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ABSTRACT: BACKGROUND: Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice. METHODS: The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos - 7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT. RESULTS: DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4 T cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals. CONCLUSION: This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.
Genetic Vaccines and Therapy 08/2012; 10(1):7. · 2.10 Impact Factor
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ABSTRACT: 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.
Parasite Immunology 06/2011; 33(9):495-505. · 2.60 Impact Factor
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ABSTRACT: Gastrointestinal nematode parasites undergo several developmental stages within their mammalian host, each presenting different antigenic challenges to the immune system. To examine the expression of different immune mediators over time, biopsy samples were collected from the cannulated abomasum (true stomach) of immune sheep at several times after a challenge infection with Haemonchus contortus L3s. IL-5 and IL-13 mRNA expression levels were significantly increased above saline-challenged control levels at 5 and 7 days post challenge, while IL-4 showed an earlier peak at day 2 post challenge. IL-5, IL-13 and IL-4, as well as IFN-γ mRNA levels, peaked at 7 days before decreasing to non-significant levels at 28 days post challenge. TNF-α followed a similar profile while there was a slight increase in TGF-β in both control and challenged sheep. There was a significant increase in galectin-14 mRNA in the L3 challenged compared with the saline challenged group at 7 days while both galectin-11 protein and mRNA levels increased significantly by day 3 post challenge, peaking at 5-7 days post challenge. Distinct correlations were observed between these immune parameters at different times after L3 challenge. The galectin-14 protein level at day 2 post challenge was the only measured mediator significantly negatively correlated with worm burden. These studies highlight the dynamic nature of the immune response during parasite infection and the need to consider the different life cycle stages involved.
International journal for parasitology 04/2011; 41(5):487-93. · 3.39 Impact Factor
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ABSTRACT: Canaria Hair Breed (CHB) sheep are more resistant than Canaria sheep (CS) to experimental Haemonchus contortus infection. Protective responses appear effective against the adult stage of the parasite, not as commonly reported in other breeds against the larval stages. In this study we have quantified several abomasal immune cells and correlated these with parasitological variables for each breed. A significant negative correlation between CD4+ T cell numbers and worm burden or length at 28 dpi was seen only in CS sheep. Significant negative correlations for both abomasal eosinophils and γδ/WC1+ T cells, and fecundity of the adult worms were observed only in the resistant CHB sheep breed. Tissue eosinophils and γδ/WC1+ T cells were positively correlated in CHB sheep. We suggest that the two sheep breeds have disparate immune responses following infection with the parasite and that γδ+ T cells in association with eosinophils may play a hitherto unrecognised role in modulating fecundity in H. contortus adult female parasites.
Veterinary Parasitology 01/2011; 178(3-4):286-92. · 2.58 Impact Factor
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ABSTRACT: In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.
Veterinary Parasitology 01/2011; 178(3-4):264-72. · 2.58 Impact Factor
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ABSTRACT: With a rising world population and economic development, the global demand for meat, milk and other animal products is increasing dramatically. Controlling parasitic diseases in livestock, in particular helminth infections, could rapidly improve productivity and resource utilization. There is a growing interest in indigenous ruminant breeds because these animals have adapted to survive with minimal maintenance in the presence of high exposure to parasite infection. Recent findings on the mechanisms of parasite resistance in indigenous breeds are discussed, and the possibility that such studies may lead to new insight into the immunity and control of parasites proposed. These findings have important implications for the preservation of poorly characterized local indigenous breeds.
Trends in Parasitology 12/2010; 26(12):568-73. · 5.14 Impact Factor
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ABSTRACT: Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue gammadelta TCR+ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R+) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by gammadelta T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.
Veterinary Research 02/2010; 41(4):37. · 4.06 Impact Factor
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Simone A Beckham, David Piedrafita,
Carolyn I Phillips,
Nirma Samarawickrema,
Ruby H P Law,
Peter M Smooker,
Noelene S Quinsey,
James A Irving,
Deanne Greenwood,
Steven H L Verhelst,
Matthew Bogyo,
Boris Turk,
Theresa H Coetzer,
Lakshmi C Wijeyewickrema,
Terry W Spithill,
Robert N Pike
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ABSTRACT: The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.
The international journal of biochemistry & cell biology 08/2009; 41(7):1601-12. · 4.89 Impact Factor
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ABSTRACT: Liver flukes produce cathepsin B and cathepsin L in their excretory-secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L's are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.
Veterinary Parasitology 12/2008; 160(3-4):230-6. · 2.58 Impact Factor
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ABSTRACT: The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.
Journal of Parasitology 09/2008; 95(2):450-5. · 1.40 Impact Factor
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ABSTRACT: This study compares the susceptibility to Haemonchus contortus infection in two breeds of sheep endemic to the Canary Islands, the Canaria Hair Breed sheep and the Canaria sheep. Sheep were experimentally infected with 20,000 larvae of H. contortus and animals killed on days 7 and 28 post-infection. No difference between sheep breeds were detected in immature worm counts at days 7 or 28 post-infection. However, in comparison to the Canaria sheep breed, the Canaria Hair Breed sheep showed lower mean faecal egg counts, lower adult worm counts, lower number of eggs in utero and female worm stunting. Overall, these data suggest that the Canaria Hair Breed sheep has a greater resistance to H. contortus infection than Canaria sheep, and that this resistance may act at the level of the adult parasite.
