I. Navarrete

Analysis of the humoral immune response against total and recombinant antigens of Leishmania infantum: correlation with disease progression in canine experimental leishmaniasis.

Leishmaniasis by Leishmania infantum in the Mediterranean Basin constitutes an important problem in both human and veterinary medicine. Based in both the importance of canids as reservoirs for the human disease and the fact that the canine disease may be an excellent model for the human condition, the present work has been conducted to analyze clinical and immune mechanisms associated with canine experimental leishmaniasis. Six-month-old mixed-breed dogs were intravenously infected with L. infantum promastigotes and the infection course was monitored along a 343 days-period. On day 75 post-infection (p.i.), amastigotes were observed in the lymph nodes of all dogs. The analysis of the humoral response against total L. infantum antigens by both ELISA and Western blotting evidenced a correlation between the levels of IgG isotypes (IgG1 and IgG2) and disease progression. It was observed that in those animals showing either a regressive or an oligosymptomatic form of the disease, the anti-Leishmania IgG1 antibodies were undetectable whereas those animals developing active disease showed high levels of anti-Leishmania IgG1 antibodies. Additionally, the time-course of antibody production against L. infantum recombinant antigens in the experimentally infected dogs has been analyzed. The present data suggest that reactivity against the heat-shock protein 70 (HSP70) may be used as diagnostic marker of early steps of infection, and that the appearance of anti-histone antibodies is associated with progression of infection to disease status.

Pathological changes in kidneys of dogs with natural Leishmania infection.

A study was made of the nephropathy in canine leishmaniasis produced in ten adult dogs naturally infected with Leishmania infantum. Renal function analyses were performed (uraemia, creatinaemia, plasma proteins, biochemistry and urinary sediment), the humoral immune response (fluorescent antibodies and levels of serum IgG, IgM and IgA) was assessed and histopathological studies were carried out. Correlation of the results showed acute renal insufficiency which was reversible in two animals (endotheliomesangial glomerulonephritis) and irreversible in four cases corresponding to glomerulonephritis in its Type I and Type II proliferative forms; extensive increase in the glomerular basal membrane, proliferation of mesangial cells and growth of the mesangial matrix were observed, as was a widespread incidence of immune complex deposits. Two animals showed chronic renal insufficiency. Lack of renal changes (minimal-changes glomerulonephritis) in two dogs was accounted for in one animal by an almost complete absence of symptoms and in the other by chronic viscerocutaneous symptoms; neither showed more than a slight immunoglobulin response.

Meningeal leishmaniosis induced by Leishmania infantum in naturally infected dogs

We report two cases of meningitis caused by Leishmania infantum in naturally infected dogs. In both of these dogs the typical phenotypic features of granulomatous meningitis were observed with important lympho-plasma-cellular infiltrates and the presence of large numbers of parasites inside and outside macrophages. The immunological study of the cerebrospinal fluid of both animals showed that a large number of protein bands were recognized by those fluids and that they were similar to the ones recognized by the sera from the same animals. To our knowledge, this is the first description of meningitis associated to leishmaniosis.

Sylvatic trichinellosis in southwestern Spain.

The epidemiology of Trichinella spp. in their main sylvatic hosts, wild boar (Sus scrofa ferus and red fox (Vulpes vulpes), in Extremadura (southwestern Spain) was studied. We examined 88 Trichinella spp.-positive wild boar muscle-tissue samples from a total of 29,333 killed animals, referred to the Veterinary Parasitology Department (University of Extremadura, Spain) by the Extremadura Veterinary Service. Additionally, 227 red foxes killed during the hunting season and thus not subject to veterinary controls were examined for trichinellosis. Trichinella spp. larvae were found in six (3%) of the red foxes. All samples were examined using direct diagnostic techniques, including trichinoscopy and artificial digestion. The mean intensity of infection was 74.8 larvae/g (LPG) of muscle tissue in wild boars, compared to 30.6 LPG in foxes. Trichinella spiralis (sensu stricto) predominated over T. britovi in wild boars. Random amplified polymorphic DNA (RAPD) and alloenzyme typing showed that 74% of infected wild boars had only T. spiralis, 21% had only T. britovi, and 5% showed mixed infections. In contrast, 33% of infected foxes were infected only with T. spiralis, while 67% had T. britovi, suggesting a clear predominance of the latter in foxes. We suspect the existence of a paranthropic or sylvatic cycle in large areas of this region; given the ease of transfer between sylvatic and domestic or semi-domestic animals, this implies a high epidemiological risk.

Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis.

Babesia ovis isolated in Extremadura (Spain) was the subject of a serological study in experimentally inoculated sheep. The first antibody titres, determined by the indirect fluorescent antibody (IFA) test, were observed 7-8 days post infection (p.i.) in all animals except the splenectomized group, in which the only animal that survived showed antibody response 10 days p.i. A faster response following challenge was observed in sheep which were seropositive before inoculation, which suggests the existence of an antigen memory. The highest titres were reached 16-25 days p.i., and subsequently began to fall, reaching minima at the end of the experimental period (330 days p.i.). The chronic carrier state in experimental B. ovis infection had a duration of at least 2 years. Passive transmission of antibodies from experimentally infected mothers to newborn lambs was also detected. Antibody levels were observed for a period not longer than 2 months after birth.

Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme‐linked immunosorbent assay

Abstract.  A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linné) was examined. The enzyme‐linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory‐secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P < 0.001) was found between the number of O. ovis larvae and the serum antibody levels using all differents antigens, except L3 CE. In Spain, where a long favourable period exists for the evolution and development of the different stage larvae between March and November, the ELISA test using L1 ESP antigen during winter and L2 ESP antigen in summer may be used for ovine oestrosis immunodiagnosis.

Influence of infection intensity on predilection sites in swine trichinellosis

The muscular distribution of Trichinella spiralis or T. britovi was studied by digestion in 59 experimentally infected pigs and seven wild boars. Crus muscle was the predilection site in 89.3% of 28 heavily infected swine with 146-3634 larvae per gram (lpg), but in 51.6% of middle to light infections (0.005-59 lpg) the basis of the tongue showed higher larval densities than the crus muscle. The basis of the tongue was also the predilection site in 71.4% of wild boars. Highest counts in other muscles were found only in lightly infected pigs. The influence of intensity of infection, host species, and Trichinella species on muscle distribution is discussed.

Immunohistochemical distribution of antigens in liver of infected and immunized pigs with Ascaris suum

In the present work, we carry out an immunopathological study of the swine ascariosis, under different conditions (control, infection and immunization). Twenty-one Iberian pigs were used and divided in seven groups. Groups 1 and 2 were the uninfected and challenged controls, respectively. Groups 3 and 4 were weakly infected with increasing doses of Ascaris suum eggs and treated with pyrantel (Group 4). Groups 5-7 were immunized with 14, 42 and 97 kDa proteins from the parasite, respectively. Groups 2-7 were challenged with 10,000 infective eggs 7 days before the sacrifice. The focal parasitic granulomata with eosinophils and lymphocytes were the main histopathological lesions in the liver of reinfected pigs, while more marked cellular infiltrate and abundant connective tissue were seen in the livers of immunized animals. There were important deposits of antigens in the livers of immunized and infected pigs. Antigens were mainly located in the connective tissue, with positive staining detection of the somatic larvae antigen, the body wall from the adult worms and the 14-, 42- and 97-kDa proteins. However, cholangiols, biliary ducts and macrophages presented an immunohistochemical positive stain against excretory-secretory and somatic antigens from the larvae and the body fluid antigen from the adult parasite. The detection of A. suum antigens in the liver of infected pigs improves the diagnosis of swine ascariosis. It may be possible to apply these procedures for diagnosis of human ascariosis in liver biopsies since A. suum from swine have been previously used as a substitute for the study of the human parasite Ascaris lumbricoides.

Trichinella strain, pig race and other parasitic infections as factors in the reliability of ELISA for the detection of swine trichinellosis.

An enzyme-linked immunosorbent assay (ELISA) using crude worm extracts (CWE) and mixtures of these as antigens of five Spanish isolates (P, C, B1, B2 and W) was developed for detecting homologous and heterologous experimental infections with these isolates between-14 and 82 days post-infection (p.i.) in white and Iberian pigs. A total of 243 pigs (Iberian or cross-bred with this race) with numerous parasitic infections were also screened for the presence of antibodies to a mixture of CWE of C, B1 and B2 isolate. The test showed a specificity of 93.1-98.9% depending on the cut-off values and a maximum sensitivity of 92.8-100% between days 34 and 82 p.i. A low grade of infectivity was shown in the T3 isolates compared to the T1 isolates (P, C, B1 and B2) but high cross-reactions were observed between all the isolates with minor differences between P and W isolates. The highest antibody response was found in P infections and the lowest in pigs infected with the W isolate. A clear association between the presence of several parasitic infections and false positive reactions was not found, but an important relation was shown between high background levels and the Iberian race in experimentally and conventionally raised pigs.

Seroprevalence of Oestrus ovis (Diptera, Oestridae) Infestation and Associated Risk Factors in Ovine Livestock from Southwestern Spain

Abstract This survey was carried out to determine the seroprevalence of nasal infestation by sheep bot fly, Oestrus ovis L., and to identify the risk factors associated with the disease in flocks in southwestern Spain. In total, 5,878 sera samples of adult sheep were collected at random in 551 farms from four provinces in the southwestern Spain: Badajoz, Cáceres, Córdoba, and Sevilla. Sera were tested by enzyme-linked immunosorbent assay for O. ovis antibodies, by using a crude L2 larval as antigen. The seropositive mean prevalence was 69.30%, and mean percentage of optical densities was 61.83%. There were significant differences between the provinces studied; Córdoba and Sevilla were the provinces with more infested animals and higher seroprevalences. The correlation between seroprevalence and percentage of antibodies by farms was significant. There were only 18 farms free of seropositive animals, and 115 of the total 551 farms had all sampled animals seropositive, an indication of the high importance of this parasitosis in the investigated areas. Altitude, latitude, flock size, and ovine population density were the potential risk factors associated with the detection of O. ovis antibodies. Those animals breeding in regions located at low altitudes (<500 m), meridian latitudes (<39.5° N), and on farms with medium-to-large flock size (>250 sheep) and high ovine population density (>100 sheep per km2) were more likely to be seropositive. These findings confirm that these studied factors should be considered as potential risk factors to the presence of O. ovis in ovines from southwestern Spain.