Detection and differentiation of Babesia banethi and Babesia vulpes in foxes and potential tick vectors by a duplex real-time PCR assay

The increasing interactions among wildlife, domestic animals, vectors, and humans are reshaping the epidemiology of vector-borne diseases, including babesiosis. Among piroplasmids infecting wild canids, Babesia vulpes is widely distributed in red foxes, whereas many scientific aspects of the recently described Babesia banethi (e.g., epidemiology, host range, tissue tropism, and potential tick vectors) remain poorly understood. To support future investigations on B. vulpes and B. banethi , a duplex quantitative real-time PCR (dqPCR) targeting the 18S rDNA gene was developed for their simultaneous detection and differentiation in host- and tick-derived DNA samples. The assay employed generic primers and species-specific TaqMan-MGB probes, and was validated using blood, organ, and tick-DNA samples from naturally infected red foxes, other vertebrate hosts, as well as tick species. The dqPCR showed high analytical specificity and sensitivity (i.e., up to 10⁰ plasmid copies/reaction), with no cross-reactivity with other haemoparasites infecting carnivores and successfully detected both Babesia spp., either individually or in simulated mixed infections. The assay also detected B. banethi DNA in an engorged Ixodes kaiseri tick and B. vulpes DNA in Haemaphysalis erinacei , supporting its applicability for vector investigations. Overall, the dqPCR assay developed represents a rapid, sensitive, and species-specific diagnostic tool for large-scale epidemiological studies, screening of candidate vertebrate hosts and tick vectors, assessment of parasite burden in tissues, and detection of mixed infections involving these Babesia species.