Genetic differentiation of the G6/7 cluster of Echinococcus canadensis based on mitochondrial marker genes.

Among the genotype/species causing cystic echinococcosis, the taxonomic status of Echinococcus canadensis is only partially resolved. Within E. canadensis, four genotypes (G6, G7, G8 and G10) have been described based on short mitochondrial sequences, of which G6 and G7 (the 'camel' and the 'pig' strain, respectively) are closely related and variously regarded as microvariants of a single strain G6/7. Globally, this G6/7 cluster is the second most important agent of human cystic echinococcosis and is the predominant Echinococcus taxon in large parts of sub-Saharan Africa. To add data on the internal structure and the geographical distribution of this cluster, we analysed diversity and population structure of 296 isolates of E. canadensis from sub-Saharan Africa, the Middle East and Europe using the complete mitochondrial cytochrome c oxidase subunit 1 (cox1) (1,608bp) and NADH dehydrogenase subunit 1 (nad1) (894bp) gene sequences. Polymorphism of the mtDNA loci gave 51 (cox1), 33 (nad1) and 73 (cox1-nad1 concatenated) haplotypes. African and Middle Eastern isolates mainly grouped in a star-like structure around a predominant haplotype, while the European isolates produced more diversified networks. Although the cox1 diagnostic sequence for G6 is frequent in the African/Middle Eastern sub-cluster, and that for G7 is common in the European isolates, numerous intermediate variants prevent a clear distinction into 'G6' or 'G7', and the entire taxon is best treated as a common haplotype cluster G6/7. Meanwhile, the G6/7 cluster is clearly distinct from sequences of wildlife isolates of G8 and G10 from the northern hemisphere, and sequences of the latter genotypes were remarkably distant from each other. It is clear from the present study that, based on mitochondrial data, G6/7 is a coherent genotypic entity within E. canadensis that retains substantial intraspecific variance, and sub-populations share common ancestral polymorphisms and haplotypes. This study provides the basis for wider biogeographic comparison and population genetics studies of this taxon.

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa.

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.