Figure 3

Oocysts frozen in vitrification cassettes are viable and exhibit in vitro infectivity. (a) C. parvum oocysts at 1, 6 and 12 weeks of age were bleached (20%), dehydrated in 1.6 M trehalose and then incubated with 30% DMSO for 5 min and additional 50% DMSO for 1 min. Oocysts were then immediately loaded into cassettes and submerged in liquid nitrogen for 10 min to achieve vitrification. Oocysts were thawed by quickly transferring the cassette from liquid nitrogen to a 40 °C water bath. Viability and infectivity were quantified after CPA removal. (b) Oocyst viability was determined by PI exclusion, both pre-freeze and after thawing of oocysts at different ages. Horizontal lines indicate mean and error bars standard deviation (n = 4). There were no differences in pre-freeze and thawed (post-freeze) viability between oocysts of different ages (Kruskal–Wallis test, pre-freeze: p = 0.64; post-freeze: p = 0.06), except a slight decrease in viability after thawing for 12- in comparison to 1-week-old oocysts (Mann–Whitney test, *p = 0.03). (c) In vitro oocyst infectivity was determined using the MDBK infection assay. Thawed oocysts were co-incubated with MDBK cells for 24 h at MOI 1:1. Establishment of intracellular stages was detected using FITC-labeled Vicia villosa lectin and imaged under × 200 magnification. Scale indicates 50 µm. Additional images are available in Supplementary Fig. S4.