F.J. Martínez-Moreno

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.

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

Abstract 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.

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.

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.

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.

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.

Comparison of the use of secretory and somatic antigens in an ELISA for the serodiagnosis of hypodermosis

An antigen was prepared from metabolic products which were produced by maintaining first instar larvae of Hypoderma lineatum in RPMI tissue culture medium for 48 h. Three major proteins were identified in the secretory products and were characterised in terms of their molecular weights and iso-electric points. The antigen compared favourably with a soluble extract of larvae when used in an enzyme-linked immunosorbent assay (ELISA) against a panel of control sera and 2000 bovine sera collected from farms in Andalucia, Spain.

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.