Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from similar to 10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite. Author summaryEchinococcus multilocularis is a tiny tapeworm whose adults live in the intestines of wild and domestic canids and shed their eggs with the host feces. If accidentally ingested by people, eggs can cause human alveolar echinococcosis (AE), a highly lethal parasitic disease. Despite Em being common in North America, only two locally acquired AE human cases were reported in North America until 2013. However, in the last 6 years, 14 new cases have been detected in Alberta, Canada, and recent findings showed that the European strain of this parasite is now common in wildlife in this province, which could potentially increase the risk of infection in humans and domestic animals. In this study, we genetically characterized Em in coyotes and red foxes to assess the extent of the European-type strain distribution in three Canadian provinces. We used deep amplicon sequencing, an innovative approach that allows us to detect co-infections and the presence of less prevalent genetic variants by sampling a larger proportion of the parasite subpopulation per host. Our study showed an overwhelming prevalence of the European strain over the native North American strain in both hosts, and a limited geographical distribution of some European genetic variants, likely due to multiple introduction events of this invasive strain.