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The control of gastrointestinal nematodes among ruminants maintained in zoological parks remains difficult due to infective stages develop in the soil. For the purpose to improve the possibilities of the control of gastrointestinal nematodes (genera Trichostrongylus, Nematodirus, Chabertia and Haemonchus) affecting wild captive bovidae ruminants belonging to the subfamilies Antilopinae, Caprinae, Bovinae and Reduncinae, commercial pelleted feed enriched with a blend of 104–105 spores of both filamentous fungi Mucor circinelloides + Duddingtonia flagrans per kg meal was provided for a period of 3.5 years. All animals were dewormed at the beginning of the trial and also when exceeding a cut-off point of 300 eggs per gram of feces (EPG). The anthelmintic efficacy ranged between 96 and 100%. The need for repeating the administration of parasiticide treatment disappeared at the 24th month of study in the Antilopinae individuals, and at the 8th month in the Caprinae, Bovinae and Reduncinae. No side-effects were observed on the skin or in the digestive, respiratory or reproductive system. It was concluded that this strategy provides a sustainable tool for preventing the contamination of paddocks where captive ruminants are maintained, decreasing the risk of infection by gastrointestinal nematodes and consequently the need of frequent deworming.

Parasiticidal fungi have been used in several in vivo experiments in livestock farms worldwide, constituting an effective tool for the biocontrol of gastrointestinal parasites in grazing animals. In the first year of study, two groups of eight first-season pasturing ewe lambs infected by strongyles were dewormed with albendazole, and then, the test group received an oral dose of 106 chlamydospores of Mucor circinelloides and 106&nbsp, Duddingtonia flagrans individually and thrice a week from mid-September to May (FS1), while the control group remained without fungi (CT1). In the second year, two new groups of first-season grazing ewe lambs were treated with ivermectin and subjected to the same experimental design (FS2 and CT2, respectively). The anthelmintic efficacy was 96.6% (CT1), 95.6% (FS1), 96.1% (CT2), and 95.1% (FS2). The counts of strongyle egg output increased in the control groups (CT1 and CT2) throughout the study and reached numbers higher than 600 eggs per gram of feces (EPG), while in FS1 and FS2, they were &lt, 250 EPG. The values of red blood cell parameters registered for CT1 and CT2 were lower than those of the reference standards, while a significant increment was recorded in FS1 and FS2, and values within the physiological range were attained. It is concluded that integrating efficient anthelminthic deworming with rotational pasturing and the regular intake of chlamydospores of M. circinelloides and D. flagrans provides a helpful strategy for maintaining low levels of strongyle egg output in first-season grazing ewe lambs and improves their health status.

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.

Two different formulations containing a blend of spores of the filamentous fungi Mucor circinelloides and Duddingtonia flagrans have been assayed against trichostrongylids in wapitis captive in a grassy parcel from a zoological park and dewormed with fenbendazole. One approach (sprayed-on pellets) consisted of providing them, during 10 months, nutritional pellets sprayed the spores just before to be ingested, and the other (formulated pellets) involved the administration of pellets industrially manufactured with the spores for an identical period. Data collected on wapitis treated with fenbendazole without receiving spores during a 10-months interval were considered as controls. The effect of these strategies was evaluated through the analysis of feces collected directly from the soil, by estimating the reduction of the counts of eggs of trichostrongylids per gram of feces (EPG). The possibility of side effects in the digestive tract, respiratory apparatus or on the skin was assessed. The coprological analyses showed that, despite the successfulness of fenbendazole, the values of trichostrongylids egg-output increased four months later above 300 EPG. Through the administration of pellets sprayed spores, a significant EPG reduction of 69% (30–100) was recorded, and 71% (36–100) by giving the wapitis pellets industrially enriched with the spores. No unfavorable effects were observed among the wapitis taking the spores. It is concluded that ingestion of a blend of spores of M. circinelloides and D. flagrans every two days affords a beneficial long-term effect on controlling the infection by trichostrongylids in wild captive animals.

