Simple SummaryParatuberculosis is a chronic inflammatory disease leading to emaciation and production losses in ruminants. Important aspects of disease control are the detection of infected herds and environmental areas where contact between animals and the causative pathogenMycobacterium aviumsubsp.paratuberculosis(MAP) is possible. Bedding, dust, feed, and water samples were collected from the barn of a paratuberculosis-infected dairy goat herd and analyzed for culturable MAP and MAP DNA. Cultivation was successful in 28 bedding and one dust sample, while MAP DNA was detected in 117 out of 256 samples from all materials. Samples collected from areas of high animal traffic, such as the milking parlor, were more likely to reveal positive results than adult and young goat areas. Positive culture results were also obtained from kidding pens, constituting this barn area as a possible infection site. Overall, environmental sampling may be suitable for the detection of MAP in goat herds and the identification of potential critical areas for pathogen transmission. These results should be taken into account for paratuberculosis control programs in goat herds to improve their efficiency and, thus, animal welfare.AbstractEnvironmental samples are often used to classify the paratuberculosis status of cattle herds. The disease is caused byMycobacterium aviumsubsp.paratuberculosis(MAP), predominantly through oral ingestion during infancy. In this explorative study, the presence of MAP was determined in the barn environment of a paratuberculosis-infected vaccinated dairy goat herd. A total of 256 bedding, dust, feed, and water samples were collected at eight time points and examined using culture and qPCR. Detection rates of both methods were compared, and factors determining MAP confirmation were identified. MAP was cultured from 28 bedding and one dust sample, while MAP DNA was detected in all materials (117/256). Samples from high animal traffic areas and those collected during the indoor season were more likely to yield positive culture and qPCR results. Cultivation of MAP from kidding pens indicated this area as a possible infection site. Dust proved to be the most suitable material for detecting MAP DNA, as bedding was for MAP culture. Environmental sampling was demonstrated to be an effective way to detect MAP in a dairy goat herd. qPCR results could confirm herd infection, while culture results provided insight into crucial areas for MAP transmission. These findings should be considered when designing farm-specific paratuberculosis control plans.