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).

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.

Pathological, immunological and parasitological study of sheep vaccinated with the recombinant protein 14-3-3z and experimentally infected with Fasciola hepatica.

In this study, the immunogenicity and protective capacity of a new recombinant vaccine candidate, the rFh14-3-3z protein was analysed in sheep experimentally challenged with Fasciola hepatica, in terms of fluke burden, faecal egg counts, hepatic damage and humoral immune response. Three groups of 8 animals each were used for study, group 1 was immunised with the rFh14-3-3z in Montanide adjuvant, whereas group 2 and 3 remained as adjuvant control and infection control groups, respectively. The parasitological analysis showed that no significant reduction in fluke burden, fluke size and faecal egg counts was detected. The extent of hepatic damage was very similar between groups. Nonetheless, animals immunised with the rFh14-3-3z protein induced the development of specific IgG1 and IgG2, being the IgG1 the predominant antibody; which confirms the immunogenicity of this protein in sheep. This is the first report of the 14-3-3z proteins as vaccine against the infection with F. hepatica.

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.

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.

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.

Humoral immune response in goats immunised with cathepsin L1, peroxiredoxin and Sm14 antigen and experimentally challenged with Fasciola hepatica.

The humoral immune response was analysed in goats immunised with FhCL1, FhPrx, Sm14, and experimentally challenged with Fasciola hepatica. All immunised animals developed significant levels of anti-fluke specific antibodies and those immunised with FhCL1 showed the highest antibody titre. After experimental infection, an increase in the antibody level was detected only in goats immunised with FhCL1. In the adjuvant-control animals, the experimental challenge induced significant production of specific antibodies against FhCL1, FhPrx and Sm14. While liver fluke specific humoral responses were seen in all groups, no significant protection in any of the vaccinated groups was found.

Evaluation of hepatic damage and local immune response in goats immunized with native glutathione S-transferase of Fasciola hepatica.

Worm burden, hepatic damage and local cellular and humoral immune responses were assessed in goats immunized with glutathione-S-transferase and challenged with Fasciola hepatica. Infected but unimmunized and uninfected control groups were also studied. Hepatic damage was evaluated grossly and microscopically. Local immune response was evaluated by (1) microscopical examination of hepatic lymph nodes (HLNs); (2) analysis of the distribution of CD2(+), CD4(+), CD8(+), T-cell receptor gammadelta(+) lymphocytes and immunoglobulin (Ig) G(+) plasma cells; and (3) investigation of the distribution of cells expressing interleukin (IL)-4 and interferon (IFN)-gamma in the hepatic inflammatory infiltrates and HLNs. Immunized animals did not have significant reduction in fluke number, but there was significant (P<0.05) reduction of fluke size relative to the control groups. The lesions in the two infected groups were similar and consisted of fibrous perihepatitis and white tortuous tracts, mainly involving the left hepatic lobe. Microscopical lesions were similar in both infected groups and were typical of chronic fascioliosis. These included portal fibrosis, inflammatory infiltration with plasma cells, formation of lymphoid follicles, accumulation of haemosiderin-laden macrophages and granulomatous foci. Both infected groups had a marked local immune response characterized by infiltration of CD2(+), CD4(+) and CD8(+) T lymphocytes, and IgG(+) plasma cells in hepatic lesions and in HLNs. There was no expression of IL-4 or INF-gamma by cells in the hepatic inflammatory infiltrate, but expression of INF-gamma in HLNs was much lower than that of IL-4, suggesting an immune response dominated by T helper 2 cells.

Immune response of goats immunised with glutathione S-transferase and experimentally challenged with Fasciola hepatica.

Glutathione S-transferase (FhGST) purified from Fasciola hepatica adult worms was used to immunise goats against F. hepatica in an experimental infection; the level of protection, in terms of fluke burden, faecal egg counts and hepatic damage was determined, as well as the humoral and cellular immune response elicited. Animals were allocated into three groups of six animals each: group 1 (immunised with FhGST and infected), group 2 (unimmunised and infected), and group 3 (unimmunised and uninfected). There was no significant reduction of fluke burden (9.3%) or faecal egg counts; hepatic damage was also similar in both infected groups. However, immunisation with FhGST induced the development of a well-defined immune response, characterized by the production of specific-FhGST antibodies as well as the appearance of circulating IL-4.

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen.

The nature of the local immune response was assessed studying the distribution of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes, IgM(+) B cells, IL-4(+) and IFN-gamma(+) cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2(+), CD4(+), CD8(+), gammadelta(+) T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-gamma(+) lymphoid cells was absent or very occasional in HLN where the number of IL-4(+) cells was higher than that of IFN-gamma, suggesting a polarized Th2 response in immunised and in infected control group.

A study of the liver of goats immunized with a synthetic peptide of the Sm14 antigen and challenged with Fasciola hepatica.

