Gastrothylax Crumenifer Research Articles

Anthelmintic Activity of Foeniculum vulgare Seeds and Meta-Analysis of Some Other Medicinal Plants from Pakistan

Helminthiasis diseases caused by parasitic worms in animals have been a significant challenge worldwide, resulting in substantial economic losses and decreased productivity in the agricultural sector. The use of synthetic drugs to treat these infections has several drawbacks, including the development of drug resistance, high costs, and potential toxicities to both animals and humans. Therefore, researchers looked for other ways to solve the problem and found that medicinal plants might have anthelmintic activities because they have many secondary metabolites. These metabolites have demonstrated the potential to act as natural anthelmintics, making them attractive options for developing novel drugs. Firstly, an experiment was done to test the effectiveness of Foeniculum vulgare seed extract against Gastrothylax crumenifer. Subsequently, we employed a meta-analysis approach to identify plant species and compounds with the most promising anthelmintic activities. The methodology will involve a comprehensive search of various databases to identify relevant research articles on the anthelmintic activity of medicinal plants. The study found that Foeniculum vulgare seed extract was effective against Gastrothylax crumenifer. The study also identified the most active and promising medicinal plants that warrant further investigation for their potential to eradicate parasites. Furthermore, a meta-analysis revealed anthelmintic activity in 34 plants, with the Musaceae, Solanaceae, and Asteraceae families receiving the most extensive research. The findings have implications for developing cost-effective and safe anthelmintic treatments for animals.

Multi-detection for paramphistomes using novel manually designed PAR-LAMP primers and its application in a lateral flow dipstick (LAMP-LFD) system

Loop-mediated isothermal amplification (LAMP) has been applied for the detection of various parasites, and its application in lateral flow dipstick (LFD) can improve the convenience of point-of-care diagnosis. A novel PAR-LAMP probe and primers were designed by manual selection from a region of low variation in the ITS-2 DNA sequence. Up to six species of rumen fluke were detected by LAMP and LAMP-LFD in this study. Target specificity and sensitivity were tested, revealing a high target specificity (accuracy) and a low limit of detection (sensitivity). Different target sensitivities of paramphistome were presented, including 5 pg for Gastrothylax crumenifer and Carmyerius sp.; 1 pg for Fischoederius elongatus, Orthocoelium parvipapillatum, and O. dicranocoelium; and 0.1 pg for Paramphistomum epiclitum. LAMP-LFD can detect a paramphistome egg even in contaminated in feces that was spiked with the egg under laboratory conditions. In addition, natural paramphistome infection in cattle from Surat Thani and Khon Kaen provinces, Thailand, was evaluated by detection of egg contamination in fecal specimens using PAR-LAMP primers. The PAR-LAMP detection result was also statistically evaluated by microscopic examination of feces. This study presents the application of novel manually designed primers in a LAMP-LFD system for improving performance in detection and diagnosis assays for paramphistomosis.

Assessment of antioxidant enzymes during the development of the digenetic trematode parasite Gastrothylax crumenifer, infecting the rumen of Indian water buffalo, Bubalus bubalis.

The presence of antioxidant enzymes in helminth parasites is well known. These enzymes help the parasites to survive in their hosts by detoxifying host-generated reactive oxygen species (ROS). The literature survey reveals that most of the studies related to antioxidant enzymes in helminth parasites are restricted to the adult stage while the larval stages are neglected. The present investigation is designed to evaluate the level of antioxidant enzymes in the adult and larval stages of rumen-infecting paramphistome parasites, Gastrothylax crumenifer. The larval stages include 0-day eggs, 4-day eggs, and eggs containing mature miracidia, cercariae, and metacercariae. Antioxidant enzyme assays were performed using standard assay protocols. Our findings revealed an increasing pattern in the level of Glutathione-S-Transferase (GST), Superoxide Dismutase (SOD), Glutathione Reductase (GR), and Glutathione Peroxidase (GPx) antioxidant enzymes during the development from 0-day eggs to the adult stage. Overall analysis shows that adult worms have higher antioxidant enzyme activity as compared to the larval stages, indicating that adult flukes are more adapted to oxidative stress. It can be concluded that the miracidia, cercarial, and metacercarial developmental stages of G. crumenifer possess a considerable level of antioxidant enzymes suitable to overcome the oxidative stress encountered during the development and help them in the completion of the life cycle and survival in the definitive host.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

