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This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)