusion (n = 3). Values indicate means and error bars indicate standard deviation

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