Differentiation of amphistome species of cattle in Kerala by polymerase chain reaction - restriction fragment length polymorphism

The present study evaluated the PCR-RFLP of ITS-2+genes for species differentiation of three major amphistomes namely Gastrothylax crumenifer, Fischoederius cobboldi and Paramphistomum spp. Molecular analysis using PCR yielded amplicons of 515 bp for each species. The nucleotide differences among the sequences of the three species of amphistomes at different positions were further used for designing suitable RFLP. The amphistome parasites were distinguished taking into account the differences in the recognition sequences of TspRI on the ITS-2+ region by PCR-RFLP. The enzyme cleaved F.cobboldi at two recognition sites, CAGTG and CACTG and yielded 331, 93 and 91 bp products. Gastrothylax crumenifer had one recognition site CAGTG and resulted in 331 bp and 184 bp fragments. Since no recognition sequence was found in Paramphistomum spp., there was no cleavage. It is concluded that PCRRFLP was a promising molecular tool for species identification of amphistomes.

Serological diagnostic potential of the 38-72 kDa somatic antigen of Gastrothylax crumenifer in buffaloes using the Indirect Enzyme-Linked Immunosorbent Assay

Paramphistomosis, caused by digenetic trematodes of the superfamily Paramphistomoidea, causes heavy economic losses in terms of reduced fertility, milk and meat production in the livestock industry. In the present study, somatic and excretory secretory antigens isolated from 500 live Gastrothylax crumenifer were assessed for their diagnostic potential by using an antibody detection enzyme immunoassay. Prior to the enzyme immunoassay, the somatic and excretory/secretory (ES) antigens of G. crumenifer were subjected to SDS-PAGE and Western blot (WB) for the detection of immunogenic proteins. Indirect ELISA analysis was performed on sera from buffaloes naturally infected with G. crumenifer, with control sera of buffaloes infected with Gigantocotyle explanatum, Fasciola spp., Cotylophoron /Paramphistomum spp. The SDS-PAGE results of the somatic products of G. crumenifer identified proteins were between 10-123 kDa, showing a maximum abundance of 10, 15, 25-28, 36, 38-72, 95-123 kDa proteins. The most abundant proteins recorded in ES product were ≥95, 72, 55 and 40 kDa. The antigenic analysis of somatic proteins using WB revealed reactive polypeptides of a size between 55-70 kDa, while metabolic extracts did not show reactivity with naturally infected buffalo sera. The sensitivity and specificity of the ELISA test for 38-72 kDa somatic antigens were 85.71% and 89.74%, respectively. The cross-reactivity with other trematode sera was 16-20%. Antibodies were tested against the 38-72 kDa somatic antigens, and 19.69% (39/198) of buffaloes were found positive, while 12.1% (24/198) presented infection in the fecal/postmortem examination. The study confirmed that ELISA established for the 38-72 kDa somatic antigens of G. crumenifer had diagnostic potential against paramphistome infections.

Immunoblot analysis of excrertory/secretory antigens of Cotylophoron cotylophorum and Gastrothylax crumenifer

The study focused on purification, characterization and isolation of excretory/secretory antigens of ovine paramphistomes. A total of 5.321 and 7.170 mg of protein was obtained from 630 adult Cotylophoron cotylophorum and 290 adults Gastrothylax crumenifer, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) analysis of purified C. cotylophrum revealed 6 polypeptides in the range of 10 to 35 kDa while a prominent band at 15 kDa with G. crumenifer. Western blot analysis of purified excretory/secretory antigens of C. cotylophorum revealed 4 immuno-determinant bands in the range of 10 to 30 kDa using their specific rabbit hyperimmune serum, whereas G. crumenifer showed 2 polypeptides between 10 to 25 kDa. The common polypeptide observed at 20 kDa in both purified excretory/secretaty antigens can be used as a candidate antigen for development of serodiagnosis in the early stage of infection.

