Environmental ocurrence of Salmonella Dublin in Midwest U.S. dairy-beef operations: Associations with biosecurity and farm management

BACKGROUND: Salmonella Dublin (S. Dublin) affects both cattle health and public safety due to its host adaptation, antimicrobial resistance, and persistence in subclinical carriers. Although well documented in dairy systems, its epidemiology in U.S. dairy-origin cattle raised for beef (dairy-beef) remains understudied. OBJECTIVE: To design and validate a multiplex PCR (mPCR) assay for environmental detection of S. Dublin, apply mPCR alongside culture to estimate environmental occurrence in dairy-beef farms, and identify farm-level practices associated with detection. METHODS: Previously described S. Dublin targets were screened in silico and analytically validated in vitro. A cross-sectional study was conducted on 65 convenience-sampled Midwest dairy-beef farms. Environmental boot swabs (n = 265) were tested using mPCR and culture. Farm- and sample-level positive proportions were estimated using Bayesian beta-binomial models accounting for unequal sampling. Farm-level predictors were evaluated using multivariable negative binomial regression with an offset for total samples collected per farm. RESULTS: The mPCR assay showed high analytical specificity; only serovar Dublin isolates produced the complete three-target profile. In field samples, mPCR detected S. Dublin on 35.4% of farms versus 6.2% by culture. The overall proportion of mPCR-positive environmental samples across the sampled operations was 11.1% (95% CrI: 3.8-20.6%). Detection was significantly higher on farms with frequent calf introductions and where employees had regular contact with outside livestock. CONCLUSION: In this sampled population, environmental occurrence of S. Dublin was common and associated with calf movement frequency and personnel contact patterns. The validated mPCR supports targeted environmental surveillance to inform biosecurity efforts in dairy-beef systems.