PCR-based molecular characterization and insilico analysis of food-borne trematode parasites Paragonimus westermani, Fasciolopsis buski and Fasciola gigantica from Northeast India using ITS2 rDNA.

Food-borne fluke infections/trematodiases are emerging as a major public health problem worldwide with over 40 million people affected and over 10% of world population at risk of infection. The major concentration of these infections is in Southeast Asian and Western Pacific Regions, where the epidemiological factors (including the prevalent socio-cultural food habits) are conducive for transmission of these infections. The preponderance of these infections is usually in food deficit poor communities that lack access to proper sanitary infrastructure. While targeting health for all, especially the poor rural tribal communities, it is imperative to take these infections into account. Bayesian analysis phylogeny of food-borne trematode parasites under study showed that they are closely related phylogenetic groups. To focus the control strategies at the target populations, the aim of the present study was to establish molecular methods for accurate discrimination between common food-borne trematodes parasites Paragonimus (lung fluke), Fasciolopsis (giant intestinal fluke) and Fasciola (liver fluke), the infections of which commonly prevail in NE India. In the first step, we amplified and sequenced the second internal transcribed spacer (ITS2) region of ribosomal DNA, utilizing nucleotide differences between the multiple sequence alignments of the parasites under study. Based upon the differences in nucleotide sequences of conserved regions, we designed species-specific primers that can unequivocally discriminate one species from another. ITS2 sequence motifs allowed an accurate in-silico distinction of the trematodes. The data indicate that ITS2 motifs (≤ 50 bp in size) can be considered promising tool for trematode species identification. Using molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships with various isolates of several trematode species have been discussed. The present results suggest that the ITS2 specific primers can be used for epidemiological investigations of the prevalence of trematodiasis.

Morphological and molecular identification of the metacestode parasitizing the liver of rodent hosts in bamboo growing areas of mizoram, northeast India.

In Mizoram (Northeast India), rodent outbreaks are known to occur periodically with the onset of bamboo flowering causing a tremendous destruction to food grains and as per the folk belief, often resulting in famine. In an exploratory survey of rodent pests in bamboo growing atreas for their helminth parasite spectrum, metacestodes of tapeworms were frequently encountered infecting the liver lobes and body cavity of the host. The morphological criteria were found to be closely consistent with the metacestode of Taenia species. In molecular characterization of the parasite, the ribosomal DNA (ITS1, ITS2) and mitochondrial COI were amplified and sequenced. Based upon both morphological data and molecular analysis using bioinformatic tools, the metacestode is identified as confirmed to be representing Cysticercus fasciolaris. The adult form of which (Taenia taeniaeformis) commonly occurs in felid and canid mammalian hosts.

Phylogenetic reconstruction using secondary structures and sequence motifs of ITS2 rDNA of Paragonimus westermani (Kerbert, 1878) Braun, 1899 (Digenea: Paragonimidae) and related species.

BACKGROUND: 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, not found in the primary sequence. In several mountainous regions of Northeastern India, foci of Paragonimus (lung fluke) infection reportedly involve species that are known to prevail in neighbouring countries. The present study was undertaken to demonstrate the sequence analysis of the ribosomal DNA (ITS2) of the infective (metacercarial) stage of the lung fluke collected from the edible crab hosts that are abundant in a mountain stream of the area (Miao, Changlang District in Arunachal Pradesh) and to construct its phylogeny. Using the approach of molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships of the various isolates of Paragonimus species that are prevalent in the neighbouring Near-eastern countries have been discussed. RESULTS: Initially, ten predicted RNA secondary structures were reconstructed and the topology based only on the predicted RNA secondary structure of the ITS2 region resolved most relationships among the species studied. We obtained three similar topologies for seven species of the genus Paragonimus on the basis of traditional primary sequence analysis using MEGA and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics; each data partition was allowed to have a different evolution rate. Paragonimus westermani was found to group with P. siamensis of Thailand; this was best supported by both the molecular morphometrics and combined analyses. P. heterotremus, P. proliferus, P. skrjabini, P. bangkokensis and P. harinasutai formed a separate clade in the molecular phylogenies, and were reciprocally monophyletic with respect to other species. ITS2 sequence motifs allowed an accurate in-silico distinction of lung flukes. CONCLUSION: Data indicate that ITS2 motifs (