Genetic diversity of the cestode Echinococcus multilocularis in red foxes at a continental scale in Europe

Abstract

Background: Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.

Methodology/principal findings: The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas.

Conclusions/significance: The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a "mainland-island" system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.

Figure 1. Spatial distribution of E. multilocularis sample collection.

Red lozenges represent the position of fox; polygons define a geographically restricted subregion. The red circle area indicates the historically documented E. multilocularis central endemic focus in Europe.

Figure 2. Dendrogram constructed from EmsB amplification data, achieved by hierarchical clustering analysis (Euclidean distance and unweighted pair group method).

Details of sample composition of the EmsB profiles G01 to G32 are given in Table 1. Approximately unbiased p-values are indicated in italics and in percent at each node of the tree, calculated with multiscale bootstrap (B = 1000). Worms from the same fox showing a genetic distance of less than 0.025 were pooled to simplify the dendrogram. F AUB-2: referring to three independent samples obtained from an E. multilocularis reference strains, maintained in vivo in Meriones unguiculatus during 7 months; these samples were taken to establish the genetic distance threshold of 0.08. An outgroup control is represented by E. granulosus sensu stricto (G1 strain, originating from an Algerian sheep [35]).

Figure 3. Distribution of EmsB profiles in the nine subregions in Europe.

For each profile, the red dots represent the position of one or more red foxes; n corresponds to the total number of worms found with the respective profile. Single localisations of different profiles were represented in a single map for **Switzerland: G12 (1), G13 (1), G14 (1), G15 (16), G16 (1), G24 (5), G29 (6), G30 (5) and *Czech Republic: G9 (1); these data are not shown in this figure.

Figure 4. Genetic richness and diversity (inverse Simpson index) rarefaction analysis curves for each subregion (fox as a sampling unit).

Swiss, Switzerland, canton of Zurich; NorthAu, north Austria; Swab, Jura Swab; Bav, Bavaria; Arde, Ardennes; WestCz, west Czech Republic; NorthPol, north Poland; EastSlovPol, east Slovakia and south Poland; CentSlov, central Slovakia.

Figure 5. Comparison of genetic and geographical distances as assessed by the Mantel test.

In each box plot, the red dot represents the genetic distance average, and the black bar the median value. The upper extremity of the box represents the third quartile, and the lower the first quartile. The dotted lines show ranges between upper and lower extreme genetic values. Upper bars represent the ninth decile, and the lower the first decile. The red line represents the regression line. Number of pairwise comparisons per class box plot from left to right: 1, 14,333; 2, 5,519; 3, 13,120; 4, 13,986; 5, 8,497; 6, 17,878; 7, 10,419; 8, 12,273; 9, 12,231; 10, 11,149; 11, 11,562; 12, 6,036; 13, 4,954; 14, 5,359; 15, 6,165; 16, 7,976; 17, 4,296; 18, 2,163; 19, 5,250.