A synthetic peptide of the Sm14 antigen of Schistosoma mansoni was used to immunize goats against experimental challenge with Fasciola hepatica. The goats used consisted of: group 1 (unimmunized and uninfected [controls]); group 2 (unimmunized and infected); group 3 (immunized and infected). Compared with group 2, the animals of group 3 showed at necropsy a reduction in hepatic worm burden; however, because of variability in the two groups this reduction was not statistically significant. A gross morphometric study of the hepatic changes revealed a correlation between lesions and worm burdens in group 3. No significant differences in damaged areas were found between groups 2 and 3, except that group 2 had more severe bile duct hyperplasia. A striking hepatic inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells was found in both groups 2 and 3, especially the latter.

Estimation of canine intestinal parasites in Córdoba (Spain) and their risk to public health.

The prevalence of gastrointestinal parasites in dogs was studied in the province of Córdoba (southern Spain), with special attention to those parasites that can be transmitted to man. The experiment was completed with the examination of soil samples from public parks and city gardens. The study was carried out over a population of 1800 animals entered in the Control Animal Centre (CECA) by coprological methods, and within this group, 300 dogs were sacrificed and necropsied. The prevalence of any intestinal parasitic infection was 71.33%. The following parasites of the gastrointestinal tract were recorded: Isospora canis (22%), Isospora (Cystoisospora) spp. (10.22%), Sarcocystis (2.5%), Hammondia/Neospora (1.94%), Giardia canis (1%), Dipylidium caninum (13.2%), Taenia hydatigena (7.66%), Taenia pisiformis (4%), Uncinaria stenocephala (33.27%), Toxascaris leonina (14.94%), Toxocara canis (17.72%) and Trichuris vulpis (1.66%). Related to public health, it is important to point out the presence of T. canis only in puppies younger than one year and Uncinaria, more frequent in adult dogs. Soil samples of parks revealed the presence of eggs of Toxocara, and it suggests the existence of real risk for human infection.

Oxidative responses during bacterial phagocytosis of polymorphonuclear leucocytes in primarily and secondarily Fasciola hepatica infected goats.

The polymorphonuclear leukocyte (PMN) function, in terms of oxidative response during bacterial phagocytosis, was studied using a Luminol-Dependant Chemiluminiscence (LDCL) assay in primarily and secondarily Fasciola hepatica infected goats. Polymorphonuclear leukocytes of F. hepatica infected goats displayed lower LDCL responses than naive goats. The lowest responses were observed in secondarily infected animals that had higher parasitic burdens and more prominent hepatic lesions. The reduced responses were induced by a functional defect of the circulating PMN but also by a direct involvement of serum factors. Both circulating parasite products and the non protective immune response that occurred during secondary F. hepatica infection of goats could be implied in the alteration of PMN function. These findings suggest the existence of an important mechanism for impairment of the host immune system during goat fasciolosis.

Experimental bovine hypodermosis in Spain.

An experimental survey was carried out in western Spain to investigate both the chronobiology of Hypoderma spp. and the immunoresponse of their bovine hosts. This study was initiated with a new system of obtaining Hypoderma spp. larvae directly from their host, including the eclosion of adults from their pupae, infestation under natural but controlled conditions, and confirmation of the resulting infection. This survey was carried out over 2 cattle grub seasons; it was possible to infest and reinfest the experimental animals and to monitor them by both parasitological methods and by an enzyme-linked immunosorbent assay. This method permitted the evaluation of the development of anti-Hypoderma antibodies during the experiment. The experimental design also enabled us to establish the period of detectable H. lineatum infection to be from December until the end of April with the largest number of warbles observed during March and April. After a pupal period of < 30 d, adults were seen in April and May. Hypoderma bovis (de Geer) showed a delay of 2 m.o. relative to H. lineatum (de Villiers). This study reports a completed biological life cycle of Hypoderma spp. under controlled conditions in both natural and experimental environments.

Method for collecting Hypoderma spp. (Diptera: Oestridae) larvae from cattle.

A new method for collecting 3rd-instar Hypoderma lineatum (Villers) and Hypoderma bovis (L.) is described. A procedure for carrying out chronobiological and other studies involving the life cycle of these flies is included. The larvae are collected from cattle using a dressing made of a circle of cardboard attached to a piece of tulle similar in color to the coat of the animal. These small cloth cages were attached to the animal skin with fast-drying contact glue wherever the swellings produced by 2nd- or 3rd-instar Hypoderma spp. had been previously detected. Each animal was inspected daily to detect the appearance of new larvae. This procedure allows the collection of each emerged larva without contact with other larvae and avoids friction of the cloth cages or dressing with the larvae, thus reducing larval death and increasing viability of the parasite.

Comparative scanning electron microscopy of third-instar Hypoderma spp. (Diptera: Oestridae).

