Antti Lavikainen

Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus

The northern biotype of Echinococcus granulosus occurs in North America and northern Eurasia in life-cycles involving cervids. Previously, cervid isolates of E. granulosus from North America have been characterized using molecular genetic techniques as the G8 genotype. In this study, 5 isolates of E. granulosus were collected from 4 reindeer and 1 moose in north-eastern Finland. DNA sequences within regions of mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NI)I) genes and the internal transcribed spacer 1 (ITS-1) fragment of the ribosomal DNA were analysed. The mitochondrial nucleotide sequences were identical in all isolates, but high sequence variation was found in the ITS-1 region. Mitochondrial and nuclear sequences of the Finnish cervid E. granulosus and the camel strain (G6) of E. granulosus resembled closely each other. According to phylogenetic analyses, the Finnish isolates have close relationships also with the pig (G7) and cattle (G5) strains. Although some similarities were found with the previously published North American cervid strain (G8), particularly in the NDI sequence and some of the ITS-1 clones, the Finnish E. granulosus form represents a distinct, previously undescribed genotype of E. granulosus. The novel genotype is hereby named as the Fennoscandian cervid strain (G10).

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.

A novel phylogeny for the genus Echinococcus, based on nuclear data, challenges relationships based on mitochondrial evidence

The taxonomic status of Echinococcus, an important zoonotic cestode genus, has remained controversial, despite numerous attempts to revise it. Although mitochondrial DNA (mtDNA) has been the source of markers of choice for reconstructing the phylogeny of the genus, results derived from mtDNA have led to significant inconsistencies with earlier species classifications based on phenotypic analysis. Here, we used nuclear DNA markers to test the phylogenic relationships of members of the genus Echinococcus. The analysis of sequence data for 5 nuclear genes revealed a significantly different phylogeny for Echinococcus from that proposed on the basis of mitochondrial DNA sequence data, but was in agreement with earlier species classifications. The most notable results from the nuclear phylogeny were (1) E. multilocularis was placed as basal taxon, (2) all genotypes of Echinococcus granulosus grouped as a monophyletic entity, and (3) genotypes G8 and G10 clustered together. We conclude that the analysis of nuclear DNA data provides a more reliable means of inferring phylogenetic relationships within Echinococcus than mtDNA and suggest that mtDNA should not be used as the sole source of markers in future studies where the goal is to reconstruct a phylogeny that does not only reflect a maternal lineage, but aims to describe the evolutionary history at species level or higher.

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.

A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data

The cestode family Taeniidae consists of 2 genera, Taenia and Echinococcus, which both have been the focus of intensive taxonomic and epidemiological studies because of their zoonotic importance. However, a comprehensive molecular phylogeny of this family has yet to be reconstructed. In this study, 54 isolates representing 9 Taenia species were characterized using DNA sequences in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Phylogenetic relationships within the family Taeniidae were inferred by combining cox1 and nad1 sequence data of the present and previous studies. In the phylogenetic analysis, the genus Echinococcus was shown to be monophyletic, but Taenia proved to be paraphyletic due to the position of T. mustelae as a probable sister taxon of Echinococcus. This indicates that T. mustelae should form a genus of its own. Taenia ovis krabbei was placed distant from T. ovis ovis, as a sister taxon of T. multiceps, supporting its recognition as a distinct species, T. krabbei. High intraspecific sequence variation within both T. polyacantha and T. taeniaeformis suggests the existence of cryptic sister species.

Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae): proposals for the resurrection of Hydatigera Lamarck, 1816 and the creation of a new genus Versteria.

