Artyfechinostomum sufrartyfex Trematode Infections in Children, Bihar, India.

Eating raw or insufficiently cooked mollusks is a known risk factor for human echinostomiasis. We confirmed identification of Artyfechinostomum sufrartyfex trematodes as the causative agent of disease among 170 children in northern Bihar, India. We also identified the snail Pila globosa as a potential source of infections in the study area.

Amphistomes.

Amphistomes, commonly referred to as 'stomach' or 'rumen' flukes because of the localization of these flukes in the stomach of ruminants, are digenetic trematodes distinguished by the absence of an oral sucker and the position of the ventral sucker or acetabulum at the posterior end of the body. The body is characterized by leaf-like fleshy structure, pink or red in colour with a large posterior sucker. Amphistomes are an important group of parasites since they cause 'amphistomiasis' (variously known as paramphistomosis/amphistomosis), a serious disease of great economic importance in ruminants worldwide. These parasites have a broad spectrum of definitive hosts together with a wide geographical distribution. Though, they form a continuous evolutional lineage from fishes to mammals, amphistomes mainly inhabit the rumen and reticulum of ruminant mammals, while some species occur in the large intestine or parenteric sites of ruminants, pigs, equines and man.

Biological significance of phosphoenolpyruvate carboxykinase in a cestode parasite, Raillietina echinobothrida and effect of phytoestrogens on the enzyme from the parasite and its host, Gallus domesticus.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with K mapp of 46.9 and 22.9 µ m, respectively, for phosphoenolpyruvate and K mapp of 15.4 µ m for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.

Trematode Fluke Procerovum varium as Cause of Ocular Inflammation in Children, South India.

Trematodes are recognized as a group of emerging parasites in tropical countries. We identified a trematode as a cause of ocular granulomas that developed in children who bathed in ponds or rivers in South India. DNA was isolated from patients' surgically excised granulomas and from the trematode cercariae (larvae) released by the snail Melanoides tuberculata in water in which the children bathed. Real-time and conventional PCRs were performed that targeted ribosomal DNA regions spanning the internal transcribed spacer 2 and 28S sequences of this trematode. The PCR-amplified products were subjected to bidirectional sequencing. Analysis of sequences for the granuloma samples and the trematode cercariae showed maximum sequence similarity with Procerovum varium (family Heterophyidae). Our results confirmed the etiology of the ocular infection, implicating snail vectors as environmental risk factors for ocular parasitosis.

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations.

Biocomputational analysis of phosphoenolpyruvate carboxykinase from Raillietina echinobothrida, a cestode parasite, and its interaction with possible modulators.

Phosphoenolpyruvate carboxykinase (PEPCK) involved in gluconeogenesis in higher vertebrates opposedly plays a significant role in glucose oxidation of the cestode parasite, Raillietina echinobothrida. Considering the importance of the enzyme in the parasite and lack of its structural details, there exists an urgent need for understanding the molecular details and development of possible modulators. Hence, in this study, PEPCK gene was obtained using rapid amplification of cDNA ends, and various biocomputational analyses were performed. Homology model of the enzyme was generated, and docking simulations were executed with its substrate, co-factor, and modulators. Computer hits were generated after structure- and ligand-based screening using Discovery Studio 4.1 software; the predicted interactions were compared with those of the existing structural information of PEPCK. In order to evaluate the docking simulation results of the modulators, PEPCK gene was cloned and the overexpressed protein was purified for kinetic studies. Enzyme kinetics and in vitro studies revealed that out of the modulators tested, tetrahydropalmatine (THP) inhibited the enzyme with lowest inhibition constant value of 93 nm. Taking the results together, we conclude that THP could be a potential inhibitor for PEPCK in the parasite.

Molecular phylogeny of Cyclophyllidea (Cestoda: Eucestoda): an in-silico analysis based on mtCOI gene.

