I. Francisco

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.

Intrinsic Factors Influencing the Infection by Helminth Parasites in Horses under an Oceanic Climate Area (NW Spain)

A coprological survey to determine the influence of some intrinsic factors (breed, age, and sex) on the infection by helminth parasites in equine livestock (n = 418) under an oceanic climate area (NW Spain) was conducted. Faecal samples were individually collected and analyzed by the coprological techniques. The main strongylid genera identified were Trichonema and Cyalocephalus spp (small strongyles) and Strongylus and Triodontophorus (large strongyles). The prevalence of gastrointestinal nematode was 89% (95% CI 86, 92) and 1% cestoda (0, 2). The percentage of horses with strongyloid parasites was 89% (86, 92), 11% (8, 14) for Parascaris, and 3% (1, 5) for Oxyuris. The highest prevalence for ascariosis was observed in the youngest horses (<3 years), for oxyurosis in the >10 years animals, and for strongylosis in the 3–10 years ones. Females were significantly more parasitized than males. A negative correlation between the age and the egg-excretion of ascarids and strongyles was recorded. The autochthonous and the English Pure Blood horses were the most parasitized. We concluded that the infections by helminths, especially the strongyloids, are significantly common in the region, so that greater importance should be given to this situation.

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.

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.

AN APPROACH OF THE LABORATORY TO THE FIELD: ASSESSMENT OF THE INFLUENCE OF CATTLE MANAGEMENT ON THE SEROPREVALENCE OF FASCIOLIASIS BY USING POLYCLONAL- AND RECOMBINANT-BASED ELISAs

Abstract A cross-sectional study was conducted to assess the seroprevalence of fascioliasis by immunoenzymatic probes in an endemic area (northwestern Spain). Blood samples were collected from 1,034 cattle (crossbred, Rubia Gallega, and Friesian breeds), and the diagnosis of fascioliasis was carried out by determining both the occurrence of antigenemia and the presence of specific IgG antibodies against a Fasciola hepatica recombinant protein (FhrAPS). The IgG seroprevalence was 65% (95% CI, 62–68) by the FhrAPS-ELISA, and 32% (29–35) exhibited antigenemia; the lowest percentages occurred in the Friesians, and the highest percentages were found in the crossbreds. These results confirm an elevated seroprevalence of fascioliasis that is unexpected considering that most of the cattle livestock (Friesian and Rubia Gallega) receive fasciolicide treatment. The lack of adequate measures on the environment and erratic chemotherapy seem to be responsible for the fact that control of fascioliasis has not improved in the last 10 yr in the area of study.

The efficacy of four anthelmintics against Calicophoron daubneyi in naturally infected dairy cattle

The paramphistomicidal activity of four anthelmintics in dairy cattle naturally infected by Calicophoron (Paramphistomum) daubneyi was evaluated. Seventy Friesian adult cows were treated at drying-off (19 albendazole; 23 netobimin; 13 closantel and 15 oxyclozanide), and 21 remained untreated as controls. The anthelmintic efficacy was determined by estimating the faecal egg count reduction (FECR) values for each of the anthelmintics. The reduction in the number of cows shedding eggs in the faeces was also estimated. The C. daubneyi egg-output was not fully suppressed following the administration of any of the parasiticides. The FECR values ranged from 0% to 26% in the cows receiving albendazole or netobimin, with 11-39% of cattle becoming negative after therapy. Better results were achieved with closantel and oxyclozanide, with FECR values of 97-99% and CPCR (cattle positive by coprology reduction) percentages of 85-93%. The observation of a similar efficacy with closantel and oxyclozanide against C. daubneyi led us to recommend the administration of closantel in those countries where oxyclozanide is not available.

Mixed Production of Filamentous Fungal Spores for Preventing Soil-Transmitted Helminth Zoonoses: A Preliminary Analysis

Helminth zoonoses are parasitic infections shared by humans and animals, being the soil-transmitted helminths (STHs) mainly caused by roundworms (ascarids) and hookworms. This study was aimed to assess the individual and/or mixed production of two helminth-antagonistic fungi, one ovicide (Mucor circinelloides) and other predator (Duddingtonia flagrans). Fungi were grown both in Petri plates and in a submerged culture (composed by water, NaCl, Na2HPO4 · 12 H2O, and wheat (Triticum aestivum)). A Fasciola hepatica recombinant protein (FhrAPS) was incorporated to the cultures to improve fungal production. All the cultured plates showed fungal growth, without difference in the development of the fungi when grown alone or mixed. High counts of Mucor spores were produced in liquid media cultures, and no significant differences were achieved regarding single or mixed cultures, or the incorporation of the FhrAPS. A significantly higher production of Duddingtonia spores after the incorporation of the FhrAPS was observed. When analyzing the parasiticide efficacy of the fungal mixture, viability of T. canis eggs reduced to 51%, and the numbers of third stage cyathostomin larvae reduced to 4%. It is concluded, the capability of a fungal mixture containing an ovicide (Mucor) and a predator species (Duddingtonia) for growing together in a submerged medium containing the FhrAPS offers a very interesting tool for preventing STHs.

Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to a recombinant Fasciola hepatica surface antigen in an endemic area

The utility of an enzyme-linked immunosorbent assay to determine the sensitization against the trematode Fasciola hepatica in horses from an endemic area (NW Spain) was assessed. Blood samples were collected from 536 horses and tested against a 2.9-kDa recombinant surface protein (FhrAPS) to estimate the presence of IgG antibodies. Data were analysed regarding several intrinsic (age, gender and breed) and extrinsic factors (aptitude and housing). The farm size (number of horses/farm) was also considered. Sixty percent (95% CI 56, 64) of the horses were positive to the FhrAPS-ELISA, with a significantly higher seroprevalence in the mares (67%). Foals reached the lowest percentage of sensitization against the trematode (12%), and a significant positive correlation between the seroprevalence of fasciolosis and the age of the horses was established. When considering all the factors together, the seroprevalence of fasciolosis was initially classified into two groups (nodes) regarding the age of the horses. The node composed of the horses older than 1xa0year was then divided into two other clusters according to their gender. The mares were finally classified and grouped into two nodes regarding their breed. We concluded that the FhrAPS-ELISA is very useful for the demonstration of specific equine IgG antibodies against F. hepatica. An elevated risk of exposition to this trematode in horses maintained in endemic areas was proven. The possible role of horses as reservoirs for F. hepatica infections is discussed.

A recombinant-based ELISA evaluating the efficacy of netobimin and albendazole in ruminants with naturally acquired fascioliasis.

The therapeutic efficacy of albendazole and netobimin in ruminants with naturally occurring fascioliasis was investigated using a recombinant-based ELISA. The variation in the IgG response against a 2.9-kDa recombinant protein (FhrAPS), termed efficacy index (EI) 1, and the egg-output changes, termed EI 2, were used to evaluate drug efficacy. The values of EI 1 ranged between 0% and 50% in sheep, and between 0% and 30% in cattle after treatment with albendazole and netobimin. Similar EI 2 values were observed in sheep receiving albendazole or netobimin, but the highest values were found in cattle treated with netobimin. The significant reduction in the IgG response to FhrAPS found in this study shows promise in terms of developing alternative methods for evaluating the efficacy of chemotherapy against Fasciola hepatica in grazing ruminants.