The anthelmintic potentials of medicinal plant extracts and an isolated compound (rutin, C27H30O16) from Terminalia catappa L. against Gastrothylax crumenifer

Paramphistomosis is a pathogenic disease that occurs frequently in tropical and subtropical countries including Thailand. This disease is affected in the parasites causing severe gastrointestinal disorders and death in infected animals. In the present study, we examined the anthelmintic efficacy of albendazole (ABZ) and crude plant extracts from barks of Bombax ceiba L., Diospyros rhodocalyx Kurz. and Vitex glabrata R.Br., and leaves of Terminalia catappa L. and Cassia alata L. against Gastrothylax crumenifer. The hightest anthelmintic activity on the parasites after 24 h incubation was observed in the n-butanol extract of T. catappa leaf. In this study, fractionation bioassay of n-butanol extract of T. catappa leaf was conducted to both separation and discrimination of rutin served as a new efficient compound (LC50 = 28.96; LC90 = 88.75 μg/mL) against G. crumenifer. This compound was confirmed by 1H nuclear magnetic resonance (1H NMR), 13C NMR, infrared (IR) and ultraviolet (UV) spectra as well as mass spectra data. The rutin-treated parasites with all dosages showed swift decrease of the motility and the relative motility (RM) and survival index (SI) were decreased obviously from 3 h until flukes were killed after 12 h of incubation. When observed with light microscopy, the parasites showed the earliest change in a limited region of the tegument. When observed by scanning electron microscopy, the parasites’ tegument exhibited similar sequences of surface changes after treatments with rutin and ABZ, but less severity in ABZ treatment. The sequences of changes comprised swelling of folds and ridges, formation of blebbing, rupturing of blebs, erosions, lesions and the tegument demolition. Hence, rutin could be considered as the potential anthelmintic agent for treatment of paramphistomosis.

Screening of in vitro antitrematodal activities of compounds and secondary metabolites isolated from selected Pteridophytes.

Four ferns Blechnum orientale Linn. (BO), Dicranopteris linearis (Burm.f ) Underw. (DL), Marattia fraxinea Sm. (MF), and Microlepia speluncae (L.) Moore (MS) were extracted in varied combination of organic solvents followed by the preparation of eluates and isolation of secondary metabolites using chromatography on a glass column with silica gel as the fixed phase. The chemical components were identified using HPTLC and GC-MS analysis. The in vitro anti-trematodal activities of these eluates and compounds were evaluated against the sheep trematode worm Gastrothylax crumenifer (Plagiorrchiida: Gastrothylacidae) at increasing concentrations (1 to 5 mg/mL), to find the relative motility (RM) values for 0 to 60 min of incubation as reflection of paralysis and death of the worms. Hedon-Fleig salt solution was used as negative control and Oxyclozanide® 1% as standard control. In vitro incubation study showed DL and MS extracts had strong trematodicidal activity. BO extract (5 mg/mL) produced moderate trematodicidal activity and MF (5 mg/mL) showed the least trematodicidal activity. Phytochemicals analysis revealed that the ferns are a potential source of trematodicidal compounds such as phytol isomers fern-8-ene and fern-9(11)-ene (terpenoid derivative), quercetin 7,3 ́,4 ́-trimethoxy (flavonoid derivative), etc., which offer scope for a more elaborate study for exploitation of ferns for human welfare.

Mixed course of Fasciolosis and Paramphistomatosis in sheep in Uzbekistan

The purpose of the research to study the mixed course of sheep infection, caused by pathogens of fasciolosis and paraphistomatosis, in the conditions of Samarkand and Tashkent regions. Materials and methods. The research material was fallen and forcibly killed sheep from two regions of Uzbekistan, simultaneously infected by Fasciola gigantica and Paramphistomum spp. (Gastrothylax crumenifer, Calicophoron calicophorum). The animals' liver was subjected to complete helminthological dissection, followed by the presence of young immature in the parenchyma, and in the bile ducts – sexually mature fasciola. To detect pathogens of paramphistomatosis, we examined the scar and mesh, and, if necessary, the mucous membranes of the abomasum and the initial part of the small intestine. To kill the collected F. gigantica and complete bleeding with a Paramphistomum spp., they were kept in water for one and three to four days, respectively. After they took their natural form, their type, age and number were determined, and then fixed in Barbagallo fluid. If necessary, coprological studies of sheep and malacological studies of biotopes of freshwater mollusks, intermediate hosts of the studied trematodes, were carried out. Results and discussion. In recent years, cases of a mixed course in sheep fasciolosis (F. gigantica) and paramphistomatosis in farms of Uzbekistan have become more frequent. When opening the liver of the fallen sheep, up to 278 specimens were found immature F. gigantica and up to 3994 sp. G. crumenifer. In some farms, the death of individual owners' sheep from calicophorosis caused by C. calicophorum was observed with an intensity of infection of 592 sp. of C. calicophorum.

