Bikash Ranjan Giri

Resveratrol induced structural and biochemical alterations in the tegument of Raillietina echinobothrida

The root tuber of Carex species has been used as an anthelmintic medicine for intestinal helminthic infections in the Northeast region of India. The main compound present in the root tuber of the genus Carex is resveratrol. Therefore, the present study was conducted to evaluate the anthelmintic effects of resveratrol in Raillietina echinobothrida by using motility observation, electron microscopy, histochemical and biochemical analysis. Resveratrol causes complete inactivation and flaccid paralysis of the cestode, followed by death. The treated parasites also exhibited extensive distortion of the surface fine topography and decrease in the activities of major tegumental enzymes compared to that of control parasite. Ultrastructural alterations include changes in cell organelles present in the tegument and sub-tegumental cyton. Extensive alterations in the surface topography of the treated parasites resulted in a decrease in the activities of tegumental enzyme which suggest that, resveratrol may be useful as a therapeutic agent to treat cestode parasites.

Evidence of apoptosis in Raillietina echinobothrida induced by methanolic extracts of three traditional medicinal plants of Northeast India

The therapeutic benefits of medicinal plants in terms of anthelmintic properties are known since time immemorial in India, particularly among natives of the Northeast India. However, only sporadic and scarce reports on scientific validation of these plants are available. The present study was conducted on the cestode Raillietina echinobothrida, to establish whether the anthelmintic activity of Potentilla fulgens, Alpinia nigra and Millettia pachycarpa was mediated by apoptosis or not. Light microscopic observation following MTT assay revealed the highest percentage of inhibition of viability among the worms by methanol extract of M. pachycarpa (89.33%), followed by A. nigra (65%) and P. fulgens (37%). Ultrastructural observations revealed swelling of mitochondria, disruption of mitochondrial membrane, vacuolization of mitochondria, appearance of apoptotic bodies in the cytoplasm, disintegration of nuclear membrane and nucleolus were very common throughout the tegument. DAPI stained specimens showed typical morphology of apoptosis, like nuclear condensation and fragmentation in the extracts treated parasites. A decrease in mitochondrial membrane potential was also recorded in the treated groups. Confirmatory TUNEL assay and DNA fragmentation assay of the extracts treated parasites also confirmed the apoptotic nature of cell death and is concluded to be responsible for paralysis and death of the parasite.

Ultrastructural observations on Raillietina echinobothrida exposed to crude extract and active compound of Securinega virosa.

Securinega virosa has been used traditionally by the natives of Mizoram, India, to control intestinal worm infections. In the present study, the crude ethanol extract of the plant and its active component virosecurinine were tested in vitro on Raillietina echinobothrida to evaluate its potential anthelmintic efficacy and ultrastructural changes. The test parasites were exposed to different concentrations of the plant extract, active compound virosecurinine and reference drug praziquantel. Scanning and transmission electron microscopic observations on the paralyzed worms revealed wide scale destruction of the tegument with intense vacuolization of the syncytium and swellings of the basal lamina accompanied by deformities in the cell organelles. Extensive structural alteration of tegument indicates that the plant extract and its active component alter membrane permeability of the parasite leading to paralysis and subsequent death, as confirmed by in vitro tests.

Electron microscopic observations on the alterations of tegumental surface of Raillietina echinobothrida treated with root-peel extract of Potentilla fulgens.

Potentilla fulgens, a member of the family Rosaceae, has been known since ancient times in Northeast India for its antiparasitic, antitumour and antidiabetic properties. The crude ethanolic extract of the plant was assayed against Raillietina echinobothrida, the intestinal cestode parasite of domestic fowl to authenticate the putative anthelmintic efficacy and cestocidal potential in particular of the plant. Treatment of the worm in‐vitro with 25, 50 and 100 mg of crude extract per ml of phosphate buffered saline caused paralysis leading to death taking 4.02–4.20, 3.05–3.75 and 2.05–2.83 h respectively. Scanning electron microscopic observations on treated worms revealed deformation of surface architecture, particularly in scolex and surface microtriches. Severe distortion and disorganization of the tegumental and subtegumental layer along with cytoplasmic organelle was also observed under transmission electron microscope. The possible use of the plant as a potential anthelmintic against cestode parasite is discussed. Microsc. Res. Tech., 2012.

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.

Effects of Acacia oxyphylla and Securinega virosa on functional characteristics of Raillietina echinobothrida (Phylum: Platyhelminthes; Class: Cestoidea), a poultry cestode parasite

The histochemical distribution and quantification of alkaline phosphatase, acid phosphatase, and adenosine triphosphatase have been investigated in the adult cyclophyllidean cestode, Raillietina echinobothrida after treatment with crude ethanol extract of traditionally used medicinal plants Acacia oxyphylla and Securinega virosa. Estimation of free amino acid pool and vital trace elements like calcium and magnesium were also performed to study the effect of the plant extracts on the parasite. A decrease in the levels of phosphatases and trace elements was noticed in the treated parasites when compared to control groups. At the same time, a lot of disparity was noted in the free amino acid pool as the levels of many amino acids were seen to increase or decrease in relation to the levels observed in control parasites. Role of phyto-products in the changed physiology of cestode and their possible chemotherapeutic targets are discussed.

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.

Ultrastructural and biochemical alterations in rats exposed to crude extract of Carex baccans and Potentilla fulgens.

The use of plants as a source of medicine is an important component of the health care system in rural India. Carex baccans (Cyperaceae) and Potentilla fulgens (Rosaceae) have been known since ancient times in northeast India for their antitumor, antidiabetic, and antihelmintic properties. The present study was designed to determine the subacute toxicity profile of the root tuber extract of C. baccans and root-peel extract of P. fulgens in Wistar rats. The subacute oral toxicity was conducted using sublethal doses of 40, 50, 100, 150, 200, and 400 mgkg-1 body weights. Surface topographical and ultrastructural observations of liver and intestinal microvilli showed remarkable deformation and disruption, accompanied by quantitative changes in the liver enzymes, i.e., aspartate aminotransferase and alanine aminotransferase in comparison to those of the control group. Apoptotic cell death was observed in the liver cells of rats exposed to both of the plant extracts. A significant increase in splenic lymphocyte count was also observed in rats exposed to the highest concentration of both extracts. The results showed that consumption of the plant extracts at higher doses may cause toxicological effect if treatment continues for a long time.

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.

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.