Genetic diversity of Echinococcus multilocularis on a local scale☆
Introduction
Alveolar Echinococcosis, a zoonosis which is life-threatening to humans, is caused by infection by the larval stage of the fox tapeworm Echinococcus multilocularis. The red fox (Vulpes vulpes) acts as the main definitive host species and harbors the adult stages (Thompson et al., 2006), and arvicolid rodents act as intermediate hosts (Rausch, 1995). Humans can accidentally ingest eggs released into the environment upon definitive host defecation and subsequently develop a parasitic lesion (Bresson-Hadni et al., 2005), which resembles tumor-like tissue. They primarily infiltrate the liver and secondarily may metastasize in other organs or sites. The E. multilocularis tapeworm is widespread in the Northern Hemisphere and is a serious public health problem in many endemic areas in Central Europe (Vuitton et al., 2003, Romig et al., 2006). Switzerland, France, Austria and Germany have been recognized as historical endemic countries (Eckert et al., 2000, Kern et al., 2003). In France, most human cases have been recorded in the areas of Franche-Comté, Lorraine, Auvergne and east of the Rhône-Alpes regions (Vuitton et al., 2003), and these areas have been classified as endemic (Eckert et al., 2000). A few years ago, ectopic cases appeared in regions previously considered as non-endemic, and one was the French Ardennes, which is in North-Eastern France (Piarroux et al., 2006), on the western border of the French, and European, endemic area. Here, E. multilocularis prevalence in foxes has reached 53% (Guislain et al., 2008). However, it is not clear if the occurrence of human cases in this area results from a recent western extension of the endemic area, or if low noise transmission has only recently been identified due to increased interest. Assessment of the polymorphism of the parasite in this focus could help to investigate the problem by showing either a homogeneous population of parasites, mono- or oligoclonal, which would argue in favor of the hypothesis of a recent introduction, or a genome-wide polymorphism which could reinforce the hypothesis of an old, undetected focus.
Recently, microsatellite DNA tools – small tandemly repeated sequences (1–6 bp) widely dispersed in eukaryote DNA – were isolated and characterized from Echinococcus spp. total DNA. They have exhibited a high power of discrimination in E. multilocularis samples, isolated from geographically disparate areas (Bretagne et al., 1996, Bart et al., 2003, Bart et al., 2006, Nakao et al., 2003); classical nuclear and mitochondrial targets have failed to achieve this (Haag et al., 1997). The tandemly repeated multilocus microsatellite EmsB has proved to be a highly sensitive target for investigating the genetic diversity of samples from different geographical origins, at both continental and local focus levels (Bart et al., 2006, Knapp et al., 2007).
We assessed the genetic diversity of E. multilocularis in red foxes sampled in a 900 km2 area in the south-eastern part of the French region of the Ardennes. Genotypes were studied by using the tandemly repeated multilocus microsatellite EmsB, the single-locus microsatellite NAK1 and the conventional mitochondrial target ATP6.
Section snippets
Collection of E. multilocularis samples
Intestines from 149 red fox carcasses (trapped or victims of road accidents) were examined by the Sedimentation and Counting Technique (SCT) as described by Raoul et al. (2001) between January 2001 and August 2005 in a 900 km2 (30 km × 30 km) area in the south-eastern part of the French region of the Ardennes and the neighboring Meuse region.
Fifty-three percent of foxes (ntot = 79) harbored E. multilocularis worms. Precise worm burden intensity was determined for all foxes, and a variable number of
Isolation of gravid adult worms
One hundred and forty intact gravid adult worms were isolated from 25 red foxes (part of the collection of 79 E. multilocularis-infected foxes). For red foxes with low and medium worm burden (n = 21), 5 worms were isolated and for those with a high worm burden (n = 4), 10 worms were isolated. Fox no. R03056 presented a high worm burden (18,630 worms), but only 5 worms could be isolated (Table 2). The total parasitic biomass was 175,897 worms.
The tandemly repeated multilocus microsatellite EmsB
EmsB amplification was successful for the entire sample
Discussion
The aim of our study was to assess the genetic diversity of E. multilocularis in a local range by using two recently developed typing tools, EmsB and NAK1, which have been proved to be more discriminant than those previously available (Bretagne et al., 1996, Nakao et al., 2003, Bart et al., 2006, Knapp et al., 2007). However, the E. multilocularis genome is characterized by a restricted genetic variation (Haag et al., 1997, Bart et al., 2003), which has been interpreted as resulting from the
Acknowledgements
We are very grateful to the following people for their technical help and support: Shumei Ma, Saratou Gerard and Marie-Louise Glowatzki.
This work was financially supported by the PPF Program of the University of Franche-Comté (Programme Pluri-Formations “Milieux naturels, milieux anthropisés”) and by the Swiss National Science Foundation (grant no. 31-111780/1).
We are grateful to Lois Rose for editorial assistance.
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2019, Food and Waterborne ParasitologyCitation Excerpt :In order to evaluate the genetic relationship with other previously genotyped E. multilocularis isolates, each EmsB profile identified was compared with a reference collection of profiles previously obtained by Knapp et al., 2007, Knapp et al., 2008, Knapp et al., 2009 and Umhang et al., 2017 and typed by EmsB from both European and non-European countries hereafter named the “ref collection” (Knapp et al., 2017). This collection included 49 EmsB profiles obtained from 1058 worm isolates from 269 red foxes, isolated in France (number of genotyped worms = 566; number of foxes = 162), Switzerland (84; 19), Germany (87; 18), Austria (98; 22), Slovakia (63; 14), Poland (94; 20) and Czech Republic (66, 14) (Knapp et al., 2009; Knapp et al., 2008; Knapp et al., 2007; Umhang et al., 2017; Umhang et al., 2014). The non-European collection included seven profiles from 54 worms originating from Asia (n = 13), North America (n = 14) and the archipelago of Svalbard, Norway (n = 27).