Prevalence of Paramphistomosis in Domestic Ruminants in Chittoor District of Andhra Pradesh, India

A total of 1169 dung samples from domestic ruminants were examined to record the prevalence of paramphistomosis infection. In cattle, the prevalence of infection by direct smear (DS) and sedimentation method (SD) was 17.43% and 31.19%, respectively. In sheep, the prevalence rates were recorded higher than cattle (26.09% and 33.18%). In goats, the prevalence of paramphistomosis was 20.66% and 30.52%. Out of 109 cattle carcasses, 47 were found with flukes in rumen, reticulum and bile duct during slaughterhouse examination (SH). In sheep and goat, the prevalence rates were 42.15% and 40.85%, respectively by SH. Age-wise the highest prevalence was recorded in cattle of 2-4 years (50.0%) by SH. In goats, the prevalence of infection was lower in the age group oflessthan1 year, when compared to their counterparts in sheep. In contrast, the higher prevalence was noticed in >1-2 years old goats (63.27%) than sheep of the same age group (36.85%). Sex-wise, statistically no significant difference was observed between male and female animals. Morphologically identified, Cotylophoron cotylophorum, Paramphistomum cervi, Gastrothylax crumenifer, Fischoederius elongatus and Gigantocotyle spp. In conclusion, an overall prevalence of 24.29%, 32.51% and 42.0% of paramphistomosis infection was recorded in cattle, sheep and goats by DS, SD and SH examinations, respectively.

Monoclonal antibody against Fasciola gigantica glutathione peroxidase and their immunodiagnosis potential for fasciolosis

Glutathione peroxidases (GPx), major antioxidant enzymes, secreted by Fasciola spp., are important for the parasite evasion and protection against the host’s immune responses. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica glutathione peroxidase (rFgGPx) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgGPx. This MoAb (named 7B8) is IgG1 with κ light chains, and it reacted specifically with rFgGPx at a molecular weight 19 kDa as shown by immunoblotting, and reacted with the native FgGPx in the extracts of whole body (WB), metacercariae, newly excysted juveniles (NEJs), 4 week-old juveniles and adult F. gigantica as shown by indirect ELISA. It did not cross react with antigens in WB fractions from other adult trematodes, including Fischoederius cobboldi, Paramphistomum cervi, Setaria labiato-papillosa, Eurytrema pancreaticum, Gastrothylax crumenifer and Gigantocotyle explanatum. By immunolocalization, MoAb against rFgGPx reacted with the native protein in the tegument, vitelline cells, and eggs of adult F. gigantica. In addition, the sera from mice experimentally infected with F. gigantica were tested positive by this indirect sandwich ELISA. This result indicated that FgGPx is an abundantly expressed parasite protein that is secreted into the tegumental antigens (TA), therefore, FgGPx and its MoAb may be used for immunodiagnosis of both early and late fasciolosis gigantica in animals and humans.

