Urusa Thaenkham

Families Opisthorchiidae and Heterophyidae: are they distinct?

Superfamily Opisthorchioidea Looss, 1899 consists of three well-known families, Opisthorchiidae, Heterophyidae, and Cryptogonimidae, with basic similarities in morphology and life-cycles. Many species in the first two of these families are human pathogens, such as Opisthorchis viverrini, O. felineus, Clonorchis sinensis, Haplorchis spp. and Metagonimus spp. Recently, a molecular phylogenetic study on the classification of Digenea revealed a paraphyletic relationship between Opisthorchiidae and Heterophyidae. For our study, we gathered and analyzed all available data in GenBank, together with new data of nuclear 18S ribosomal DNA and ribosomal internal transcribed spacer 2 (ITS2) sequences of the families within the Opisthorchioidea. Maximum likelihood and Bayesian inference analyses suggested that families Opisthorchiidae and Heterophyidae are inseparable from each other, with the former nested within the latter. Groupings in molecular trees are generally consistent with morphological features used in taxonomy.

The second outbreak of trichinellosis caused by Trichinella papuae in Thailand

A human trichinellosis outbreak caused by Trichinella papuae occurred in the Uthai Thani Province of Thailand in September 2007. A total of 34 villagers suffering at least one of the symptoms suggestive of trichinellosis, or those who were asymptomatic but had a history of ingesting raw wild pig meat, were enrolled in the study. Twenty-two villagers had ingested undercooked pork from a hunted wild pig (Sus scrofa). One patient with a severe clinical picture was hospitalised and more than 80 non-encapsulated larvae were detected in the muscle biopsy. The larvae were identified as T. papuae by molecular analyses of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the expansion segment 5 (ES5) of the large subunit rRNA. Of the 34 suspected cases, 27 agreed to be subjected to haematological and serological tests. Immunoblot analysis using crude antigens from T. spiralis muscle larvae revealed anti-Trichinella IgG in 20 of the 26 serum samples (1 serum sample could not be analysed). All infected people were successfully treated with mebendazole; the one patient with severe symptomatology was treated successfully with prednisolone.

Confirmation of the paraphyletic relationship between families Opisthorchiidae and Heterophyidae using small and large subunit ribosomal DNA sequences

Opisthorchiidae and Heterophyidae are classified into different families based on morphological identification. However, recent molecular phylogenetic studies suggested the possible paraphyletic relationship between these two families. In this study, the paraphyletic relationship between these two families was confirmed further by maximum likelihood and Bayesian inference analyses using the combined sequences of SSU and LSU rDNA.

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region.

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.

Monophyly of Opisthorchis viverrini populations in the lower Mekong Basin, using mitochondrial DNA nad1 gene as the marker.

The liver fluke, Opisthorchis viverrini, causes serious public-health problems in the Lower Mekong Basin. This study aimed to clarify whether O. viverrini populations may be genetically divided into sub-specific taxa. We collected 6 populations of O. viverrini from different places in Cambodia, Lao PDR, and Thailand, along both sides of the Mekong River, and analyzed the population structure of these using the mitochondrial nad1 gene as a marker. The results of the DNA polymorphism measurements, by theta-w (thetaw) and -pi (thetapi) values, neutrality tests, and mismatch distribution, suggested that the population of O. viverrini has expanded under the influence of purifying selection and selective sweep. The analysis of molecular variance (AMOVA) test revealed no significant genetic differences among the O. viverrini populations on opposite sides of the Mekong River. O. viverrini haplotypes occurred in multiple populations, and no distinct geographical clade. The star-like haplotype network confirmed a demographic expansion of the O. viverrini population. Overall, the genetic data from these populations suggested that the postulated existence of an O. viverrini species complex should be rejected. The bio-geographical diversity of O. viverrini populations should be explored further, using other appropriate markers and a wider range of samples from geographically different areas.

Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): A New Record of Small Liver Flukes In the Greater Mekong Sub-Region

Abstract Metacercariae, morphologically similar to those of small liver flukes, were found to parasitize red-tailed snakehead fish, Channa limbata, collected from the city of Vientiane, Lao Peoples Democratic Republic. Adult worms that were recovered from experimentally-infected hamsters showed characteristics distinctly different from Opisthorchis viverrini, but closely similar to Opisthorchis lobatus, which was first reported in poultry (Anas sp.) from Pakistan. The present study aimed to redescribe O. lobatus based on the adult worms recovered from experimentally-infected hamsters. Additionally, it aimed to document the genetic relationships among O. lobatus and other opisthorchiid liver flukes using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the internal transcribed spacer 2 (ITS2) region. DNA alignment of the O. lobatus and O. viverrini COI partial sequences (330 bp) showed 3.03% fixed differences (2.72% of amino acids changed) while the ITS2 region (350 bp) indicated a 0.86% difference for nucleotides. Species boundaries between the 2 parasites were determined by neighbor-joining analysis using the molecular sequence data. The phenogram confirmed that O. lobatus was distinctly different from O. viverrini, representing the first reported instance of O. lobatus in the Greater Mekong Sub-region (GMS) and the first record of C. limbata as the second intermediate host of a small liver fluke. Questions regarding human infection and the extent of the geographic distribution of these species should be investigated further.