Soil-transmitted helminths (STHs) are parasites transmitted through contact with soil contaminated with their infective eggs/larvae. People are infected by exposure to human-specific species or animal species (zoonotic agents). Fecal samples containing eggs of Ascaris suum or Lemurostrongylus sp. were sprayed with spores of the soil saprophytic filamentous fungi Clonostachys rosea (CR) and Trichoderma atrobrunneum (TA). The antagonistic effect was assessed by estimating the viability of eggs and their developmental rate. Compared to the controls (unexposed to fungi), the viability of the eggs of A. suum was halved in CR and decreased by two thirds in TA, while the viability of the eggs of Lemurostrongylus sp. was reduced by one quarter and one third in CR and TA treatments, respectively. The Soil Contamination Index (SCI), defined as the viable eggs that attained the infective stage, reached the highest percentages for A. suum in the controls after four weeks (66%), with 21% in CL and 11% in TA. For Lemurostrongylus sp., the values were 80%, 49%, and 41% for control, CR and TA treatments, respectively. We concluded that spreading spores of C. rosea or T. atrobrunneum directly onto the feces of animal species represents a sustainable approach under a One Health context to potentially reduce the risk of zoonotic STHs in humans This research was funded by the Research Projects CTM2015-65954-R and RYC-2016-21407 (Ministerio de Economía y Competitividad, Spain; FEDER), and ED431F 2018/03 (Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, Spain). María Sol Arias Vázquez is recipient of a Ramón y Cajal (Ministerio de Economía y Competitividad, Spain) contract SI

In order to provide up-to-date information about the prevalence of infectious and parasitic agents in Spanish roe deer (Capreolus capreolus), samples from 93 animals hunted from January 2013 to April 2015 were collected and analysed by parasitological, serological and molecular techniques. Sampled animals came from four roe deer populations corresponding to Oceanic, Continental, Mediterranean and Mountainous ecosystems of Spain. Data regarding sex, age and year were also considered. A high percentage of roe deer (95.7%) resulted positive for at least one agent. Sarcocystis spp. was the most frequently diagnosed genus (88.8%), followed by gastrointestinal nematodes (62.9%) and Schmallenberg virus (53.5%). Varestrongylus capreoli (38%), Anaplasma phagocitophylum (34.2%), Eimeria spp. (29.2%), Toxoplasma gondii (25%) and Cephenemyia stimulator (23.8%) displayed medium prevalences and, finally low percentages were registered for Moniezia spp. (6.7%), Dictyocaulus noerneri (2.4%) and Mycobacterium avium subsp. paratuberculosis (1.5%). No infections by Neospora caninum, Bovine herpesvirus, pestivirus or Coxiella burnetii were found. Climate was significantly associated with the prevalence of T. gondii, C. stimulator and A. phagocitophylum, with higher prevalences in animals from Oceanic and Mediterranean areas. Our results suggest that infections affecting Spanish roe deer, especially those of parasitic aetiology, represent one of the causes of the descent in the abundance of this ungulate in the last years. Moreover, the high prevalence of zoonotic agents such as T. gondii and A. phagocitophylum could also have a great relevance in the environmental and/or Public Health. This research was supported by a Programme for consolidating and structuring competitive research groups (GRC2015/003, Xunta de Galicia) and by the Research Project ‘RUMIGAL: Rede de estudo multidisciplinar dos ruminantes en Galicia’ (R2014/005, Xunta de Galicia, Spain) SI

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 (1 mg 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.

The ability of the spores of the ovicide fungus Mucor circinelloides to resist the industrial manufacturing of pelleted feed and retain their biological and parasitological activities has been tested. Firstly, survival of M. circinelloides spores at elevated temperatures was in vitro assayed. In a second assay, the spores of M. circinelloides were added in the mixing phase of the industrial pelleting of livestock (calves and horses) feed. The biological development (mycelium growth rates and sporogenesis) and the ovicidal activity on eggs of the parasites Fasciola hepatica and Parascaris equorum eggs were measured in plates. In the in vitro assay, a similar level of biological development in all the conditions except by heating the spores at 72 °C for 10 min were observed. Viability of F. hepatica eggs reduced to 55–60%, and 56–70% that of P. equorum eggs. After the addition of the spores to the meal previous to the pelletization phase, percentages of reduction of 54–58% viability F. hepatica eggs and 61–67% P. equorum eggs were recorded. It is concluded that the spores of M. circinelloides maintain their antagonistic effect against eggs of the parasites F. hepatica and P. equorum in industrially manufactured pellets, providing thus a very helpful tool to prevent infection by trematodes or ascarids among pasturing livestock.

