An insulin-like signalling pathway model for Fasciola gigantica.

BACKGROUND: The insulin/insulin-like signalling (IIS) pathway is common in mammals and invertebrates, and the IIS pathway is unknown in Fasciola gigantica. In the present study, the IIS pathway was reconstructed in F. gigantica. We defined the components involved in the IIS pathway and investigated the transcription profiles of these genes for all developmental stages of F. gigantica. In addition, the presence of these components in excretory and secretory products (ESPs) was predicted via signal peptide annotation. RESULTS: The core components of the IIS pathway were detected in F. gigantica. Among these proteins, one ligand (FgILP) and one insulin-like molecule binding protein (FgIGFBP) were analysed. Interestingly, three receptors (FgIR-1/FgIR-2/FgIR-3) were detected, and a novel receptor, FgIR-3, was screened, suggesting novel functions. Fg14-3-3ζ, Fgirs, and Fgpp2a exhibited increased transcription in 42-day-old juveniles and 70-day-old juveniles, while Fgilp, Fgigfb, Fgsgk-1, Fgakt-1, Fgir-3, Fgpten, and Fgaap-1 exhibited increased transcription in metacercariae. FgILP, FgIGFBP, FgIR-2, FgIR-3, and two transcription factors (FgHSF-1 and FgSKN-1) were predicted to be present in FgESPs, indicating their exogenous roles. CONCLUSIONS: This study helps to elucidate the signal transduction pathway of IIS in F. gigantica, which will aid in understanding the interaction between flukes and hosts, as well as in understanding fluke developmental regulation, and will also lay a foundation for further characterisation of the IIS pathways of trematodes.

Immune modulation of goat monocytes by Fasciola gigantica Legumain-1 protein (Fg-LGMN-1).

Legumains belonging to C_13 peptidase family of proteins, and are ubiquitously disseminated among all vertebrate and invertebrate organisms, and have been implicated in innumerable biological and cellular functionality. Herein, we characterized and evaluated immunoregulatory characteristics of Legumain-1 from Fasciola gigantica (Fg-LGMN-1) during its interaction with host immune cells. The isopropyl-ß-d-thiogalactopyranoside (IPTG) stimulated RFg-LGMN-1 protein was positively detected by rat serum containing anti-RFg-LGMN-1 polyclonal antibodies. Furthermore, the uptake of RFg-LGMN-1 by goat monocytes was successfully confirmed using Immunofluorescence Assay (IFA). The immunohistochemical analysis revealed the native localization of LGMN-1 protein on the periphery and internal structures such as suckers, pharynx, and genital pore of the adult parasite, thereby validating its presence in excretory-secretory (ES) products of F. gigantica. The RFg-LGMN-1 co-incubated with concanavalin-A (Con-A) stimulated the increase of interleukin 2 (IL-2), IL-10, and IL-17 in monocytes derived from peripheral blood mononuclear cells (PBMCs) in the concentration-dependent manner. However, the IL-4 cytokine in response to the RFg-LGMN-1 protein declined. These results illuminated the role of LGMN-1 during the parasite-host interface. Our findings elaborated additional evidence that Legumain protein play a role in the manipulating host immune responses during parasite infections. However, further evaluation of RFg-LGMN-1 protein in context of its immunomodulatory roles should be conducted to enhance our understandings of the mechanisms employed by F. gigantica to evade host immune responses.

Intra- and interspecies variation and population dynamics of Fasciola gigantica among ruminants in Sudan.

