Fasciola Gigantica Research Articles

Photoactivated chlorophyllin and acetylcholinesterase/ cytochrome oxidase activity in Fasciola gigantica cercaria larvae

Objective: The effect of chlorophyllin (CHL), CHL + freeze dried cow urine (FCU) and FCU on acetylcholinesterase (AChE) and cytochrome oxidase activity in the nervous tissue of the host snail Lymnaea acuminata, and parasitic cercaria larvae of Fasciola gigantica were studied. Methods: Whole cercaria larvae and snail nervous tissue was homogenized and supernatant was used as an enzyme source. Enzyme activity was measured in 10-mm path length cuvette using incubation mixtures and change in optical density was recorded. Results: Sublethal 4 h LC50 40% and 80% of these formulations in sunlight and laboratory conditions caused no significant change in AChE activity in the nervous tissue of host snail. It was noted that sublethal in vivo 8 h exposure to 40% and 80% of 4 h LC50 of single and binary combination of CHL+FCU, CHL and FCU significantly altered the AChE and cytochrome oxidase activity in the cercaria larvae. Binary combinations of CHL+FCU in sunlight were more effective in inhibiting both the enzymes. Conclusion: The present study reported that enzymes AChE and cytochrome oxidase were inhibited by chlorophyllin formulations and thus causing the larval death inside the snail body without affecting the snail itself which is an important bioindicator of aquatic ecosytem.

Helminths of veterinary and zoonotic importance in Nigerian ruminants: a 46-year meta-analysis (1970\u20132016) of their prevalence and distribution

BackgroundThe livestock industry plays a vital role in the economy of Nigeria. It serves as a major source of income and livelihood for majority of Nigerians who are rural settlers and contributes about 5.2% to the National Gross Domestic Product (GDP). Helminths however, cause economic losses due to reductions in milk production, weight gain, fertility and carcass quality. Zoonotic helminths of livestock origin cause health problems in humans.MethodsUsing the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines, the prevalence and distribution of helminths of veterinary and zoonotic importance in Nigerian ruminants were determined in a meta-analysis of data published between 1970 and 2016. Data were stratified based on regions, hosts, study periods, sample sizes and study types while helminths were phylogenetically grouped into cestodes, nematodes and trematodes.ResultsData from 44 studies reported across 19 Nigerian states revealed an overall pooled prevalence estimate (PPE) of 7.48% (95% CI: 7.38–7.57) for helminths of veterinary and zoonotic importance from a total of 320 208 ruminants. We observed a significant variation (P < 0.001) between the PPEs range of 1.90% (95% CI: 1.78–2.02) and 60.98% (95% CI: 58.37–63.55) reported across different strata. High heterogeneity (99.78, 95% CI: 7.38–7.57) was observed. Strongyloides papillosus was the most prevalent (Prev: 32.02%, 95% CI: 31.01–33.11), while, Fasciola gigantica had the widest geographical distribution.ConclusionsHelminths of veterinary and zoonotic importance are prevalent in ruminants and well distributed across Nigeria. Our findings show that helminths of ruminants may also be possible causes of morbidity in humans and economic losses in the livestock industry in Nigeria. High heterogeneity was observed within studies and the different strata. Good agricultural practices on farms, standard veterinary meat inspection and adequate hygiene and sanitation in abattoirs, farms and livestock markets need to be implemented in Nigeria in order to reduce the economic, public health and veterinary threats due to these helminths.

Do triclabendazole medicated molasses blocks have a role in control of Fasciola gigantica in smallholder cattle production in Lao PDR?

