Donald P. McManus

Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing

The pattern of species and strain variation within the genus Echinococcus is complex and controversial. In an attempt to characterise objectively the various species and strains, the sequence of a region of the mitochondrial cytochrome c oxidase subunit I (CO1) gene was determined for 56 Echinococcus isolates. Eleven different genotypes were detected, including 7 within Echinococcus granulosus, and these were used to categorise the isolates. The 4 generally accepted Echinococcus species were clearly distinguishable using this approach. In addition, the consensus view of the strain pattern within E. granulosus, based on a variety of criteria of differentiation, was broadly upheld. Very little variation was detected within Echinococcus multilocularis. Remarkable intra-strain homogeneity was found at the DNA sequence level. This region of the rapidly evolving mitochondrial genome is useful as a marker of species and strain identity and as a preliminary indication of evolutionary divergence within the genus Echinococcus.

Towards a taxonomic revision of the genus Echinococcus

Echinococcus remains a significant public health problem worldwide and, in several regions, the aetiological agents of cystic hydatid disease/echinococcosis are extending their range. The taxonomy of Echinococcus has been a controversial issue for decades, but the outcome of recent molecular epidemiological studies has served to reinforce proposals made ten years ago to revise the taxonomy of Echinococcus. A formal nomenclature is essential for effective communication, and provides the stability that underpins epidemiological investigations. It will also serve to recognize the contribution of early taxonomists.

Prevention and control of cystic echinococcosis

Human cystic echinococcosis (hydatid disease) continues to be a substantial cause of morbidity and mortality in many parts of the world. Elimination is difficult to obtain and it is estimated that, using current control options, achieving such a goal will take around 20 years of sustained efforts. Since the introduction of current (and past) hydatid control campaigns, there have been clear technological improvements made in the diagnosis and treatment of human and animal cystic echinococcosis, the diagnosis of canine echinococcosis, and the genetic characterisation of strains and vaccination against Echinococcus granulosus in animals. Incorporation of these new measures could increase the efficiency of hydatid control programmes, potentially reducing the time required to achieve effective prevention of disease transmission to as little as 5-10 years.

A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes

Taxonomic revision by molecular phylogeny is needed to categorize members of the genus Echinococcus (Cestoda: Taeniidae). We have reconstructed the phylogenetic relationships of E. oligarthrus, E. vogeli, E. multilocularis, E. shiquicus, E. equinus, E. ortleppi, E. granulosus sensu stricto and 3 genotypes of E. granulosus sensu lato (G6, G7 and G8) from their complete mitochondrial genomes. Maximum likelihood and partitioned Bayesian analyses using concatenated data sets of nucleotide and amino acid sequences depicted phylogenetic trees with the same topology. The 3 E. granulosus genotypes corresponding to the camel, pig, and cervid strains were monophyletic, and their high level of genetic similarity supported taxonomic species unification of these genotypes into E. canadensis. Sister species relationships were confirmed between E. ortleppi and E. canadensis, and between E. multilocularis and E. shiquicus, regardless of the analytical approach employed. The basal positions of the phylogenetic tree were occupied by the neotropical endemic species, E. oligarthrus and E. vogeli, whose definitive hosts are derived from carnivores that immigrated from North America after the formation of the Panamanian land bridge. Host-parasite co-evolution comparisons suggest that the ancestral homeland of Echinococcus was North America or Asia, depending on whether the ancestral definitive hosts were canids or felids.

Rapid Discrimination of Echinococcus Species and Strains Using a Polymerase Chain Reaction-Based Rflp Method

Echinococcus species and genetically distinct strains of Echinococcus granulosus can be rapidly and reliably identified using a polymerase chain reaction (PCR)-linked restriction fragment length polymorphism (RFLP) method which surveys the sequence of a rapidly evolving region of the ribosomal DNA (rDNA) unit. Internal transcribed spacer 1 (ITS1) of the rDNA repeat was amplified from various isolates and the product was digested with one of a number of 4-base cutting restriction enzymes. Characteristic patterns were produced when samples within various species and strain groups were analysed. This method offers an objective, simple, highly sensitive and rapid approach for the discrimination of Echinococcus isolates and for study of other parasite complexes.

