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.

In vitro cestocidal activity of Persicaria hydropiper (L.) Delarbre, a traditionally used anthelmintic plant in India.

Persicaria hydropiper is a medicinal plant used for the treatment of helminth infections among the Naga people of India. To verify the traditional claim of the plant, an in vitro anthelmintic efficacy of the methanol extract of P. hydropiper leaves was investigated based on the paralytic and mortality effects on Raillietina echinobothrida, an intestinal cestode parasite of domestic fowl, following exposure to 10, 20 and 30 mg/ml concentrations of extract. The effects of extract were also studied on the body surface of the parasites by scanning electron microscopy (SEM). The in vitro results showed an efficacy that was dose-dependent. At the highest dose (30 mg/ml), mortality of parasites occurred in 4.79 ± 0.17 h, in comparison to control, where the parasites survived till 45.63 ± 0.18 h. The SEM observations of extract-treated parasites revealed notable impairment in scolex, with distorted suckers and eroded spines. Also, the tegument was observed to be shrunken with impaired microtriches. The results indicate that P. hydropiper leaves possess noteworthy anthelmintic efficacy and justify their use in traditional medicine against intestinal worms.

A kaempferol derivative isolated from Lysimachia ramosa (Wall ex. Duby) induced alteration of acetyl cholinesterase and nitric oxide synthase in Raillietina echinobothrida.

Lysimachia ramosa has been used as a traditional medicine among the tribal population of Meghalaya, northeast India, for the control of helminthosis. The anthelmintic efficacy of L. ramosa has been documented earlier. In the present study, the active compound from L. ramosa has been isolated and identified using mass and NMR spectra. It's in vitro anthelmintic activity was evaluated against Raillietina echinobothrida, one of the most pathogenic cestode of domestic fowl. The isolated active compound was characterized to be a kaempferol derivative which showed potent anthelmintic activity against R. echinobothrida by changing surface ultrastructure and also inhibiting the activity of two neurotransmitter enzymes: acetyl cholinesterase (AChE) and nitric oxide synthase (NOS), both of which are known to perform dynamic roles in the intracellular communication mediated through neuromuscular system. Motility reduction, deformation in the surface architecture, extensive ultrastructural alterations and reduced histochemical stain intensity in both AChE and NOS was observed in the treated parasites. Biochemical result also revealed alteration in the enzyme activities in the treated parasites. Further, depletion in the nitric oxide (NO) production in the bioactive component exposed tissues of R. echinobothrida was also detected. The results provided evidence that the bioactive compound could be further explored to control helminthosis at a large scale.

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.

Characterization of Clinostomum metacercariae using microscopic and molecular approaches

One of the fundamental aspects in understanding the biology, diversity and epidemiology of a parasite lies in its proper identification. In the present study, morphological and molecular characterization of Clinostomum metacercariae recovered from an ornamental fish, Trichogaster fasciata, was carried out in order to ascertain its identity. To serve the purpose, scanning electron micrographs and gene sequences for two commonly used molecular markers, i.e., nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) were obtained. The sequences were further used for generating similarity index matrix as well as inferring phylogenies. Light and electron microscopic observations on metacercariae of the parasite revealed that it belongs to the genus Clinostomum. Identification of the same up to the level of species was made possible through sequence and phylogenetic analyses. The ITS2 sequence analyses of our species (KX758630) showed similarity to unidentified Clinostomum sp. reported from Nigeria (KY865625) and China (KP110579), and C. tilapiae recorded from South Africa (KX034048) and Nigeria (KY649353). However, the CO1 gene analyses suggested it to be highly identical to C. philippinense and the same was also corroborated in the phylogenetic analysis. Thus, morphological and molecular characterization revealed that the recovered metacercariae belong to the species C. philippinense. Additionally, a brief description of secondary structures of ITS2 of various species of Clinostomum has also been discussed.

Molecular identification of two cestodes species parasitizing freshwater fishes in India.

In the present study, molecular identification of two species of cestodes, Lytocestus indicus and Senga lucknowensis infecting freshwater fishes Clarias magur and Channa punctata, respectively in Manipur is carried out. To ascertain the taxonomic status of these helminth parasites, 18S gene marker was used. Phylogenetic analysis of 18S of Lytocestus sp. showed that it claded with L. indicus from Indian Isolate with a sequence similarity index of 99%. In case of Senga sp., the phylogenetic analysis revealed that it formed a separate clade with S. lucknowensis and Senga vishakapatnamensis, and the sequence similarity index showed maximum homogeneity with S. lucknowensis i.e., 99.8%. Thus, molecular characterization revealed that the two species of cestodes belong to L. indicus and S. lucknowensis.

