Cristiana Filipa Cazapal-Monteiro

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.

Infection by Paramphistomidae trematodes in cattle from two agricultural regions in NW Uruguay and NW Spain.

The analysis of infection by Paramphistomidae trematodes was conducted in two agricultural regions with different knowledge on this parasitosis. Faecal and blood samples were collected from 374 cattle in Salto (NW Uruguay) where there is a lack of information about paramphistomosis. A total of 429 cattle from Galicia (NW Spain), an area with previous records of infection by gastric flukes, were sampled. Diagnostics of trematodosis was developed by using a copromicroscopic probe and an ELISA with excretory/secretory antigens collected from adult Calicophoron daubneyi (Paramphistomidae) specimens. Results were evaluated according intrinsic and extrinsic factors. In the Uruguay, the percentage of cattle passing Paramphistomidae-eggs by faeces was 7% (95% Confidence Interval 5, 10). A significantly higher prevalence of paramphistomosis in the Hereford × Angus cattle (OR=3.5) was recorded, as observed for the oldest ruminants (>3.5 years). An overall seroprevalence of 29% (25, 34) was obtained by ELISA, with the highest values in the Friesians (OR=3), the youngest bovines (<2.5 years) and dairy cattle (Friesians). Twenty-six percent (22, 30) of the cattle from Spain passed eggs by faeces, and cattle aged 2.5-7 years reached significant highest prevalences. By means of the ELISA, a percentage of 55 cattle (50, 59) had antibodies against the gastric fluke, and the highest seroprevalence was observed among the bovines under 6 years. It is concluded that paramphistomosis is on the increase in cattle from NW Spain, partly due to the absence of an effective treatment against the trematode. There is a need for reducing the risk of infection by Paramhistomidae spp. in cattle from Uruguay, especially by improving their management to avoid exposure to the gastric trematode. Further studies are in progress for identifying the species of Paramphistomidae affecting ruminants in Uruguay.

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.

Potential use of Mucor circinelloides for the biological control of certain helminths affecting livestock reared in a care farm

A preliminary study to evaluate the possibilities of biological control procedures against parasites affecting livestock reared in a care farm has been conducted. Adults with mental disabilities were involved in spreading the spores of the filamentous fungus Mucor circinelloides directly onto the faeces, or as a food additive. In the first assay, the spores were sprayed directly onto the faeces of piglets and calves parasitised by roundworms (Ascaris suum) and liver flukes (Fasciola hepatica), respectively. In the second assay, the spores were mixed with on-farm mash feed. Participation of the adults in the experiments was fully satisfactory. In the manure sprayed Mucor spores, the viability of eggs of roundworms and flukes reduced by 53% and 74%, respectively. Significant reductions of viability of eggs of Ascaris (60%) and Fasciola (67%) in the faeces of piglets and calves given mash feed-added Mucor spores were achieved, which demonstrates their ability to survive in the digestive tract of the animals. It is concluded that biological control of parasites could be helpful to decrease the risk of infection in animals reared by intellectually disabled adults in a care farm, and it could motivate them to accomplish new tasks.

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.

Isolation of Ovicidal Fungi from Fecal Samples of Captive Animals Maintained in a Zoological Park

There are certain saprophytic fungi in the soil able to develop an antagonistic effect against eggs of parasites. Some of these fungal species are ingested by animals during grazing, and survive in their feces after passing through the digestive tract. To identify and isolate ovicidal fungi in the feces of wild captive animals, a total of 60 fecal samples were taken from different wild animals kept captive in the Marcelle Natureza Zoological Park (Lugo, Spain). After the serial culture of the feces onto Petri dishes with different media, their parasicitide activity was assayed against eggs of trematodes (Calicophoron daubneyi) and ascarids (Parascaris equorum). Seven fungal genera were identified in the feces. Isolates from Fusarium, Lecanicillium, Mucor, Trichoderma, and Verticillium showed an ovicidal effect classified as type 3, because of their ability to adhere to the eggshell, penetrate, and damage permanently the inner embryo. Penicillium and Gliocladium developed a type 1 effect (hyphae attach to the eggshell but morphological damage was not provoked). These results provide very interesting and useful information about fungi susceptible for being used in biological control procedures against parasites.