Order Cyclophyllidea (of cestode platyhelminths) has a rich diversity of parasites and includes many families and species that are known to cause serious medical condition in humans and domestic and wild animals. Despite various attempts to resolve phylogenetic relationships at the inter-family level, uncertainty remains. In order to add resolution to the existing phylogeny of the order, we generated partial mtCO1 sequences for some commonly occurring cyclophyllidean cestodes and combined them with available sequences from GenBank. Phylogeny was inferred taking a total 83 representative species spanning 8 families using Bayesian analysis. The phylogenetic tree revealed Dilepididae as the most basal taxon and showed early divergence in the phylogenetic tree. Paruterinidae, Taeniidae and Anoplocephalidae showed non-monophyletic assemblage; our result suggests that the family Paruterinidae may represent a polyphyletic group. The diverse family Taeniidae appeared in two separate clades; while one of them included all the members of the genus Echinococcus and also Versteria, the representatives of the genera Taenia and Hydatigera clubbed in the other clade. A close affinity of Dipylidiidae with Taenia and Hydatigera was seen, whereas existence of a close relationship between Mesocestoididae and Echinococcus (of Taeniidae) is also demonstrated. The crown group comprised the families Anoplocephalidae, Davaineidae, Hymenolepididae and Mesocestoididae, and also all species of the genus Echinococcus and Versteria mustelae; monophyly of these families (excepting Anolplocephalidae) and the genus Echinococcus as well as its sister-taxon relation with V. mustelae is also confirmed. Furthermore, non-monophyly of Anoplocephalidae is suggested to be correlated with divergence in the host selection.

Differential diagnosis and molecular characterization of Hymenolepis nana and Hymenolepis diminuta (Cestoda: Cyclophyllidea: Hymenolepididae) based on nuclear rDNA ITS2 gene marker.

Given the widespread distribution and medical implication of members of the genus Hymenolepis, specific identification of the aetiological agent becomes imperative. For precise diagnosis of the species, molecular techniques such as PCR and RFLP of the nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) gene marker were carried out. The results showed distinct restriction patterns for both Hymenolepis nana and Hymenolepis diminuta when digested with either of the enzymes RsaI, HaeIII or HhaI. The annotated rDNA-ITS2 sequences from the two species revealed differences in the length; the folded secondary structure also depicted clear demarcation between the two species with variations in length of the helices, pyrimidine-pyrimidine mismatches and sites where motifs occur. In phylogenetic analysis of the evolutionary relationship between the two species as well as with other members of the family Hymenolepididae, the species causing human hymenolepiasis were found to be distantly related as they diverged independently from the ancestral lineage.

Differential kinetics at PK/PEPCK branch point in the cestode, Raillietina echinobothrida.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.

Secondary Structure Analysis of Fasciola from Semi-wild Ruminants of Northeast India.

PURPOSE: The objective of this study is to study the secondary structure analysis of Fasciola flukes from a rare mithun host from Manipur. Fascioliasis, a neglected tropical trematodiasis, is poorly studied in India and is widely believed to be predominantly caused by F. gigantica. Through this study, we want to assess the flukes from the rare semi-wild ruminants of Northeast India. This study is important as the mithun population is semi-wild and its population is declining in Manipur. METHODS: Sample collected from the difficult and challenging terrain of Northeast India. The sample was collected from mithun and observed under the microscope. DNA was isolated, sequenced, and analyzed using various bioinformatics tools. The secondary structure analysis of the Internal Transcribed Spacer 2 (ITS2) region was also performed. RESULTS: The secondary structure species tree corroborated the Bayesian inference and, hence, strengthened the phylogeny reconstructed. The annotated ITS2 sequence and RNA secondary of the Manipur isolate displayed the typical four-helix or four-domain model. Helix III reveals the presence of the UGGU motif with other deviations like UGG and GGU. CONCLUSION: This is an in-depth analysis of the secondary structure of Fasciola species. The present study has demonstrated the usefulness of ITS2 and its secondary structures for characterizing parasites. The information on fascioliasis in the mithun's population presents itself useful with regards to their conservation strategy as their populations in both Manipur and Nagaland are dwindling.

Utility of divergent domains of 28S ribosomal RNA in species discrimination of paramphistomes (Trematoda: Digenea: Paramphistomoidea).

Among the digenetic trematodes, paramphistomes are known to be the causative agent of "amphistomiasis" or the stomach fluke disease of domestic and wild animals, mainly ruminants. The use of 28S (divergent domains) and 18S rRNA for phylogenetic inference is significantly warranted for these flukes since it is as yet limited to merely the exploration of the second internal transcribed spacer (ITS2) region. The present study intended to explore the divergent domains (D1-D3) of 28S rRNA and simultaneously equate the phylogenetic information with 18S rRNA in paramphistomes. Divergence of the 28S rRNA domains was evident amongst the divergent (D) domains, where D1 domain emerged as the most variable and D2, the most robust domain, since the latter could provide a higher resolution of the species. D2 was the only domain that comprised compensatory mutations in the helices of its structural constraints; this domain is thus well suited for species distinction and may be considered a potential DNA barcode complementary to mitochondrial DNA. 28S (D1 + D2 + D3) rRNA provided a significant resolution of the taxa corroborating with the taxonomy of these flukes and thus proved to be more robust as a phylogenetic marker for lower levels than 18S rRNA. Phylogenetic inferences of paramphitomes are still scarcely explored; additional data from other taxa belonging to this family may estimate better the biodiversity of these flukes.

