Study of the helminth fauna in eagle owl (Bubo bubo) in the south of Spain.

In this study we show the results of the eagle owls' (Bubo bubo) helminthfauna found in Andalusia. A total number of 50 specimens have been analysed in a period of 10 years (from 2011 to 2020). Prevalence ( P % ), mean intensity (IM) and mean abundance (AM) of parasitation have been obtained. The percentage of parasitation in the total sample was 80% (40 out of 50 eagle owls): 78% nematodes, 8% trematodes, 6% cestodes and 4% acantocephalans. 7 species of helminths were identified: 6 nematodes, and 1 trematode. In the case of cestodes and acantocephalans it was not possible to determine species and only the genus was identified. The intestinal nematode Capillaria tenuissima ( P %  = 58% (44-71.2); IM = 11,52 (5.83-28.9)) was the core species whereas Synhimantus laticeps (P% = 16 (7.5-28.8); IM = 4 (1.75-7.25)) and Hartertia hispanica (P% = 16 (7.5-28.8); IM = 1,5 (1-2)) were the secondary species. The remainder species were considered satellite species, with low prevalence and average abundance. Likewise, descriptive parameters of the helminth community were determined: species richness, 1.56 (1.29-1.94), total abundance, 12 (7.24-26.40), Brillouin's diversity index, 0.18 (0.10-0.29) and Berger-Parker dominance index, 0.88 (0.81-0.93). The data from this study show a non-diverse helminthic community, without species dominance with C. tenuissima as the central species, followed by S. laticeps and H. hispanica as secondary species. Worth mentioning is the presence of H. hispanica, which is considered an endemic species in Spain and specifically in Andalusia. To the authors' knowledge, this is the largest population sample taken in parasitological studies about helminths of this raptor in Europe and the first one carried out in the south of Spain (Andalusia).

Lipidomics: An omics discipline with a key role in nutrition.

The importance of lipidomics to unveil crucial aspects in the nutrition field is afforded in this article. With this aim, historical facts such as demonization of fats, enthronization of carbohydrates, and subsequent changes in the food pyramid are first discussed. After considering basic and analytical aspects of lipidomics and the upstream information this omics provides, its key role in personalized nutrition (PN), and the importance of lipids as nutrition biomarkers are critically discussed by appropriate examples. Pendent challenges to clarify the role of lipidomics in nutrition are overcome limiting factors, design of new lipidomics-based biomarkers, unveil mechanisms involved in lipidomics processes, and integrate lipidomics with other omics for a more complete and validated information useful in PN. The conclusions of this study also include the scant role of analytical chemists in the lipidomics-nutrition binomial, basically supported on analytical data.

Microscopical Techniques to Analyze the Hepatic and Peritoneal Changes Caused by Fasciola hepatica Infection.

The helminth parasite Fasciola hepatica modulates the host immune response at early stages of infection (Rodríguez et al., PLoS Negl Trop Dis 9:e0004234, 2015; Vukman et al., J Immunol 190:2873-2879, 2013). Nevertheless, little is known about the cell composition of the peritoneal fluid at these early stages of infection.In this chapter, we describe a method to perform peritoneal lavages and to recover peritoneal fluid from sheep experimentally infected and noninfected with F. hepatica at early stages of infection. In addition, with the aim to characterize the peritoneal fluid immune cell phenotype, we describe a procedure to obtain the total leukocyte count, the differential leukocyte count and the preparation and storage of peritoneal fluid smears, together with the application of an immunocytochemical technique and an automatic method to count the immunoreactive cells. Finally, the present protocol describes the evaluation of the gross and the histopathological lesions together with the immunohistochemical analysis of the hepatic tissue.

The associations between menopausal symptoms and sleep quality in Spanish postmenopausal women.

