Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of echinococcus multilocularis

Abstract

Alveolar echinococcosis (AE)--caused by the cestode Echinococcus multilocularis--is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

FIG. 1.

Electrophoregrams of EmsJ (152 and 155 bp) and EmsK (248 and 250 bp) loci, performed with the automatic sequencer ABI Prism 3100 (Applied Biosystems, Foster City, CA). A, European genotype; B, Asian, Alaskan, and Canadian genotypes; C, Alaskan genotype (found for only one rodent).

FIG. 2.

Electrophoregrams of the NAK1 (192 to 201 bp) locus, performed with the automatic sequencer ABI Prism 3100 (Applied Biosystems, Foster City, CA). Genotypes A to G are shown, and a representative sample of each genotype is indicated in parentheses: A, Chinese samples (2PRC-r); B, Alaskan, Canadian, and Swiss samples (2AL-r); C, Alaskan, Japanese, Swiss, German, Austrian, Czech, and Dutch samples (14CH-r); D, Japanese and European samples (116D-F); E, Swiss samples (CH5-h); F, Alaskan, Swiss, and Austrian samples (32A-h); and G, one Polish sample (392PL-F).

FIG. 3.

Comparison between electrophoregrams performed with the automatic sequencers Beckman CEQ 8000 (a) and ABI Prism 3100 (b) for three isolates arbitrarily selected from the present sample collection: 1AL-r, Alaskan rodent isolate; 31CH-m, Swiss zoo monkey isolate; and 116D-F, German red fox isolate. The Pearson correlation coefficient was determined for results obtained on the two systems and was 0.99 for AL-r, 0.96 for 31CH-m, and 0.99 for 116D-F (P < 0.001).

FIG. 4.

E. multilocularis genetic classification according to EmsB results. On the left side (a), an example of electrophoregrams of EmsB loci (209 bp to 241 bp), obtained using the automatic sequencer ABI Prism 3100. On the right side (b), a dendrogram based on EmsB genotypic data, constructed by hierarchical clustering analysis (Euclidian distance, average link clustering method), with pvclust, under the R Project. The approximately unbiased P values (numbers on nodes, in percent) were calculated with a multiscale bootstrap (B = 1,000). The three solid-line boxes show St. Lawrence Island's samples (upper box), the Asian-North American samples (middle box), and the European samples (lower box). The electrophoregrams correspond to (A) the St. Lawrence Island profile in the genetic tree and the dotted-line boxes refer to (B) the Chinese rodent profile, (C) the Japanese fox profile, and (D through H) the main European profiles. Samples 500, 501, and 502 represent a single isolate maintained in vivo by several passages in Meriones unguiculatus. Sample 116 originating from a Mauritanian camel and sample 539 originating from an Algerian sheep were E. granulosus samples and were included as outgroup controls.