First Detection of $Cytauxzoon$ spp. DNA in Questing $Ixodes$ $ricinus$ Ticks

Feline cytauxzoonosis is an emerging tick-borne disease in Europe. While infections have been reported in different European countries, the tick vector remains unknown. This study investigated 665 ticks collected in 2019 (n = 160), 2022 (n = 7), and 2024 (n = 498) in a $Cytauxzoon$ spp. hotspot region in central Switzerland (62 ticks from cats; 603 ticks from vegetation). Ticks were morphologically characterized, pooled by origin and life-stage, and screened for $Cytauxzoon$ spp. 18S rRNA by qPCR and conventional PCR, and positive samples confirmed by sequencing. All ticks belonged to Ixodes ricinus (50 males, 83 females, 532 nymphs). Four tick pools from 2019 tested $Cytauxzoon$ spp. positive: one pool of 3 non-engorged male ticks from two cats and three pools of 5–6 nymphs each from vegetation. All ticks collected in 2022 and 2024 tested negative. Amplification of the almost full-length (1535 bp, one pool) or partial (140–219 bp, three pools) 18S rRNA gene revealed a sequence identity of 98.6–100% with $Cytauxzoon$ spp. previously detected in cats from this area. The detection of $Cytauxzoon$ spp. in questing I. ricinus nymphs suggests a potential role of this tick species in the parasites’ transmission cycle in Central Europe and raises the possibility of transstadial or potentially transovarial transmission. Mitochondrial gene sequencing was unsuccessful, but the detected $Cytauxzoon$ spp. likely represent $Cytauxzoon$ $europaeus$. Discrepancies between qPCR and conventional PCR results point to possible amplification of tick endosymbionts, highlighting the importance of confirmatory sequencing, particularly when testing tick-derived DNA. Thus, the 18S rRNA qPCR assay used appears suboptimal for screening tick samples, as its specificity in this matrix was limited. In conclusion, this is the first report of $Cytauxzoon$ spp. in questing I. ricinus ticks in Europe. Our findings underscore the need for further research to confirm vector competence and clarify transmission dynamics.