Correlating early evolution of parasitic platyhelminths to Gondwana breakup.

Investigating patterns and processes of parasite diversification over ancient geological periods should involve comparisons of host and parasite phylogenies in a biogeographic context. It has been shown previously that the geographical distribution of host-specific parasites of sarcopterygians was guided, from Palaeozoic to Cainozoic times, mostly by evolution and diversification of their freshwater hosts. Here, we propose phylogenies of neobatrachian frogs and their specific parasites (Platyhelminthes, Monogenea) to investigate coevolutionary processes and historical biogeography of polystomes and further discuss all the possible assumptions that may account for the early evolution of these parasites. Phylogenetic analyses of concatenated rRNA nuclear genes (18S and partial 28S) supplemented by cophylogenetic and biogeographic vicariance analyses reveal four main parasite lineages that can be ascribed to centers of diversity, namely Australia, India, Africa, and South America. In addition, the relationships among these biogeographical monophyletic groups, substantiated by molecular dating, reflect sequential origins during the breakup of Gondwana. The Australian polystome lineage may have been isolated during the first stages of the breakup, whereas the Indian lineage would have arisen after the complete separation of western and eastern Gondwanan components. Next, polystomes would have codiverged with hyloid sensu stricto and ranoid frog lineages before the completion of South American and African plate separation. Ultimately, they would have undergone an extensive diversification in South America when their ancestral host families diversified. Therefore, the presence of polystome parasites in specific anuran host clades and in discrete geographic areas reveals the importance of biogeographic vicariance in diversification processes and supports the occurrence and radiation of amphibians over ancient and recent geological periods.

Secondary structure analysis of ITS2 in the rDNA of three Indian paramphistomid species found in local livestock.

Of paramphistomid trematodes, three species viz., Homalogaster paloniae, Calicophoron calicophorum and Orthocoelium streptocoelium are commonly prevalent in bovine hosts in Northeast India. The aim of the present study was to genetically characterise these species using rDNA second internal transcribed spacer (ITS2) so as to supplement the morphological criteria substantiated by molecular findings. The annotated ITS2 region from H. paloniae, C. calicophorum and O. streptocoelium were found to be 289 bp, 288 bp and 288 bp long, respectively. On comparison, the Indian isolates of the three species were observed to have a maximum identity of 99% with each of their respective counterparts from Japan. The secondary structure models were inferred using minimum free energy modelling algorithms. The paramphistomes displayed the typical four helix ITS2 secondary structure and differed from each other due to minor nucleotide differences. The consensus ITS2 secondary structure model revealed the presence of conservative motifs GACGAGGGUG and GCGGUAGAGUC in helix III. Monophyly is well supported for a clade consisting of the Japanese and Indian paramphistomes with significant bootstrap values.

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 (

De novo genome and transcriptome analyses provide insights into the biology of the trematode human parasite Fasciolopsis buski.

Many trematode parasites cause infection in humans and are thought to be a major public health problem. Their ecological diversity in different regions provides challenging questions on evolution of these organisms. In this report, we perform transcriptome analysis of the giant intestinal fluke, Fasciolopsis buski, using next generation sequencing technology. Short read sequences derived from polyA containing RNA of this organism were assembled into 30,677 unigenes that led to the annotation of 12,380 genes. Annotation of the assembled transcripts enabled insight into processes and pathways in the intestinal fluke, such as RNAi pathway and energy metabolism. The expressed kinome of the organism was characterized by identifying all protein kinases. A rough draft genome assembly for Fasciolopsis buski is also reported herewith with SRA accessions for crosschecking the findings in the analyzed transcriptome data. Transcriptome data also helped us to identify some of the expressed transposable elements. Though many Long Interspersed elements (LINEs) were identified, only two Short Interspersed Elements (SINEs) were visible. Overall transcriptome and draft genome analysis of F. buski helped us to characterize some of its important biological characteristics and provided enormous resources for development of a suitable diagnostic system and anti-parasitic therapeutic molecules.