Fasciola gigantica is a widespread parasite that causes neglected disease in livestock worldwide. Its high transmissibility and dispersion are attributed to its ability to infect intermediate snail hosts and adapt to various mammalian definitive hosts. This study investigated the variation and population dynamics of F. gigantica in cattle, sheep, and goats from three states in Sudan. Mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes were sequenced successfully to examine intra and interspecific differences. ND1 exhibited higher diversity than COI, with 15 haplotypes and 10 haplotypes, respectively. Both genes had high haplotype diversity but low nucleotide diversity, with 21 and 11 polymorphic sites for ND1 and COI, respectively. Mismatch distribution analysis and neutrality tests revealed that F. gigantica from different host species was in a state of population expansion. Maximum likelihood phylogenetic trees and median networks revealed that F. gigantica in Sudan and other African countries had host-specific and country-specific lineages for both genes. The study also indicated that F. gigantica-infected small ruminants were evolutionarily distant, suggesting deep and historical interspecies adaptation.

Fasciola gigantica vaccine construct: an in silico approach towards identification and design of a multi-epitope subunit vaccine using calcium binding EF-hand proteins.

Continuous attempts have been made to pinpoint candidate vaccine molecules and evaluate their effectiveness in order to commercialise such vaccines for the treatment of tropical fascioliasis in livestock. The pathophysiology of fascioliasis can be related to liver damage brought on by immature flukes that migrate and feed, as well as immunological reactions to chemicals produced by the parasites and alarm signals brought on by tissue damage. Future research should, in our opinion, concentrate on the biology of invasive parasites and the resulting immune responses, particularly in the early stages of infection. The goal of the current study was to use the calcium-binding proteins from F. gigantica to create a multi-epitope subunit vaccine. The adjuvant, B-cell epitopes, CTL epitopes, and HTL epitopes that make up the vaccine construct are all connected by certain linkers. The antigenicity, allergenicity, and physiochemical properties of the vaccine construct were examined. The vaccine construct was docked with toll-like receptor 2, and simulations of the molecular dynamics of the complex's stability, interaction, and dynamics were run. After performing in silico cloning and immunosimulation, it was discovered that the construct was suitable for further investigation. New vaccination technologies and adjuvant development are advancing our food safety procedures since vaccines are seen as safe and are accepted by the user community. This research is also applicable to the F. hepatica system.

Reconstruction of the TGF-ß signaling pathway of Fasciola gigantica.

In the present study, we reconstructed the transforming growth factor beta (TGF-ß) signaling pathway for Fasciola gigantica, which is a neglected tropical pathogen. We defined the components involved in the TGF-ß signaling pathway and investigated the transcription profiles of these genes for all developmental stages of F. gigantica. In addition, the presence of these components in excretory and secretory products (FgESP) was predicted via signal peptide annotation. The core components of the TGF-ß signaling pathway have been detected in F. gigantica; classical and nonclassical single transduction pathways were constructed. Four ligands have been detected, which may mediate the TGF-ß signaling pathway and BMP signaling pathway. Two ligand-binding type II receptors were detected, and inhibitory Smad7 was not detected. TLP, BMP-3, BMP-1, and ActRIb showed higher transcription in 42-day juvenile and 70-day juvenile, while ActRIIa, Smad1, ActRIIb, Smad8, KAT2B, and PP2A showed higher transcription in egg. TLM, Ski, Smad6, BMPRI, p70S6K, Smad2, Smad3, TgfßRI, Smad4, and p300 showed higher transcription in metacercariae. Four ligands, 2 receptors and 3 Smads are predicted to be present in the FgESP, suggesting their potential extrinsic function. This study should help to understand signal transduction in the TGF-ß signaling pathway in F. gigantica. In addition, this study helps to illustrate the complex mechanisms involved in developmental processes and F. gigantica - host interaction and paves the way for further characterization of the signaling pathway in trematodes.

Efficacy of flukicides on Fasciola gigantica, a food-borne zoonotic helminth affecting livestock in Bangladesh.

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 µg mL−1 was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

Superoxide dismutase inhibits cytotoxic killing of Fasciola gigantica newly excysted juveniles expressed by sheep invitro.