The cestode family Taeniidae generally consists of two valid genera, Taenia and Echinococcus. The genus Echinococcus is monophyletic due to a remarkable similarity in morphology, features of development and genetic makeup. By contrast, Taenia is a highly diverse group formerly made up of different genera. Recent molecular phylogenetic analyses strongly suggest the paraphyly of Taenia. To clarify the genetic relationships among the representative members of Taenia, molecular phylogenies were constructed using nuclear and mitochondrial genes. The nuclear phylogenetic trees of 18S ribosomal DNA and concatenated exon regions of protein-coding genes (phosphoenolpyruvate carboxykinase and DNA polymerase delta) demonstrated that both Taenia mustelae and a clade formed by Taenia parva, Taenia krepkogorski and Taenia taeniaeformis are only distantly related to the other members of Taenia. Similar topologies were recovered in mitochondrial genomic analyses using 12 complete protein-coding genes. A sister relationship between T. mustelae and Echinococcus spp. was supported, especially in protein-coding gene trees inferred from both nuclear and mitochondrial data sets. Based on these results, we propose the resurrection of Hydatigera Lamarck, 1816 for T. parva, T. krepkogorski and T. taeniaeformis and the creation of a new genus, Versteria, for T. mustelae. Due to obvious morphological and ecological similarities, Taenia brachyacantha is also included in Versteria gen. nov., although molecular evidence is not available. Taenia taeniaeformis has been historically regarded as a single species but the present data clearly demonstrate that it consists of two cryptic species.

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.

Molecular identification of unilocular hydatid cysts from domestic ungulates in Ethiopia: Implications for human infections

To identify the etiologic agents of cystic echinococcosis in Ethiopia, unilocular hydatid cysts were collected from 11 sheep, 16 cattle and 16 camels slaughtered in abattoirs of Aweday, Jijiga, Haramaya and Addis Ababa during June 2010 to February 2011. A PCR-based DNA sequencing of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) was conducted for 40 cysts. The majority of cysts (87.5%) were identified as Echinococcus granulosus sensu stricto and the rest as Echinococcus canadensis. The fertile cysts of E. granulosus s.s. were found only from sheep, although it occurred in all the host species. The predominance of E. granulosus s.s. has important implications for public health since this species is the most typical causative agent of human cystic echinococcosis worldwide. The major cox1 haplotype of E. granulosus s.s. detected in Ethiopia was the same as that has been reported to be most common in Peru and China. However, a few cox1 haplotypes unique to Ethiopia were found in both of the two Echinococcus species. The present regional data would serve as baseline information in determining the local transmission patterns and in designing appropriate control strategies.

Taenia arctos n. sp. (Cestoda: Cyclophyllidea: Taeniidae) from its definitive (brown bear Ursus arctos Linnaeus) and intermediate (moose/elk Alces spp.) hosts

Taenia arctos n. sp. (Cestoda: Cyclophyllidea: Taeniidae) is described from the brown bear Ursus arctos Linnaeus (definitive host) and moose/elk Alces spp. (intermediate hosts) from Finland (type-locality) and Alaska, USA. The independent status of the new species and the conspecificity of its adults and metacestodes have been recently confirmed by the mtDNA sequence data of Lavikainen et al. (2011; Parasitology International, 60, 289–295). Special reference is given to morphological differences between the new species and T. krabbei Moniez, 1879 (definitive hosts primarily canines for the latter), both of which use the moose/elk (Alces spp.) as intermediate hosts (the latter also uses Rangifer and perhaps other northern ruminants), and between the new species and T. ursina Linstow, 1893, both of which use the brown bear U. arctos as a definitive host. New morphological data are also provided for adults and cysticerci of T. krabbei. The analysis includes potentially useful morphometric features that have not been previously applied to Taenia spp.

Molecular identification of Taenia spp. in wolves (Canis lupus), brown bears (Ursus arctos) and cervids from North Europe and Alaska

Taenia tapeworms of Finnish and Swedish wolves (Canis lupus) and Finnish brown bears (Ursus arctos), and muscle cysticerci of Svalbard reindeer (Rangifer tarandus platyrhynchus), Alaskan Grants caribou (Rangifer tarandus granti) and Alaskan moose (Alces americanus) were identified on the basis of the nucleotide sequence of a 396 bp region of the mitochondrial cytochrome c oxidase subunit 1 gene. Two species were found from wolves: Taenia hydatigena and Taenia krabbei. The cysticerci of reindeer, caribou and one moose also represented T. krabbei. Most of the cysticercal specimens from Alaskan moose, however, belonged to an unknown T. krabbei-like species, which had been reported previously from Eurasian elks (Alces alces) from Finland. Strobilate stages from two bears belonged to this species as well. The present results suggest that this novel Taenia sp. has a Holarctic distribution and uses Alces spp. as intermediate and ursids as final hosts.