First detection of Cytauxzoon spp. DNA in questing Ixodes ricinus nymphs

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= 658) 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, 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 4 non-engorged male ticks from 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, 1 pool) or partial (219-140 bp, 3 pools) 18S rRNA gene revealed a sequence identify of 98.6-100% with Cytauxzoon spp. previously detected in cats from this area. The detection of Cytauxzoon spp. in questing Ixodes ricinus nymphs suggests a potential role of this tick species in the parasites transmission cycle in Central Europe and raises the possibility of 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. 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.