Objectives: Around the menopause, sleep disturbances frequently occur or worsen and are associated with decreased health quality and physical and psychological problems. The aim of this study was to analyze sleep quality and its association with the impact of menopausal symptoms in Spanish postmenopausal women. Methods: A total of 278 postmenopausal women (age 60.95 ± 8.01 years) participated in this cross-sectional study. The Medical Outcomes Study Sleep Scale (MOS-SS) and the Menopause Rating Scale (MRS) were used to analyze sleep quality and severity of menopausal symptoms, respectively. Anxiety and depression were measured using the Hospital Anxiety and Depression Scale. Results: The linear regression showed that a greater impact of menopausal symptoms (MRS total score) was associated with worse scores regarding sleep adequacy (p < 0.001, R2 = 0.056), snoring (p = 0.020, R2 = 0.036), awaken short of breath (p < 0.001, R2 = 0.089), and quantity of sleep (p < 0.001, R2 = 0.075) domains. Anxiety (p < 0.001) and worse somatic symptoms (p = 0.001) were related to greater sleep disturbances (R2 = 0.164). We also found relationships of heightened psychological symptoms (p < 0.001) and low physical activity level (p = 0.003) with increased daytime somnolence (R2 = 0.064). Finally, higher MRS total score and anxiety levels were associated with worse sleep quality assessed by MOS-SS Sleep Problems Index I (R2 = 0.179, p < 0.001 and p = 0.001, respectively) and Sleep Problems Index II (R2 = 0.146, p < 0.001 and p = 0.011, respectively). Conclusions: Anxiety and severity of menopausal symptoms were associated with poorer sleep quality. Furthermore, low physical activity level and worse psychological symptoms in menopause were predictors for increased somnolence. Therefore, screening for these factors in postmenopausal women is important, since they may be susceptible for intervention.

Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola hepatica.

The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in hepatic lymph nodes (HLN) and livers from sheep infected with Fasciola hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-ß, TNF-α and IL-1ß genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1ß during chronic stages of infection with F. hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. hepatica vaccinated and infected sheep.

Study of peritoneal macrophage immunophenotype in sheep experimentally infected with Fasciola hepatica.

During Fasciola hepatica infection, the parasite has the capability to modulate the host immune response towards a non-protector Th2 type instead of Th1. This type of immune response is closely related to the alternative activation of macrophages (M2 profile) as has been shown in vivo in murine models. In this study, an experiment was carried out in order to evaluate the expression of CD68, CD14, CD206 and iNOS in cells present in the peritoneal fluid of sheep during early stages of infection with F. hepatica (1, 3, 9 and 18 days post-infection, dpi) by immunocytochemistry. To the authors' knowledge, this is the first report that studies the in vivo immunophenotype of macrophages from the peritoneal fluid of sheep infected with F. hepatica. Throughout the experiments the absolute number of leucocytes progressively increased, reaching its highest value at 18 dpi, mainly due to the increase of eosinophils. This immunocytochemical study had two purposes: 1) CD68 expression was assessed with Hansel counterstaining, to optimally identify peritoneal macrophages, eosinophils and lymphocytes; 2) expression of CD14, CD206 and iNOS was evaluated to identify alternative or classical pathways of macrophage activation. The results showed a significant increase in CD14 from day 3 dpi compared with the non-infected group. CD206 expression at all time-points showed a significant and dramatic increase in comparison with the uninfected group. On the other hand, iNOS expression showed little variation, and was significantly decreased at 18 dpi in comparison with the uninfected group. These results suggest that F. hepatica induces an alternative activation of peritoneal macrophages of sheep from the first day post-infection, which may facilitate parasite survival. This is the first report describing M2 activation of peritoneal macrophages in ruminants infected with F. hepatica.

Th1/Th2 balance in the liver and hepatic lymph nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola hepatica.

The expression of IFNγ and IL4 was quantified using q-PCR in the liver and hepatic lymph nodes (HLN) of sheep during early stages of infection with Fasciola hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.

Apoptosis of peritoneal leucocytes during early stages of Fasciola hepatica infections in sheep.