Effect of isoflavone from Flemingia vestita (Fabaceae) on the Ca2+ homeostasis in Raillietina echinobothrida, the cestode of domestic fowl.

The alcoholic crude root-peel extract of Flemingia vestita and its major isoflavone, genistein, have been shown to have a vermifugal/vermicidal effect by causing a flaccid paralysis accompanied by alterations in the structural architecture of the tegumental interface and metabolic activity in Raillietina echinobothrida, the cestode of domestic fowl. In the present study, the crude root-peel extract and pure genistein were tested in vitro with respect to Ca2+ homeostasis and the occurrence of some metal ions was detected in the parasite. Live cestodes were incubated in pre-defined concentrations of the crude root-peel extract, genistein and praziquantel (as reference drug), till the paralysis time with simultaneous maintenance of respective controls. In the parasite tissue, a significant amount of Ca2+ (approximately 400 microg/g dry tissue wt) was found to be present besides magnesium, iron, zinc, lead and chromium, whilst manganese, cadmium and nickel were below the level of detection. The Ca2+ concentration was decreased significantly by 39%-49%, in the parasite tissue exposed to the test materials in comparison to the respective controls. There was also an increase in Ca2+ efflux by 91%-160% into the culture medium under similar treatments. The changes in Ca2+ homeostasis may be related to the rapid muscular contraction and consequent paralysis in the parasite due to the anthelmintic stress caused by the phytochemicals of F. vestita.

Helminth parasite spectrum of fishes in Meghalaya, Northeast India: a checklist.

Fish constitute a major component of diet for the people of Northeast India and they are extensively used as a protein-rich food for human consumption. The present studies incorporate the spectrum, composition and diversity of the parasitic species in freshwater fishes in Meghalaya, Northeast India, with a view to identifying the species recovered by morphological criteria based on light microscopy. The collection sites included sixteen foci from eleven districts of Meghalaya for parasites occurring in the common food fishes (Cypriniformes, Channiformes, Silurformes, Symbranchiformes and Anguilliformes). The helminth parasite spectrum recovered from the various piscine host species in the study area comprised of a total of 19 taxa: 2 monogenean, 8 trematode (4 adult and 4 metacercarial stages), 12 cestode (11 adult and a metacestode stage), 6 nematode (3 adult and 3 larval stages) and a single acanthocephalan species. A checklist of the parasite species with short remarks for each is provided herein.

Utilizing ribosomal DNA gene marker regions to characterize the metacercariae (Trematoda: Digenea) parasitizing piscine intermediate hosts in Manipur, Northeast India.

Freshwater fishes in Manipur, Northeast India frequently harbour several types of metacercariae, which based on morphological criteria were identified as Clinostomoides brieni, Euclinostomum heterostomum (Clinostomidae) and Polylekithum sp. (Allocreadiidae). Molecular techniques utilizing PCR amplification of rDNA regions of larger subunit (LSU or 28S), smaller subunit (SSU or 18S) and inter transcribed spacers (ITS1, 2) were used for molecular characterization of these types. Sequences generated from the metacercariae were compared with their related sequences available in public databases; an analysis of the identity matrices and phylogenetic trees constructed was also carried out, which confirmed their identification. Similarly, the sequences generated from Polylekithum sp. were found to be highly similar to the species of the same genus. The rDNA ITS2 secondary structure provided additional confirmation of the robustness of the molecular marker as a tool for taxon-specific characterization.

Animal Fascioliasis: Perspectives from high altitudinal regions.

The parasitic flukes of the genus Fasciola (Platyhelminthes: Trematoda: Digenea) cause fascioliasis or liver-rot disease in ruminant livestock in tropical and sub-tropical regions of the world. Classically, two species of Fasciola- F. hepatica and F. gigantica, are universally recognized as taxonomically valid species. Our survey studies on ovid and bovid animals including yak and mithun from high altitudinal mountainous regions in Northeast India revealed the occurrence of Fasciola gigantica and also Fasciola sp.- an intermediate form, at altitudes between 5000 and 14,085 feet above sea level (asl). Two morphotypes- F. hepatica - like and F. gigantica - like, of Fasciola species were reported from the high altitudinal areas of Northeast India; most of these locales constitute new-locality and first records for the occurrence of these liver flukes.