Isolation, Characterization, and Epizootiology of Clostridioides cuniculi from Immunodeficient Mice with Enteric Disease

Mouse strains deficient in adaptive and innate immune functions, such as NSG, NSG-SGM3, and NBSGW, are highly susceptible to opportunistic infections. Over a period of 7 mo, 1,193 mice from the above 3 strains in an SPF barrier were observed with mild loose stool (LS). Affected mice had minimal weight loss and mortality. Histopathology revealed erosion of the jejunal villi with neutrophilic inflammation and Gram-positive bacterial rods adhering to the cecal mucosa with varying degrees of mucosal hyperplasia, epithelial vacuolation, and apoptosis. Anaerobic culture revealed a clostridial species that could not be speciated using standard biochemical phenotyping. Further, Clostridioides difficle and Clostridioides perfringens ELISA on intestinal contents were negative for toxins. We performed a challenge study by exposing naïve NSG mice to dirty bedding from affected cages; metagenomics on pre- and postchallenge feces identified and associated the etiopathogenesis to Clostridioides cuniculi. Whole genome sequencing and phylogenetic analysis confirmed the identity of C. cuniculi. The isolate was sensitive to trimethoprim-sulfamethoxazole (TMS). TMS was effective in abrogating signs of LS and clearing infection in mice in studies. A probe-based real-time PCR specific for C. cuniculi was established. This assay was used to screen environmental and fomite contamination and potential use in rack-level screening. We traced the source of the outbreak to a NBSGW breeding colony. However, in our observation, spontaneous C. cuniculi-induced disease was only seen in the presence of an irradiated diet in the breeding NBSGW strain and not in the breeding colonies of NSG or NSG-SGM3 strains. Interestingly, we observed that exposure to infected feces from NBSGW-induced LS in both NSG and NSG-SGM3 mice. This investigation provides insights into the etiopathogenesis and probable source of sporadic clostridial infections in immunodeficient mice and lays the groundwork for its prevention and surveillance in immunodeficient mouse colonies.