Tetsuya Yanagida

Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae)

Echinococcosis is a serious helminthic zoonosis in humans, livestock and wildlife. The pathogenic organisms are members of the genus Echinococcus (Cestoda: Taeniidae). Life cycles of Echinococcus spp. are consistently dependent on predator-prey association between two obligate mammalian hosts. Carnivores (canids and felids) serve as definitive hosts for adult tapeworms and their herbivore prey (ungulates, rodents and lagomorphs) as intermediate hosts for metacestode larvae. Humans are involved as an accidental host for metacestode infections. The metacestodes develop in various internal organs, particularly in liver and lungs. Each metacestode of Echinococcus spp. has an organotropism and a characteristic form known as an unilocular (cystic), alveolar or polycystic hydatid. Recent molecular phylogenetic studies have demonstrated that the type species, Echinococcus granulosus, causing cystic echinococcosis is a cryptic species complex. Therefore, the orthodox taxonomy of Echinococcus established from morphological criteria has been revised from the standpoint of phylogenetic systematics. Nine valid species including newly resurrected taxa are recognised as a result of the revision. This review summarises the recent advances in the phylogenetic systematics of Echinococcus, together with the historical backgrounds and molecular epidemiological aspects of each species. A new phylogenetic tree inferred from the mitochondrial genomes of all valid Echinococcus spp. is also presented. The taxonomic nomenclature for Echinococcus oligarthrus is shown to be incorrect and this name should be replaced with Echinococcus oligarthra.

State-of-the-art Echinococcus and Taenia: phylogenetic taxonomy of human-pathogenic tapeworms and its application to molecular diagnosis.

The taxonomy of tapeworms belonging to the family Taeniidae has been controversial because of the paucity of adult phenotypic characters and the great plasticity of larvae in intermediate hosts. The family consists of the medically important two genera Echinococcus and Taenia, which are closely related to each other. Cladistic approaches using the molecular data of DNA and the numerical data of morphologic characters are clarifying phylogenetic relationships among the members of these genera. The nucleotide data of worldwide taeniid parasites accumulated in public DNA databases may provide a basis for the development of molecular diagnostic tools, and make it possible to identify the parasites, at least the human Taenia spp. by non-morphologists. Furthermore, the detection of intraspecific genetic variations prompts evolutionary and ecological studies to address fundamental questions of parasite distributional patterns. Here, we introduce the recent advances of taeniid phylogeny and its application to molecular diagnosis.

Phylogenetic relationships within Echinococcus and Taenia tapeworms (Cestoda: Taeniidae): an inference from nuclear protein-coding genes.

The family Taeniidae of tapeworms is composed of two genera, Echinococcus and Taenia, which obligately parasitize mammals including humans. Inferring phylogeny via molecular markers is the only way to trace back their evolutionary histories. However, molecular dating approaches are lacking so far. Here we established new markers from nuclear protein-coding genes for RNA polymerase II second largest subunit (rpb2), phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold). Bayesian inference and maximum likelihood analyses of the concatenated gene sequences allowed us to reconstruct phylogenetic trees for taeniid parasites. The tree topologies clearly demonstrated that Taenia is paraphyletic and that the clade of Echinococcus oligarthrus and Echinococcusvogeli is sister to all other members of Echinococcus. Both species are endemic in Central and South America, and their definitive hosts originated from carnivores that immigrated from North America after the formation of the Panamanian land bridge about 3 million years ago (Ma). A time-calibrated phylogeny was estimated by a Bayesian relaxed-clock method based on the assumption that the most recent common ancestor of E. oligarthrus and E. vogeli existed during the late Pliocene (3.0 Ma). The results suggest that a clade of Taenia including human-pathogenic species diversified primarily in the late Miocene (11.2 Ma), whereas Echinococcus started to diversify later, in the end of the Miocene (5.8 Ma). Close genetic relationships among the members of Echinococcus imply that the genus is a young group in which speciation and global radiation occurred rapidly.

Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences

The genetic polymorphisms of Echinococcus spp. in the eastern Tibetan Plateau and the Xinjiang Uyghur Autonomous Region were evaluated by DNA sequencing analyses of genes for mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear elongation factor-1 alpha (ef1a). We collected 68 isolates of Echinococcus granulosus sensu stricto (s.s.) from Xinjiang and 113 isolates of E. granulosus s. s., 49 isolates of Echinococcus multilocularis and 34 isolates of Echinococcus shiquicus from the Tibetan Plateau. The results of molecular identification by mitochondrial and nuclear markers were identical, suggesting the infrequency of introgressive hybridization. A considerable intraspecific variation was detected in mitochondrial cox1 sequences. The parsimonious network of cox1 haplotypes showed star-like features in E. granulosus s. s. and E. multilocularis, but a divergent feature in E. shiquicus. The cox1 neutrality indexes computed by Tajimas D and Fus Fs tests showed high negative values in E. granulosus s. s. and E. multilocularis, indicating significant deviations from neutrality. In contrast, the low positive values of both tests were obtained in E. shiquicus. These results suggest the following hypotheses: (i) recent founder effects arose in E. granulosus and E. multilocularis after introducing particular individuals into the endemic areas by anthropogenic movement or natural migration of host mammals, and (ii) the ancestor of E. shiquicus was segregated into the Tibetan Plateau by colonising alpine mammals and its mitochondrial locus has evolved without bottleneck effects.

Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.

Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East

Echinococcus granulosus sensu stricto is a cosmopolitan parasite causing cystic echinococcosis in humans and livestock. Recent molecular phylogeographic studies suggested the rapid dispersal of the parasite by the anthropogenic movement of domestic animal hosts. In the present study, genetic polymorphism of E. granulosus s. s. in the Middle East, where the domestication started, was investigated to validate the dispersal history of the parasite. Thirty-five and 26 hydatid cysts were collected from Iran and Jordan, respectively, and mitochondrial cytochrome c oxidase subunit I (cox1) gene was sequenced. Chinese and Peruvian specimens were also analyzed for comparison. Haplotype network analysis demonstrated the existence of a common haplotype EG01 in all populations. Although EG01 and its one-step neighbors were the majority in all regions, most of the neighboring haplotypes were unique in each locality. Haplotype diversity was high but nucleotide diversity was low in Iran, Jordan and China. Both diversities were lowest and only a few haplotypes were found in Peru. Neutrality indices were significantly negative in Iran, Jordan and China, and positive but not significant in Peru. Pairwise fixation index was significant for all pairwise comparisons, indicating genetic differentiation among populations. These results suggest a evolutionary history of E. granulosus s. s. in which a genetic subgroup including EG01 was selected at the dawn of domestication, and then it was rapidly dispersed worldwide through the diffusion of stock raising. To approach the origin of the ancestral strain, extensive sampling is needed in many endemic regions. To evaluate the hypothetical evolutionary scenario, further study is needed to analyze specimens from diverse host species in wider regions.

Mitochondrial phylogeny of the genus Echinococcus (Cestoda: Taeniidae) with emphasis on relationships among Echinococcus canadensis genotypes

The mitochondrial genomes of the genus Echinococcus have already been sequenced for most species and genotypes to reconstruct their phylogeny. However, two important taxa, E. felidis and E. canadensis G10 genotype (Fennoscandian cervid strain), were lacking in the published phylogeny. In this study, the phylogeny based on mitochondrial genome sequences was completed with these taxa. The present phylogeny highly supports the previous one, with an additional topology showing sister relationships between E. felidis and E. granulosus sensu stricto and between E. canadensis G10 and E. canadensis G6/G7 (closely related genotypes referred to as camel and pig strains, respectively). The latter relationship has a crucial implication for the species status of E. canadensis. The cervid strain is composed of two genotypes (G8 and G10), but the present phylogeny clearly suggests that they are paraphyletic. The paraphyly was also demonstrated by analysing the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) of E. canadensis genotypes from various localities. A haplotype network analysis using the short cox1 sequences from worldwide isolates clearly showed a close relatedness of G10 to G6/G7. Domestic and sylvatic life cycles based on the host specificity of E. canadensis strains have been important for epidemiological considerations. However, the taxonomic treatment of the strains as separate species or subspecies is invalid from a molecular cladistic viewpoint.

Evaluation of a loop-mediated isothermal amplification method using fecal specimens for differential detection of Taenia species from humans.

ABSTRACT We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.

Genetic diversity of Echinococcus spp. in Russia

In Russia, both alveolar and cystic echinococcoses are endemic. This study aimed to identify the aetiological agents of the diseases and to investigate the distribution of each Echinococcus species in Russia. A total of 75 Echinococcus specimens were collected from 14 host species from 2010 to 2012. Based on the mitochondrial DNA sequences, they were identified as Echinococcus granulosus sensu stricto (s.s.), E. canadensis and E. multilocularis. E. granulosus s.s. was confirmed in the European Russia and the Altai region. Three genotypes, G6, G8 and G10 of E. canadensis were detected in Yakutia. G6 was also found in the Altai region. Four genotypes of E. multilocularis were confirmed; the Asian genotype in the western Siberia and the European Russia, the Mongolian genotype in an island of Baikal Lake and the Altai Republic, the European genotype from a captive monkey in Moscow Zoo and the North American genotype in Yakutia. The present distributional record will become a basis of public health to control echinococcoses in Russia. The rich genetic diversity demonstrates the importance of Russia in investigating the evolutionary history of the genus Echinococcus.

Multiplex PCR for Differential Identification of Broad Tapeworms (Cestoda: Diphyllobothrium) Infecting Humans

ABSTRACT The specific identification of broad tapeworms (genus Diphyllobothrium) infecting humans is very difficult to perform by morphological observation. Molecular analysis by PCR and sequencing represents the only reliable tool to date to identify these parasites to the species level. Due to the recent spread of human diphyllobothriosis in several countries, a correct diagnosis has become crucial to better understand the distribution and the life cycle of human-infecting species as well as to prevent the introduction of parasites to disease-free water systems. Nevertheless, PCR and sequencing, although highly precise, are too complicated, long, and expensive to be employed in medical laboratories for routine diagnostics. In the present study we optimized a cheap and rapid molecular test for the differential identification of the most common Diphyllobothrium species infecting humans (D. latum, D. dendriticum, D. nihonkaiense, and D. pacificum), based on a multiplex PCR with the cytochrome c oxidase subunit 1 gene of mitochondrial DNA.