PCR conditions and primers.

<div><p>Background</p><p>Potentially zoonotic pathogens have been previously detected in bat-associated ticks, but their role in disease transmission and their frequency of feeding on non-bat hosts is poorly known.</p><p>Methodology/Principal findings</p><p>We used molecular blood meal analysis to reveal feeding patterns of the bat-associated tick species <i>Ixodes ariadnae</i>, <i>I</i>. <i>simplex</i>, and <i>I</i>. <i>vespertilionis</i> collected from cave and mine walls in Central and Southeastern Europe. Vertebrate DNA, predominantly from bats, was detected in 43.5% of the samples (70 of 161 ticks) but in these ticks we also detected the DNA of non-chiropteran hosts, such as dog, <i>Canis lupus familiaris</i>, wild boar, <i>Sus scrofa</i>, and horse, <i>Equus caballus</i>, suggesting that bat-associated ticks may exhibit a much broader host range than previously thought, including domestic and wild mammals. Furthermore, we detected the zoonotic bacteria <i>Neoehrlichia mikurensis</i> in bat ticks for the first time, and other bacteria, such as <i>Bartonella</i> and <i>Wolbachia</i>.</p><p>Conclusions/Significance</p><p>In the light of these findings, the role of bat ticks as disease vectors should be urgently re-evaluated in more diverse host systems, as they may contribute to pathogen transmission between bats and non-chiropteran hosts.</p></div>