Simple SummaryThis study aimed to investigate the occurrence of paratuberculosis (Johne’s disease), caused byMycobacterium aviumsubsp.paratuberculosis(MAP), in Austrian sheep and goats by testing 22,019 blood samples. Furthermore, detailed investigations in five MAP-infected goat herds were carried out to detect the disease in blood and faecal samples. The detected animal MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Herds with a more intensive production system had a significantly higher risk of being infected, as well as farms with frequent trading of animals or where other ruminant species were kept on the same premise. In the five goat farms investigated, 21.8% (11.7%–28.0%, calculated true seroprevalence 38.6%) of the animals were found to be infected and 12.3% (5.0%–24.7%) of the animals were shedding the bacterium with their faeces. It was further possible to identify the bacterium using boot swab samples from the stable environment in each of the five herds. The results indicated a moderate paratuberculosis infection rate in small ruminants in Austria.AbstractThis study aimed to investigate the prevalence ofMycobacterium aviumsubsp.paratuberculosis(MAP) in small ruminants in Austria by testing 22,019 serum samples with ELISA for the presence of specific antibodies. Furthermore, detailed investigations in five MAP-infected goat herds were carried out by ELISA, qPCR and bacterial culture. The found animal-level apparent MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Significant risk factors for seropositivity in goat herds were: herd size, animal trading, farmed as a dairy herd, Animal Health Service membership and cohabitation with farmed game. For sheep flocks, seroprevalence was significantly higher in flocks with animal trading and where cattle or goats were kept in the flock, respectively. The overall apparent within-herd MAP seroprevalence in the five goat farms investigated was 21.8% (11.7%–28.0%, calculated true seroprevalence 38.6%) and an overall rate of MAP shedding of 12.3% was detected (5.0%–24.7%). It was possible to identify MAP by culture using boot swab samples in each herd. The results indicated a moderate MAP infection rate in small ruminants in Austria.