Clostridioides difficile in canine puppies: relevance and riskfactors for infection

Title: Clostridioides difficile in canine puppies: relevance and risk factors for infection Mirjam Duijvestijn1*, Miriam Koene2, Ed Kuijper3, Lapo Mughini-Gras1,4, Jaap Wagenaar1,2 1Utrecht University, Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Yalelaan 1, 3584 CL Utrecht, The Netherlands. 2Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands. 3Department of Medical Microbiology, Centre for Infectious Diseases, Leiden University Medical Centre, Leiden, the Netherlands 4National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), PO Box 1 - 3720 BA Bilthoven, The Netherlands. * [email protected] Objective(s): To determine prevalence, clinical relevance and risk factors for Clostridioides difficile (CD) in faecal samples of canine puppies (<1 year). Materials and Methods: 49 Dutch veterinary practices submitted faecal samples from puppies ≤1 year, with (n=104) and without (n=47) acute diarrhoea (≤10 days) for bacteriological culture, together with epidemiological data on age, clinical signs, living conditions and antibiotic use (2009-2011). The samples were heat shock treated, and after enrichment for 7 days plated on two CD selective media (CDSM and Brazier). Gram-staining and a gluD-PCR were used as confirmation tests. Additionally, CD ribotyping (PCR 16s rRNA, 23s rRNA) and PCR tests targeting toxin genes (TcdA, TcdB CdtA and CdtB) were performed. Multivariate logistic regression was used to identify determinants of CD infection. Results: Twenty four samples were positive for CD; 17% (8/47) in clinically healthy control puppies and 15% (16/104) in puppies with acute diarrhoea (p=0.799)1. Ribotype 010 was most common (14x), followed by ribotype 039 (4x). Ribotype 09, 012, 031 and 045 were found once. Two strains were undifferentiated. Ribotype 012 was TcdA and TcdB positive, ribotype 045 was TcdA,TcdB, CdtA and CdtB positive. Younger mean age (88 days vs.122 days (p=0,005) spring /autumn season (p=0,036/ p= 0,046), and antibiotic use at sampling (p=0,000) were associated with increased CD detection. (Combinations with) metronidazole were most frequently mentioned antibiotics. Conclusion: In this study detection of CD was not associated with diarrhoea in puppies. CD was significantly more often found in younger animals, in spring and autumn, or when antibiotics were used at sampling, as is also reported in other animals and humans. Toxigenic ribotypes were found in only two samples, one of which was ribotype 045, a type that can also be found in humans. A possible zoonotic potential needs further research. Acknowledgements: Michael Visser, Caroline de Leeuw References [1] Duijvestijn M, Mughini-Gras L, Schuurman N, Schijf W, Wagenaar JA, Egberink H. Enteropathogen infections in canine puppies: (Co-)occurrence, clinical relevance and risk factors. Vet Microbiol. 2016 Nov 15;195:115-122. doi: 10.1016/j.vetmic.2016.09.006.