Genetic diversity of Echinococcus multilocularis on a local scale

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Abstract

Echinococcus multilocularis is the causative agent of human Alveolar Echinococcosis (AE), and it is one of the most lethal zoonotic infections in the Northern Hemisphere. In France, the eastern and central regions are endemic areas; Franche-Comté, Lorraine and Auvergne are particularly contaminated. Recently, several human cases were recorded in the French Ardennes area, a region adjacent to the western border of the E. multilocularis range in France. A previous study in this focus described a prevalence of over 50% of the parasite in red foxes. The present study investigated the genetic diversity of adult worms collected from foxes in a 900 km2 area in the Ardennes. Instead of a conventional mitochondrial target (ATP6), two microsatellite targets (EmsB and NAK1) were used. A total of 140 adult worms isolated from 25 red foxes were genotyped. After hierarchical clustering analyses, the EmsB target enabled us to distinguish two main assemblages, each divided into sub-groups, yielding the differentiation of six clusters or assemblage profiles. Thirteen foxes (52% of the foxes) each harbored worms from at least two different assemblage profiles, suggesting they had become infected by several sources. Using the NAK1 target, we identified 3 alleles, two found in association with the two EmsB assemblages. With the NAK1 target, we investigated the parasite breeding system and the possible causes of genetic diversification. Only one fox harbored hybrid worms, indicative of a possible (and rare) occurrence of recombination, although multiple infections have been observed in foxes. These results confirm the usefulness of microsatellite targets for assessing genetic polymorphism in a geographically restricted local range.

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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    Nucleotide sequence data reported in this the paper are available in the DDBJ/EMBL/GenBank databases under accession numbers AY680860, AB100031, EU044715, EU044716, EU044717, AB018440 and AB027557.

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