ABSTRACTThe efficiency of direct steam injection (DSI) at 105°C for 3 s to inactivateMycobacterium aviumsubsp.paratuberculosisin milk at a pilot-plant scale was investigated. Milk samples were artificially contaminated withM. aviumsubsp.paratuberculosisand also with cow fecal material naturally infected withM. aviumsubsp.paratuberculosis. We also tested milk artificially contaminated withMycobacterium smegmatisas a candidate surrogate to compare thermal inactivation betweenM. smegmatisandM. aviumsubsp.paratuberculosis. Following the DSI process, no viableM. aviumsubsp.paratuberculosisorM. smegmatiswas recovered using culture methods for both strains. For pureM. aviumsubsp.paratuberculosiscultures, a minimum reduction of 5.6 log10was achieved with DSI, and a minimum reduction of 5.7 log10was found withM. smegmatis. The minimum log10reduction for wild-typeM. aviumsubsp.paratuberculosisnaturally present in feces was 3.3. In addition, 44 dairy and nondairy powdered infant formula (PIF) ingredients used during the manufacturing process of PIF were tested for an alternate source forM. aviumsubsp.paratuberculosisand were found to be negative by quantitative PCR (qPCR). In conclusion, the results obtained from this study indicate that a >7-fold-log10reduction ofM. aviumsubsp.paratuberculosisin milk can be achieved with the applied DSI process.IMPORTANCEM. aviumsubsp.paratuberculosisis widespread in dairy herds in many countries.M. aviumsubsp.paratuberculosisis the causative agent of Johne's disease in cattle, and infected animals can directly or indirectly (i.e., fecal contamination) contaminate milk. Despite much research and debate, there is no conclusive evidence thatM. aviumsubsp.paratuberculosisis a zoonotic bacterium, i.e., one that causes disease in humans. The presence ofM. aviumsubsp.paratuberculosisor its DNA has been reported in dairy products, including pasteurized milk, cheese, and infant formula. In light of this, it is appropriate to evaluate existing mitigation measures to inactivateM. aviumsubsp.paratuberculosisin dairy products. The work conducted in this study describes the efficacy of direct steam injection, a thermal process commonly used in the dairy industry, to eliminateM. aviumsubsp.paratuberculosisand a surrogate bacterium in milk, thus ensuring the absence ofM. aviumsubsp.paratuberculosisin dairy products subject to these process conditions.