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Clostridium difficile is an important spore-forming, opportunistic pathogen in animal husbandry and health care. In pig farming, only neonatal piglets are affected, and diarrhoea and necrotising lesions are common symptoms leading to dehydration and in some cases death. This study aimed at the assessment of the quantitative development of C. difficile colonisation in neonatal piglets by determining the shedding of spores and C. difficile toxins A (TcdA) and B (TcdB) concentrations in sow (n = 5-6) and piglet pen faeces (n = 5-6) at different time points. Spores were quantified on selective agar plates and toxins using ELISA method. C. difficile was not detected in the faeces of all but one sow during the perinatal period. Faeces of 2- and 4-day-old piglets contained 0.65 log cells/g and 5.88 log cells/g of C. difficile, respectively. Toxins were detected on day 4 at a concentration of 2.13 log ng/g (TcdA) and 2.06 log ng/g (TcdB). On day 6, concentration of C. difficile reached 6.14 log CFU/g and toxins 2.02 log ng/g (TcdA) and 2.20 log ng/g (TcdB). Two-week-old piglets showed 4.72 log CFU/g of C. difficile but toxins could not be detected. At 21 days of age, both C. difficile and toxins were undetectable. The concentration and the prevalence of C. difficile were positively associated with the prevalence of toxins in piglets. A very short time window for colonisation by C. difficile, including toxin-producing strains can be observed in neonatal piglets. The significance for animal health and the risk of a carrier status need to be addressed in future studies.