Smallholder cattle production in many developing tropical countries including Laos is compromised by widespread endoparasitism, with limited farmer knowledge of parasites and facilities for administration of therapeutics. We report a pilot study examining the potential for triclabendazole provided in medicated molasses blocks offered to control Fasciola gigantica in smallholder cattle production. This study involved 241 cattle allocated into three groups: (1) triclabendazole (as Fasinex®, Novartis Animal Health Australia, Pty Ltd) medicated molasses blocks (MMB) with each tonne of MMB containing 0.5 kg triclabendazole; (2) unmedicated molasses blocks; and (3) a Control group. Data and faecal samples were obtained at Weeks 1, 4, 8 and 12 for faecal egg counts (FEC) determination. Reductions in FEC in the MMB group of 90.48% and a mean FEC of 4 ± 17 eggs per gram of faeces at 12 weeks post-treatment was observed, with liveweight increasing from 174.60 (±3.35) kg to 191.50 (±3.69) kg in Weeks 1 and 12, respectively (P = 001) and an average daily weight gain of 201 g/day. Reduction in FEC in the unmedicated molasses blocks group was also observed, by 28.78% and 18.96%, with liveweight increasing from 179.50 (±3.35) kg to 189.90 (±6.05) kg in Weeks 1 and 12 respectively (P = 0.3), with an average daily gain of 124 g/day. This study suggests that productivity was enhanced when triclabendazole was added to the blocks, delivering parasite suppression or potentially therapeutic doses on ad libitum feeding of the MMB. Although further work is required to establish the therapeutic potential of MMB, the use of MMB may offer a parasite management and nutritional supplementation strategy for smallholder farmers, particularly in Laos and other countries where unmanaged Fasciola spp. infestations reduce ruminant productivity and facilities for animal restraint to enable delivery of oral anthelmintics, are largely non-existent.

Cross-sectional prevalence of Fasciola gigantica infections in beef cattle in Botswana.

A cross-sectional study was carried out to determine the prevalence and distribution of Fasciola gigantica infections in communally grazed and ranch-grazed beef cattle through coprological examination. A total of 8646 cattle (4618 adults, 2843 weaners and 1185 calves) faecal samples were collected during the 24months of study. Results from this study indicated that only 64 (0.74%; 95% CI 0.57, 0.94%) of the samples were positive for F. gigantica eggs. The positive samples were detected in one (Central) of the six study districts and was restricted to the Tuli Block (commercial) farms in Machaneng village in eastern Botswana. The prevalence of fluke eggs was significantly higher in adult cattle (12.85%; 95% CI 9.72, 16.54%) than weaners (6.49%; 95% CI 3.40, 11.06%) and calves (0.79%; 95% CI 0.02, 4.31%), (χ2(2) = 19.01, p < 0.001). Thus, adults (OR = 18.57; 95% CI 2.54, 135.81%) were approximately 20 times more likely to be infected than calves. By taking liver inspection as a gold standard for diagnosis of fasciolosis, the sensitivity of the sedimentation technique was found to be 72.41% and the specificity 100%, with moderate relationship (κ = 0.53; 95% CI 0.31-0.75) between the two methods. This study has demonstrated that infection of cattle from Botswana, with F. gigantica, was low and distribution of the fluke appeared to be linked to suitable environmental conditions for the intermediate host snail. However, detailed surveillance studies, involving more cattle and areas, are required to verify the true prevalence. Such information would assist in the design of appropriate, strategic and effective fluke control programmes.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis.The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.

A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

BackgroundThe molecular structure of Fasciola gigantica 14-3-3 protein has been characterized. However, the involvement of this protein in parasite pathogenesis remains elusive and its effect on the functions of innate immune cells is unknown. We report on the cloning and expression of a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and testing its effects on specific functions of goat peripheral blood mononuclear cells (PBMCs).MethodsrFg14-3-3e protein was expressed in Pichia pastoris. Western blot and immunofluorescence assay (IFA) were used to examine the reactivity of rFg14-3-3e protein to anti-F. gigantica and anti-rFg14-3-3e antibodies, respectively. Various assays were used to investigate the stimulatory effects of the purified rFg14-3-3e protein on specific functions of goat PBMCs, including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis, and apoptotic capabilities. Potential protein interactors of rFg14-3-3e were identified by querying the databases Intact, String, BioPlex and BioGrid. A Total Energy analysis of each of the identified interaction was performed. Gene Ontology (GO) enrichment analysis was conducted using Funcassociate 3.0.ResultsSequence analysis revealed that rFg14-3-3e protein had 100% identity to 14-3-3 protein from Fasciola hepatica. Western blot analysis showed that rFg14-3-3e protein is recognized by sera from goats experimentally infected with F. gigantica and immunofluorescence staining using rat anti-rFg14-3-3e antibodies demonstrated the specific binding of rFg14-3-3e protein to the surface of goat PBMCs. rFg14-3-3e protein stimulated goat PBMCs to produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), corresponding with low levels of IL-4 and interferon gamma (IFN-γ). Also, this recombinant protein promoted the release of NO and cell apoptosis, and inhibited the proliferation and migration of goat PBMCs and suppressed monocyte phagocytosis. Homology modelling revealed 65% identity between rFg14-3-3e and human 14-3-3 protein YWHAE. GO enrichment analysis of the interacting proteins identified terms related to apoptosis, protein binding, locomotion, hippo signalling and leukocyte and lymphocyte differentiation, supporting the experimental findings.ConclusionsOur data suggest that rFg14-3-3e protein can influence various cellular and immunological functions of goat PBMCs in vitro and may be involved in mediating F. gigantica pathogenesis. Because of its involvement in F. gigantica recognition by innate immune cells, rFg14-3-3e protein may have applications for development of diagnostics and therapeutic interventions.

