J. Pedreira

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.

Feeding horses with industrially manufactured pellets with fungal spores to promote nematode integrated control

The usefulness of pellets industrially manufactured with spores of parasiticide fungi as a contribution to integrated nematode control was assessed in grazing horses throughout sixteen months. Two groups of 7 Pura Raza Galega autochthonous horses (G-T and G-P) were dewormed pour-on (1mg Ivermectin/kg bw) at the beginning of the trial, and other group (G-C) remained untreated. The G-P was provided daily with commercial pellets to which was added a mixture of fungal spores during the industrial manufacturing (2×106 spores of Mucor circinelloides and same dose of Duddingtonia flagrans/kg), and G-T and G-C received pellets without spores. The efficacy of the parasiticidal strategy was assessed by estimating the reduction in the faecal egg counts (FECR) and in the number of horses shedding eggs in the faeces (PHR), and also the egg reappearance periods (ERP). Blood analyses were performed to identify the changes in the red and white cell patterns. To ascertain if horses developed an IgG humoral response against the fungi, antigenic products collected from M. circinelloides and D. flagrans were exposed to the horse sera by using an ELISA. The faecal elimination of eggs of Parascaris equorum and strongyles ceased 2 weeks after treatment in G-T and G-P, thus the values of FECR and PHR were 100%. No P. equorum-eggs were detected later, and the strongyle egg reappearance period was 28 weeks in G-P, and 8 weeks in G-T. Strongyle egg-output values remained lower than 300 eggs per gram of faeces in the G-P, whereas numbers between 330 and 772 in G-C and G-T were recorded. Normal values for the erythrocytes, haemoglobin and haematocrit in horses consuming pellets with spores were recorded, and lower than normal in the other groups. Sensitization of horses to the fungal species was disproven. It is concluded that feeding horses with pellets industrially manufactured with fungal spores represents a very useful tool to implement an integrated control of helminths affecting horses. This strategy allows a decrease in their risk of infection, aids in reducing the frequency of anthelmintic treatment.

Effect of the Filamentous Fungus Mucor circinelloides On The Development of Eggs of the Rumen Fluke Calicophoron daubneyi (Paramphistomidae)

Abstract Ruminants infected by Paramphistomidae flukes shed eggs in the feces, which pass through different stages in the environment until the infective stages (metacercariae) are reached. The activity of the soil fungus Mucor circinelloides on the development of eggs of the rumen fluke Calicophoron daubneyi was presently tested with 3 probes, i.e., in petri plates, feces, and an aqueous environment (tubes). The effect of the fungus was assessed by recording the numbers of undeveloped, nonviable, and embryonated eggs. Nonviable eggs were considered when vacuolization occurred, the inner structures were not clearly observed, the eggshell was broken, or the embryo inside was destroyed. By considering the ability of hyphae of M. circinelloides to develop in the presence of C. daubneyi eggs, attach to their surface, and penetrate and destroy the inner embryo, this ovicidal effect was classified as type 3. After a period of 50 days, the percentage of undeveloped eggs in the feces of infected cattle was 40%; furthermore, 27% of the eggs were nonviable, and 33% were embryonated (1 miracidium inside). The addition of 4 doses of M. circinelloides spores directly onto the feces resulted in 9–31% undeveloped eggs, 38–60% nonviable eggs, and 9–21% embryonated eggs, and no statistical significances were obtained among the different doses. Placing the eggs of C. daubneyi into an aqueous solution containing 107 spores of M. circinelloides/ml for 29 days resulted in 43% undeveloped eggs, 40% nonviable eggs, and 17% embryonated eggs, whereas in the controls, the percentages were 48%, 12%, and 40%, respectively. These data demonstrate the usefulness of the spores of the fungus M. circinelloides in limiting the development of the eggs of the trematode C. daubneyi.