Several immunomodulatory properties have been described in Fasciola hepatica infections. Apoptosis has been shown to be an effective mechanism to avoid the immune response in helminth infections. The aim of the present work was to study apoptosis in peritoneal leucocytes of sheep experimentally infected with F. hepatica during the early stages of infection. Five groups (n=5) of sheep were used. Groups 2-5 were orally infected with 200 metacercariae (mc) and sacrificed at 1, 3, 9 and 18days post-infection (dpi), respectively. Group 1 was used as the uninfected control (UC). Apoptosis was detected using three different methods 1) immunocytochemistry (ICC) with a polyclonal antibody anti-active caspase-3; 2) an annexin V flow cytometry assay using the Annexin V-FITC/propidium iodide (PI); and 3) transmission electron microscopy (TEM). The differential leucocyte count revealed that the majority of peritoneal granulocytes were eosinophils, which increased significantly at 9 and 18 dpi with respect to the uninfected controls. The ICC study revealed that the percentage of caspase-3+ apoptotic peritoneal leucocytes increased significantly from 3 dpi onwards with respect to the uninfected controls. The flow cytometry annexin V assay detected a very significant (P<0.001) increase of apoptotic peritoneal macrophages, lymphocytes and granulocytes, which remained higher than in the UC until 18 dpi. Transmission electron microscopy studies also confirmed the presence of apoptosis in peritoneal eosinophils at 18 dpi. This is the first report of apoptosis induced by F. hepatica in the peritoneal leucocytes of sheep in vivo. The results of this work suggest the importance of apoptosis induction for the survival of the juvenile parasites in the peritoneal migratory stages of infection.

Distribution of Foxp3+ T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica.

Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3+ and CD3+ T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3+ and CD3+ T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.

Eosinophilic Enteritis in Horses with Motor Neuron Disease.

BACKGROUND: Equine motor neuron disease (EMND) is a neuromuscular disorder that affects adult horses. Although EMND has been linked to vitamin E deficiency, its etiopathogenesis is poorly understood. OBJECTIVES: To describe clinical features, laboratory results, and postmortem findings in a series of young horses with motor neuron disease (MND). ANIMALS: A herd of 15 young Andalusian horses with weakness, weight loss, muscle atrophy, and muscle fasciculations related to restricted intake of green forage. METHODS: A case series is presented in which horses were subjected to a clinical examination and plasma vitamin E measurement. Five severely affected horses were euthanized for detailed postmortem examination. Muscle specimens were taken from the M. sacrocaudalis dorsalis medialis and the M. gluteus medius for histopathologic and morphometric evaluation. RESULTS: MND was diagnosed in 5 horses based on clinical signs, low serum levels of vitamin E (0.11 ± 0.05 mg/dL; normal range,: 0.3-1.5 mg/dL), changes in muscle histopathology (neurogenic atrophy), and spinal cord lesions (neuronal chromatolysis in ventral horns). An unexpected postmortem finding was the presence of intestinal inflammation (catarrhal enteritis, edema, and eosinophilic infiltrate) associated with the presence of giant ciliated protozoa in all of the horses. CONCLUSIONS: Although a mechanistic link could not be established, it is hypothesized that intestinal inflammation may have been involved in the decreased absorption of vitamin E, thus favoring the development of MND.

Fasciola hepatica induces eosinophil apoptosis in the migratory and biliary stages of infection in sheep.

The aim of the present work was to evaluate the number of apoptotic eosinophils in the livers of sheep experimentally infected with Fasciola hepatica during the migratory and biliary stages of infection. Four groups (n=5) of sheep were used; groups 1-3 were orally infected with 200 metacercariae (mc) and sacrificed at 8 and 28 days post-infection (dpi), and 17 weeks post-infection (wpi), respectively. Group 4 was used as an uninfected control. Apoptosis was detected using immunohistochemistry with a polyclonal antibody against anti-active caspase-3, and transmission electron microscopy (TEM). Eosinophils were identified using the Hansel stain in serial sections for caspase-3, and by ultrastructural features using TEM. At 8 and 28 dpi, numerous caspase-3(+) eosinophils were mainly found at the periphery of acute hepatic necrotic foci. The percentage of caspase -3(+) apoptotic eosinophils in the periphery of necrotic foci was high (46.1-53.9) at 8 and 28 dpi, respectively, and decreased in granulomas found at 28 dpi (6%). Transmission electron microscopy confirmed the presence of apoptotic eosinophils in hepatic lesions at 8 and 28 dpi. At 17 wpi, apoptotic eosinophils were detected in the infiltrate surrounding some enlarged bile ducts containing adult flukes. This is the first report of apoptosis induced by F. hepatica in sheep and the first study reporting apoptosis in eosinophils in hepatic inflammatory infiltrates in vivo. The high number of apoptotic eosinophils in acute necrotic tracts during the migratory and biliary stages of infection suggests that eosinophil apoptosis may play a role in F. hepatica survival during different stages of infection.