Molecular epidemiology of cystic echinococcosis

Echinococcus granulosus exhibits substantial genetic diversity that has important implications for the design and development of vaccines, diagnostic reagents and drugs effective against this parasite. DNA approaches that have been used for accurate identification of these genetic variants are presented here as is a description of their application in molecular epidemiological surveys of cystic echinococcosis in different geographical settings and host assemblages. The recent publication of the complete sequences of the mitochondrial (mt) genomes of the horse and sheep strains of E. granulosus and of E. multilocularis, and the availability of mt DNA sequences for a number of other E. granulosus genotypes, has provided additional genetic information that can be used for more in depth strain characterization and taxonomic studies of these parasites. This very rich sequence information has provided a solid molecular basis, along with a range of different biological, epidemiological, biochemical and other molecular-genetic criteria, for revising the taxonomy of the genus Echinococcus. This has been a controversial issue for some time. Furthermore, the accumulating genetic data may allow insight to several other unresolved questions such as confirming the occurrence and precise nature of the E. granulosus G9 genotype and its reservoir in Poland, whether it is present elsewhere, why the camel strain (G6 genotype) appears to affect humans in certain geographical areas but not others, more precise delineation of the host and geographic ranges of the genotypes characterised to date, and whether additional genotypes of E. granulosus remain to be identified.

A Molecular Phylogeny of the Genus Echinococcus

Three nucleotide data sets, two mitochondrial (COI and ND1) and one nuclear (ribosomal ITS1), have been investigated in order to resolve relationships among species and strains of the genus Echinococcus. The data have some unusual properties in that mitochondrial heteroplasmy was detected in one strain of E. granulosus, and more than one class of ITS1 sequence variant can occur in a single isolate. The data failed to support the hypothesis that E. granulosus, as it is currently viewed, is a single valid species. Rather, the strains of E. granulosus seem to comprise at least three evolutionarily diverse groups, the sheep strain group, bovine strain group and horse strain group. Molecular distances between them are comparable to, or greater than, molecular evolutionary distances observed between recognized species. The affinities of the cervid strain of E. granulosus are unclear because of ambiguous data, but this strain does not appear to be ancestral to others. E. multilocularis may not be distinct from E. granulosus. However, the remaining two species. E. vogeli and E. oligarthrus appear distinct and rather distant from the first two. Based on the results presented here, taxonomic revision of the genus is clearly warranted.

Molecular genetic analysis of human cystic hydatid cases from Poland: identification of a new genotypic group (G9) of Echinococcus granulosus

We have used nuclear (ribosomal ITS1) and mitochondrial (ND1) sequences to characterize human and pig isolates of Echinococcus granulosus collected by fine needle aspiration biopsy (FNAB) in Poland. The data indicate clearly that the Polish patients were not infected with the common sheep strain (G1 genotype) of E. granulosus, normally associated with human cystic hydatid infection. Instead, the hydatid parasite infecting the Polish patients shares very similar ND1 sequence with the previously characterized pig (G7) genotype but it also exhibits some clear differences. In particular, E. granulosus DNA from the Polish patients amplified a single ITS1 fragment in PCR and distinct ITS1-RFLP patterns were obtained after restriction digestion. The form of hydatid isolated from the Polish patients appears, therefore, to represent a distinct, previously undescribed genotype (designated G9) of E. granulosus.

Mitochondrial genomes of parasitic flatworms

Complete or near-complete mitochondrial genomes are now available for 11 species or strains of parasitic flatworms belonging to the Trematoda and the Cestoda. The organization of these genomes is not strikingly different from those of other eumetazoans, although one gene (atp8) commonly found in other phyla is absent from flatworms. The gene order in most flatworms has similarities to those seen in higher protostomes such as annelids. However, the gene order has been drastically altered in Schistosoma mansoni, which obscures this possible relationship. Among the sequenced taxa, base composition varies considerably, creating potential difficulties for phylogeny reconstruction. Long non-coding regions are present in all taxa, but these vary in length from only a few hundred to approximately 10000 nucleotides. Among Schistosoma spp., the long non-coding regions are rich in repeats and length variation among individuals is known. Data from mitochondrial genomes are valuable for studies on species identification, phylogenies and biogeography.

Genetic variation and epidemiology of Echinococcus granulosus in Argentina

Polymerase chain reaction-ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the carnel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.