Fasciola gigantica cases in ruminants from Samsun Province, Turkey.

Animal fasciolosis occurs every year as an occasional problem in Samsun Province, especially of grazing animals in the Kizilirmak and Yesilirmak Deltas and surrounding areas. On occasions, dead animals or internal organs, including livers, lungs and hearts, are submitted to the Parasitology Department of the Faculty of Veterinary Medicine at Ondokuz Mayis University for diagnostic purposes. Upon diagnosis of fasciolosis, the owners and veterinarians are informed about prevention measures and treatment of the disease. In that context, the livers and gallbladders of one sheep, one cow and two water buffalo, suspected of having died as a result of fasciolosis, were submitted to the parasitology laboratory of the faculty in 2015 and 2016. All organs were examined thoroughly and 38, 1076, 456 and 714. F. gigantica were collected from the sheep, cow and buffaloes' livers and gallbladders, respectively. In addition to these cases, according to information received from animal breeders and some veterinarians, at least 5 sheep and 3 cattle had also died due to fasciolosis in the previous two years. Because of the re-occurrence of F. gigantica cases in recent years, it is pertinent to discuss the past and current situation surrounding this parasite in Turkey.

Evaluation of Echinococcus granulosus recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens in the diagnosis of cystic echinococcosis in buffaloes

Three recombinant proteins of Echinococcus granulosus including two antigen B sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein 1 (EPC1) were expressed in prokaryotic expression vectors. The diagnostic potential of these three recombinant proteins was evaluated in the detection of cystic echinococcosis in buffaloes in IgG-ELISA. The EgAgB8/1 and EgAgB8/2 recombinant proteins reacted fairly with the hydatid infected buffaloes with sensitivity of 75.0% and 78.6%, respectively and specificity of 75.8% while EPC1 recombinant protein showed higher sensitivity (89.3%) but lower specificity (51.5%). Cross-reactivity of these three antigens was assayed with buffalo sera naturally infected with Explanatum explanatum, Paramphistomum epiclitum, Gastrothylax spp., Fasciola gigantica and Sarcocystis spp. EgAgB8/1 and EPC1 antigens cross-reacted with all these sera while EgAgB8/2 showed no cross-reaction with Sarcocystis spp. and reacted with some of the E. explanatum infected buffalo sera. This study explores the potential of three hydatid antigens viz. EgAgB8/1, EgAgB8/2 and EPC1 for their diagnostic potential in buffaloes positive for cystic echinococcosis.

Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs.

Background:Fasciolosis is an important zoonotic disease affecting the productive performance of farm animals in Egypt.Aim:The aim of the present study was comparing the ovicidal effect of different extracts as an alcoholic (Methanolic and Ethanolic) and aqueous Moringa oleifera leaf extracts on Fasciola gigantica non-embryonated and developed eggs.Materials and Methods:Tested concentrations of extracts ranged from 12.5 to 800 mg/ml. Nitroxynil was used as reference drug with a dose of 100 mg/ml.Results:M. oleifera alcoholic and aqueous extracts showed a concentration-dependent ovicidal effect on F. gigantica non-embryonated and developed eggs. Based on LC50 values, water extract showed the highest ovicidal activity since it registered the lowest values of 2.6 mg/ml on non-embryonated eggs. Non-embryonated eggs were more susceptible to aqueous extract than developed eggs. On the other hand, the developed eggs were more susceptible to ethanolic extract than non-embryonated eggs even the lowest LC50 (12.38 mg/ml).Conclusion:M. oleifera leaf extracts especially aqueous extract could be a promising step in the field of controlling fascioliasis. Further, in vivo studies are needed to enlighten the therapeutic potential of M. oleifera extracts in treating F. gigantica infection.

