Keeseon S. Eom

A PHYLOGENETIC HYPOTHESIS FOR SPECIES OF THE GENUS TAENIA (EUCESTODA: TAENIIDAE)

Cladistic analysis of a numerical data matrix describing 27 characters for species of Taenia resulted in 4 most parsimonious phylogenetic trees (174 steps; consistency index = 0.28; homoplasy index = 0.72; retention index = 0.48). Monophyly for Taenia is diagnosed by the metacestode that is either a cysticercus or a form derived from a bladder-like larva; no other unequivocal synapomorphies are evident. Tree structure provides no support for recognition of a diversity of tribes or genera within the Taeniinae: Fimbriotaeniini and Taeniini have no phylogenetic basis. Hydatigera, Fimbriotaenia, Fossor, Monordotaenia, Multiceps, Taeniarhynchus, Tetratirotaenia must be subsumed within Taenia as synonyms. Taenia saginata and Taenia asiatica are sister species and distantly related to Taenia solium. Cospeciation with respect to carnivorous definitive hosts and Taenia appears to be limited. Although felids are putative ancestral hosts, contemporary associations appear to have resulted from extensive host-switching among felids, canids, hyaenids, and others. In contrast, relationships with herbivorous intermediate hosts are indicative of more pervasive coevolution; rodents as intermediate hosts are postulated as ancestral for the Taeniidae, Taenia + Echinococcus. Patterns appear consistent with rapid shifts between phylogenetically unrelated carnivores but among those that historically exploited a common prey resource within communities in specific biogeographic regions.

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.

A common origin of complex life cycles in parasitic flatworms: evidence from the complete mitochondrial genome of Microcotyle sebastis (Monogenea: Platyhelminthes)

BackgroundThe parasitic Platyhelminthes (Neodermata) contains three parasitic groups of flatworms, each having a unique morphology, and life style: Monogenea (primarily ectoparasitic), Trematoda (endoparasitic flukes), and Cestoda (endoparasitic tapeworms). The evolutionary origin of complex life cyles (multiple obligate hosts, as found in Trematoda and Cestoda) and of endo-/ecto-parasitism in these groups is still under debate and these questions can be resolved, only if the phylogenetic position of the Monogenea within the Neodermata clade is correctly estimated.ResultsTo test the interrelationships of the major parasitic flatworm groups, we estimated the phylogeny of the Neodermata using complete available mitochondrial genome sequences and a newly characterized sequence of a polyopisthocotylean monogenean Microcotyle sebastis. Comparisons of inferred amino acid sequences and gene arrangement patterns with other published flatworm mtDNAs indicate Monogenea are sister group to a clade of Trematoda+Cestoda.ConclusionResults confirm that vertebrates were the first host for stem group neodermatans and that the addition of a second, invertebrate, host was a single event occurring in the Trematoda+Cestoda lineage. In other words, the move from direct life cycles with one host to complex life cycles with multiple hosts was a single evolutionary event. In association with the evolution of life cycle patterns, our result supports the hypothesis that the most recent common ancestor of the Neodermata giving rise to the Monogenea adopted vertebrate ectoparasitism as its initial life cycle pattern and that the intermediate hosts of the Trematoda (molluscs) and Cestoda (crustaceans) were subsequently added into the endoparasitic life cycles of the Trematoda+Cestoda clade after the common ancestor of these branched off from the monogenean lineage. Complex life cycles, involving one or more intermediate hosts, arose through the addition of intermediate hosts and not the addition of a vertebrate definitive host. Additional evidence is required from monopisthocotylean monogeneans in order to confirm the monophyly of the group.

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

BackgroundThe orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.ResultsFor this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomiasiamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.ConclusionThe phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

IDENTIFICATION OF TAENIA ASIATICA IN CHINA: MOLECULAR, MORPHOLOGICAL, AND EPIDEMIOLOGICAL ANALYSIS OF A LUZHAI ISOLATE

Multiple analysis has characterized a recently described tapeworm of people, Taenia asiatica, in mainland China. Six adult tapeworms collected from people of the Zhuang minority residing in the southern part of China (Luzhai isolate) were comparatively analyzed with other tapeworms from people: T. asiatica (n = 2, South Korea), T. saginata (n = 1, Poland; n = 1, Korea), and T. solium (n = 1, Peoples Republic of China). Experimental infections with eggs from the Luzhai isolate in pigs and cattle produced cysticerci, each with a hookletless scolex and with wartlike formations on the external surface of the bladder wall. There were rostellar protrusions in the scolices of adult worms. Random amplified polymorphic DNA analysis using 3 arbitrary primers produced bands identical to those of the Korean T. asiatica. Conversely, T. saginata and T. solium exhibited different banding patterns. Phylogenetic relationships inferred from the complete nucleotide sequences of the internal transcribed spacer 2 placed the Chinese tapeworms consistently within the T. asiatica clade by 96% bootstrapping value in the maximum likelihood analysis, 96% in maximum parsimony, and 100% in neighbor joining. These collective data demonstrate that T. asiatica is sympatrically distributed with the other 2 species of Taenia in the human host in mainland China.