Scanning electron microscope study of third-instar larvae of four species of Hypoderma revealed differences among species in the pattern of spination, spine morphology and morphology of the spiracular plates. These observations identify characters that enable the differentiation of Hypoderma actaeon and H. diana, parasitizing red deer (Cervus elaphus) in Europe, and provide additional characters for differentiating H. bovis and H. lineatum parasitizing cattle.

Humoral and cellular immune responses to experimental Fasciola hepatica infections in goats.

Humoral and cellular immune responses to Fasciola hepatica excretory-secretory products (ESPs) in primary and secondary experimental infections in goats were studied. Primary infection induced the development of chronic subclinical fascioliasis that did not affect the establishment of flukes coming from the secondary infection, as the same percentages of recovered flukes were found in both groups. The specific IgG response to F. hepatica ESPs was similar in primary and secondary infections; challenge flukes did not induce any modification in the IgG response. The specific lymphocyte response to F. hepatica ESPs was absent in most of the infected goats, both primarily and secondarily infected. A modulation of the nonspecific cellular responses to mitogens was also observed. All infected goats showed a reduced proliferative response to concanavalin A and phytohemagglutinin. According to our results, humoral and cellular responses to F. hepatica ESPs in goats have no protective effect on the establishment of flukes and the development of disease in either primary or secondary infections.

Triclabendazole treatment in experimental goat fasciolosis: anthelmintic efficacy and influence in antibody response and pathophysiology of the disease.

A controlled test of the efficacy of triclabendazole against all stages (early immature, late immature and mature) of Fasciola hepatica has been performed in experimentally infected goats. The influence of triclabendazole treatment on the pathophysiology of the disease, in terms of haematological parameters and serum enzyme levels, and in the dynamics of production of specific antibodies to excretory/secretory products (ESP) of F. hepatica were also examined. Goats were orally infected with 200 viable metacercarie and treated at 4, 8 and 16 weeks postinfection (PI) with triclabendazole at the dose rate of 10 mg kg-1 body weight. The drug can be regarded as highly effective against mature (100%) and late immature (99.2%) flukes and effective against early immature flukes (94.9%). A moderate anaemia was found associated with the presence of late immature and mature flukes in bile ducts. Treatment with triclabendazole, by eliminating most of these flukes, largely reduced haematological alterations. Serum levels of the enzymes aspartate aminotransferase, lactate dehydrogenase and gamma-glutathione transferase reflected hepatic damage during goat fasciolosis. Early treatment (at 4 weeks PI) prevents the development of both parenchyma and bile ducts lesions; treatment at 8 weeks PI only prevents bile ducts lesions and treatment at 16 weeks PI has no appreciable effect on the development of the main hepatic lesions. The antibody response to F. hepatica ESP, as measured by enzyme-linked immunosorbent assay, was also affected by treatment with triclabendazole. In all treated animals a peak in antibody levels was observed between weeks 9 and 13, followed by a drop whose magnitude depended on the efficacy of treatment. In those animals in which triclabendazole was highly effective, antibody levels fell back to negative values similar to those recorded preinoculation at 18-21 weeks PI.

Deer hypodermosis.

Hypodermyiasis is a common myiasis which affects a wide variety of deer species in many parts of the world. In this article a great review of the disease is made, treating the different species of flies that cause the myiasis in the different species of hosts. Biologies of deer and flies are also studied, with special dedication to the chronobiology of the life cycle of the parasite. Prevalence and intensity of parasitization in many parts of Europe are recorded like expression of the importance of hypodermyiasis in deer. Finally, some remarks about treatment and control are included.

Humoral and cell-mediated immunity in natural and experimental canine leishmaniasis.

This paper describes immunological and clinicopathological findings in dogs naturally and experimentally infected with progressive visceral leishmaniasis. Eight dogs were intravenously inoculated with 5 x 10(7) stationary phase promastigotes of Leishmania infantum (LEM 2002, ZMON-1). A further eight naturally infected dogs were diagnosed by parasitological and serological methods and selected according to their clinical and immunological condition. Clinical, hematological, pathological and parasitological examinations, including parasite burden and distribution, were included in the study. Antibody production was estimated by means of enzyme-linked immunosorbent assay and immunofluorescence assay techniques; the cellular immune response was studied by means of the skin test and the lymphocyte proliferation test. Experimentally infected dogs developed a chronic and progressive disease with the same clinical signs shown by naturally infected dogs. Both naturally and experimentally infected dogs developed the same histopathological reaction, but to differing degrees. Parasite burden and distribution were related to the extent of lesions, and were consequently less pronounced in experimentally infected dogs. The main feature of the immune response in experimental and natural infection was the lack of specific T-cell response to leishmanial antigen. Non-specific responses to mitogens were normal (i.e. as compared with healthy dogs) throughout the experimental infection, but were partially suppressed (65.3%) in naturally infected animals. A remarkable humoral response was evident in both natural and experimental infection: IgG-isotype antibodies were detected in experimental infection at 50-70 days post infection, and their production increased during the course of the infection. However, high titers were observed only in naturally infected dogs.