Investigation of the combined efficacy of two Haemonchus contortus vaccines in weaner Merino sheep.

This study was conducted to investigate whether co-administration of Barbervax® (Bvax) with Haemonchus contortus surface larval antigen (HcsL3) would increase the protective efficacy and duration of protection against H. contortus infection in weaner Merino sheep. A total of 132 10-month-old weaned Merino ewe lambs were randomly allocated into six treatment groups (n = 22). Sheep were vaccinated four times with either Barbervax® (Bvax), H. contortus L3 surface larval antigen (HcsL3), combined vaccination (Bvax + HcsL3), Bvax + AlOH, HcsL3 + Saponin or remained as unvaccinated controls. Aluminium hydroxide (AlOH) and saponin adjuvants were included in HcsL3 and Bvax vaccines respectively. The first three vaccinations were given at 4 week intervals and the fourth vaccination provided as booster, 9 weeks later. All animals were treated with Zolvix™ (monepantel 25 mg/mL, Elanco) at the third vaccination and commencing two weeks later, artificially trickle infected with H. contortus L3. Worm egg count (WEC), packed cell volume (PCV), antibody titre and bodyweight were measured throughout the study as was specific antibody directed against each antigen using ELISA. The administration of Bvax and HcsL3, alone or in combination, induced an antibody response against HcsL3 but only the Bvax and the combined treatment elicited an antibody response to the Bvax antigen. The targeting of HcsL3 by each vaccine was confirmed by immunofluorescence staining of H. contortus L3. However, only the booster vaccination in the Bvax treatments reduced WEC to levels below untreated controls. The HcsL3 vaccine did not reduce WEC in this experiment and co-administration with Bvax did not improve the efficacy and duration of protection against H. contortus infection.

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

The composition and stability of the faecal microbiota of Merino sheep.

AIMS: To determine the composition and temporal stability of the gut (faecal) microbiota of sheep (Ovis aries). METHODS AND RESULTS: Microbial population dynamics was conducted using ARISA (28 sheep) and 16S rRNA sequencing (11 sheep). Firmicutes and Bacteroidetes were the predominant bacterial phyla, constituting ~80% of the total population. The core faecal bacterial microbiota of sheep consisted of 67 of 136 detected families and 91 of 215 detected species. Predominant microbial taxa included Ruminococcaceae, unassigned families in Bacteroidales and Clostridiales, Verrucomicrobiaceae and Paraprevotellaceae. Diversity indices and core microbiota composition demonstrated the stability of the core microbiota over 2-4 weeks. The core microbiota remained similar over ~5 months. CONCLUSIONS: Temporal stability of the sheep microbiota is high over 2-4 weeks in the absence of experimental variables. The core microbiota of Merino sheep shares taxa found in other breeds of sheep and other ruminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerous studies seek to investigate the impact of experimental variables on gut microbiota composition. To do so, knowledge of the innate stability (or instability) of the microbiota over an experimental time course is required, independent of other variables. We have demonstrated high stability of the gut microbiota in sheep over 3-4 weeks, with moderate stability over ~5 months.

Potential role for mucosal IgA in modulating Haemonchus contortus adult worm infection in sheep.

Haemonchus contortus (H. contortus) is a haematophagous parasite which causes important economic losses in small ruminants. On the island of Gran Canaria, two sheep breeds coexist which differ in their susceptibility to the infection with H. contortus; the resistant Canaria Hair Breed (CHB) sheep and the susceptible Canaria Sheep (CS) breed. The major target of resistance mechanisms in CHB sheep are directed to the adult parasite stage, reducing the worm burden, and decreased length and fecundity of surviving worms. Mucosal IgA (mIgA) has been shown to be an important regulator of immunity in Haemonchus and Teladorsagia infections; through correlations with larval stages where such mechanisms as antibody-dependent cell cytotoxicity and enzyme inhibition may mediate resistance. Here for the first time, we demonstrate a significant negative correlation between mIgA and adult worm length and fecundity only in the resistant CHB sheep. In contrast, and as reported in other sheep breeds, mIgA was only negatively correlated against the larval stage in the more susceptible CS breed. This study suggests mIgA may play a role in resistance to both larval and adult stages.

