Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer

Abstract

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5) oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

Figure 1. Pattern of oocyst shedding (oocyst/g feces) of Dex-treated SCID mice.

Experimental groups were inoculated with intended doses of 1, 10, 100 and 10⁵ oocysts. Each point represents one mouse, the lines being the geometric means of oocyst shedding per group. Only animals with oocyst shedding at a precise moment of the day are represented. None of mice infected with one oocyst released parasites until day 15 (see material and methods for oocyst shedding assessment).

Figure 2. Gastro-intestinal neoplastic lesions in Dex-treated SCID mice infected with one oocyst of C. parvum.

A. Invasive adenocarcinoma of the antropyloric region (arrows) in a mouse 100 days P.I.. Bar = 1250 µm (hematoxylin and eosin). B. Invasive adenocarcinoma of the antropyloric region in a mouse 100 days P.I. showing parasites inside the glands (arrows). Bar = 50 µm (hematoxylin and eosin). C. Adenoma of the ileo-caecal region in a mouse 45 days P.I.. Bar = 625 µm. D. High grade intraepithelial neoplasia in the ileo-caecal region in a mouse 45 days P.I. with numerous parasites (arrow) inside the glands. Bar = 25 µm (hematoxylin and eosin).