Strain-resolved metagenomic analysis and qPCR validation suggest <i>Clostridium cuniculi</i> is the etiologic agent for infectious diarrhea in severely immunodeficient mice

In late 2020, the mouse barrier facility at Stanford University experienced an outbreak of diarrhea in adult mice and sudden deaths in mid-lactation females. Affected strains were immunodeficient, carrying either the <i>Prkdc</i>^scid or <i>Rag</i>^null mutations and the <i>Il2rγ</i>^null mutation, predominantly NOD.Cg-<i>Prkdc^scid</i> <i>Il2rγ^tm1Wjl</i>/SzJ (NSG) mice. The diarrhea was transmissible to naïve NSG mice by co-housing or gavage of intestinal homogenates from diarrheic mice, suggesting the involvement of an infectious agent and thus was given the name "infectious diarrhea." Conventional testing failed to identify an etiology. Strain-resolved metagenomic analyses using DNA from diarrheic and control fecal samples yielded the genome sequence of an enterotoxin-encoding <i>Clostridium cuniculi</i> strain, a candidate factor underlying the diarrhea outbreak. We hypothesized that the presence of <i>C. cuniculi</i> and its enterotoxin in fecal samples could serve as biomarkers. Quantitative real-time PCR (qPCR) assays using specific primers for <i>C. cuniculi</i> and its enterotoxin were generated and validated. We analyzed fecal samples from 111 NSG or NSG-related mice that were healthy, 37 that had clinical signs of infectious diarrhea, and 28 that had diarrhea attributable to known causes. Positive qPCR results for <i>C. cuniculi</i> and its enterotoxin only occurred in feces from mice with infectious diarrhea. All positive samples contained both <i>C. cuniculi</i> and its enterotoxin. Our data suggest that infectious diarrhea in these cases is mediated, at least in part, by the transmission of <i>C. cuniculi</i> and its enterotoxin. Our novel qPCR assays for <i>C. cuniculi</i> and its enterotoxin are effective tools for the detection of infectious diarrhea in NSG mice.