Immunity to Haemonchus contortus and Vaccine Development.

Sheep are capable of developing protective immunity to Haemonchus contortus through repeated exposure to this parasite, although this immune protection is the result of a complex interaction among age, gender, physiological status, pregnancy, lactation, nutrition and innate and adaptive immunity in the host animal. There are multiple effectors of the protective immune response, which differ depending on the developmental stage of the parasite being targeted, and our understanding of the effector mechanisms has developed considerably in the 2000s. The rational design of vaccines based on 'natural' or 'exposed' antigens depends on an understanding of this exposure-induced immunity. However, the most effective current vaccines rely on protection via the induction of high circulating antibody levels to 'hidden' gut antigens of H. contortus. The success of this latter strategy has resulted in the launch of a vaccine, which is based on extracts of the parasite's gut, to aid in the control of Haemonchus in Australia. The development of recombinant subunit vaccines based on the components of the successful native vaccine has not yet been achieved and most of the recent successes with recombinant subunit vaccines have focussed on antigens unrelated to the gut antigens. The future integration of an understanding of the immunobiology of this parasite with advances in antigen identification, expression (or synthesis) and presentation is likely to be pivotal to the further development of these recombinant subunit vaccines. Recent progress in each of the components underpinning this integrated approach is summarized in this review.

Galectin-11: A novel host mediator targeting specific stages of the gastrointestinal nematode parasite, Haemonchus contortus.

Galectin-11 is released from epithelial cells of the gastrointestinal tract, specifically following infection with gastrointestinal parasites including the highly pathogenic nematode, Haemonchus contortus. The function(s) of galectin-11 are currently unknown but seem to be associated with the development of immunity by the host. The aim of the present study was to examine the interaction of galectin-11 with the different parasitic life cycle stages of H. contortus and determine any effects on parasite development. The results of this study showed that galectin-11 binds to the surface of the L4 and adult stages of the parasite but not to the exsheathed L3 stage. In addition, at a lower concentration, binding to the L4 was specifically localised to the pharynx region. Subsequent in vitro assays demonstrated significant inhibition of larval growth and development in the presence of recombinant galectin-11. These results indicate, to our knowledge for the first time, a functional role for galectin-11 in gastrointestinal nematode infection of ruminants and a mechanism of action of galectin-11, targeting the development and growth of the L4 and possibly the adult parasite stage.

Field vaccination of sheep with a larval-specific antigen of the gastrointestinal nematode, Haemonchus contortus, confers significant protection against an experimental challenge infection.

The availability of effective vaccines would add a valuable tool to the management of gastrointestinal nematode infections in livestock. While some experimental vaccines have shown protection in laboratory trials, few have been tested in the field. In the present study, eight month old sheep kept on pasture were treated with anthelmintic 8 weeks before vaccination with a larval surface antigen of the nematode parasite, Haemonchus contortus, under a commercially acceptable protocol, i.e. 2 immunizations using a commercial adjuvant; they were then given a controlled challenge infection 4 weeks later in indoor pens. Vaccination of sheep with 4 increasing doses of antigen resulted in significant reductions of 61% and 27% in cumulative faecal egg counts in the two highest dose groups, and a 69% reduction in worm burden in the highest dose group. Blood loss, as determined by packed cell volume, was also significantly reduced in the highest dose group of sheep. One outlier sheep showed an unusual increase in egg count without a concomitant increase in worm burden compared to the control sheep, indicating a vaccine-induced stress response. Antigen-specific serum antibody levels steadily increased in sheep while on pasture and decreased when transported to indoor pens. No difference in antibody levels could be detected between vaccinated and unvaccinated sheep, but all showed increased antibody levels compared to uninfected control sheep kept in indoors pens for 2-3 months, suggesting sheep were sensitized to the larval antigen either from low dose pasture contamination or cross reaction with pasture-related antigens. The results of these studies confirm the protective properties of the larval surface antigen and its protective effect when vaccinations are performed in the field.

