Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum

Abstract

In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

FIG. 1.

Dose-response curves for five genotype 2 isolates of C. parvum in CD-1 mice (A), HCT-8 cell monolayers (B), Caco-2 cell monolayers (C), and MDCK cell monolayers (D). The ID₅₀ is the point at which the regression line crosses the logit response 0 axis. Infectivity was measured for the Iowa (○, ——), Moredun (□, - - — - -), TAMU (▵, — — —), Maine (⋄, - - - - -), and Glasgow (∗, — - —) isolates. Each data point is the logit of the mean proportional infectivity (P_inf) for three to six replicate cages of mice (9 to 12 mice per cage) or for two to seven replicate sets of cell cultures (6 to 10 monolayers per set) at each dose of oocysts.

FIG. 2.

Infectivities of the Iowa (○) (R² = 0.57) and Moredun (□) (R² = 0.62) isolates of C. parvum in HCT-8 cell monolayers based on individual logit calculations for each oocyst challenge dose. The ID₅₀s based on these dose-response curves were 106 oocysts for the Iowa isolate and 30 oocysts for the Moredun isolate.

FIG. 3.

Infectivity of the TAMU isolate as measured by CD-1 mouse (□), HCT-8 cell culture RT-PCR (○), Caco-2 cell culture RT-PCR (⋄), and MDCK cell culture IFA (▵) assays based on logit calculations from average percentages of infectivity with oocyst doses of 5 to 25 oocysts.

FIG. 4.

Overall comparison of infectivity measurements obtained with mice and cell cultures for untreated fresh oocysts of all isolates combined. Symbols: ○, CD-1 mice versus HCT-8 cells (R² = 0.77); □, CD-1 mice versus Caco-2 cells (R² = 0.67); ▵, CD-1 mice versus MDCK cells (R² = 0.67). Each data point represents the average for all replicate analyses performed at each dose for the various isolates. All lines are best-fit linear regressions.

FIG. 5.

Comparison between infectivity in CD-1 mice and infectivity in the HCT-8 cell culture RT-PCR assay (R² = 0.80) (solid regression line) for oocysts of the Iowa, TAMU, and Moredun isolates exposed to various doses of UV (•) and ozone (○). The horizontal and vertical error bars indicate the average standard deviations for all replicate analyses with oocysts exposed to disinfectants for CD-1 mice and HCT-8 cells, respectively. The dashed line indicates the theoretical 1:1 correlation. The same oocyst challenge doses were inoculated into CD-1 mice and HCT-8 monolayers for each dose of disinfectant.