Chasing intermediate hosts of Echinococcus multilocularis at the southern edge of its European distribution using red fox stomach content analysis

<i>Echinococcus multilocularis</i> (<i>Em</i>) is a Taeniidae cestode circulating among canids (definitive hosts) and among voles (intermediate hosts). Humans can develop alveolar echinococcosis (AE) following egg ingestion. In Italy, <i>Em</i> is present in red foxes (<i>Vulpes vulpes</i>) from the Trentino-Alto Adige/Südtirol Region, with reports in canids from Liguria (wolf, <i>Canis lupus</i>, and domestic dog, <i>Canis lupus familiaris</i>) and Tuscany (wolf and red fox) Regions. The first autochthonous case of human AE was recently confirmed in the Province of Bolzano. Despite its relevance, the intermediate hosts maintaining the <i>Em</i> life cycle in this area have never been identified. This study aimed to fill this knowledge gap by collecting ingested rodents from the stomachs of 148 legally culled or found dead foxes across the Province of Bolzano. For 142 prey items, species was ascertained from tissue DNA using a PCR of a 350 bp fragment of <i>cytb</i> mitochondrial gene. Positivity to <i>Em</i> was investigated by RT-PCR and conventional PCR on organ tissues from 97 rodents targeting a 69 bp fragment of <i>cob</i> and a 126 bp fragment of <i>nad2</i> mitochondrial genes, respectively. <i>Microtus arvalis</i> was the most common prey rodent in terms of both frequency in foxes (20.8 %; 30/144) and total rodent prey items (81.7 %; 116/142). Other prey species included nine <i>Arvicola amphibius</i>, five <i>Microtus lavernedii</i>, two <i>M</i>. <i>subterraneus</i>, one <i>M</i>. <i>liechtensteini</i>, six <i>Clethrionomys glareolus</i>, one <i>Apodemus flavicollis</i> and two <i>Ap</i>. <i>sylvaticus</i>. Only 3/97 rodents were <i>Em</i>-positive (<i>M. arvalis</i>; 3/85) in two <i>Em</i>-positive red foxes. Prevalence in <i>M. arvalis</i> was estimated at 0.035 (95 % CI: 0.008-0.103). Further research is required to explain why <i>Em</i> distribution is limited to this area in the eastern Italian Alps despite the spatial contiguity to hyperendemic foci.