Differential diagnosis of Taenia asiatica using multiplex PCR

Taenia asiatica and T. saginata are frequently confused tapeworms due to their morphological similarities and sympatric distribution in Asian regions. To resolve this problem, a high-resolution multiplex PCR assay was developed to distinguish T. asiatica infections from infection with other human Taenia tapeworms. For molecular characterization, the species specificity of all materials used was confirmed by sequencing of the cox1 gene. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China, Korea, and the Philippines; 24 samples of T. saginata from Belgium, Chile, China, Ethiopia, France, Indonesia, Korea, Laos, the Philippines, Poland, Taiwan, Thailand, and Switzerland; and 10 samples of T. solium from Cape Verde, China, Honduras, and Korea. The diagnostic quality of the results obtained using PCR and species-specific primers designed from valine tRNA and NADH genes was equal to that based on the nucleotide sequencing of the cox1 gene. Using oligonucleotide primers Ta4978F, Ts5058F, Tso7421F, and Rev7915, the multiplex PCR assay was useful for the differentially diagnosing T. asiatica, T. saginata, and T. solium based on 706-, 629-, and 474-bp bands.

Geographical Distribution of Taenia asiatica and Related Species

Geographical information of Taenia asiatica is reviewed together with that of T. solium and T. saginata. Current distribution of T. asiatica was found to be mostly from Asian countries: the Republic of Korea, China, Taiwan, Indonesia, and Thailand. Molecular genotypic techniques have found out more countries with T. asiatica from Japan, the Philippines, and Vietnam. Specimens used in this paper were collected from around the world and mostly during international collaboration projects of Korean foundations for parasite control activities (1995-2009) in developing countries.

Fishborne trematode metacercariae detected in freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR.

Freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR were examined by the muscle compression and artificial digestion methods to know the infection status with trematode metacercariae. In the fish from Savannakhet, 2 species of metacercariae, Opisthorchis viverrini and Haplorchis taichui, were detected. O. viverrini metacercariae were found in 6 species of fish, Puntius brevis, Hampala dispar, Esomus metallicus, Mystacoleucus marginatus, Puntioplites falcifer, and Cyclocheilichthys armatus. H. taichui metacercariae were detected in 3 species of fish, P. brevis, P. falcifer, and M. marginatus. In the fish from Vientiane, 4 species of metacercariae, O. viverrini, H. taichui, Haplorchis yokogawai, and Centrocestus formosanus, were detected. Among them, O. viverrini metacercariae were found in 7 species of fish, Onychostoma elongatum, C. armatus, H. dispar, P. brevis, Cyclocheilichthys repasson, Osteochilus hasseltii, and Hypsibarbus lagleri. The metacercariae of H. taichui were detected in 6 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, and Cirrhinus molitorella. The metacercariae of H. yokogawai were found in 9 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, O. hasseltii. C. molitorella, Hypsibarbus wetmorei, and H. lagleri. The metacercariae of C. formosanus were detected in 4 species of fish, C. repasson, P. brevis, O. hasseltii, and C. molitorella. From these results, it is confirmed that fishborne trematode metacercariae, i.e. O. viverrini, H. taichui, H. yokogawai and C. formosanus, are prevalent in various species of freshwater fish from Savannakhet Province and Vientiane Municipality, Lao PDR.

The mitochondrial genome sequence of Enterobius vermicularis (Nematoda: Oxyurida) - an idiosyncratic gene order and phylogenetic information for chromadorean nematodes.

The complete mitochondrial genome sequence was determined for the human pinworm Enterobius vermicularis (Oxyurida: Nematoda) and used to infer its phylogenetic relationship to other major groups of chromadorean nematodes. The E. vermicularis genome is a 14,010-bp circular DNA molecule that encodes 36 genes (12 proteins, 22 tRNAs, and 2 rRNAs). This mtDNA genome lacks atp8, as reported for almost all other nematode species investigated. Phylogenetic analyses (maximum parsimony, maximum likelihood, neighbor joining, and Bayesian inference) of nucleotide sequences for the 12 protein-coding genes of 25 nematode species placed E. vermicularis, a representative of the order Oxyurida, as sister to the main Ascaridida+Rhabditida group. Tree topology comparisons using statistical tests rejected an alternative hypothesis favoring a closer relationship among Ascaridida, Spirurida, and Oxyurida, which has been supported from most studies based on nuclear ribosomal DNA sequences. Unlike the relatively conserved gene arrangement found for most chromadorean taxa, E. vermicularis mtDNA gene order is very unique, not sharing similarity to any other nematode species reported to date. This lack of gene order similarity may represent idiosyncratic gene rearrangements unique to this specific lineage of the oxyurids. To more fully understand the extent of gene rearrangement and its evolutionary significance within the nematode phylogenetic framework, additional mitochondrial genomes representing a greater evolutionary diversity of species must be characterized.

Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda).

In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.