Free-Ranging Wolves (Canis lupus) are Natural Reservoirs of Intestinal Microeukaryotes of Public Health Significance in Southwestern Europe

Abstract

Introduction: Intestinal microeukaryote parasites are major contributors to the burden of diarrhoea in humans and domestic animals, but their epidemiology in wildlife is not fully understood. We investigated the frequency, genetic diversity and zoonotic potential of protists of animal and public health significance in free-ranging grey wolf (Canis lupus) populations in south-western Europe. Methods: Individually formed faecal samples collected from necropsied wolves or scat trails in Italy (n=47), Portugal (n=43) and Spain (n=225) during the period 2011¿2023 were retrospectively analysed using molecular (PCR and Sanger sequencing) methods. Complementary epidemiological data were gathered when available. Results: Giardia duodenalis was the most frequent microeukaryote found (40.3%, 127/315; 95% CI: 34.9¿46.0), followed by Cryptosporidium spp. (3.5%, 11/315; 95% CI: 1.8¿6.2), Enterocytozoon bieneusi and Encephalitozoon spp. (1.6%, 5/315; 95% CI: 0.5¿3.7 each). Blastocystis was not identified in any of the faecal samples analysed. Sequence analyses confirmed the presence of canine-adapted assemblage D within G. duodenalis (n=7). Three Cryptosporidium species were identified, namely canineadapted C. canis (n=9), zoonotic C.parvum (n=1) and primarily anthroponotic C.hominis (n=1). Genotyping tools enabled the identification of subtype family XXe2 within C. canis. Among microsporidia, the canine-adapted genotype PtEb IX was identified within E. bieneusi. Two samples were confirmed as Enc. intestinalis and three more as Enc. cuniculi genotype IV. This is the first record of Enc. intestinalis and Enc. cuniculi in the grey wolf globally. Conclusions: Silent carriage of intestinal microeukaryotes seems common in free-ranging grey wolves in southwestern Europe. Wolves can contribute to environmental contamination through the transmission stages (cysts, oocysts, spores) of species/genotypes potentially infective to humans. Individuals in close contact with wolf carcasses or their faecal material may be at potential risk of infection by microeukaryotic pathogens.