Gastrointestinal parasitic infections in cattle and swamp buffalo of Guwahati, Assam, India

The objective of the present study was to determine the prevalence of gastrointestinal (G.I.) parasitic infections in cattle and swamp buffalo of Assam, India. A total of 3597 fecal samples of cattle (2339) and buffaloes (1258) were examined for presence of parasitic infections. Examinations revealed an overall prevalence of 58.35% and 29.80% infections in cattle and buffalo, respectively. In cattle, Strongyle sp. (18.76%) was predominant followed by Eimeria sp. (11.97%), Amphistome sp. (8.72%), Strongyloides sp. (3.76%), Moniezia sp. (2.65%), Toxocara vitulorum (1.32%), Buxtonella sulcata (0.81%), Trichuris sp. (0.72%), Fasciola gigantica (0.47%) and Bunostomum sp. (0.38%). In buffalo, Amphistome sp. (8.90%) was predominant followed by T. vitulorum (5.64%), Strongyle sp. (5%), Eimeria sp. (4.53%), Strongyloides sp. (1.35%) and Trichuris sp. (0.47%). Mixed infections with more than one species were recorded in 8.76% and 3.89% cattle and buffalo, respectively. The egg per gram (EPG) of feces in cattle and buffalo was 582.44±8.13 and 475.86±11.29, respectively while oocyst per gram (OPG) of feces was 425.00±20.37 and 251.75±22.35, respectively. Correlation of rainfall and temperature with EPG/OPG of cattle and buffalo was highly significant (P less than 0.01) while correlation of relative humidity (Av.) with EPG of cattle and buffalo was significant (P less than 0.05). Multiple linear regressions explained about 98.10% of total variance in EPG of cattle with environmental variables. 89.10% of total variance in EPG of buffalo was explained in multiple linear regressions due to environmental variables. This shows that the climate in this region is exclusively conducive for the development and propagation of parasites.

Comparison of early detection of Fasciola hepatica in experimentally infected Merino sheep by real-time PCR, coproantigen ELISA and sedimentation

Fasciolosis due to infection with Fasciola hepatica, Fasciola gigantica or their hybrids is a significant global cause of livestock production loss. Infection is commonly diagnosed by a labour-intensive sedimentation and faecal egg count (FEC), which has limited throughput and is only applicable after completion of the 8–12 week pre-patent period (PPP). A commercially-available ELISA for the detection of coprological antigen (coproELISA) enables detection prior to the completion of the PPP and is suitable for diagnosis of larger sample sizes, although the sensitivity reported under experimental infection settings can be difficult to replicate in the field, particularly in cattle. A recently-published real-time PCR workflow for the sensitive detection of Fasciola spp. DNA in faecal samples provides increased sample throughput, although the point at which this technique is first able to diagnose infection remains unknown. Other tools for the molecular diagnosis of fasciolosis, such as conventional PCR and loop-mediated isothermal amplification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal samples were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. Samples were first considered positive at 6–8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of samples were positive by all three methods. To evaluate the capacity of the real-time PCR approach to detect infection prior to completion of the PPP, two methods of sample preparation were compared at 2 WPI: (i) 150 mg raw faecal samples and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of sample preparation yielded positive results at 2 WPI suggesting that DNA amplification by real-time PCR is associated with faecal egg load.

Long Noncoding RNA Profiling from Fasciola Gigantica Excretory/Secretory Product-Induced M2 to M1 Macrophage Polarization

Background/Aims: Long noncoding RNAs (lncRNAs) are well known regulators of gene expression that play essential roles in macrophage activation and polarization. However, the role of lncRNA in Fasciola gigantica excretory/secretory products (ESP)-induced M2 polarization into M1 macrophages is unclear. Herein, we performed lncRNA profiling of lncRNAs and mRNAs during the ESP-induced macrophage polarization process. Methods: F. gigantica ESP was used to induce peritoneal cavity M2 macrophages in BALB/c mice (5-6 weeks old) in vivo, and these cells were subsequently isolated and stimulated with IFN-γ + LPS to induce M1 cells in vitro. LncRNA and mRNA profiling was performed via microarray at the end of both polarization stages. Results: In total, 2,844 lncRNAs (1,579 upregulated and 1,265 downregulated) and 1,782 mRNAs (789 upregulated and 993 downregulated) were differentially expressed in M2 macrophages compared to M1 macrophages, and six lncRNAs were identified during polarization. We selected 34 differentially expressed lncRNAs and mRNAs to validate the results of microarray analysis using quantitative real-time PCR (qPCR). Pathway and Gene Ontology (GO) analyses demonstrated that these altered transcripts were involved in multiple biological processes, particularly peptidase activity and carbohydrate metabolism. Furthermore, coding and non-coding gene (CNC) and mRNA-related ceRNA network analyses were conducted to predict lncRNA expression trends and the potential target genes of these lncRNAs and mRNAs. Moreover, we determined that four lncRNAs and four mRNAs might participate in F. gigantica ESP-induced M2 polarization into M1 macrophages. Conclusions: This study illustrates the basic profiling of lncRNAs and mRNAs during F. gigantica ESP-induced M2 polarization into M1 macrophages and deepens our understanding of the mechanism underlying this process.

