The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum

Abstract

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against C. parvum and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10(7) for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.

FIG. 1

GKO mice (C57 background) (•) and control C57 mice (○) were each infected with 5,000 oocysts 28 days after observation was begun. At all times after infection, the log-transformed oocyst shedding score was significantly greater in GKO mice than in control C57 mice (P < 0.001 at all time points). The overall oocyst shedding score (log mean ± standard deviation) for the GKO group was 1.38 ± 0.08, while that for the C57 control group was −0.26 ± 0.02 (P < 0.001). CI, confidence interval.

FIG. 2

Groups of seven GKO mice were infected with 0 (○), 10 (▸), 100 (◂), 1,000 (■), or 5,000 (•) oocysts. The time to peak patency was related to the inoculum, and before mice began to die of the infection, oocyst excretion was temporally related to the initial inoculum. Each order-of-magnitude fall in the inoculum led to a 2- to 3-day delay to the time to peak patency. In the displayed as well as a confirmatory experiment, only 6 of 14 mice infected with 5,000 oocysts were alive on day 14, whereas 11 of 14 mice infected with 100 oocysts were alive on day 14 (χ² = 3.743, P = 0.053). All mice given 10 oocysts became infected. On day 12, mouse no. 3 and 4 were positive; on day 14, mouse no. 2 and 4 (but not no. 3) were positive; on day 16, mouse no. 1, 2, 5, 6, and 7 (but not no. 3 or 4) were positive; and on day 19, all mice were positive. Thus, when a 10-oocyst inoculum is used, shedding may initially be at the limits of detection, yet it still rises to the same level as in mice infected with a larger inoculum. CI, confidence interval.

FIG. 3

GKO mice were infected with 5,000 oocysts, and treatment with 0 (○), 500 (◂), 1,000 (■), or 2,000 (•) mg of paromomycin/kg/day began on day 5 of infection. There was a highly significant inverse relationship between fecal shedding of oocysts and the treatment dose administered to the mice (P < 0.0001). On day 9, 2,000 mg of paromomycin/kg/day decreased the fecal shedding of oocysts by ∼2.5 log units. Oocyst shedding was very highly significantly decreased by paromomycin in a dose-dependent fashion (r² = 0.5142, 0.7215, 0.7752, and 0.6703 on days 7, 9, 12, and 15, respectively; P < 0.0001). In multiple regression analysis, the dose of paromomycin was very highly significant (P < 0.0001) whereas the day of the experiment was not significant (P = 0.2060). CI, confidence interval.

FIG. 4

The effect of paromomycin treatment  (0 [○], 500 [◂], 1,000 [■], or 2,000 [•] mg/kg/day) on oocyst shedding was assessed in mice that had been infected with 100 oocysts. Treatment was begun on day 8 of infection. By day 11 and thereafter, there was a dose-related decrease in oocyst shedding in all groups receiving paromycin treatment. This relationship was highly significant on all days  (F = 235.35, P < 0.001, regression analysis). On day 18, there were no mice in the untreated group because all  (7 of 7) had died, whereas in the treated groups only 1 of 21 had died  (by Fisher’s exact test, P < 0.001 for differences in survival). CI, confidence interval.

FIG. 5

The weights of uninfected GKO mice  (□) and of GKO mice infected with 100 oocysts on day 0 and treated with paromomycin  (0 [○], 500 [◂], 1,000 [■], or 2,000 [•] mg/kg/day) on day 8 and thereafter are displayed. Four days after infection, there were no differences in weight among the treatment groups. In contrast, by 11 days after inoculation and thereafter, there was a highly significant difference in the weights of the uninfected control mice and the untreated  (placebo) control group  (means ± standard deviations, 21.31 ± 0.38 g and 14.25 ± 0.57 g, respectively; P < 0.001). The infected, untreated mice weighed only 67% as much as the uninfected mice. Paromomycin treatment very significantly blunted this decrease in weight in a dose-dependent fashion  (F = 25.881, P < 0.001). CI, confidence interval.