Epidemiology of cattle amphistomiasis in Gilan province, north of Iran

Amphistomiasis is a widespread disease of ruminants in many parts of the world including Iran. The aim of this study was to determine the prevalence and intensity of amphistomiasis in cattle from Gilan province, north of Iran in association with sex, age, breed, and season. This investigation was carried out over a 12-month period in Gilan. Through seasonal sampling and visual examination for amphistomiasis, the rumens and reticulum of 406 cattle, slaughtered in Rasht abattoir, Gilan province, were collected and relevant data on sex, age, and breed were recorded. The total number of harvested amphistomes, which was present in each organ was collected, counted, and preserved in a 10% formaldehyde solution. After preparation and identification, our findings revealed that Carmyerius spatiosus, Paramphistomum gotoi, Gastrothylax crumenifer, and Calicophoron calicophorum were present in this study. The overall prevalence of infection was 19.70% with a mean intensity of 764.70 (ranged from 10 to 6200). The relation between prevalence and season (P 0.05). On the other hand, the relative mean intensity had a positive relation with the breed (P = 0.050) and age (P < 0.01), but not with season (P = 0.108) and sex (P = 0.344). The results of this study can be used to plan for the control and prevention of this disease.

Expression and characterization of glutathione peroxidase of the liver fluke, Fasciola gigantica.

Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as an anti-oxidant that helps to protect parasites from damage by free radicals released from the host immune cells, it has both diagnostic as well as vaccine potential against fasciolosis. In this study, we have cloned, characterized, and detected the expression of the GPx protein in Fasciola gigantica (Fg). FgGPx (582bp) was cloned by polymerase chain reaction (PCR) from complementary DNA (cDNA) from an adult fluke. Its putative peptide has no signal sequence and is composed of 168 amino acids, with a molecular weight of 19.1kDa, and conserved sequences at NVACKUG, FPCNQFGGQ, and WNF. Phylogenetic analysis showed that GPx is present from protozoa to mammals and FgGPx was closely related to Fasciola hepatica GPx. A recombinant FgGPx (rFgGPx) was expressed in Escherichia coli BL21 (DE3) and used for immunizing mice to obtain polyclonal antibodies (anti-rFgGPx) for immunoblotting and immunolocalization. In immunoblotting analysis, the FgGPx was expressed in all stages of F. gigantica (eggs, metacercariae, newly excysted juveniles (NEJ), 4-week-old juveniles, and adults). This mouse anti-rFgGPx reacted with the native FgGPx at a molecular weight of 19.1kDa in adult whole body (WB) and tegumental antigens (TA) as detected by immunoblotting. The FgGPx protein was expressed at a high level in the tegument, vitelline glands, and eggs of the parasite. Anti-rFgGPx exhibited no cross-reactivity with the other parasite antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. The possibility of using rFgGPx for immunodiagnosis and/or as a vaccine for fasciolosis in animals of economic importance will be explored in the future.

Morphological and molecular characterization of Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae) infecting rumen of Capra hircus in Chandigarh (India)

Morphological and molecular characterization of the amphistomid trematode, Gastrothylax crumenifer (Creplin, 1847) Poirier 1883 collected from Capra hircus goat in Chandigarh was carried out using Gower's carmine stain and by investigations on the nucleotide sequences of 28S and internal transcribed spacer 2 (ITS2). A total of 10 guts of C. hircus were examined for infection with trematodes. All the worms collected were morphologically identified as G. crumenifer by comparing the arrangement of median sagittal sections of adult amphistomes. The BLAST analysis of 28S gene of the study specimen G. crumenifer (KY786331) included 100 hits and showed maximum homogeneity of 99% with G. crumenifer as only 7 gaps in the case of G. crumenifer (JX518971, JX518960, JX518959, JX518970); 8 gaps in the case of G. crumenifer (JX518969) and 18 gaps in case of G. crumenifer (KJ559529). Phylogenetic tree revealed close relatedness of the present specimens with G. crumenifer infecting Bos indicus and Bos frontalis from India with 0.01 and 0.0 (nil) evolutionary divergence, respectively. The BLAST analysis of ITS2 gene of the study species G. crumenifer (KY786332) included 100 hits and showed maximum homogeneity of 97% to 99% with G. crumenifer as only 1 gap in the case of G. crumenifer (HM159382) and 5 gaps in the case of G. crumenifer (KU530204) were found between them. Phylogenetic tree revealed close relatedness of the present specimens of Gastrothylax with G. crumenifer infecting Bos indicus and Capra hircus from India with 0.01 and 0.0 (nil) evolutionary divergence, respectively. The molecular results revealed ITS2 as the better gene marker as it formed clear monophyly of the present species with G. crumenifer but in the case of 28S, the tree was somewhat more towards the polyphyly of Gastrothylacidae. Thus on the basis of morphological and molecular data based on 28S and ITS2, the study species was identified as G. crumenifer

Preparation and characterization of excretory and secretory antigen of Cotylophoron cotylophorum and Gastrothylax crumenifer.

