# Clostridioides difficile in equidae necropsied in Northwestern France, between 2019 and 2021

## TL;DR

This study found that 20% of horses in France had harmful C. difficile strains, some of which could spread to humans, highlighting a One Health concern.

## Contribution

The study provides new insights into C. difficile prevalence, diversity, and virulence in French equidae, emphasizing zoonotic potential.

## Key findings

- 20% of necropsied equidae had toxigenic C. difficile strains, including ribotype 017, which caused fatal infections.
- Four animals showed free toxins and signs of CDI, with ribotype 017 being the sole identified cause in two cases.
- Common pathogenic ribotypes like 078 and 017 were found, suggesting possible cross-species transmission.

## Abstract

Clostridioides difficile, an anaerobic, spore-forming enteropathogen, is less studied in animals than in humans despite 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, from 2019 to 2021. We systematically recovered all cecal contents and any watery 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 20 had a toxigenic strain, including one animal co-colonized by a non-toxigenic strain. Toxigenic isolates belonged to eight already described ribotypes: (i) 078 and 126 (toxin genes of wild-type size: tcdA+ tcdB+ cdt+), (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 displayed common pathogenic C. difficile, and ribotype 017 was highly virulent. These findings are of concern from a One Health perspective.

C. 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 horse breeding region in France revealed that 20% harbored pathogenic strains. These belonged to clinically important ribotypes, raising the possibility of cross-species, zoonotic or anthropo-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.