Immunohistochemical characterisation of parasitic pneumonias of dolphins stranded in the Canary Islands.

This is the first work that applies immunohistochemistry in the characterisation of the inflammatory infiltrate of verminous bronchopneumonia associated with lungworm parasites in stranded dolphins. Samples from three different species (Stenella coerulealba, Stenella frontalis and Delphinus delphis) stranded in the Canary Islands were used. The most common findings found in these animals varied from bronchitis to verminous bronchopneumonia with different degree of severity. The immunohistochemical study showed variable expressions of Lysozyme, MHC-II, iNOS and IgG. The main population presenting in the inflammatory infiltrates were CD3(+) lymphocytes. However, moderate number of Foxp3(+) lymphocytes was found in lymph nodes even though no Foxp3(+) cells were found in lung lesions in any of the samples analysed. This study revealed that lung lesions showed a chronic inflammatory infiltrate mainly composed by lymphocytes CD3(+). Deeper studies are needed in order to provide a more complete scope about the infiltrates involved in these types of lesions.

Mucoperiosteal exostoses in the tympanic bulla of African lions (Panthera leo).

Mucoperiosteal exostoses (MpEs) of the tympanic bulla (TB), also referred as middle-ear otoliths, have been occasionally described in dogs and cats in association with clinical signs of otitis media or as an incidental finding, but they have not been recorded in other species. In this report, we describe the radiographic, gross, and histopathologic features of MpEs in 8 African lions (Panthera leo). All animals (5 males and 3 females) were adults that had been kept in captivity and had their skeletons conserved as part of an anatomic academic collection. A radiographic study revealed mineralized structures in the TB consistent with MpEs in 7 of the 16 examined TB; a computed tomography study identified MpEs in 12 of the 16 TB. Six TB from 4 lions were sectioned, and several MpEs were demineralized for histopathologic analysis. Grossly, MpEs appeared variable in number and shape. Some were globular structures that were loosely attached to the mucosal surface of the TB; others were isolated to coalescent bone spicules extending from the mucoperiosteum. Position was also variable, but MpEs frequently developed in the hypotympanum, especially on the ventromedial aspect of the TB wall. Microscopically, MpEs were composed of osteonal bone growing from the periosteum and not by dystrophic calcification of necrotic tissue debris, as is hypothesized in dogs.

Biliary cirrhosis caused by Campula spp. in a dolphin and four porpoises.

Biliary cirrhosis produced by Campula spp. is described in 1 striped dolphin Stenella coeruleoalba and 4 harbour porpoises Phocoena phocoena. The hepatic lesions consisted of severe proliferation of fibrous connective tissue with loss of the lobular pattern, nodular regeneration of the hepatic tissue, bile duct hyperplasia and severe inflammatory infiltrate composed of eosinophils, macrophages, lymphocytes and plasma cells. These lesions were associated with severe infestation by Campula spp. Although inflammatory and degenerative hepatic lesions are frequently found in stranded dolphins, biliary cirrhosis has not been previously reported in cetaceans. Massive infestation by these parasites should be included as a cause of hepatic failure resulting in stranding of marine mammals.

Immunopathological study of parasitic cholangitis in cetaceans.

This paper describes the immunophenotype of cellular inflammatory infiltrates in chronic cholangitis in six common dolphins (Delphinus delphis), four striped dolphins (Stenella coeruleoalba), three Atlantic spotted dolphins (Stenella frontalis) and one pygmy sperm whale (Kogia Breviceps) found stranded along the coasts of the Canary Islands (Spain). A panel of 5 antibodies previously tested in dolphins (anti-CD3, -IgG, -MHC class II, -S100 protein and -lysozyme) were used. The present work also reports cross reactivity with dolphin antigens of two antibodies not used to date in dolphins (anti-mouse iNOS and anti-mouse Foxp3). The most common type of cholangitis found was chronic granulomatous cholangitis, associated with the presence of the parasite Campula spp., or its eggs in bile ducts. The cellular composition of the hepatic inflammatory infiltrate associated to chronic parasitic cholangitis was closely similar to that found in the cortex of control lymph nodes, including the presence of S100(+) and MHC class II(+) dendritic-like cells in lymphoid follicles and interfollicular areas. Only occasional macrophages expressed iNOS, whereas Foxp3(+) lymphocytes were not found in any of the lesions described in the different types of cholangitis.

