Adaptation and Validation of a Droplet Digital PCR Assay for the Detection of Cryptosporidium spp. and Giardia duodenalis in Buffalo Milk

Cryptosporidium spp. and Giardia duodenalis are major protozoan enteropathogens of public health and food-safety concern. Their detection in dairy matrices remains challenging due to the lack of standardized analytical protocols and the limited sensitivity of conventional diagnostic methods. In this study, we developed and validated a droplet digital PCR (ddPCR) assay for the detection of Cryptosporidium spp. and G. duodenalis in buffalo milk. The method showed satisfactory recovery efficiencies (61% for G. duodenalis and 55% for Cryptosporidium spp.) and excellent analytical performance, including a limit of detection of 1 gc/μL, 100% specificity, and strong intra- and inter-day repeatability. The operator-independent robustness of the dd-PCR platform further supports its suitability for routine diagnostics in complex dairy matrices. Application of the validated assay to 44 buffalo milk samples collected from two dairy farms in the Campania Region (southern Italy) revealed prevalences of 31.8% for G. duodenalis and 18.2% for Cryptosporidium spp. A higher number of positive samples was observed in May, suggesting a possible seasonal influence; however, this indication is preliminary and cannot be interpreted as a confirmed seasonal pattern. Overall, the findings demonstrate that the proposed dd-PCR assay is a highly sensitive and reliable tool for protozoan surveillance in buffalo milk and highlight the need for strengthened monitoring during highrisk seasonal periods.