Abstract
Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.
Highlights
Four species have been recognized within the genus Echinococcus: E. multilocularis, E. oligarthrus, E. vogeli and E. granulosus [1]
E. granulosus became considered as a complex consisting of four species: E. granulosus sensu stricto (G1/G2/G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–G10)
Infective eggs are shed with the feces into the environment and are orally ingested by intermediate hosts where they develop into the metacestode stage, causing cystic echinococcosis (CE) in humans and livestock
Summary
Four species have been recognized within the genus Echinococcus: E. multilocularis, E. oligarthrus, E. vogeli and E. granulosus [1]. The poorly characterized strain G9 is closely related to E. canadensis (G7) [7] and the existence of G9 as a separate genotype remains still controversial [8,9]. New data obtained from phylogenetic analysis have shown an even more pronounced genetic divergence between these ten E. granulosus genotypes [5,10]. The phylogenetic relations within the latter group remain unresolved and are still under controversial discussion, since the E. canadensis cluster was proposed to be divided into the two species E. canadensis (G8/ G10) and E. intermedius (G6/G7) [14,15].
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