Circulation of Species and Their Exposure to Humans through .

Human babesiosis in Europe has been attributed to infection with and, to a lesser extent, with and which are all transmitted to humans through a bite of . These species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these species, their presence in reservoir hosts and in was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of species were used to test 25,849 questing . Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. was found in two widespread rodent species: and , whereas was detected in the geographically restricted and , and occasionally in free-ranging . was detected in the ubiquitous , and occasionally in free-ranging . pecies-specific PCR revealed co-infections in and resulting in higher prevalence of and than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic species found were , sp. deer clade and badger-associated species. The infection rate of zoonotic species in questing ticks was higher for clade I (2.6%) than clade X (1.9%). Co-infection of with sensu lato and in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of and in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.