Biochemical and ultrastructural changes in Raillietina echinobothrida in vitro exposed to extract of Lysimachia ramosa.

Lysimachia ramosa (Primulaceae) is a traditionally used medicinal plant, leaves extract of which is being widely used by the Jaintia tribes of Meghalaya, India for controlling helminthiasis. Preliminary investigation carried out on helminth parasites revealed that the crude extract of the plant causes deformity in the surface topography leading to death of the parasites. Therefore, the present study was conducted to identify the specific fraction of the crude leaf extract of the plant responsible for cestocidal efficacy, through biochemical and ultrastructural studies in Raillietina echinobothrida exposed to crude extract and its different fractions namely hexane, chloroform, ethyl acetate and n-butanol. A dose dependent efficacy, with highest rate of mortality among n-butanol exposed parasites was recorded. The treated parasites exhibited complete erosion of microtriches from the tegument, disintegration of muscle bundles, cellular organelles, plasma membrane, nuclear membrane, nucleolus and vacuolization of mitochondria was also observed. Observations on histochemical distribution of some important tegumental enzymes like adenosine triphosphatase (ATPase), alkaline phosphatase (AlkPase), acid phosphatase (AcPase) and 5'Nucleotidase (5'-Nu) revealed a marked diminished stain intensity in the tegument of R. echinobothrida exposed to the crude extract and n-butanol fraction of the crude extract compared to the control. Highest reduction (77.93%) in the activity of ATPase was observed when the parasites exposed to 6 mg n-butanol fraction/ml of PBS. The results suggest that these enzymes act as target for anthelmintic stress caused by the phytochemicals present in the plant.

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.

Status of tick infections among semi-wild cattle in Arunachal Pradesh, India.

UNLABELLED: To explore the seasonal prevalence of tick infections in mithun (Bos frontalis) and yak (B. grunniens) a study was conducted in Papum Pare district, Kurung-Kumey district and West Kameng district of Arunachal Pradesh, India from December 2012 to November 2014. Adult ticks were collected from both the animals during the month of December 2012 to November 2014. Ectoparasites were identified on the basis of their morpho-anatomical features observed through light microscope. A total of nine different species of ectoparasites recovered belong to the family Ixodidae are Rhipicephalus (B.) microplus, R. (B.) geigy, Haemaphysalis davisi, H. darjeeling, H. longicornis, H. bispinosa, Ixodes acutitarsus, I. ricinus and Rhipicephalus sanguineus. Out of 129 mithun examined during the survey R. (B.) microplus and H. davisi showed 100% prevalence of infections throughout the year followed by H. longicornis (35.65%), H. bispinosa (30.23%), R. (B.) geigy (25.58%), I. acutitarsus (20.93%), H. darjeeling (7.75%), I. ricinus (1.55%) and Rhipicephalus sanguineus (0.77%). Similarly, all 21 yak examined revealed to be infected with R. (B.) microplus (100%) followed by I. acutitarsus (51.14%) and H. davisi (33.33%). In mithun the rate of infection due to all the nine ectoparasites rose to a peak during June to August. Except for the winter season, R. (B.) geigy occurred throughout the year. I. acutitarsus and H. darjeeling showed their occurrence throughout the year except during spring, however, I. ricinus and R. sanguineus occurs only during rainy season. KEY WORDS: Bos frontalis, B. grunniens, ecto-parasite, Arunachal Pradesh, India.

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.

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.

Praziquantel induced oxidative stress and apoptosis-like cell death in Raillietina echinobothrida.

Praziquantel (PZQ) is an anthelmintic drug used against trematode and cestode parasites of humans and veterinary animals. Since praziquantel was introduced as a broadspectrum anthelmintic, numerous studies described its successful use against helminth parasites, but its exact mechanism of action is feebly understood. Therefore, the present study was carried out to evaluate the possible role of PZQ induced oxidative stress in apoptosis-like cell death in the poultry tapeworm Raillietina echinobothrida. Parasite viability assay revealed a time-dependent reduction in the worm viability compared to the control. Transmission electron microscopy showed typical apoptotic features like condensed nucleus, damaged nuclear envelope and altered mitochondrial membrane in PZQ exposed parasites. Results revealed chromatin condensation and DNA fragmentation in PZQ exposed parasites. There was a notable decline in the level of glutathione and glutathione-s-transferase activity leading to the augmented generation of reactive oxygen species. This led to the alterations in the mitochondrial membrane potential with increased active caspase-3/7, confirms the involvement of mitochondria in the event. The present study suggests that PZQ exerts oxidative stress leading to apoptosis-like events in the parasites resulting their death.

