Real-Time PCR–Based Detection of Mycoplasma agalactiae in Sheep Bulk Tank Milk to Support Flock-Level Epidemiology

This study presents the development and large-scale field application of a real-time PCR assay designed for the detection of Mycoplasma agalactiae in bulk tank milk from dairy sheep farms. The assay demonstrated high analytical specificity and sensitivity, enabling reliable identification of low levels of target DNA typically present in bulk milk samples. Following analytical validation, the test was used to estimate within-herd prevalence and was applied in a regional surveillance program encompassing more than 900 sheep farms across Sardinia, Italy. The findings reveal widespread but often low-prevalence circulation of M. agalactiae within Sardinian flocks. The real-time PCR assay proved effective for identifying infected herds through a non-invasive, cost-efficient sampling matrix, underscoring its value as a practical tool for early detection and herd-level surveillance. This approach provides a scalable framework for monitoring contagious agalactia in areas with high densities of small-ruminant farms and supports the establishment of sustainable milk-based surveillance systems.