C. cuniculi diarrhea in NSG mice
In late 2020, the high-barrier  mouse facility at Stanford University experienced an outbreak of diarrhea in adult mice and acute deaths in mid-lactation females. Affected mouse strains were immunodeficient, carrying either the SCID (Prkdcscid) or RAGnull mutations and the IL2rgnull mutation (predominantly NSG mice). The diarrhea was transmissible to naïve NSG mice via gavage of intestinal homogenates from diarrheic mice or by co-housing, suggesting an infectious agent. Conventional diagnostics failed to identify an etiology. An in-depth metagenomic analysis of DNA samples from diarrheic and control fecal samples identified Clostridium cuniculi and an enterotoxin as candidate factors underlying the diarrheal outbreak. We hypothesized that the presence of C. cuniculi and its enterotoxin in fecal samples could serve as biomarkers for the infectious diarrhea. Real-time quantitative PCR (qPCR) diagnostic assays using selective specific primers for C. cuniculi were generated. We analyzed 37 fecal samples from NSG or NSG-related mice with clinical signs of infectious diarrhea without a known cause, 111 fecal samples from healthy NSG mice, and 28 fecal samples from NSG or NSG-related mice with diarrhea due to other known causes. C. cuniculi and its enterotoxin were identified only in feces from mice with infectious diarrhea of unknown etiology. Healthy NSG mice, NSG mice with diarrhea due to other known causes, and non-NSG strains with diarrhea were negative for C. cuniculi by qPCR assays. All positive samples contained both C. cuniculi and its enterotoxin. Healthy NSG mice co-housed with NSG mice with infectious diarrhea or housed on their soiled bedding resulted in experimental mice expressing C. cuniculi and enterotoxin DNA markers within 24 hours .  This study suggests that infectious diarrhea is mediated, at least in part, by the transmission of C. cuniculi and its enterotoxin. The novel qPCR assays for C. cuniculi and its enterotoxin are effective diagnostic tools for detection and prevention of the spread of infectious diarrhea in NSG mice.