Clostridioides difficilein Equidae necropsied in Normandie, France from 2019 to 2021: common carriage of toxigenic strains and virulence of emerging ribotype 017

Background Clostridioides difficile is an anaerobic, spore-forming entero-pathogen, causing diarrhea in humans and animals. We aimed to evaluate C. difficile occurrence, diversity, virulence and putative circulation among French Equidae. Methods We took advantage of necropsies of Equidae in Normandie, France, from 2019 to 2021 (n=100), to systematically recover the caecal content and, if watery and possibly diarrheal, any intestinal content. We searched for C. difficile and characterized isolates for their toxin gene profile and PCR-ribotype according to the WEBRIBO database. We performed free toxin detection to reach a high-standard diagnosis of infection. Results Twenty-seven Equidae were positive for C. difficile and twenty had a toxigenic strain, including one animal also colonized by a non-toxigenic strain. Toxigenic isolates belonged to eight ribotypes: 078, 126 (three-toxin profile), 005, 012, 020, 181, AI-53 (two-toxin profile), 017 (one-toxin profile), and non-toxigenic isolates to ribotypes 009, 035 and 439. 017 and 009 were the predominant ribotypes (n=5). In two premises, few Equidae shared the same ribotype, either 020 or 009, suggesting two potential transmission clusters. Free toxin detection demonstrated C. difficile infection in four animals displaying post-mortem signs of diarrhea and toxigenic isolates of ribotype 126 (n=1) or 017 (n=3), which were the least susceptible to vancomycin. The infection by ribotype 017 caused animal death in two cases. Conclusions French Equidae represented an underestimated reservoir of C. difficile and ribotype 017 was highly virulent in these animals, both preoccupying findings in a One Health perspective.