R. Sánchez-Andrade

Use of a sandwich-enzyme-linked immunosorbent assay (SEA) for the diagnosis of natural Fasciola hepatica infection in cattle from Galicia (NW Spain) ☆

An indirect-enzyme immune-linked immunosorbent assay (IEA) was compared to a sandwich-enzyme-linked immunosorbent assay (SEA) to diagnose fasciolosis in naturally infected cattle. By means of a rabbit polyclonal IgG antibody to F. hepatica antigens, captured circulating antigens were detected by an SEA technique. A total of 85.1% of the samples examined was considered positive by using the IEA-test, whereas this percentage was of 37.3% by the SEA test. Using the two enzymatic probes, only the 38.3% resulted positive to both immunoassay probes. Our data indicate that circulating antigen detection assay in combination with results of antibody detection may offer a more reliable diagnostic technique than classic coprologic ones, with results very appropriate for epidemiologic studies carried out on a wide area.

Diagnosis of Parasitic Zoonoses by Immunoenzymatic Assays—Analysis of Cross‐Reactivity Among the Excretory/Secretory Antigens of Fasciola hepatica, Toxocara canis, and Ascaris suum

Several parasitic infections such fasciolosis, toxocariosis or ascariosis are important zoonoses. During the infection with Fasciola hepatica, Toxocara canis and Ascaris suum, an important intraorganic phase in their hosts takes place, releasing antigens responsible for a humoral immune response, which enables the diagnosis of that parasitosis. A study to identify the existence of cross‐reactivity among the excretory/secretory antigens of F. hepatica, T. canis and A. suum was developed. One group of Sprague–Dawley rats was infected with 20 metacercariae of F. hepatica and another group remained uninfected as control. By means of an Indirect‐ELISA, the rat humoral immune response (IgG and IgM) against the excretory/secretory antigens of F. hepatica was analysed and measured for cross reactivity with T. canis and A. suum. IgM cross‐reaction was mainly observed in the first 10 weeks post‐infection. IgG cross‐reaction was observed throughout the study, and was maximal at the 2–3 weeks and 3–6 weeks post‐infection, which corresponds to the intraorganic migratory phase of these parasites. The western‐blot showed that the rat IgG recognised three proteins of 190, 160 and 33 kDa in the antigens from F. hepatica, T. canis and A. suum. The existence of cross‐reactivity among these antigens seems to demonstrate also the presence of structural similarities, such as tegumental proteins. These results should be consider when immunoassay probes are used in the diagnosis of parasitic infections.

Influence of Age and Breed on Natural Bovine Fasciolosis in an Endemic Area (Galicia, NW Spain)

An analysis was undertaken of the effect of breed and age on bovine fasciolosis using antibody and antigen detection ELISAs. A total of 84.3% of the 1284 serum samples examined had positive antibody values and 20.4% exhibited antigenaemia. The seroprevalence of antibodies in crossbred cattle was higher than that in autochthonous Rubia Gallega, Friesian or Brown Swiss cows. The highest percentage antigenaemia occurred in the Brown Swiss cattle, but the breed differences were not statistically significant. Cattle aged 3–5 years had the highest antibody and circulating antigen prevalence and the age differences were significant. It was concluded that the apparent influence of breed was probably closely associated with the husbandry system. The autochthonous Rubia Gallega may be better adapted to fasciolosis as its percentage of antigenaemia was the lowest.

Ability of the fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydospores

The analysis of the capability of the nematode trapping-fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output in horses was evaluated. Fecal samples from 196 pasturing autochthonous Pura Raza Galega horses were collected from the rectum and then divided according to the egg-output into three groups: ≤ 300, 310-800 and >800 eggs per gram feces. Four doses of chlamydospores (0.1, 0.2, 0.4 and 0.8 × 10(6)/100g feces) were directly spread onto fecal pats on the ground, remaining one without treatment as control. Fecal pats confirmed the presence of gastrointestinal nematode larvae belonging to strongylid cyathostomins (Cyathostomum and Gyalocephalus spp). An overall 94% (95% CI 91, 97) percentage of reduction was obtained, and an increase in the activity of the trapping-fungi simultaneously to the rising in the number of cyathostomin eggs and larvae in the coprocultures was detected. A significantly highest reduction of the cyathostomin L3 in the coprocultures with more than 800 EPG was found, which indicates that Df trapping activity is larvae nematode density-dependant. The present research showed the high biological activity of D. flagrans against nematode larvae can adjust to the cyathostomin egg-output, and underlines its efficacy as a practical method for the control of these parasites in grazing horses.

Field Evaluation for Anthelmintic-Resistant Ovine Gastrointestinal Nematodes by In Vitro and In Vivo Assays

A coprological survey to analyze the presence of flock resistance to benzimidazoles (BZ) and macrocyclic lactones (ML) was performed in sheep under field conditions. Fecal samples were collected from 2,625 sheep in 72 commercial farms from Galicia (NW Spain). The in vitro (FECRT, fecal egg count reduction test) and in vivo (EHA, egg hatch assay, and LFIA, larval feeding inhibition assay) tests were used to assess the efficacy of these anthelmintics. Coprocultures were also developed to obtain knowledge on the main genera of trichostrongylid nematoda prior to, and after, the administration of the anthelmintics. By using the FECRT, BZ resistance was observed in 13 (18%) flocks, whereas ML resistance was only detected in 2 (3%) farms. The number of resistant flocks to BZ was 21 (29%) by using the EHA and 7 (10%) by means of the LFIA. None of the flocks used in this study showed simultaneous resistance to both employed anthelmintics. The results from the in vitro and in vivo tests revealed that 92% of the flocks FECRT resistant to BZ were also resistant with the EHA. The LFIA confirmed all the farms resistant to ML by using the in vivo test. After the administration of BZ, nematode larvae belonging to Teladorsagia circumcincta (32.2%), Trichostrongylus spp. (29%), Nematodirus spp. (6.5%), and Chabertia ovis (3.2%) were identified. In the flocks receiving ML, only T. circumcincta was identified (57%). We recommend the use of in vitro tests because they are more efficient. As the use of macrocyclic lactones is increasing in this region, further investigation is needed for detecting resistance to the anthelmintic family compounds by the LFIA.

