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

Abstract

Clostridioides difficile is an anaerobic, spore-forming enteropathogen, which is much more extensively studied in humans than animals, despite growing evidence supporting its importance in One Health. We evaluated C. difficile occurrence, diversity, circulation and virulence in French Equidae (n=100) after their necropsy in northwestern France (Normandy), from 2019 to 2021. We systematically recovered all cecal contents and any watery (possibly diarrheal) intestinal contents. We isolated C. difficile strains, determined their toxin gene profile by PCR and established their PCR-ribotype according to the WEBRIBO database. We also performed free toxin detection. 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 common ribotypes: i) 078 and 126 (tcdA+ tcdB+ cdt+ according to multiplex PCR, i.e. able to produce toxin A, toxin B and binary toxin), ii) 005, 012, 020, AI-53 and FR227 (tcdA+ tcdB+), and iii) 017 (tcdB+). Non-toxigenic isolates were of ribotypes 009, 035 and 439. Ribotypes 017 and 009 were predominant (n=5). In two cases, Equidae of the same premises shared the same ribotype, either 020 or 009. Free toxins were detected in four animals: they displayed signs of diarrhea and a C. difficile of ribotype 126 (n=1) or 017 (n=3) as the only detected enteropathogen, suggesting a C. difficile infection (CDI). Three of them had received antibiotics. Two had died from an entero-toxic infection, for which C. difficile ribotype 017 was the only identified cause. French Equidae were found to display common pathogenic C. difficile, and ribotype 017 was highly virulent. These findings are of concern from a One Health perspective. ImportanceC. difficile, a major enteropathogen widely disseminated in the environment, is a One Health issue. Animals are raising concern as human contamination sources. Equidae are in close contact with humans and also develop post-antibiotic and healthcare-associated CDIs. The systematic survey of Equidae necropsied from 2019 to 2021 in the leading region for horse breeding in France, revealed that 20% harbored pathogenic strains. These belonged to clinically important ribotypes, raising the possibility of cross-species, possibly zoonotic transmission. Free toxins, which are rarely tested in animals, were detected in four animals with signs of diarrhea and a toxigenic C. difficile as the only identified enteropathogen, suggesting CDI. In two of them, C. difficile ribotype 017 was the only identified cause of entero-toxic disease and death. French Equidae could play a role in the dissemination of pathogenic C. difficile and notably ribotype 017. They should be surveilled carefully from a One Health perspective.