Veterinary Parasitology 06/2008; 153(3-4):374-8. · 2.58 Impact Factor
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Veterinary Parasitology 06/2008; · 2.58 Impact Factor
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ABSTRACT: Fasciola hepatica and Fasciola gigantica are trematode parasites responsible for fasciolosis, a disease of ruminant animals which is also increasingly recognised as a disease in humans. By biochemical and in silico methods, we have cloned and characterised the 70 kDa heat-shock proteins (HSP70s) of F. hepatica and F. gigantica. The nucleotide and protein sequences for HSP70 were found to be 98% and 99% identical between liver fluke species, respectively, and to encode conserved amino acid motifs that are of putative functional importance. Western blot analysis demonstrated that HSP70 proteins were expressed at a higher level in F. gigantica recovered from sheep relative to F. hepatica, but HSP70 was not detected in the excretory-secretory products of these liver fluke samples. Real-time reverse-transcriptase PCR analysis of HSP70 expression in parasites from sheep, but not cattle, showed HSP70 expression to be higher in F. gigantica than F. hepatica. These results suggest that hosts refractory to F. gigantica are associated with higher HSP70 expression by this parasite and that HSP70 expression may represent a biochemical marker of the stress response of F. gigantica.
Experimental Parasitology 05/2008; 118(4):536-42. · 2.12 Impact Factor
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ABSTRACT: Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.
Infection and Immunity 05/2007; 75(4):1954-63. · 4.16 Impact Factor
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ABSTRACT: DNA vaccines have proven to be an efficient means of inducing immune responses in small laboratory animals; however, their efficacy in large out-bred animal models has been much less promising. In addressing this issue, we have investigated the ability of ovine cytotoxic lymphocyte antigen 4 (CTLA-4) mediated targeting and ruminant specific CpG optimised plasmids, both alone and in combination, to enhance immune responses in sheep to the pro cathepsin B (FhCatB) antigen from Fasciola hepatica. In this study, CTLA-4 mediated targeting enhanced the speed and magnitude of the primary antibody response and effectively primed for a potent memory response compared to conventional DNA vaccination alone, which failed to induce a detectable immune response. While the CpG-augmentation of the CTLA-4 targeted construct did not further enhance the magnitude or isotype profile of the CTLA-4 induced antibody titres, it did result in the induction of significant antigen-specific, lymphocyte-proliferative responses that were not observed in any other treatment group, showing for the first time that significant cellular responses can be induced in sheep following DNA vaccination. In contrast, CpG-augmentation in the absence of CTLA-4 mediated targeting failed to induce a detectable immune response. This is the first study to explore the potential adjuvant effects of ruminant specific CpG motifs on DNA vaccine induced immune responses in sheep. The ability of CpG-augmented CTLA-4 mediated targeting to induce both humoral and cellular immune responses in this study suggests that this may be an effective approach for enhancing the efficacy of DNA vaccines in large out-bred animal models.
Vaccine 03/2006; 24(7):970-9. · 3.77 Impact Factor
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ABSTRACT: The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.
Infection and Immunity 01/2004; 71(12):6921-32. · 4.16 Impact Factor
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ABSTRACT: The development of subunit vaccines against most parasitic helminth infections will require a better understanding of the different components of a natural rejection process including (1) recognition of parasite antigens; (2) induction of protective immune response phenotypes; and (3) activation of appropriate immune effector mechanisms. While novel technologies have allowed significant progress to be made in the identification of candidate vaccine antigens, the large scale production of these antigens and their presentation to the host with appropriate adjuvant systems remains a major problem in vaccine research. Identification of the molecular interactions involved in the innate immune response to helminth infections and the application of new genomic and proteomic technologies are likely to lead to major advances in these research fields. Gastrointestinal nematode parasites and liver fluke are the most important helminth parasites of production animals. In recent years, a lot of new knowledge has been gathered on the immunobiology of the host-parasite interactions in these two infection systems, which has allowed new vaccination strategies to be considered. Functional genomic technologies such as gene expression analysis by microarrays, promise to further advance our understanding of the molecular pathways leading to protection against parasite infections. This will not only have implications for vaccine research, but also provide novel targets for drug development and genetic selection.
International Journal for Parasitology 10/2003; 33(11):1285-90. · 3.39 Impact Factor
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ABSTRACT: 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.
Parasite Immunology 05/2000; 22(6):287 - 295. · 2.60 Impact Factor
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ABSTRACT: Canaria Hair Breed (CHB) sheep are more resistant than Canaria sheep (CS) to experimental Haemonchus contortus infection. Protective responses appear effective against the adult stage of the parasite, not as commonly reported in other breeds against the larval stages. In this study we have quantified several abomasal immune cells and correlated these with parasitological variables for each breed. A significant negative correlation between CD4+ T cell numbers and worm burden or length at 28 dpi was seen only in CS sheep. Significant negative correlations for both abomasal eosinophils and γδ/WC1+ T cells, and fecundity of the adult worms were observed only in the resistant CHB sheep breed. Tissue eosinophils and γδ/WC1+ T cells were positively correlated in CHB sheep. We suggest that the two sheep breeds have disparate immune responses following infection with the parasite and that γδ+ T cells in association with eosinophils may play a hitherto unrecognised role in modulating fecundity in H. contortus adult female parasites.
Veterinary Parasitology.
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ABSTRACT: In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.
Veterinary Parasitology.
