S. Hernández

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.

Detection of antibodies to 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.

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.

Experimental haemonchosis in goats: effects of single and multiple infections in the host response

Histopathological changes and the distribution of T lymphocytes (CD3), B cells (CD79alpha) and IgG secreting plasma cells were recorded in the abomasum and abomasal lymph nodes of goats during early and late post-infection stages with one to four doses of Haemonchus contortus L3. The infiltration of eosinophils, mast cells, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells in the abomasal mucosa increased dramatically from 10dpi onwards, whereas globule leukocytes were observed only during chronic infection. In late post-infection stages abomasal infiltration of globule leukocytes, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was significantly higher (P<0.05) in reinfected (groups 6-8) than in primarily infected goats (group 5). In the abomasal lymph nodes, marked hyperplasia of lymphoid follicles and medullary cords, with increase of CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was recorded from 10dpi (group 3) onwards. Worm burdens and the severe abomasal response during the late post-infection stages suggests that a rapid expulsion of nematodes did not occur. The prolonged time required for generating globule leukocytes suggested that immune mechanisms dependent of this cell type are of crucial importance in the protective immunity against H. contortus in goats.

Cellular phenotypes in the abomasal mucosa and abomasal lymph nodes of goats infected with Haemonchus contortus.

The distribution of T-cell subsets (CD2, CD4, CD8, and gammadelta) and B cells (IgM) was examined at 3, 6, 10 and 13 days post-infection (dpi) in the abomasal mucosa and abomasal lymph nodes of goats primarily infected with Haemonchus contortus. In the abomasal mucosa a mild (3 and 6 dpi) or marked (10 and 13 dpi) increase of T cells, particularly CD4+ and gammadelta+ lymphocytes, was observed, whereas the increase in CD8+ cells was less pronounced. B cells and IgG+ plasma cells also showed a marked increase in the abomasal mucosa at 10 and 13 dpi. The abomasal lymph nodes showed an increase in size, particularly at 10 and 13 dpi, and a decrease in the proportion of T cells, particularly CD8+ lymphocytes, due to the increased proportion of B cells. The proportion of CD4+ and gammadelta+ lymphocytes did not change significantly during the infection in the abomasal lymph nodes, but their absolute numbers were augmented as a result of the enlargement of the nodes. The results revealed a strong cellular and humoral immune response during the early post-infection stages. However, as indicated by the worm burdens, this rapid host response was unable to induce larval expulsion.

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.

Ultrastructural colocalization of phosphorylcholine and a phosphorylcholine-associated epitope in first-stage larvae ofTrichinella spiralis

Although the presence of phosphorylcholine (PC) inTrichinella is well established, the structures of the TSL-4 antigens that bear this epitope are unknown. A subset of TSL-4 antigens (TSL-8 antigens) has been reported to be absent from the surface of first-stageT. spiralis larvae. We report experiments with a monoclonal antibody (mAb US2) developed in mice with a relative inability to produce antibodies to PC. In immunoblotting, mAb US2 and anti-PC mAb (BH8) showed apparently identical binding patterns. In addition, we used an immunogold double-labeling technique to study the anatomical distribution of the epitopes recognized by these mAbs; the results obtained indicate close colocalization of epitopes for BH8 and US2 in tissues ofT. spiralis first-stage larvae. On the basis of these results, we suggest that US2 probably binds to allT. spiralis TSL-4 antigens, including TSL-8 antigens. We also clarify some conflicting previous reports on the distribution of PC immunoreactivity in first-stage larvae ofT. spiralis.

Acuaroid nematodes in the common kestrel (Falco tinnunculus) in the south of Spain.

The prevalence, intensity and abundance of acuaroid nematodes were determined in the common kestrel (Falco tinnunculus) in Andalusia, Spain. Acuaroid nematodes were present in 26/41 (63.4%) of birds examined. The most common species belonged to the genus Synhimantus subgenus Synhimantus (56%): S. (S.) laticeps (36.5%), S. (S.) robertdollfusi (24.3%) and a single specimen of a third, unknown, Synhimantus (S.) spp., unlike any other described previously (2.4%). Other species identified were Synhimantus (Dispharynx) spp. (2.4%), S. (D.) nasuta (4.8%), Desportesius spinulatus (9.7%) and Skrjabinoclava spp. (2.4%). This is the first record of these three species in F. tinnunculus, but the latter two are considered to be accidental parasites in birds of prey.

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.