Fasciola gigantica faces a series of threats from various free radicals produced by the host immune system during its invasion through the abdominal cavity and establishment in the bile duct of ruminants, limiting the fluke viability. The role of the superoxide radical produced by Muzaffarnagari sheep immune effector cells against F. gigantica newly excysted juveniles (NEJs) is highlighted in this study, as is the critical role of superoxide dismutase enzyme (SOD) in dismutation of superoxide radicals derived from host immune effector cells in vitro. Three concentrations of the ovine immune effector cells viz. 2.5, 5, and 10 × 106 cells were tested for their ability to induced cytotoxic killing of the parasite. All the three cell concentrations caused significant (p < 0.01) cytotoxic killing of NEJs in comparison to the control groups. Also, reduction of the immune effector cell concentration directly correlates with the NEJs killing. Attachment of immune effector cells to the parasite tegument in the presence of anti-F. gigantica antibodies was found to be critical in inducing NEJs killing via antibody-dependent cell-mediated cytotoxicity (ADCC). However, the addition of SOD greatly inhibits cytotoxic killing of NEJs, demonstrating the importance of SOD enzyme in fluke survival and parasite evasion of the host immunity. Thus, F. gigantica SOD warrants a promising candidate for immunoprophylactic studies in ruminants against the tropical liver fluke.

A global phosphoproteomics analysis of adult Fasciola gigantica by LC-MS/MS.

Protein phosphorylation plays key roles in a variety of essential cellular processes. Fasciola gigantica is a tropical liver fluke causing hepatobiliary disease fascioliasis, leading to human health threats and heavy economic losses. Although the genome and protein kinases of F. gigantica provided new insights to understand the molecular biology and etiology of this parasite, there is scant knowledge of protein phosphorylation events in F. gigantica. In this study, we characterized the global phosphoproteomics of adult F. gigantica by phosphopeptide enrichment-based LC-MS/MS, a high-throughput analysis to maximize the detection of a large repertoire of phosphoproteins and phosphosites. A total of 1030 phosphopeptides with 1244 phosphosites representing 635 F. gigantica phosphoproteins were identified. The phosphoproteins were involved in a wide variety of biological processes including cellular, metabolic, and single-organism processes. Meanwhile, these proteins were found predominantly in cellular components like membranes and organelles with molecular functions of binding (51.3%) and catalytic activity (40.6%). The KEGG annotation inferred that the most enriched pathways of the phosphoproteins included tight junction, spliceosome, and RNA transport (each one contains 15 identified proteins). Combining the reports in other protozoa and helminths, the phosphoproteins identified in this work play roles in metabolic regulation and signal transduction. To our knowledge, this work performed the first global phosphoproteomics analysis of adult F. gigantica, which provides valuable information for development of intervention strategies for fascioliasis.

Identification and expression of a transforming growth factor beta (TGF-ß) homologue in the tropical liver fluke Fasciola gigantica.

Liver flukes, Fasciola spp., are veterinary and medically important parasites infecting numerous species of economically important animals in addition to humans on a global scale. The components of transforming growth factor beta (TGF-ß) signalling are widely distributed throughout the animal kingdom and are considerably conserved. Through shared common signal transduction mechanisms, crosstalk of TGF-ß signalling between a host and the parasite during infection is possible. Herein, we have identified and undertaken the molecular characterisation of a putative TGF-ß homologue from the tropical liver fluke F. gigantica (FgTLM). A FgTLM cDNA was 3557 bp in length, it encoded for 620 amino acid polypeptide which consisted of 494 amino acids of prodomain and 126 amino acids comprising the mature protein. FgTLM displayed characteristic structures of mammalian TGF-ß ligands that were unique to the inhibin-ß chain, monomer of activin. A phylogenetic analysis revealed the high degree of conservation with TGF-ß molecules from trematode species. Interestingly, the sequence of amino acid in the active domain of FgTLM was completely identical to FhTLM from F. hepatica. FgTLM expressed throughout the lifecycle of F. gigantica but was highly expressed in developmental active stages. The dynamics of expression of FgTLM during the developmental stages of F. gigantica was comparable to the pattern of TGF-ß expression in F. hepatica. Our findings demonstrated that FgTLM exhibits a high level of similarity to FhTLM in the context of both amino acid sequence and the life stage expression patterns. These similarities underline the possibility that the FgTLM molecule might have the same properties and functions as FhTLM in biological processes of the immature parasites and host immune evasion. Consequently, the specific biological functions of FgTLM on either parasite or relevant hosts need to be defined experimentally.