Fasciola hepatica - Convoluted life forms and names.

Infection of ruminants and humans with Fasciola gigantica is attracting increasing attention due to its economic impact and public health significance. However, little is known of innate immune responses during F. gigantica infection. Here, we investigated the expression profiles of genes involved in Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways in buffaloes infected with 500 F. gigantica metacercariae. Serum, liver and peripheral blood mononuclear cell (PBMC) samples were collected from infected and control buffaloes at 3, 10, 28, and 70 days post infection (dpi). Then, the levels of 12 cytokines in serum samples were evaluated by ELISA. Also, the levels of expression of 42 genes, related to TLRs and NLRs signaling, in liver and PBMCs were determined using custom RT2 Profiler PCR Arrays. At 3 dpi, modest activation of TLR4 and TLR8 and the adaptor protein (TICAM1) was detected. At 10 dpi, NF-κB1 and Interferon Regulatory Factor signaling pathways were upregulated along with activation of TLR1, TLR2, TLR6, TLR10, TRAF6, IRF3, TBK1, CASP1, CD80, and IFNA1 in the liver, and inflammatory response with activated TLR4, TLR9, TICAM1, NF-κB1, NLRP3, CD86, IL-1B, IL-6, and IL-8 in PBMCs. At 28 dpi, there was increase in the levels of cytokines along with induction of NLRP1 and NLRP3 inflammasomes-dependent immune responses in the liver and PBMCs. At 70 dpi, F. gigantica activated TLRs and NLRs, and their downstream interacting molecules. The activation of TLR7/9 signaling (perhaps due to increased B-cell maturation and activation) and upregulation of NLRP3 gene were also detected. These findings indicate that F. gigantica alters the expression of TLRs and NLRs genes to evade host immune defenses. Elucidation of the roles of the downstream effectors interacting with these genes may aid in the development of new interventions to control disease caused by F. gigantica infection.

Prepatent RT-LAMP Detection of Fasciola gigantica in Snails (Lymnaea spp.) and Goats (Capra hircus) Targeting Cathepsin B3 gene

Cathepsins are cysteine proteases secreted in different stages of Fasciola species of flukes. Cathepsin B is predominantly found in eggs, metacercariae and the newly excysted juvenile (NEJ) stages of the fluke but not in adults. Cathepsin B3 was identified in F. gigantica NEJs that facilitates the migration of the excysted parasite to the liver. Here, RT-LAMP was utilized in the detection of F. gigantica during the prepatent period or the period between infection and the demonstration of the parasite i.e. from the egg to the NEJ stages. We investigated the feasibility of identifying the cathepsin B3 gene in various samples including snails, as the intermediate host, the plasma, sera and feces of goats as the definitive host animal targeting the Cathepsin B3 gene. Out of 90 snails, 61% were detected positive using RT-LAMP, 26% (32/122) in fecal samples, 4% (5/122) in sera and 70% (86/122) in plasma of the suspected infected goats. These findings provide further indication for early diagnosis of fasciolosis and a novel approach for the improved disease management and control.