Peripheral blood lymphocyte subsets in Fasciola hepatica infected and immunised goats.

The proportions of CD4(+), CD8(+) and WC1+ T lymphocytes from peripheral blood using flow cytometry were investigated in goats infected with Fasciola hepatica and previously immunised with recombinant Cathepsin-L1 (rCL1) and Glutathione-S-transferase sigma class (GST). The immunisation trial did not induce protective responses, and no significant differences were recorded between immunised and non-immunised groups. However, there was a significant decrease in the proportion of CD4(+) T lymphocytes in the infected groups both at 5 weeks post-infection (wpi), coinciding with the migratory stage of the infection, and at 12 wpi in the biliary stage of the infection. The proportional decrease in this circulating population may be related to the recruitment of CD4(+) T cells in liver and hepatic lymph nodes and also to the immunomodulatory effect of the parasite through the interaction of F. hepatica excretory-secretory products (FhESP) with this cell population. To date, this is the first report about the effect of F. hepatica infection in peripheral lymphocyte subsets in goats.

Early hepatic and peritoneal changes and immune response in goats vaccinated with a recombinant glutathione transferase sigma class and challenged with Fasciola hepatica.

Changes and local immune response were evaluated in the peritoneal cell populations, duodenal lamina propria and liver from goats immunized with recombinant glutathione transferase sigma class (rFhGST-S1) during early stages of infection with Fasciola hepatica. Group 1 (n=7) was unimmunized and uninfected; group 2 (n=10) was immunized with adjuvant Quil A and infected; group 3 (n=10) was immunised with rFhGST-S1 and infected. Three goats from each group were killed at 7-9 days post-infection (dpi) to evaluate early changes and immune response. The remaining goats were killed at 15 weeks post-infection (wpi). rFhGST-S1 vaccination induced variable response: three goats showed low fluke burden at 15 wpi and two goats showed low hepatic damage at early infection stages. This response was associated to a severe infiltrate of eosinophils in peritoneal fluid and hepatic necrotic foci, high iNOS expression in peritoneal cells and abundant infiltrate of eosinophils surrounding hepatic migrating flukes. T lymphocyte subsets were found in the vicinity of necrotic areas but they were absent in the vicinity of migrating larvae. No significant variation for T cell subsets, except for CD4 and γδ T lymphocytes, that were higher in the Quil A group compared to the rFhGST-S1 group. Expression of IL4 and IFN-γ in the hepatic inflammatory infiltrates was very occasional.

Early and late peritoneal and hepatic changes in goats immunized with recombinant cathepsin L1 and infected with Fasciola hepatica.

The aim of the present study was to study peritoneal and hepatic changes during early [7-9 days postinfection (dpi)] and late [15 weeks postinfection (wpi)] infection of goats immunized with recombinant F. hepatica pro cathepsin L1 (rCL1) in Quil A and challenged with Fasciola hepatica. Despite finding no significant reduction in fluke burdens between the control and immunized group, at 15 dpi the rCL1-vaccinated group showed significantly higher weight gain and reduced severity of hepatic lesions compared with the control group that received only Quil A. In the rCL1-vaccinated group, two of three goats sacrificed at 7-9 dpi had little hepatic damage and had a higher percentage of peritoneal eosinophils and elevated induced nitric oxide synthase (iNOS) expression in peritoneal cells than the goats from the control group. Moreover, while these two goats showed a heavy infiltration of eosinophils surrounding migrating flukes, the remaining animals examined at 7-9 dpi had no inflammatory infiltration surrounding migrating flukes. Two out of seven goats in the rCL1-vaccinated group had low fluke burdens and little hepatic damage at 15 wpi, suggesting an effective protective response in some of the vaccinated goats. This protective response did not correlate with peripheral eosinophilia or with serum titres of anti-rCL1 immunoglobulin (Ig) G. The results of the present work suggest that an eosinophil-mediated immune response plays a crucial role in the early effective host response against F. hepatica in goats. Adjuvants designed to increase cell-mediated immunity should be tested in future vaccine trials against F. hepatica.