Cysticercus fasciolaris infection induced oxidative stress and apoptosis in rat liver: a strategy for host-parasite cross talk.

Parasitic helminths have developed various strategies to induce or inhibit apoptosis in the cells of their host, thereby modulating the host's immune response and aiding dissemination to the host. Cysticercus fasciolaris, the larval form of Taenia taeniaeformis, parasitized different intermediate hosts like rats, rabbits, etc. and is cosmopolitan in distribution. In the present study, we have investigated host-parasite interactions and the resulting effect of C. fasciolaris in the liver of rat. Histology of the infected livers showed dilation and damages of hepatic cells near the parasite. Infected liver cells showed an increase in DNA fragmentation and chromatin condensation compared to the normal liver. Acridine orange and ethidium bromide dual staining revealed the presence of apoptotic cells in the infected liver. The decline in the mitochondrial membrane potential in the infected liver suggested that the observed apoptosis is mitochondria mediated. Occurrence of an elevated level of active executioner caspases 3/7 in the infected rat liver further confirms the occurrence of apoptosis. Different antioxidant enzymes were also evaluated and revealed a notable decline in the level of glutathione and glutathione-S-transferase activity leading to the augmented generation of reactive oxygen species. Results of the present study revealed that C. fasciolaris infection leads to apoptosis in the liver of rats which may be a surviving strategy for the parasitic larvae.

α-Viniferin and resveratrol induced alteration in the activities of some energy metabolism related enzymes in the cestode parasite Raillietina echinobothrida.

α-Viniferin (AVF) and its monomer resveratrol (RESV) are natural phytostilbenes produced by several plants in response to injury or under the influence of pathogens such as bacteria or fungi. Our earlier studies have revealed that both the compounds exert anthelmintic activity through alterations of cestode tegument and its associated enzymes. The present study investigates the effects of these phytochemicals on some energy metabolism related enzymes in the fowl tapeworm, Raillietina echinobothrida. The phytostilbenes AVF, RESV and the reference drug praziquantel (PZQ) were tested against some selected enzymes i.e., phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of R. echinobothrida. Exposure of the tapeworm to AVF, RESV and PZQ causes reduction in activity of PEPCK to the extent of 40.57/41.96, 24.58/23.75 and 41.11/13.47%, respectively, and LDH up to 48.95/16.25, 38.31/38.42 and 45.67/41.87%, respectively, at the time of paralysis. Whereas activity of MDH decreased by 34.22/37.7, 39.1/35.24 and 28.83/19.26%, respectively. Decrease in activities of LDH and MDH was also visible through histochemical observations. The results suggest that both the phytochemicals interfere with the energy transducing pathways by inhibiting the studied energy metabolism related enzymes of the parasite.

The seasonal incidence of parasitic helminth infection among the walking catfish, Clarias batrachus of Tripura, India

In the present study, a total of 868 walking catfish, Clarias batrachus, were collected during the period of April, 2012 to March, 2015, to evaluate the prevalence of helminth parasites from different parts of Tripura, Northeast India. Of these, 606 fish were found to be infected with one genus of trematode viz., Astiotrema, two genera of cestode viz., Lytocestus and Djombangia and one genus of nematode parasite viz., Anisakis. Of the three groups of parasites recovered, the major group infecting the host were found to be cestodes, followed by nematodes and trematodes. Seasonal studies show that the overall prevalence of the collected helminth parasites was highest during the postmonsoon season, followed by the monsoon and pre-monsoon seasons.

Filarial infections in semi-wild cattle of Northeast India: first record of Setaria digitata (von Linstow 1906) in Bos frontalis (Lambert 1804).

The occurrence of adult Setaria digitata, a filarial nematode of bovine host is reported for the first time from mithun (Bos frontalis) of Arunachal Pradesh, India. During December 2011 to March 2012, a thorough survey was undertaken to evaluate the incidence and intensity of helminth infections among semi-wild cattle in Northeast India. During the course of investigation, the relatively uncommon species of Setaria was encountered among two out of eleven mithun examined. Through light and scanning electron microscopy, the nematode is identified as S. digitata.

Apoptosis like cell death in Raillietina echinobothrida induced by resveratrol.