Prevalence of Fasciola gigantica infestation in cattle slaughtered at Minna metropolitan abattoir in North Central Nigeria

The study was conducted to provide a baseline data for the prevalence of Fasciola gigantica infestation in cattle slaughtered at Minna metropolitan abattoir in Chanchaga Local Government area of Niger State, Nigeria. The study was conducted for 90 days. Liver and gall bladder tissue samples were obtained from slaughtered cattle at abattoir. Ten cattle were sampled per week which translated to 120 animals. The liver and gall bladder tissue samples were processed at the State Veterinary Centre, Minna for worm recovery and identification. Liver was examined visually for the presence of Fasciola species by making length wise incision on the ventral aspect of the liver by cutting the bile duct and gall bladder open. Data were subjected to t-test statistics. Results showed that there were significant differences (p<0.05) between the sex of Fasciola gigantica and the prevalence of Fasciola gigantica obtained in the organs. This study established the presence of Fasciola gigantica in cattle slaughtered in Minna Metropolis. It is recommended that grazing of cattle should be restricted to lesser snail infested areas particularly around rivers and streams.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase.

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure-based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein-ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (-91.479 KJ · mol-1 ), having -152.657, -141.219, and -92.931 kJ · mol-1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

RNA interference in Fasciola gigantica: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke.

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170–223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / μl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.

Expression profiles of genes involved in TLRs and NLRs signaling pathways of water buffaloes infected with Fasciola gigantica

Infection of ruminants and humans with Fasciola gigantica is attracting increasing attention due to its economic impact and public health significance. However, little is known of innate immune responses during F. gigantica infection. Here, we investigated the expression profiles of genes involved in Toll-like receptors (TLRs) and NOD-like receptors (NLRs) signaling pathways in buffaloes infected with 500F. gigantica metacercariae. Serum, liver and peripheral blood mononuclear cell (PBMC) samples were collected from infected and control buffaloes at 3, 10, 28, and 70days post infection (dpi). Then, the levels of 12 cytokines in serum samples were evaluated by ELISA. Also, the levels of expression of 42 genes, related to TLRs and NLRs signaling, in liver and PBMCs were determined using custom RT2 Profiler PCR Arrays. At 3 dpi, modest activation of TLR4 and TLR8 and the adaptor protein (TICAM1) was detected. At 10 dpi, NF-κB1 and Interferon Regulatory Factor signaling pathways were upregulated along with activation of TLR1, TLR2, TLR6, TLR10, TRAF6, IRF3, TBK1, CASP1, CD80, and IFNA1 in the liver, and inflammatory response with activated TLR4, TLR9, TICAM1, NF-κB1, NLRP3, CD86, IL-1B, IL-6, and IL-8 in PBMCs. At 28 dpi, there was increase in the levels of cytokines along with induction of NLRP1 and NLRP3 inflammasomes-dependent immune responses in the liver and PBMCs. At 70 dpi, F. gigantica activated TLRs and NLRs, and their downstream interacting molecules. The activation of TLR7/9 signaling (perhaps due to increased B-cell maturation and activation) and upregulation of NLRP3 gene were also detected. These findings indicate that F. gigantica alters the expression of TLRs and NLRs genes to evade host immune defenses. Elucidation of the roles of the downstream effectors interacting with these genes may aid in the development of new interventions to control disease caused by F. gigantica infection.

Anthelmintic Activity of Ocimum sanctum Linn. Leaves Ethanol Extract Against Fasciola gigantica in vitro

The aims of this research are to know the concentration, exposure time and interaction between concentration and exposure time of Ocimum sanctum Linn. leaves ethanol extract which cause the most mortality toward Fasciola gigantica. Also to know its value of LC50 and LC90. The research was completely randomized design. There were five treatments. Each treatment was done in four replications and used 10 Fasciola gigantica. The observation and recording of dead Fasciola gigantica were done at 0, 2, 4, 6, 8 and 10 hours. Fasciola gigantica were declared dead if there was no movement when disturbed by anatomy tweezers and when dipped in slightly warm water (50ºC). The obtained data was analyzed using ANOVA Factorial and continued with Duncan Multiple Range Test. The result was 10% concentration and exposure time for 10 hours caused the most mortality toward Fasciola gigantica. However, the interaction between concentration and exposure time resulted that 5% concentration for 8 hours already caused the most mortality of Fasciola gigantica. Probit analysis was used to calculate the LC50 and LC90. The results were LC50 of Ocimum sanctum Linn. leaves ethanol extract was 7.9% at 4 hours, 3.7% at 6 hours, 1.8% at 8 hours and 0.8% at 10 hours and the LC90 was 8.4% at 10 hours. Key words: Ocimum sanctum Linn. leaves, Fasciola gigantica, ethanol extract, in vitro.