Excretory and secretory antigen of adult Cotylophoron cotylophorum and Gastrothylax crumenifer was prepared and characterized by SDS-PAGE. Adult paramphistomes were collected in PBS from rumen of sheep slaughtered at Perambur and Villivakkam slaughter houses, Chennai, Tamil Nadu. Excretory/Secretory (E/S) antigens of G. crumenifer and C. cotylophorum were prepared by incubating in PBS at 28.3°C overnight in a shaking incubator and centrifuged. The culture supernatant was used as antigen. The protein concentration of E/S antigens of C. cotylophorum and G. crumenifer ranged from 0.4 to 7.1mg/ml and 3.3 to 5.9mg/ml, respectively. SDS-PAGE analysis of E/S antigen of C. cotylophorum revealed two polypeptide bands from the range of 32 and 40kDa. Whereas, SDS-PAGE analysis of E/S antigen of G. crumenifer revealed only one polypeptide band of 35kDa. Excretory/secretory antigen may be used for serodiagnosis of paramphistomosis infection in sheep.

Partial purification and characterization of glutathione S-transferase from the somatic tissue of Gastrothylax crumenifer (Trematoda: Digenea).

Aim:Aim of the present study was to carry out the partial purification and biochemical characterization of glutathione S-transferase (GST) from the somatic tissue of ruminal amphistome parasite, Gastrothylax crumenifer (Gc) infecting Indian water buffalo (Bubalus bubalis).Materials and Methods:The crude somatic homogenate of Gc was subjected to progressive ammonium sulfate precipitation followed by size exclusion chromatography in a Sephacryl S 100-HR column. The partially purified GST was assayed spectrophotometrically, and the corresponding enzyme activity was also recorded in polyacrylamide gel. GST isolated from the amphistome parasite was also exposed to variable changes in temperature and the pH gradient of the assay mixture.Results:The precipitated amphistome GST molecules showed maximum activity in the sixth elution fraction. The GST subunit appeared as a single band in the reducing polyacrylamide gel electrophoresis with an apparent molecular weight of 26 kDa. The GST proteins were found to be fairly stable up to 37°C, beyond this the activity got heavily impaired. Further, the GST obtained showed a pH optima of 7.5.Conclusion:Present findings showed that GST from Gc could be conveniently purified using gel filtration chromatography. The purified enzyme showed maximum stability and activity at 4°C.

Incidence of amphistome infection in sheep from Mhow, Madhya Pradesh.

A total of 445 faecal samples and 128 rumens of sheep collected from Slaughter house, Mhow (Madhya Pradesh) were examined for a period of 1year from February 2011 to January 2012. Faecal sample examination based, incidence of amphistomes was found to be 23.37% from the study area. The incidence of amphistomes was significantly higher (p<0.01) during summer (36.75%) than winter (27.74%) and monsoon (3.52%). Age-wise, non-significantly higher infection was observed in ≥1year-old animals (23.61%) than <1year-old animals (23.11%). Non-significantly higher rate of infection was recorded in case of females (25.63%) than that of males (19.64%). Out of 128, 49 (38.28%) rumens were found positive for amphistomes. The prevalence of Paramphistomum cervi (27.34%) was found to be significantly (p<0.01) higher than Gastrothylax crumenifer (10.94%).

