Cryopreservation of infectious Cryptosporidium parvum oocysts

Abstract

Cryptosporidiosis in an enteric infection caused by Cryptosporidium parasites and is a major cause of acute infant diarrhea in the developing world. A major bottleneck to research progress is the lack of methods to cryopreserve Cryptosporidium oocysts, thus requiring routine propagation in laboratory animals. Here, we report a method to cryopreserve C. parvum oocysts by ultra-fast cooling. Cryopreserved oocysts exhibit high viability and robust in vitro excystation, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to what we observe with unfrozen oocysts. Oocyst viability and infectivity is not visibly changed after several weeks of cryogenic storage. Cryopreservation will facilitate the sharing of oocysts from well-characterized isolates and transgenic strains among different laboratories.

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

Fig. 2

Vitrified C. parvum oocysts are viable and excyst. a To ensure vitrification, bleached oocysts were dehydrated in 1 M trehalose and then suspended in 0.5 M trehalose/30% DMSO. Oocysts were then immediately loaded into microcapillaries and plunged into liquid nitrogen for 5 min, or incubated with CPA solution for 20 min followed by loading of microcapillaries and freezing. Oocysts were thawed by quickly transferring the microcapillary from liquid nitrogen to a 37 °C water bath. Viability was quantified after CPA removal based on PI exclusion and quality of excysted sporozoites. b Oocyst viability was determined by PI exclusion, both pre-freeze and after cryogenic storage. Values indicate means and error bars indicate standard deviation (n = 12). c DIC micrographs show excysted sporozoites. Sporozoite viability was assessed on basis of their shape, structure and observed motility after excystation in 0.75% taurocholic acid at 37 °C. Scale indicates 5 µm

Fig. 3

Cryopreserved C. parvum oocysts are infectious to IFN-γ knockout mice. Dehydrated oocysts were vitrified using either the 0 min or 20 min DMSO incubation protocol. IFN-γ knockout mice (n = 2–3) were inoculated orally with 5000 PI^- thawed or unfrozen control oocysts. Intensity of fecal shedding was quantified daily by microscopic enumeration of oocysts in 30 fields of acid-fast stained fecal smears under 1000× magnification. Positive (unfrozen) and negative controls (heat-inactivated) were included as matched controls. To determine whether oocyst age has effect on the success of the cryopreservation protocol, a 1- b 6- and c 12-week-old oocysts were studied. Values indicate means of log transformed oocyst counts and error bars indicate standard deviation. Viability of oocysts was determined by PI exclusion prior to inoculation and was as follows: (a) 43.1% and 76.7% (b) 45.2% and 80.6% (c) 53.1% and 72.4%, for 20 min and 0 min incubations, respectively. d Micrographs of hematoxylin and eosin–stained jejunal sections from IFN-γ mice infected with fresh, cryopreserved or heat-inactivated 12-week-old oocysts are shown. Arrows indicate intracellular stages of the parasite located in the apical region of intestinal epithelial cells. Scale indicates 20 µm. Data regarding infectivity of each individual mouse can be found in Supplementary Fig. 6

Fig. 4

Response of C. parvum to dehydration and CPA uptake prior to freezing. The shrink-swell response of bleached oocysts to a challenge with 1 M trehalose for 10 min, with or without subsequent addition of DMSO for 0 min or 20 min (at final concentration of 0.5 M trehalose/30% DMSO) was measured by image analysis and compared to the PBS control. Loss of 57.2 ± 5.2% of oocyst volume related to dehydration in trehalose is partly restored by addition of DMSO. This is likely related to a decreased trehalose gradient after immediate addition of DMSO (0 min) or intra-oocyst uptake of DMSO during 20 min incubation. Values indicate means and error bars indicate standard deviation (n = 3). DIC micrographs demonstrate volumetric changes of oocysts. Scale indicates 5 µm

Fig. 5

Long-term cryogenic storage does not decrease infectivity of cryopreserved oocysts. Bleached C. parvum oocysts (5%, 1 min) were frozen using the ultra-fast cooling protocol following dehydration in trehalose (10 min) and 0 min incubation in 0.5 M trehalose/30% DMSO solution. Oocysts were stored in microcapillaries in liquid nitrogen and thawed after 1, 4, and 12 weeks. Positive (unfrozen) and negative controls (heat-inactivated) were included as matched controls. IFN-γ knockout mice (n = 3) were inoculated orally with 5000 PI^- oocysts. Intensity of fecal shedding was quantified daily by microscopic enumeration of oocysts in 30 fields of acid-fast stained fecal smears under 1000× magnification. Values indicate means of log transformed oocyst counts and error bars indicate standard deviation. Mice inoculated with cryopreserved oocysts developed similar infection at 6 dpi regardless of the storage length, as evidenced by fecal shedding of oocysts. Viability of oocysts was determined by PI exclusion prior to inoculation and was as follows: 82.6, 79.1 and 80% for 1, 4 and 12 weeks of storage respectively. Data regarding the infectivity of each individual mouse can be found in Supplementary Figure 7