Geography and ecology of invasive Pseudosuccinea columella (Gastropoda: Lymnaeidae) and implications in the transmission of Fasciola species (Digenea: Fasciolidae) - a review.

Pseudosuccinea columella is considered invasive and has become an important intermediate host of both Fasciola species in many regions of the world. This systematic review assessed the geographical distribution of P. columella, and its implications in the transmission of Fasciola hepatica and Fasciola gigantica, globally. A literature search was conducted on Google Scholar, JSTOR and PubMed databases using Boolean operators in combination with predetermined search terms for thematic analysis. Results show that P. columella has been documented in 22 countries from Europe (3), Africa (8), Oceania (2), North America (3) and South America (6). Furthermore, this snail species has shown to adapt to and inhabit a vast array of freshwater bodies including thermal lakes and ditches with acidic soils. Studies showed that P. columella transmits F. hepatica, with natural and experimental infections documented in sub-Saharan Africa, Europe, South America and North America. Experimental infection studies in Cuba showed the presence of P. columella populations resistant to F. hepatica infection. Furthermore, some populations of this invasive snail collected from F. hepatica endemic locations in Brazil, Venezuela, Australia, South Africa, Colombia and Argentina were found without Fasciola infection. As a result, the role played by this snail in the transmission of Fasciola spp. in these endemic areas is still uncertain. Therefore, further studies to detect natural infections are needed in regions/countries where the snail is deemed invasive to better understand the veterinary and public health importance of this snail species in Fasciola-endemic areas and determine the global dispersion of resistant populations of P. columella.

Complementary transcriptomic and proteomic analyses reveal the cellular and molecular processes that drive growth and development of Fasciola hepatica in the host liver.

BACKGROUND: The major pathogenesis associated with Fasciola hepatica infection results from the extensive tissue damage caused by the tunnelling and feeding activity of immature flukes during their migration, growth and development in the liver. This is compounded by the pathology caused by host innate and adaptive immune responses that struggle to simultaneously counter infection and repair tissue damage. RESULTS: Complementary transcriptomic and proteomic approaches defined the F. hepatica factors associated with their migration in the liver, and the resulting immune-pathogenesis. Immature liver-stage flukes express ~ 8000 transcripts that are enriched for transcription and translation processes reflective of intensive protein production and signal transduction pathways. Key pathways that regulate neoblast/pluripotent cells, including the PI3K-Akt signalling pathway, are particularly dominant and emphasise the importance of neoblast-like cells for the parasite's rapid development. The liver-stage parasites display different secretome profiles, reflecting their distinct niche within the host, and supports the view that cathepsin peptidases, cathepsin peptidase inhibitors, saposins and leucine aminopeptidases play a central role in the parasite's destructive migration, and digestion of host tissue and blood. Immature flukes are also primed for countering immune attack by secreting immunomodulating fatty acid binding proteins (FABP) and helminth defence molecules (FhHDM). Combined with published host microarray data, our results suggest that considerable immune cell infiltration and subsequent fibrosis of the liver tissue exacerbates oxidative stress within parenchyma that compels the expression of a range of antioxidant molecules within both host and parasite. CONCLUSIONS: The migration of immature F. hepatica parasites within the liver is associated with an increase in protein production, expression of signalling pathways and neoblast proliferation that drive their rapid growth and development. The secretion of a defined set of molecules, particularly cathepsin L peptidases, peptidase-inhibitors, saponins, immune-regulators and antioxidants allow the parasite to negotiate the liver micro-environment, immune attack and increasing levels of oxidative stress. This data contributes to the growing F. hepatica -omics information that can be exploited to understand parasite development more fully and for the design of novel control strategies to prevent host liver tissue destruction and pathology.

