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 (θw) and -π (θπ) 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.     

Sero-epidemiological survey of gnathostomiasis in Lao PDR

While human gnathostomiasis cases have been reported sporadically in Lao PDR since 1975, little is known about the disease in this country. We aimed to investigate sero-prevalence of gnathostomiasis and Gnathostoma species in Lao PDR. One village each in the north, central and south regions of Lao PDR was selected as the study sites. Overall, 125 (29.8%) of 420 sera from the randomly selected participants were sero-positive by immunoblot technique, with anti-Gnathostoma IgG antibody against the 24 kDa fraction. The sero-prevalence was high in the central (47.1%) and south (38.6%), but low (3.6%) in the north. Risk factor analyses revealed that the consumption of raw/undercooked fish was significantly associated with Gnathostoma sero-positivity (95% CI 1.05–17.05, P = 0.042). The sero-positivity significantly increased with the age of the participants. Several fish, swamp eels, and frogs collected from central and southern Lao were infected with G. spinigerum advanced 3rd-stage larvae. Channa limbata (red-tailed snakehead fish) was identified as a natural second intermediate host of G. spinigerum. Eggs of G. spinigerum were found in dog feces collected in the south. Gnathostomiasis is endemic in central and southern Laos, so that preventive measures should be introduced for people living in these regions.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.     

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.     

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.     

Discovery of Opisthorchis lobatus (Trematoda: Opisthorchiidae): a new record of small liver flukes in the Greater Mekong Sub-region

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 People's 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.     

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.