Molecular characterization of Opisthorchis noverca (Digenea: Opisthorchiidae) based on nuclear ribosomal ITS2 and mitochondrial COI genes.

Opisthorchiasis is a public health problem in South-East Asian countries and Eastern Europe. The infection implicates mainly two species of Opisthorchis, namely O. viverrini and O. felineus, that occur mostly in fish-eating mammals and humans, although there are rare reports of human cases involving two other species, O. noverca and O. guayaquilensis. Opisthorchis noverca has been reported frequently in dogs and pigs from the Indian subcontinent, with rare reports from cattle and human subjects. With a view to supplementing morphology-based identification of this species, the present study aimed to provide molecular characterization of O. noverca, using rDNA internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase I (mt COI) markers so as to determine its genetic correlation with other species of Opisthorchiidae, and also to generate a taxon-specific molecular marker based on the ITS2 region. The phylogenetic relationship between O. noverca and other species of the genus was determined using molecular sequence data. To strengthen the result, secondary structure sequence analyses of ITS2 with hemi-compensatory base changes (hCBCs), and amino acid sequence analyses, were also evaluated. Our results confirm that O. noverca is a distinct and valid species.

Crustacean-borne infections with microphallid metacercariae (Digenea: Microphallidae) from focal areas in Meghalaya, north-east India.

During a survey of edible Crustacea for recovery of infective stages (metacercariae) of potential helminthozoonoses of trematode origin in north-east India, the crab species Barytelphusa lugubris mansoniana, collected from suspected foci of lungfluke infection in Meghalaya and Assam, was found to harbour metacercarial cysts that were different from the earlier reported infection, in which the lungfluke Paragonimus was confirmed to be implicated. Using morphological criteria, this metacercaria was identified as Microphallus indicus Mukherjee & Ghosh, 1967 of the trematode family Microphallidae. The present study extends the previous work by providing molecular characterization of this parasite using ribosomal internal transcribed spacer regions (rDNA ITS1 and ITS2) and the partial large ribosomal subunit DNA, lsr. These target regions were amplified by polymerase chain reaction (PCR) using trematode universal primers and sequenced. In BLAST analysis the query sequences were found close to members of Microphallidae and closest to the genus Microphallus.

Use of sequence motifs as barcodes and secondary structures of internal transcribed spacer 2 (ITS2, rDNA) for identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae).

Most phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics that give "morphological" information which is not found in the primary sequence. Also DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat are useful for identification of trematodes. The species of liver flukes of the genus Fasciola (Platyhelminthes: Digenea: Fasciolidae) are obligate parasitic trematodes residing in the large biliary ducts of herbivorous mammals. While Fasciola hepatica has a cosmopolitan distribution, the other major species, i.e., F. gigantica is reportedly prevalent in the tropical and subtropical regions of Africa and Asia. To determine the Fasciola sp. of Assam (India) origin based on rDNA molecular data, ribosomal ITS2 region was sequenced (EF027103) and analysed. NCBI databases were used for sequence homology analysis and the phylogenetic trees were constructed based upon the ITS2 using MEGA and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics and revealed a close relationship with isolates of F. gigantica from China, Indonesia and Japan, the isolate from China with significant bootstrap values being the closest. ITS2 sequence motifs allowed an accurate in silico distinction of liver flukes. The data indicate that ITS2 motifs (

Molecular identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae) based on the ribosomal internal transcribed spacer regions.

The species of liver flukes of the genus Fasciola (Platyhelminthes: Digenea: Fasciolidae) are obligate parasitic trematodes residing in the large biliary ducts of herbivorous mammals. While Fasciola hepatica has a cosmopolitan distribution, the other major species, i.e., Fasciola gigantica is reportedly prevalent in the tropical and subtropical regions of Africa and Asia. To determine the phylogenic location of Fasciola sp. of Assam (India) origin based on rDNA molecular data, ribosomal ITS regions were sequenced and compared with other species of trematodes in the family Fasciolidae. NCBI databases were used for sequence homology analysis using BLAST and ClustalW programs. The phylogenetic trees constructed based upon the ITS (1 and 2) sequences revealed a close relationship with isolates of F. gigantica from China, Indonesia, Japan, Egypt, and Zambia, the isolate from China with significant bootstrap values being the closest. Using the novel approach of molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships of the various isolates of fasciolid species have also been discussed. While comparing ITS1, the sequence of another Indian isolate designated as F. gigantica (accession no. EF198867) showed almost absolute match with F. hepatica. Hence, this particular isolate should be identified as F. hepatica and not F. gigantica.