Molecular cloning and characterisation of ovine dual oxidase 2.

The dual oxidases (DUOX1 and DUOX2) are NADPH-dependent hydrogen peroxide-producing enzymes that are reported to function in a physiological capacity and as a component of the mucosal immune response. We have previously reported increased expression of the DUOX2 gene in the gut mucosa of sheep in response to gastrointestinal nematode (GIN) challenge. In this paper, we report the cloning of the full-length ovine DUOX2 transcript, using a PCR based strategy. The ovine DUOX2 transcript includes an ORF of 4644 bases, and encodes a protein with 97% identity to the bovine sequence. We also cloned a fragment of DUOX1 (encompassing nucleotides 2692-2829), and the proximal promoter sequence of DUOX2. Through analysis of sequence data we have confirmed that DUOX1 and DUOX2 are co-located in a head to tail arrangement conserved across many species. Alignment of the sequences to the ovine genome predicts a location of this gene cluster on ovine chromosome 7. We quantified the expression of ovine DUOX1 and DUOX2 transcripts in 24 different sheep tissues, and discovered tissue specific expression signatures. DUOX2 was found to be most highly expressed in tissues of the gastrointestinal tract, while expression of DUOX1 predominated in the bladder. Rapid amplification of cDNA ends (RACE) analysis identified the existence of multiple 5' UTR variants in DUOX2, ranging in size from 32 to 242 nucleotides, with 3 distinct transcribed regions. Real time PCR quantification of the DUOX2 UTR variants revealed that these were differentially expressed between tissues, and at various stages of the response to GIN parasite infection. The collective evidence suggested a complex regulation of DUOX2, prompting a bioinformatic analysis of the proximal promoter regions of ovine DUOX2 to identify potential transcription factor binding sites (TFBS) that may explain the differences in the observed expression of the transcript variants of DUOX2. Possible transcription factor families that may regulate this process were identified as Kruppel-like factors (KLF), ETS-factors, erythroid growth receptor factors (EGRF) and myogenic differentiation factors (MYOD).

Novel immunomic technologies for schistosome vaccine development.

Schistosomiasis remains one of the most common human helminthiases, despite the availability of an effective drug against the causative parasites. Drug treatment programmes have several limitations, and it is likely that a vaccine is required for effective control. While decades of vaccine development have seen the discovery and testing of several candidate antigens, none have shown consistent and acceptable high levels of protection. The migrating larval stages are susceptible to immunity, however few larval-specific antigens have been discovered. Therefore, there is a need to identify novel larval-specific antigens, which may prove to be more efficacious than existing targets. Immunomics, a relatively new field developed to cope with the recent large influx of biological information, holds promise for the discovery of vaccine targets, and this review highlights some immunomic approaches to schistosome vaccine development. Firstly, a method to focus on the immune response elicited by the important and vulnerable larval stage is described, which allows a targeted study of the immunome at different tissue sites. Then, two high-throughput arrays are discussed for the identification of protein and carbohydrate antigens. It is anticipated that these approaches will progress vaccine development against the schistosomes, as well as other parasites.

Resistance to liver fluke infection in the natural sheep host is correlated with a type-1 cytokine response.

