Advances in Fasciola hepatica research using ‘omics’ technologies

The liver fluke Fasciola hepatica is an economically important pathogen of livestock worldwide, as well as being an important neglected zoonosis. Parasite control is reliant on the use of drugs, particularly triclabendazole, which is effective against multiple parasite stages. However, the spread of parasites resistant to triclabendazole has intensified the pursuit for novel control strategies. Emerging 'omics' technologies are helping advance our understanding of liver fluke biology, specifically the molecules that act at the host-parasite interface and are central to infection, virulence and long-term survival within the definitive host. This review discusses the technological sequencing advances that have facilitated the unbiased analysis of liver fluke biology, resulting in an extensive range of ‘omics’ datasets. In addition, we highlight the ‘omics’ studies of host responses to F. hepatica infection that, when combined with the parasite datasets, provide the opportunity for integrated analyses of host-parasite interactions. These extensive datasets will form the foundation for future in-depth analysis of F. hepatica biology and development, and the search for new drug or vaccine interventions.     

Liver fluke vaccines in ruminants: strategies,progress and future opportunities

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30–54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.     

Progress in development of liver fluke vaccines

Infection of ruminants by Fasciola spp continues to cause large economic losses worldwide. Recent results from several laboratories have demonstrated that animals can be significantly protected against infection by vaccination with defined Fasciola antigens. Apart from reducing fluke burdens, some vaccines can elicit a concurrent reduction in parasite egg production. The expectation of a commercially feasible vaccine that might also reduce parasite transmission in the field is now realistic, although major hurdles still exist. Here, Terry Spithill and John Dalton review the results of several recent vaccine trials and discuss the future prospects for vaccine development.     

Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro

Sandeman R. M. and Howell M. J. 1982. Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro. International Journal for Parasitology12: 467–471. The role of sheep antibodies which precipitate with surface antigens of Fasciola hepatica is unclear. In an attempt to clarify their function these antibodies were characterized as to their immunoglobulin class and ability to affect the survival of fluke in rats. The ability of fluke antigens complexed with sheep antibody to vaccinate rats against infection was also tested. IgM antibodies were involved in precipitate formation on the teguments of fluke 3 weeks after infection but IgG₁ predominated at later stages of infection. The decreased survival of fluke in rats after culture with increasing levels of sheep antibodies suggests that the antibodies exert some deleterious effect on the fluke in vitro. The fluke antigen-sheep antibody complex failed to immunize rats against infection. Since sheep antibodies to F. hepatica can impair the ability of fluke to resist further attack in rats but not sheep, it is suggested that some effector mechanism other than antibody is defective in the latter.     

The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Fasciolicidal potential of proline analogues and proline biosynthesis inhibitors

Sheers Marion, Campbell Anne J., Beames D. J., Edwards S. R., Moore R. J. and Montague P. E. 1982. Fasciolicidal potential of proline analogues and proline biosynthesis inhibitors. International Journal for Parasitology12: 47–52. Hydroxylamine HCl and thiazolidine-4'-carboxylic acid, known inhibitors of important enzymes of proline biosynthesis, inhibited to a similar extent the arginine-dependent proline production by the liver fluke Fasciola hepofica; in vitro there appeared to be no correlation between inhibition of proline synthesis and deterioration of the fluke. Another known inhibitor, thiosemicarbazide, had no effect on arginine-dependent proline production in vitro. None of these compounds was effective in vivo either as a flukicidal agent per se or in the prevention of the establishment of fluke in the bile duct of rats. A variety of proline analogues was also tested for flukicidal activity in vitro and in vivo as well as for their ability to inhibit the establishment of fluke in the bile duct of rats. Only one was effective in vitro and none was effective in vivo. Also continuous administration of l-azetidine-2-carboxylic acid failed to prevent the establishment of an infection of liver fluke in the bile duct. It is concluded that there is little prospect of a successful approach to chemotherapy of fascioliasis in this area.     

An approach to the production of helminth antigens in vitro: The formation of hybrid cells between Fasciola hepatic a and a rat fibroblast cell line

Using polyethylene glycol, hybrid cells were formed between rat fibroblasts lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT, B.C. 2.4.2.8) and cells of the liver fluke Fasciola hepatica. The hybrid cells survived in a medium containing hypoxanthine, aminopterin and thymidine (HAT) indicating that the enzyme deficiency of the parental rat cells had been corrected. Isoelectric focusing in agarose gels showed that the HGPRT activity in the hybrids was of F. hepatica rather than rat origin. F. hepatica chromosomes could not be identified with certainty in hybrids; and fluke antigens, other than HGPRT, could not be detected in them or in culture medium in which they had grown.     

The chemical nature of the egg-shell of helminths. I. Absence of quinone tanning in the egg-shell of the liver fluke, Fasciola hepatica

Ramalingam K. 1973. The chemical nature of the egg-shell of helminths—I. Absence of quinone tanning in the egg-shell of the liver fluke, Fasciola hepatica. International Journal for Parasitology, 3: 67–75. The mode of stabilization of the egg-shell protein of Fasciola hepatica has been studied. It was observed that quinone tanning is absent in the egg-shell. Unlike quinone-tanned proteins, egg-shell proteins show auto-fluorescence. Investigation on the fluorescent compounds present in the egg-shell protein shows the presence of di-tyrosine. In addition to the presence of di-tyrosine, the egg-shell protein is stabilized by —S—S—bonding. The resistant properties of the egg-shell of F. hepatica are attributed to the presence of the above types of cross-links.     

Glutathione S-transferases in Fasciola hepatica

Glutathione S-transferases (GST's) are widespread in the tissues of the liver fluke, Fasciola hepatica, and consist of multiple isozymes. Following purification to apparent homogeneity by affinity chromatography on glutathione agarose, fluke GST's were shown to comprise 2 components with molecular weights of about 25,000. Fluke GST's were immunogenic to rats, but when used as a vaccine conferred no protection on the animals against a challenge infection with F. hepatica metacercariae.     

Fasciola hepatica: immunisation of rats by intraperitoneal injection of adult fluke antigen in Freund's adjuvant

Intraperitoneal injections of rats with freeze-dried, adult Fasciola hepatica material, which had been resuspended in phosphate-buffered saline and emulsified in Freund's incomplete adjuvant, reduced fluke burdens by 48 to 81% following oral infection. The addition of Bordetella pertussis to the adjuvant antigen emulsion enhanced the protection slightly (but not to a statistically significant degree); fluke antigens with B. pertussiss alone induced no protection.     

Towards delineating functions within the fasciola secreted cathepsin l protease family by integrating in vivo based sub-proteomics and phylogenetics

Background

Fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family.

Methodology/Principal Findings

The sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples.

Conclusions/Significance

We have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.     

Molecular Characterization of a Tetraspanin from the Human Liver Fluke, Opisthorchis viverrini

Background

The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens.

Methodology/Principal Findings

The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls.

Conclusions/Significance

This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis

Background

Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.

Results

We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.

Conclusions

This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.     

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.     

The Diterpenoid 7-Keto-Sempervirol,Derived from Lycium chinense,Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

BackgroundTwo platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.

Methodology/ Principle Findings

Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD₅₀ = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD₅₀ = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.

Conclusions/ Significance

7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.