Relationships of Forms within the Bactrocera tau (Walker) (Diptera: Tephritidae) Taxon Based on Heat Shock Protein 70 Cognate Sequences

Abstract A new molecular marker has been developed to clarify the status and systematic relationships of forms within the Bactrocera tau (Walker) taxon. The B. tau taxon, previously described as a widely distributed species, has recently been subdivided into forms A, B, C, D, E, F, G, and I based on host-plant preferences, cytological differences and external morphologies. This new molecular marker is derived from the sequence of the heat shock protein 70 cognate gene Bthsc1. Fragments of this gene were analyzed from B. tau individuals representing each of the different forms. Patterns of sequence variation revealed that the average genetic distance measurement within the B. tau form A is significantly smaller compared with the other B. tau forms. Unweighted pair-group method with arithmetic average and neighbor joining analyses both indicated that B. tau form A individuals derived from various geographical populations may be reliably separated from other forms B, C, D, E, F, G, and I. Our results also show that the Bthsc1 marker may successfully resolve other relationships among these forms. For example the B. tau forms B, F, and G, which are monophagous on related host plants, also cluster together as a closely related group.

A molecular phylogeny of Asian species of the genus Metagonimus (Digenea)—small intestinal flukes—based on representative Japanese populations

Metagonimus Katsurada, 1912 is a genus of small intestinal parasites. The genus comprises eight species, primarily from far-eastern Asia, with two exceptions reported from Europe. Metagonimus yokogawai, the most widespread species, is the main agent responsible for the intestinal disease, metagonimiasis, in Japan and some other East Asian countries. On the basis of the ratio of the size of the ventral and oral suckers, Metagonimus has traditionally been morphologically divided into two groups; however, the genus has not been extensively studied using molecular data. To reveal phylogenetic relationships within Metagonimus based on molecular data, we analyzed six of the seven species present in Asia using samples collected in central Japan. Maximum likelihood and Bayesian analyses of a combined 28S ribosomal DNA (rDNA), internal transcribed spacer 2 (ITS2), and mitochondrial cox1 gene sequence dataset separated the six species into two well-supported clades. One clade comprised M. yokogawai, M. takahashii, M. miyatai, and M. hakubaensis, whereas the other consisted of M. otsurui and M. katsuradai. Genetic distances calculated from 28S rDNA and ITS2 nucleotide sequences and a comparison of the predicted amino acid sequences of cox1 gene suggested that M. otsurui and M. katsuradai may have diverged recently. None of the four main morphological characters used to delimit species of Metagonimus (i.e., sucker ratio, positions of the uterus and testes, and distribution of vitelline follicles) was consistent with the distribution of species in the molecular tree.

Is Opisthorchis viverrini an avian liver fluke

Recently, in the Journal of Helminthology (May 2013), Dao et al. reported that Opisthorchis viverrini-like flukes were found in the bile duct of domestic ducks in Vietnam. They stated that this is the first record of Opisthorchis sp. in birds in Vietnam. However, three Opisthorchis species--O. cheelis, O. longissimus and O. parageminus--in birds in Vietnam were described by Le in 2000. Amongst these, O. parageminus was first reported, by Oshmarin in 1970, as a new Opisthorchis species found in domestic ducks (Anas platyrhynchos) in Vietnam. Morphologially O. viverrini-like flukes described by Dao et al. are much more similar to O. parageminus than to O. viverrini. The phylogenetic trees of internal transcribed spacer 2 (ITS2) and cytochrome oxidase 1 (CO1) gene sequences also showed that the O. viverrini-like liver flukes from domestic ducks were closer to O. lobatus than to O. viverrini. Therefore, O. viverrini-like liver flukes reported by Dao et al. (2013) are most likely to be O. parageminus.

Cryptic lineage diversity in the zoonotic pathogen Angiostrongylus cantonensis

Delimitation of species is still a necessity among parasitic pathogens especially where morphological characters provide limited discernibility. Identification of cryptic lineages (independently evolving lineages that are morphologically similar) is critical as there could be lineage-specific traits that are of epidemiological importance. Angiostrongylus cantonensis is a zoonotic pathogen that can cause eosinophilic meningitis in humans. Recent reports of single marker sequence divergence hint at the potential for cryptic diversity in this lungworm. However, to definitively address if single marker divergence corresponds to independent evolving lineages, a multilocus approach is necessary. Using multilocus data, our goal was to determine if there were cryptic lineages within Thailand, a country plagued by several outbreaks and isolated cases of A. cantonensis infection. We analyzed the genetic structure of A. cantonensis samples collected from snails, Achatina fulica, across provinces in Thailand. Multilocus data (mitochondrial sequence data and 12 nuclear microsatellites) and individual based analyses were used to test for cryptic lineages. We found strong linkage disequilibrium patterns between mitochondrial haplotypes and nuclear-identified genetic clusters. There were clearly two divergent and independent clades. Moreover, within each clade, the data suggested additional substructure where individual provinces were likely to harbor unique genetic clusters. The results indicate there are at minimum two and possibly up to eight cryptic lineages within the assumed single species of A. cantonensis. Importantly, the two main clades do not show geographic affiliation and can be found in sympatry. With recent studies highlighting A. cantonensis strain diversity in pathogenicity and infectivity, it will be important to determine if these critical epidemiological traits are associated with specific lineages.