Effect of Caging on Cryptosporidium parvum Proliferation in Mice

Abstract

Cryptosporidiosis is an enteric infection caused by several protozoan species in the genus Cryptosporidium (phylum Apicomplexa). Immunosuppressed mice are commonly used to model this infection. Surprisingly, for a pathogen like Cryptosporidium parvum, which is readily transmitted fecal-orally, mice housed in the same cage can develop vastly different levels of infection, ranging from undetectable to lethal. The motivation for this study was to investigate this phenomenon and assess the association between the severity of cryptosporidiosis and the fecal microbiota. To this aim, the association between severity of cryptosporidiosis and caging (group caged vs. individually caged) and between the microbiota taxonomy and the course of the infection was examined. In contrast to mice caged in groups of four, a majority of mice caged individually did not excrete a detectable level of oocysts. Microbiota α diversity in samples collected between three days prior to infection and one day post-infection was negatively correlated with the severity of cryptosporidiosis, suggesting a causal negative relationship between microbiota diversity and susceptibility to C. parvum.

Keywords: Cryptosporidium; constrained ordination; cryptosporidiosis; dysbiosis; microbiota.

Figure 1

Oocyst shedding by mice housed in groups of four and in individually housed mice by experiment. Black lines indicate groups; purple lines indicate individual caging. Experiment 37 shedding curves are not shown because the grouped mice were not monitored individually. Selected error bars represent the standard deviation of four replicate counts. Counts of zero were artificially offset by two, three or four counts, depending on the Y axis scale, to reveal overlapping lines.

Figure 2

Effect of caging on microbiota β diversity between individual mice. Distribution of weighted UniFrac distances indicates that co-housing does not lead to microbiota convergence among mice. In experiment 35 the effect was the opposite, whereas in experiment 37 and 38, caging had no significant effect. Black bars indicate caged mice; purple bars indicate individually caged mice. Y axes show raw counts. Y axes are not drawn to scale.

Figure 3

Mice in different experiments are populated with different intestinal microbiota. Mice for experiment 35 were purchased in July 2020, whereas experiments 37 and 38 were performed in February and March 2021. Each datapoint represents one fecal sample, regardless of the day of collection. Color indicates experiment; turquoise, experiment 35; olive, experiment 37; beige, experiment 38.

Figure 4

Temporal profile of relative abundance of OTUs significantly associated with low- and high-infection phenotypes in individually caged mice from experiment 37. Two OTUs shown in blue and light blue, respectively, are significantly more abundant in mice with low or no infection. Red and pink shows the relative abundance of sequences belonging to two OTUs significantly over-represented in heavily infected g5 and g8 mice. Tick marks on the x axis represent for each mouse day −one, one, three and 17 post-infection as indicated for mouse g3 and g8. The y axis scale represents the number of 16S sequences assigned to each OTU.

Figure 5

Severity of cryptosporidiosis is negatively correlated to α diversity. Oocysts were enumerated on acid-fast stained fecal smears as described in Materials and Methods and the intensity of the infection quantified by averaging oocysts counts over the number of observations. A total of 26 individually caged mice from experiment 35 (turquoise), experiment 37 (olive); and experiment 38 (beige) are included. Each datapoint represents one fecal DNA sample.