Clostridium cuniculi is associated with chronic high-morbidity low-mortality diarrhea in NSG and NSG-related mouse strains

In October 2020, adult male and female NSG (NOD. Cg-Prkdc ^scid Il2rg ^tm1Wjl /Sz) mice were reported for diarrhea within a mouse barrier facility. Other immunodeficient strains harboring the SCID (Prkdc ^scid ) or Rag (Rag ^null ) mutations together with the IL2rg (Il2rg ^null ) mutation were affected. At its peak, over 20 laboratories in 10/16 (62.5%) barrier rooms were affected. Mortality was rare except in lactating females (≥ P11). Grossly, nonlactating adult female and male mice (n = 16) had mild to moderate, small and large intestinal distension with corresponding individual cell death and sloughing of superficial enterocytes in the cecocolonic mucosa. Lactating NSG dams (n=6) had moderate to severe gastrointestinal distension and/or segmental, dark red to gray, small intestinal discoloration. In addition to the same histologic lesions seen in nonlactating female NSG mice, lactating NSG dams often had severe ulcerative inflammation affecting the jejunum, ileum, cecum, and colon. Traditional ancillary diagnostic tests including aerobic and anaerobic cultures (blood, liver, spleen, and intestines), fecal PCR, and fecal floatation failed to yield a causative organism. Further cohousing and oral gavage studies determined neither immunocompetent CD1 (Crl:CD1 [ICR]) mice nor immunodeficient NOD scid (NOD.Cg-Prkdc ^scid /J) and Rag2 KO (C57BL/6. Cg-Rag2 ^tm1.1Cgn /J) mice were susceptible to clinical disease. Extensive control barriers were implemented including a veterinary-managed NSG breeding barrier, alterations in husbandry practices, and strategic environmental disinfection, allowing for continuity of experimental studies while avoiding widespread depopulation of the barrier. Subsequent strain-resolved metagenomics and qPCR assay development identified Clostridium cuniculi and its enterotoxin exclusively within diarrheic mice.