Due to increased travel, climatic, and environmental changes, the incidence of 
tick-borne disease in both humans and animals is increasing throughout Europe. 
Therefore, extended surveillance tools are desirable. To accurately screen 
tick-borne pathogens (TBPs), a large scale epidemiological study was conducted 
on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the 
Netherlands using a powerful new high-throughput approach. This advanced 
methodology permitted the simultaneous detection of 25 bacterial, and 12 
parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, 
Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and 
Theileria genus) across 94 samples. We successfully determined the prevalence of 
expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia 
helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia 
venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) 
pathogens in the three European countries. Moreover we detected Borrelia 
spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the 
first time in Danish ticks. This surveillance method represents a major 
improvement in epidemiological studies, able to facilitate comprehensive testing 
of TBPs, and which can also be customized to monitor emerging diseases.