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.

Echinococcus multilocularis is a frequent parasite of red foxes (Vulpes vulpes) in Latvia

“…Genetic diversity in parasites was higher in the historically documented central European endemic zone (0.082 % of polymorphic sites in examined gene fragments) than in Latvia (0.052% of polymorphic sites) which is presumed to be invaded with the tapeworm more recently. Coupled with the commonly distributed transversal profiles of sequencing patterns even in apparently newly endemic parts of Europe, our data support a view of Knapp et al (2007) "ns" -non-synonymous substitution; "s" -synonymous substitution; "-" not examined at given locus ancestral foci supplied hitherto non-endemic areas by dispersal generated by fox mobility and migration.…”

Phylogenetic Pattern, Evolutionary Processes and Species Delimitation in the Genus Echinococcus

“…These heterozygote deficiencies cannot be accounted for by unobserved population structuring (Wahlund effect), at least for the Australian samples (see Lymbery et al, 1997), and the most likely explanation is predominant self-fertilisation. Cross-fertilisation must also occur, however, because heterozygous genotypes have been found, not only in E. granulosus Lymbery et al, 1990Lymbery et al, , 1997Badaraco et al, 2008), but also in E. multilocularis (Nakao et al, 2003;Knapp et al, 2007) and E. vogeli (Santos et al, 2012). Furthermore, Haag et al (1999;2011) found no evidence of heterozygote deficiencies in populations of E. granulosus in Brazil in two separate studies.…”

The genomic Echinococcus microsatellite EmsB sequences: from a molecular marker to the epidemiological tool

“…(3) Is the marker useful to study the spatial structure and the dynamic activity of the parasite at different geographical ranges/levels ? The power of the EmsB marker in the search for E. multilocularis genetic polymorphism was realized with a global collection composed of 76 isolates from humans, monkeys, rodents and foxes, originating from North America (Saint Lawrence island in Alaska and Canada), China (Tibetan plateau), Japan (Hokkaido island) and Europe (Switzerland, France, Germany, Austria, Poland, Czech Republic, Slovakia and the Netherlands) (Knapp et al 2007). Finally with the EmsB marker, 29 genotypes were described ( Fig.…”

“…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 an out-group control (redrawn from (Knapp et al 2007)). The Echinococcus microsatellite EmsB 445…”

“…Despite substantial efforts to develop and apply valuable genetic tools for E. multilocularis genotyping (Table 1) (Bowles et al 1992 ;Bowles and McManus, 1993 ;Gasser and Chilton, 1995 ;Bretagne et al 1996 ;Haag et al 1997 ;Rinder et al 1997 ;van Herwerden et al 2000 ;Nakao et al 2003 ;Yang et al 2005 ;Knapp et al 2007 ;Okamoto et al 2007), a high discriminatory power was not really achieved except when using microsatellite targets. Overall, an important homology was described within the species E. multilocularis, which could probably be explained first by an almost unique reproduction pattern at the adult stage by homogamy (Haag et al 1997(Haag et al , 1998 and secondly by the recent report of E. multilocularis and the evolutionary history of the genus (Bowles Table 1.…”