Indonesian thin-tail (ITT) sheep can resist infection with Fasciola gigantica but not F. hepatica and presents an ideal model to investigate the mechanisms of liver fluke resistance in a natural host. This study examines the local and systemic immune responses of sheep during Fasciola infection and demonstrates that different anatomical tissues display distinct cytokine profiles consistent with liver fluke migration. The study also reveals a significant difference in the cytokine and antibody profiles of ITT sheep infected with F. gigantica compared with F. hepatica, with a higher ratio of IL-4/IFN-γ mRNA expression and specific IgG1/IgG2 antibodies strongly correlating with pathology. Interestingly, the significant type-1 cytokine profile occurred in the lymph node closest to the site of infection at a time when the effective immune response against F. gigantica liver flukes is thought to occur. When the same F. gigantica infection in the resistant ITT sheep was compared with the susceptible Merino breed, the resistant type-1 phenotype against liver fluke infection was only observed in the ITT sheep. These studies provide the first evidence to suggest that the induction of an early type-1 immune response in this natural sheep host may be responsible for the ability to resist liver fluke infection.

Dual oxidase 2 and glutathione peroxidase gene expression are elevated in hyperimmunized sheep challenged with Haemonchus contortus.

A sequential biopsy sampling method was used to investigate oxidant and antioxidant gene responses in resistant sheep challenged with Haemonchus contortus larvae or a sham saline challenge. The expression of key sheep oxidant and antioxidant producing genes were measured in sequential samples removed from the abomasums at days 0, 1, 2, 3, 5, 7 and 28 post challenge. Gene expression levels at each time point were compared to expression at day 0, and levels of the various genes were also correlated to other markers of infection including immune cell counts and cytokine gene expression. The early response to larval challenge infection in resistant animals was marked by a divergence of two groups of host oxidant producing genes: the dual oxidase group (DUOX2/DUOXA2) showing increases in expression to day 7, while members of the phagocytic NADPH oxidase (PHOX) group showed significant decreases in expression. The change in DUOX2 expression between days zero and seven, when host resistance to infection is mediated, was negatively correlated to final worm burden suggesting NADPH oxidase expression may play a role in parasite expulsion. Expression of the DUOX group oxidants was positively correlated to expression of the Th2 cytokine IL4. Changes in host antioxidant pathways between different members of the glutathione peroxidase family (intestinal and plasma GPX) and genes involved in glutathione metabolism were also observed. This first study of the putative roles of oxidant production by the dual oxidase group, antioxidant glutathione pathways, immune cell populations, and cytokine profiles, in the development of resistance to infection by hyperimmune sheep are discussed.

Immune mechanisms of resistance to gastrointestinal nematode infections in sheep.

Infections with gastrointestinal nematode parasites are a major problem for the sheep industry in Australia and New Zealand and have been the subject of intensive research to define mechanisms of resistance. The ability to take continuous biopsy samples of infected organs and cannulate both afferent and efferent lymphatics of draining lymph nodes has been particularly useful in illuminating the kinetics of immune responses at the site of infection. Distinct localized immune responses were shown to occur within and between sheep breeds at different sensitization regimes, as well as at different developmental stages of the parasite within the host. Using localized antibodies derived from mucus and lymph nodes, two major antigens have been identified on the infective L3 stage, which may be responsible for inducing protection and have potential as vaccine targets. Recent advances in sheep genomics also offer the potential of gaining further insight into the underlying genetics of resistance to nematode infections.

Improving animal and human health through understanding liver fluke immunology.

Sheep, goats and cattle represent the most numerous and economically important agricultural species worldwide used as sources for milk, fibre and red meat. In addition, in the developing world, these species often represent the sole asset base for small-holder livestock farmers and cattle/buffaloes often provide the majority of draught power for crop production. Production losses caused by helminth diseases of these animals are a major factor in extending the cycle of poverty in developing countries and a major food security issue for developed economies. Fasciola spp. are one of the most important zoonotic diseases with a global economic impact in livestock production systems and a poorly defined but direct effect on human health. Improvements in human and animal health will require a concerted research effort into the development of new accurate and simple diagnostic tests and increased vaccine and drug development against Fasciola infections. Here, the use of definitive natural host breeds with contrasting resistance to Fasciola infections is discussed as a resource to contrast parasite-host interactions and identify parasite immune evasion strategies. Such studies are likely to boost the discovery of new vaccine, drug and diagnostic candidates and provide the foundation for future genetic selection of resistant animals.

