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.

Toxoplasma gondii in ruminant species (cattle, sheep, and goats) from southern Spain.

Seroprevalences of Toxoplasma gondii antibodies were assessed with the use of a commercial indirect ELISA in 1,501 domestic ruminants in southern Spain. Antibodies against T. gondii were detected in 420 (83.3%) of 504 cattle, 248 (49.3%) of 503 sheep, and 124 (25.1%) of 494 goats. The herd seroprevalence was 100% (72/72), 84.7% (61/72), and 72.2% (52/72) for cattle, sheep, and goats, respectively. Seropositivity was significantly higher in herds with a low density of animals (P < 0.001). Significant differences (P < 0.05) among municipalities were also found. The seroprevalence observed in the present study indicates a widespread exposure to T. gondii in livestock in southern Spain.

Pathological and immunohistochemical study of the liver and hepatic lymph nodes in goats infected with one or more doses of Fasciola hepatica.

Lesions produced by Fasciola hepatica in the liver, gall-bladder and hepatic lymph nodes (HLNs) of four groups of five goats are described; in addition, the distribution of CD3+ T lymphocytes and IgG-lambda light chain-bearing cells was analysed in the hepatic lesions and HLNs. One group of goats received a single oral dose of metacercariae, but the other four groups received four or five doses at different intervals over a period of 11 weeks. Animals that survived were killed 53-55 weeks after the first infective dose. Goats were more susceptible to multiple doses than to a single dose, even when the total number of metacercariae was the same. This greater susceptibility was manifested by the occurrence of deaths and the severity of hepatic lesions. CD3+ lymphocytes were sparse in the infiltrate surrounding the acute migratory tracts, suggesting inhibition of the local cell-mediated immune response. Goats with numerous hepatic calcareous granulomas showed the most severe hepatic damage, including marked cirrhosis, with a striking infiltrate of CD3+ T lymphocytes and lambda IgG- plasma cells, replacing extensive areas of hepatic parenchyma, in which hypertrophy of the smooth endoplasmic reticulum of hepatocytes was evident. These findings were observed mainly in the goats given more than one infective dose.

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.

Detection of antibodies of Fasciola hepatica excretory--secretory antigens in experimentally infected goats by enzyme immunosorbent assay.

An ELISA with excretory-secretory (ES) antigens has been evaluated as a technique for the early detection of specific antibodies in Fasciola hepatica infections in goats. Goats were experimentally infected with 100 or 200 metacercariae of bovine origin and serum samples were taken periodically over 365 days. The ELISA test was performed with ES antigens (10 micrograms mL-1), a single dilution of sera (1:800) and anti-goat IgG conjugate (1:1000). ES specific antigens were detected in all infected goats between 15 and 30 days postinfection (PI) and maximum antibody levels were reached at 90 days PI. Positive antibody levels (significantly different from those of controls) were still found at 365 days PI. No significant differences were observed between goats infected with 100 or 200 metacercariae. In all infected goats, eggs appeared in faeces between 60 and 90 days PI. ELISA with ES antigens could be a feasible method for the early diagnosis of goat fasciolosis.

Immunological and histological study of T- and B-lymphocyte activity in canine visceral leishmaniosis.

Lymphocyte activity during canine leishmaniosis was studied by histological and immunological methods in experimental and natural infections. Eight dogs were inoculated with 5 x 10(7) promastigotes of Leishmania infantum, LEM 75, zimodeme MON-1, and maintained for 110 days post-infection. Another eight dogs with advanced natural infection were identified by parasitological and serological methods. Three techniques were used: the lymphocyte transformation test (LTT) to study T-cell activity, immunofluorescence assay and enzyme-linked immunosorbent assay to measure antibody production (B-cell activity), and methyl green-pyronin staining to assess tissue responses of lymphocytes. There was a noticeable B-cell response, reflected both histologically and immunologically. High percentages of activated lymphocytes and plasma cells were evident in lymphoid organs and production of specific antibodies was seen throughout the infection. LTT results showed a T-cell unresponsiveness during canine leishmaniosis. These same immunohistological features were observed, although to different degrees, in both experimental and natural infections.

[The seroprevalence of human toxoplasmosis in Córdoba]. / Seroprevalencia de la toxoplasmosis humana en Córdoba.

A seroprevalency study of human toxoplasmosis was carried out in Córdoba, using indirect immunofluorescence and indirect hemagglutination. The sample of people interviewed was made up of 443 serums, 356 supposedly healthy people (mainly students) and 87, considered "high risk" (patients from the "Reina Sofia" Hospital). The positiveness obtained for the total of the sample was 43.79% with IFI and 53.59% for HAI. As regards sex of the person tested, prevalence is higher in women, with 54.36% and 70.47% with IFI and HAI respectively, in men the scores were 38.43% and 44.90% for the same tests. As far as the origin of the sample was concerned, there were 31.18% with IFI and 43.25% for HAI of the normal population, in the "high risk" sample, for both tests it was 95.40%. With both tests there are significative differences in low amounts, but not in high dilutions. We conclude that human toxoplasmosis is present and widespread in the studied population.