Adaptive Radiation of the Flukes of the Family Fasciolidae Inferred from Genome-Wide Comparisons of Key Species.

Liver and intestinal flukes of the family Fasciolidae cause zoonotic food-borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place ∼90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory-secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged ∼5 Ma near the Miocene-Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size ∼10 ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance.

Extracellular vesicles from Fasciola gigantica induce cellular response to stress of host cells.

Extracellular vesicles (EVs) from parasitic helminths play an important role in immunomodulation. However, EVs are little studied in the important parasite Fasciola gigantica. Here the ability of EVs from F. gigantica to induce cellular response to stress (reactive oxygen species generation, autophage and DNA damage response) in human intrahepatic biliary epithelial cells (HIBEC) was investigated. F. gigantica-derived EVs were isolated by ultracentrifugation, and identified with transmission electron microscopy, nanoparticle size analysis and parasite-derived EV markers. Internalization of EVs by HIBEC was determined by confocal immunofluorescence microscopy and flow cytometry. ROS levels in HIBEC were detected by molecular probing. EVs-induced autophagy and DNA-damaging effects were determined by evaluating expression levels of light chain 3B protein (LC3B), phosphor- H2A.X and phosphor-Chk1, respectively. Results revealed that EVs with sizes predominately ranging from 39 to 110 nm in diameter were abundant in adult F. gigantica and contained the parasite-derived marker proteins enolase and 14-3-3, and EVs were internalized by HIBEC. Further, uptake of EVs into HIBEC was associated with increased levels of reactive oxygen species, LC3Ⅱ, phosphor-H2A.X and phosphor-Chk1, suggesting EVs are likely to induce autophagy and DNA damage & repair processes. These results indicate F. gigantica EVs are associated with modulations of host cell responses and have a potential important role in the host-parasite interactions.

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.

The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.

Newly excysted juveniles (NEJs) of Fasciola gigantica induce mice liver fibrosis and M2 macrophage-like phenotype in vivo.

Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMɑ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Fasciola hepatica and Fasciola gigantica coexistence in domestic ruminants in Nigeria: application of a PCR-based tool.

The distinction between Fasciola hepatica and Fasciola gigantica has been traditionally based on morphological criteria, although accurate recognition of the two flukes is usually difficult because of substantial variations in morphological features. The main aim of this study was to develop a PCR-based assay for discrimination between both species collected in sheep and cattle from Nigeria. A total of 47 animals, 33 cattle and 14 sheep, were sampled, and a single adult fluke was collected from each animal. DNA was extracted from flukes, and primers were designed based on mitochondrial DNA sequences to amplify a 304 bp fragment for the identification of F. hepatica and 752 bp for F. gigantica. PCR products from 12 flukes were sequenced for phylogenetic analysis. A total of 29 out of 47 flukes were identified as F. hepatica and 18 as F. gigantica. Within each host, the percentage of each fluke species was as follows: In cattle, 18/33 (54.5%) and 15/33 (45.5%) were F. hepatica and F. gigantica, respectively. In sheep, 11/14 (78.6%) were F. hepatica and 3/14 F. gigantica (21.4%). The phylogenetic analysis confirmed these results. Although the number of flukes collected in sheep was limited, it seems that F. hepatica is more prevalent in sheep than F. gigantica, whereas the percentage of each species was similar in cattle. This study confirms the presence of F. hepatica in Nigeria.

Complex and dynamic transcriptional changes allow the helminth Fasciola gigantica to adjust to its intermediate snail and definitive mammalian hosts.