Host responses during experimental infection with Fasciola gigantica and Fasciola hepatica in Merino sheep II. Development of a predictive index for Fasciola gigantica worm burden.

This study reports on the predictive relationship between serological, immunological and pathological responses following experimental inoculation with incremental doses of Fasciola gigantica in sheep. Fifty, 6-month-old, naive Merino wethers were allocated to one of 5 experimental groups, four of which received 50, 125, 225 and 400 metacercariae, respectively, whilst a 5th group acted as non-inoculated control. Strong individual correlations were observed between liver score, GLDH (glutamate dehydrogenase), GGT (gamma glutamyl transferase), CatL5 (cathepsin L5) antibody titre (IgG1, IgA), eosinophilia, and the total worm count or worm biomass. A combination of multiple indicator traits performed significantly better than any single indicator trait alone. The best predictive index accounted for up to 88% of observed worm burden (Wb) if information on inoculation dose was available. Without knowledge of inoculation dose, such as under field conditions, up to 67% of variation in worm burden could be predicted. In contrast, the best single predictor variable (liver damage score) accounted for up to 50% of worm burden, and in the absence of post-slaughter information, serum levels of anti-cathepsin IgA antibody titres accounted for 35% of predicted variation in worm burden. The utility of a predictive index under both field and experimental inoculation conditions is discussed.

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection.

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.

Identification of two allelic forms of ovine CD4 exhibiting a Ser183/Pro183 polymorphism in the coding sequence of domain 3.

The ovine CD4 cDNA sequence from four sheep sources (Australian Merino, Indonesian Thin Tail, Canadian cross bred, Prealpes du sud) predicts a protein of 455 residues with position 130 in the V2 domain exhibiting a W instead of C suggesting that, like the white whale, dog and cat sequences, sheep CD4 contains only two disulphide bonds. The sequence shows 73% amino acid identity and 83% nucleotide identity to a CD4 sequence from the white whale and significant identity to a partial sequence (314 residues) of bovine CD4 (87% amino acid identity, 93% nucleotide identity). Phylogenetic analysis showed that the ovine CD4 sequence forms a clade with the pig, white whale, dolphin, dog and cat CD4. Two forms of ovine CD4 were identified which differ by a single base pair (T/C) in their cDNA sequence at position 622. This polymorphism is also present in sheep genomic DNA in Hardy-Weinberg equilibrium, suggesting that at least two alleles of CD4 exist in the ovine genome with no selection for a particular allele. This polymorphism changes the first codon position of amino acid 183 and results in a Pro/Ser substitution in the N-terminal region of domain 3 of the CD4 protein.

Detection of coproantigens by sandwich ELISA in sheep experimentally infected with Fasciola gigantica.

Two monoclonal antibodies to Fasciola gigantica excretory/secretory (ES) antigens were used in a sandwich ELISA for the detection of Fasciola antigens in faeces of 9 sheep experimentally infected with 300 metacercariae of F. gigantica. The detection of coproantigens was found in four of the seven sheep within 5 weeks of infection, and within 7 weeks of infection coproantigens were detected in all seven of the sheep. This technique was compared to an indirect ELISA for the detection of anti-Fasciola ES antigen specific antibodies in serum. The anti-F. gigantica antibodies were detected within 3 weeks of infection in all of the infected sheep, suggesting a greater sensitivity to detect early infections. However, following anthelmintic treatment and removal of parasites, the anti-Fasciola antibody levels still remained high for at least 6 weeks when the study was terminated. In contrast, the levels of coproantigens were no longer detected in the faeces within 2 weeks of anthelmintic treatment. This study demonstrates that our sandwich ELISA for the detection of Fasciola coproantigens is able to detect immature fluke infections and more importantly, was able to detect patent infection of fasciolosis.

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni.

Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.