Determination of exposure to Fasciola hepatica in horses from Uruguay using a recombinant-based ELISA.

The risk of exposure to Fasciola hepatica in horses from Uruguay was evaluated using ELISA and a recombinant surface protein (FhrAPS). Blood samples were collected from 368 horses from different districts. Detection of antibodies and the seasonal IgG pattern against the trematode was also established. The overall seroprevalence was 54% (ranging 23% November to 93% December). Significantly higher values were observed in the Anglo-Arab horses (86%) and in those older than xix year (63%). No sex- or origin-differences were observed. Two primary risk periods for infection in Uruguay have been identified, the end of spring and autumn. Exposure to the fluke should be taken into account among those horses feeding on pastures grazed by ruminants. Using FhrAPS-ELISA, helpful information concerning the risk of horses to the infection by trematode can be obtained. This test allows the opportunity to compare the results achieved in different laboratories by minimising specific regional effects.

A combined trial of chemotherapy and biological control measures against parasites in grazing horses

Horses under grazing regime are at a high risk for infection by several parasites affecting the digestive tube, especially the strongyles. Infection occurs by the ingestion of free-living third stage larvae (also called L3) when grazing in pastures. While deworming reduces the presence of adult parasites in the horses, some action against the L3 in the pasture is needed for reducing the risk of infection. Eight adult horses exceeding a cut-off value of 300 eggs per gram of feces (EPG) were utilized in the current study. Control of parasitic strongyles consisted of the combination of two measures. Firstly, the horses were given moxidectin (Equest® oral gel, Pfizer, Madrid, Spain; 0.4 mg/kg bw dosage), and in addition, a dose of 2×106 Duddingtonia flagrans chlamydospores kg/bw was bi-weekly given to each horse. The effect of this combined procedure was assessed by estimating the reduction in the faecal egg counts. After the horses received the chemotherapy and the D. flagrans chlamydospores a significant reduction in the EPG was observed (Z=-11.391, P= 0.001). No strongyle egg-output was observed for 2 months, whereas the need for applying a new dosage of chemotherapy was established at the 6th month after the initial deworming of the equines. Our results point the efficacy of a combined strategy by giving chemotherapy and chlamydospores for reducing the frequency of antiparasitic drug administration to grazing horses. Besides the presence of free-infective larval stages can be significantly lowered and thus the risk of infection in the horses, this procedure seems a very valuable and helpful contribution to the sustainability of the environment by minimizing the effect of chemical residues dangerous for some organisms as dung beetles, essential for the natural fertilization of the soil.

Potential Usefulness of Filamentous Fungi to Prevent Zoonotic Soil-Transmitted Helminths

The usefulness of a strategy involving the biological control of soil-transmitted helminths (STHs) with zoonotic potential is tested. A blend of spores of two filamentous fungi, Mucor circinelloides and Duddingtonia flagrans, was provided to one group (G-2) of puppies (five Griffon bleu de Gascogne and five Gascon Saintongeois) shedding eggs of Toxocara canis, Toxascaris leonina, Trichuris vulpis, and Ancylostoma caninum. Other identical group (G-1) remained without spores. Fecal samples were collected daily and divided into two portions, one sample was immediately analyzed by the flotation test and the other was examined 28 days later. Viability of eggs of T. canis, T. leonina, and T. vulpis in fecal pats was ascertained by microscopic examination; the numbers of larvae of A. caninum were assessed in each group. In the feces of G-2, spores of both fungi were observed 1 day after beginning the assay. Viability of eggs of T. canis was reduced by 57%, T. leonina by 61% and T. vulpis by 53% in the fecal pats of G-2, and by 87%, 92%, and 85%, respectively, in G-1. The numbers of third-stage larvae of A. caninum were 59% lower in G-2 than that in G-1. By considering the results obtained and the absence of adverse effects in the puppies of G-2, it is concluded that the presence of the filamentous fungi in the feces has a high potential to avoid the transmission of these parasites. Further studies are in progress to determine the appropriate way of administration to pets.