BACKGROUND: The tropical liver fluke, Fasciola gigantica causes fasciolosis, an important disease of humans and livestock. We characterized dynamic transcriptional changes associated with the development of the parasite in its two hosts, the snail intermediate host and the mammalian definitive host. RESULTS: Differential gene transcription analysis revealed 7445 unigenes transcribed by all F. gigantica lifecycle stages, while the majority (n = 50,977) exhibited stage-specific expression. Miracidia that hatch from eggs are highly transcriptionally active, expressing a myriad of genes involved in pheromone activity and metallopeptidase activity, consistent with snail host finding and invasion. Clonal expansion of rediae within the snail correlates with increased expression of genes associated with transcription, translation and repair. All intra-snail stages (miracidia, rediae and cercariae) require abundant cathepsin L peptidases for migration and feeding and, as indicated by their annotation, express genes putatively involved in the manipulation of snail innate immune responses. Cercariae emerge from the snail, settle on vegetation and become encysted metacercariae that are infectious to mammals; these remain metabolically active, transcribing genes involved in regulation of metabolism, synthesis of nucleotides, pH and endopeptidase activity to assure their longevity and survival on pasture. Dramatic growth and development following infection of the mammalian host are associated with high gene transcription of cell motility pathways, and transport and catabolism pathways. The intra-mammalian stages temporally regulate key families of genes including the cathepsin L and B proteases and their trans-activating peptidases, the legumains, during intense feeding and migration through the intestine, liver and bile ducts. While 70% of the F. gigantica transcripts share homology with genes expressed by the temperate liver fluke Fasciola hepatica, gene expression profiles of the most abundantly expressed transcripts within the comparable lifecycle stages implies significant species-specific gene regulation. CONCLUSIONS: Transcriptional profiling of the F. gigantica lifecycle identified key metabolic, growth and developmental processes the parasite undergoes as it encounters vastly different environments within two very different hosts. Comparative analysis with F. hepatica provides insight into the similarities and differences of these parasites that diverged > 20 million years ago, crucial for the future development of novel control strategies against both species.

Assessing the performance of a Fasciola gigantica serum antibody ELISA to estimate prevalence in cattle in Cameroon.

BACKGROUND: Cattle rearing in Cameroon is both economically and culturally important, however parasitic diseases detrimentally impact cattle productivity. In sub-Saharan Africa bovine fasciolosis is generally attributed to F. gigantica, although understanding of Fasciola species present and local epidemiology in individual countries is patchy. Partly limited by the lack of representative surveys and understanding of diagnostic test perfromance in local cattle populations. The aims of this paper were to determine the Fasciola species infecting cattle, develop a species specific serum antibody ELISA, assess the performance of the ELISA and use it to assess the prevalence of F. gigantica exposure in two important cattle-rearing areas of Cameroon. RESULTS: A random sample of Fasciola parasites were collected and were all identified as F. gigantica (100%, CI:94.0-100%, n = 60) using RAPD-PCR analysis. A F. gigantica antibody ELISA was developed and initially a diagnostic cut-off was determined using a sample of known positive and negative cattle. The initial cut-off was used as starting point to estimate an optimal cut-off to estimate the best combination of sensitivity and specificity. This was achieved through sampling a naturally infected population with known infection status (cattle slaughtered at Bamenda abattoir, North West Region (n = 1112) and Ngaoundere abattoir, Vina Division, Adamawa Region (n = 776) in Cameroon). These cattle were tested and results analysed using a Bayesian non-gold standard method. The optimal cut-off was 23.5, which gave a sensitivity of 65.3% and a specificity of 65.2%. The prevalence of exposure to F. gigantica was higher in cattle in Ngaoundere (56.4% CI: 50.2-60.0%) than Bamenda (0.6% CI: 0.0-1.4%). CONCLUSION: Fasciola gigantica was identified as the predominant Fasciola species in Cameroon. Although the sensitivity and specificity F. gigantica antibody ELISA requires improvement, the test has shown to be a potentially useful tool in epidemiological studies. Highlighting the need for better understanding of the impact of F. gigantica infections on cattle production in Cameroon to improve cattle production in the pastoral systems of Central-West Africa. This paper also highlights that non-gold standard latent class methods are useful for assessing diagnostic test performance in naturally-infected animal populations in resource limited settings.