Northeast India is geographically nestled as one of the biodiversity hotspots, rich in traditionally used medicinal plants. Resveratrol, a naturally occurring phytoalexin found in berries, peanuts, grapes, red wine and also in numerous anthelmintic plants, has attracted wide interest because of its diverse pharmacological characteristics. Recently, anthelmintic potential of the compound is established. The present study was carried out to understand the possible mechanism of action of resveratrol on poultry tapeworm Raillietina echinobothrida. Resveratrol showed excellent cestocidal activity in a dose dependent manner as revealed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The progressive ultrastructural alterations followed by complete disruption of nuclear membrane, chromosomal condensation and in situ DNA fragmentation confirm the occurrence of apoptosis like cell death. Increased pro-apoptotic caspase activity and significant decreases in mitochondrial membrane potential in R. echinobothrida exposed to resveratrol confirm the involvement of mitochondria in the process of apoptosis.

Resveratrol- and α-viniferin-induced alterations of acetylcholinesterase and nitric oxide synthase in Raillietina echinobothrida.

Phytostilbenes, like resveratrol and α-viniferin, which occur mainly in the plants and belong to the families Cyperaceae, Vitaceae, and Gnetaceae are extensively popular for their medicinal and nutritional properties. In Northeast India, the Jaintia tribes consume these phytochemicals through aqueous extract of the medicinal plant Carex baccans to control helminthiasis. The present study aimed to investigate the inhibitory effect of the phytochemicals on neurotransmitters and its related enzymes in helminth parasite Raillietina echinobothrida. Viability of the parasites exposed to the phytostilbenes and extent of inhibition of cholinergic and nitrergic enzymes were evaluated in comparison to reference anthelmintic drug praziquantel and two known enzyme inhibitors, namely Nω-nitro-L-arginine and pyridostigmine. On exposure to resveratrol, α-viniferin, and reference drug praziquantel, the parasites ceased movement at 9.37, 11.38, and 0.24 h followed by death at 23.65, 34.13, and 1.87 h, respectively. Exposed parasites also showed a significant decrease in the activity of acetylcholinesterase (46.101, 65.935, and 63.645%) and nitric oxide synthase (61.241, 55.046, and 29.618%) in comparison to the controls. In addition, a decreased trend in nitric oxide (NO) level was also detected in the tissue of different phytochemical-exposed parasites compared to control. The present study suggests that anthelmintic potential of both the phytochemicals is mediated through inhibition of two vital enzymes which play diverse role in intracellular communications through neuromuscular system.

In vivo anthelmintic activity of Carex baccans and its active principle resveratrol against Hymenolepis diminuta.

Anthelmintic resistance against most of the commercial drugs is a great threat to humans as well as the veterinary live stocks. Hence, new treatment strategies to control helminth infections are essential at this hour. Carex baccans Nees has been traditionally used by Jaintia tribes in Northeast India to get rid of intestinal worm infections. Therefore, the present study was conducted to evaluate in vivo cestocidal activity of root tuber extract of C. baccans and its active component resveratrol against the zoonotic cestode Hymenolepis diminuta in the experimental model rat. The cestocidal activity was determined by monitoring the eggs per gram (EPG) counts in faeces of different treated groups. The result showed that the highest dose of the plant extract (50 mg/kg) and resveratrol (4.564 mg/kg body weight) has significant anthelmintic efficacy against H. diminuta. Crude extract of the plant as well as resveratrol reduced EPG count (56.012 and 46.049 %) and also resulted in decreased worm burden by 44.287 and 31.034 %, respectively. The efficacy of the crude extract and resveratrol can be compared to the reference drug praziquantel. The results exhibits considerable cestocidal potential of root tuber crude extract of C. baccans and resveratrol and justify its folklore use.

α-Viniferin-Induced Structural and Functional Alterations in Raillietina echinobothrida, a Poultry Tapeworm.

α-Viniferin, an active component of the plant Carex baccans L., is known for its anticancer, antidiabetic, and anti-inflammatory properties. In Northeast India, different tribes traditionally consume C. baccans to control intestinal helminth infections. Therefore, the present study was carried out to assess the extent of tegumental alteration caused by α-viniferin in Raillietina echinobothrida, a widely prevalent poultry helminth in northeast India. Helminths were exposed in vitro to various doses of α-viniferin (50, 100, and 200 µM/mL of physiological buffered saline) and their motility and mortality were recorded. Stereoscan observations on the parasite exposed to the active compound showed extensive distortion and destruction of the surface fine topography of the tegument compared with controls. The compound also caused extensive damage to the tegument by disintegration of microtriches, disorganization of muscle bundles, and loss of cellular organelles combined with distortion and disruption of the plasma membrane, nuclear membrane, nucleolus, mitochondrial membrane, and cristae. Histochemical and biochemical studies carried out parasites exposed to α-viniferin revealed a decline in the activity of vital tegumental enzymes like acid phosphatase, alkaline phosphatase, and adenosine triphosphatase. Extensive structural and functional alterations observed in the treated parasites are indicative of efficient cestocidal activity of the compound.