Gastrothylax crumenifer: ultrastructure and histopathology study of in vitro trematodicidal effect of Microlepia speluncae (L.) Moore

ABSTRACTAn in vitro incubation study was carried out to determine the efficacy of Microlepia speluncae (MS) fronds’ extract on the worm motility and viability, morphology and histopathological changes in/of Gastrothylax crumenifer. The methanolic extract of MS fronds was used for in vitro study at 1, 2, 3, 4 and 5 mg/ml concentration as test extracts. Oxyclozanide (OXY) at 1% (250 mg/25 ml) as standard control and Hedon–Fleig (H–F) salt solution as negative control. The solutions (25 ml), with 25 amphistomes each, distributed to 7 petriplates (90 mm diameter) and placed in an incubator with 5% CO2 at 37°C. The motility of control and treated flukes was observed at regular time intervals, 0, 10, 15, 30 and 60 min respectively with specific score 3, 2, 1, 0 for motility. Relative Motility assay and LC50 were determined by probit regression analysis. In vitro incubation study revealed death of all trematodes, lethal at 10 min incubation time at 5 mg/ml concentration, indicated RM value is 0, very much effective than Oxyclozanide, RM value was 0. 05. The LC50 was determined as 3.666. The worms subjected to in vitro trials were studied under Scanning Electron Microscope and Light Microscope for significant morphological changes.

Gastrothylax crumenifer: Ultrastructure and histopathology study of in vitro trematodicidal action of Marattia fraxinea (Sm.)

BackgroundTrematodicidal action of Marattia fraxinea (Sm.) rhizome, extracts against adult Gastrothylax crumenifer and its morphological changes under light and scanning electron microscope was studied in this work. This plant according to folk medicine has been reported to be used as antioxidant, antimicrobial and anthelmintic against ankylostomiasis.MethodsPreliminary screening involved the qualitative methods to detect the presence tannins, saponins, quinones, terpenoids, steroids, flavonoids, phenol, alkaloids, glycosides, cardiac glycosides, coumarins, anthocyanin and betacyanin. Total phenol, total terpenoids, total tannin and total flavonoids were quantitatively estimated. Total phenolic content was estimated by Folin- Ciocalteau method. Total flavonoid content was determined by the aluminium chloride colorimetric method. In-vitro incubation study of Marattia fraxinea Sm. extracts against Gastrothylax crumenifer were performed using 25 ml of Hedon-Fleig (H-F) salt solution containing various concentrations (1, 2, 3, 4 and 5 mg/ml) respectively as test extracts, positive control Oxyclozanide @ 1% (250 mg/25 ml) and negative control H-F salt solution, distributed to 7-Petri plates in an incubator with 5% CO2 at 37 °C. Twenty five amphistomes were incubated and the motility of control and test group was observed under dissection microscope at a regular time interval of (0, 10, 15, 30 and 60) min respectively. The motility response of the parasites was categorized with specific score 3, 2, 1, 0 respectively. Relative Motility (RM) value and Lethal Concentration 50 (LC50) were determined by probit regression analysis. Ultrastructure and histopathological changes on morphology of G.crumenifer were interpreted. The bioactive compounds were analysed using gas chromatography-mass spectrometry (GC-MS) instrument.ResultsPhytochemical analysis of M.fraxinea (Sm.) revealed the presence of secondary metabolites tannins, saponins, quinones, terpenoids, steroids, flavonoids, phenol, alkaloids, cardiac glycosides, coumarins, and betacyanin. Total terpenoid, total phenol, total tannin and total flavonoid content were found to be 16.00 ± 0.85 mg/g. 5.40 ± 0.71) mg GAE/g, 7.90 ± 0.24 mg TAE/g and 1.25 ± 0.41 mg QE/g respectively. GC-MS analysis showed presence of PHYTOL, a diterpenoids, fern-8-ene, a triterpenoids shown to have potent anthelmintic property. In vitro incubation study revealed death of all trematodes, lethal at 60 min incubation time at 5 mg/ml concentration, indicated Relative Motility (RM) value is 0, least effective compared to positive control Oxyclozanide, where RM value is 0 at 10 min. Lethal concentration 50 (LC50) was found to be 4.873 mg/ml. Confirmative study on gross morphology, histopathology, and ultra-structural changes showed considerable effect on suckers, teguments and internal organs and were dose dependent.ConclusionIt is indicative that presence of terpenoids, flavonoids, tannins and phenolics which are contributing factors for anthelmintic activity, producing various degree of damage to suckers, teguments and internal organs. Hence M. fraxinea (Sm.) can be a lead for synthesis of a new trematodicidal drug as alternative for other drugs and overcome anthelmintic resistance and cost effective.