Low Zoonotic Pathogen Burden in Free-Roaming Cats Revealed by 18S rRNA Metabarcoding: A Baseline Study from an Insular Natura 2000 Site in Spain

Free-roaming cats may contribute to zoonotic risk via parasites and other eukaryotic taxa, yet surveillance in protected island settings is limited and conventional coprology can miss low-intensity or degraded signals. We conducted a cross-sectional 18S rRNA metabarcoding survey to establish a baseline profile of potentially pathogenic eukaryotes in community cats from La Graciosa (Natura 2000, Canary Islands, Spain) prior to large-scale antiparasitic interventions. We analysed 152 faecal samples, including fresh samples collected during a high-throughput TNR campaign (<i>n</i> = 37) and dry environmental deposits (<i>n</i> = 115). Host amplification was reduced using a feline 18S blocking primer; libraries were sequenced with Oxford Nanopore technology; and taxonomy was assigned using SILVA-based classifiers with downstream filtering for veterinary/zoonotic relevance. After quality control, 72 eukaryotic taxa were retained and DNA from at least 24 potentially pathogenic taxa was detected. <i>Dipylidium caninum</i> was most frequent (74.3%; 113/152), and opportunistic fungi/yeasts were common (e.g., <i>Pichia kudriavzevii</i> 42.4%, <i>Diutina catenulata</i> 31.5%). Zoonotic protozoa showed low-to-moderate detection frequency (<i>Acanthamoeba castellanii</i> 13.3%, <i>Toxoplasma gondii</i> 7.9%, <i>Balamuthia mandrillaris</i> 4.6%). Overall richness did not differ between fresh and dry samples (<i>p</i> > 0.05), but fresh samples contained higher richness of potentially pathogenic taxa (<i>p</i> < 0.01).