Risk periods of infection by Calicophoron daubneyi (Digenea:Paramphistomidae) in cattle from oceanic climate areas.

From March 2001 to February 2004, 724 faecal samples of cattle under field conditions were collected in 121 farms from Lugo (NW Spain), an area with oceanic climate. The risk periods of infection by Paramphistomum trematoda in cattle was assessed by analyzing the prevalence of paramphistomosis, egg-output and climatic data. A significantly higher percentage of cattle passing trematode eggs by faeces was recorded firstly in September and secondly in March. The greatest numbers of eggs were achieved in May. Finally, the individual average prevalence was 19% and 36% of the farms had Calicophoron daubneyi-positive cattle. We conclude that the periods of high risk for C. daubneyi infection in areas with oceanic climate are those following periods with elevated rainfall, which occurs in May–June and November–December in these areas. Appropriate measures for reducing the risk for paramphistomosis are needed and the administration of chemotherapy with efficient anthelmintic in June and in November is strongly recommended.

Prevalence of natural ovine fasciolosis shown by demonstrating the presence of serum circulating antigens.

The prevalence of fasciolosis in sheep (Galicia, Northwest Spain) kept under field conditions was determined by using a sandwich-enzyme-linked immunosorbent assay (sELISA). Serum Fasciola hepatica circulating antigens were captured by means of a rabbit polyclonal IgG antibody to F. hepatica excretory/secretory products. Results were compared to those obtained by faecal sedimentation and an indirect ELISA (iELISA) and excretory/secretory antigens. Prevalences were 39.1% by sELISA, 30.4% by faecal sedimentation and 56% by iELISA; 83.3% of the sheep were positive to any one of the three tests. We observed that 59.5% of the sheep examined had active fasciolosis, 29.1% (117) had antigenaemia, 20.4% (82) passed eggs, and 40 (10%) were positive to both probes. We conclude that there is a high prevalence of fasciolosis in sheep from the studied region, and that the combination of sELISA and coprological sedimentation is extremely helpful for demonstrating current fasciolosis, so its application can be strongly recommended for epidemiological surveys.

IgG isotype specific immune response in rats infected with Fasciola hepatica

Antibody responses (IgG1, IgG2a, IgG2b, and IgG2c subclasses) against Fasciola hepatica L. in rats infected with metacercariae were analysed by ELISA. Animals of group 1 (R-1) remained infected throughout 21 weeks, and rats of group 2 (R-2) received a single oral dose of triclabendazole (Fasinex 10%, Ciba-Geigy) at 4 weeks after infection. Group C (R-C) consisted of rats left intact which served as uninfected controls. All IgG subclasses increased during the first weeks after infection, but when triclabendazole was administered IgG1 and IgG2b diminished markedly. IgG2c showed a different profile to the other antibodies evaluated, increasing earliest and decreasing profoundly from the 9-11th week after infection (w.a.i). The infected-untreated rats produced higher titres of antibodies than the rats of R-2, and these differences were statistically significant (p < 0.05) in all subclasses evaluated except in IgG2a titres.

Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy)

An indirect enzyme-linked immunosorbent assay (ELISA) with Dicrocoelium dendriticum excretory/secretory antigens was used to evaluate the presence of serum antibodies against the trematode in 738 sheep randomly chosen in Sardinia (Italy). Coprological sedimentation was used to discover egg-output. Seropositivity was detected in 86.2% tested sheep, whereas faecal prevalence was 6.7%; all that were faecal-positive also were ELISA-positive.

Silvopastoralism and autochthonous equine livestock: analysis of the infection by endoparasites.

Two groups of autochthonous Pura Raza Galega (PRG) horses, one comprising 483 animals under a silvopasturing regime, and the other comprising 72 PRG horses managed in farms, were used to analyse the effect of silvopasture on infection by endoparasites. Results were considered according to the age and the sex of the horses. Faecal samples were individually collected from each animal and analysed by the coprological flotation, sedimentation and migration techniques. Coprocultures were also done to identify the main strongylid genera affecting the horses. Eggs from the gastrointestinal nematoda Parascaris equorum, strongyles and Oxyuris equi were the only endoparasites observed in the faeces of the horses. Larvae of Trichonema and Cyalocephalus spp. (small strongyles) and Strongylus and Triodontophorus (large strongyles) were identified in the coprocultures. The silvopasturing horses had the highest prevalence of the helminth parasites. The percentage of horses passing ascarid eggs was significantly higher in pasturing horses younger than 3 years. The prevalence of strongyles was statistically greater in the oldest grazing equines. Mares reached the highest prevalence of helminth egg output. Our results showed that native horses kept under silvopasture had the highest prevalence of the ascarids, strongyles and oxyurids, possibly due to their exposure to contaminated grazing areas, lack of appropriate feeding and control of their health status. We conclude that silvopasture increases the presence of infection by gastrointestinal nematoda in wild horses, especially by strongyles. Suitable measures to control parasitic diseases affecting horses in silvopasture should be considered in those systems.