Thirty-two Friesian cattle under a leaders/followers four-day rotation and passing eggs of trematodes and gastrointestinal nematodes (GIN) were studied in two trials for the integrated control of these helminths over two years. In the first trial, the effect of rotational pasturing was assessed on a group of leaders (milking cows, G-L1) and followers (dried-off cows and heifers, G-F1) supplemented daily with commercial nutritional pellets. In the second trial, leaders (G-L2) and followers (G-F2) were maintained under a rotational pasturing regime; the cows received daily commercial pelleted feed and heifers pellets manufactured with a blend of parasiticide fungi (3 × 105 chlamydospores of both Mucor circinelloides and Duddingtonia flagrans/kg pellet). Deworming via closantel and albendazole was performed in cows in each trial at the beginning of their drying periods, and fourteen days later, the fecal egg-count reductions (FECR) of Calicophoron daubneyi and GIN were from 94 to 100% (average 98 %), while the percentages of reduction of cattle shedding eggs (CPCR) were from 50 to 100% (average 77 % and 82 %, respectively). The heifers were dewormed one time only, at the beginning of each trial, and the values of FECR and CPCR were 100 % against C. daubneyi and 96 % and 83 %, respectively, against GIN. Over a period of 24 months, significantly higher numbers of helminth egg-output were observed in G-L1, with the lowest numbers in G-F2. C. daubneyi egg output was reduced by 5 % (G-L1) and 42 % (G-F1) at the end of trial 1 and by 83 % (G-L2) and 100 % (G-F2) at the end of trial 2; the numbers of GIN egg-output decreased by 13 % (G-L1) and 18 % (G-F1) at the end of trial 1, and by 72 % (G-L2) and 85 % (G-F2) at the end of trial 2. No adverse effects were detected in cattle taking pellets enriched with fungal spores (G-F2). It is concluded that long-term ingestion of spores of M. circinelloides and D. flagrans provides a valuable tool to improve the effect of rotational grazing and to lessen the risk of infection by C. daubneyi and GIN in dairy cattle, and accordingly, the performance of integrated control programs.

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 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....

Background An approach to preventing strongyle infection in horses was tested, comprising rotational pasturing and the administration of spores of two parasiticidal fungi, Mucor circinelloides and Duddingtonia flagrans. Methods Twenty-two adult Spanish Sport Horses were dewormed with ivermectin (1 mg pour-on/kg body weight) and then randomly divided into three groups. G-1 was maintained with continuous grazing, and G-2 and G-3 were kept on a four-paddock rotation system. Commercial pelleted feed (2.5 kg/horse) was supplied to G-1 and G-2 twice a week; horses in G-3 received pellets containing 2 × 106 spores/kg of each fungus. Fecal samples were analyzed by the flotation method to estimate the reduction in the fecal egg counts (FECR), the percentage of horses shedding eggs (PHR), and the egg reappearance period (ERP). Results Third-stage larvae were identified in fecal pats as Cyathostomum (sensu lato) types A, C and D, Gyalocephalus capitatus, Triodontophorus serratus, Poteriosthomum spp., Strongylus vulgaris and S. edentatus. Two weeks after treatment, the FECR values were 100% in G-1, 96% in G-2 and 99% in G-3; the PHR values were 100% in G-1, 75% in G-2 and 88% in G-3. A strongyle ERP of 6 weeks was observed in G-1, ERP of 10 weeks was observed in G-2, and ERP of 16 weeks was observed in G-3. The counts of eggs per gram of feces (EPG) were > 300 EPG in G-1 and G-2 but remained below 250 EPG in G-3 throughout the observation period of 12 months. Conclusions These results suggest that horse strongyle infection could be decreased by combining rotational pasturing with feeding pellets containing the spores of parasiticidal fungi.

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 10

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 This research was partly supported by the Research Projects AGL2012-34355 and CTM2015-65954-R (Ministerio de Economía y Competitividad, Spain; FEDER). María S. Arias is recipient of a “Parga Pondal” postdoctoral research fellowship (Xunta de Galicia, Spain) SI

A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer.

Oestrosis is one of the most prevalent parasitosis affecting the Iberian ibex, Capra pyrenaica . To date, both the diagnosis of oestrosis and the determination of the intensity of parasitism require the use of invasive methods (necropsy), which necessarily limit research possibilities. We analyzed the immune humoral response (IgM and IgG) against Oestrus ovis L. excretory/secretory larval antigens in 32 sera taken from Iberian ibex from the Sierra Nevada Natural Space (southern Spain). Three antigens were collected: L1OES (from L1 larvae), L2OES (L2), and L3OES (L3). Necropsy was considered as the gold standard. The percentage of ibexes harboring Oestrus spp. larvae was 88%, the mean intensity of parasitism being 16.96 ± 14.96 larvae per parasitized host (range: 2-52). In our sample, first-instar larvae (L1) were found in 9% of ibexes, while 69% of hosts carried L2 larvae and 88% L3 larvae. Positive correlations between L1 and L2 numbers, and between L2 and L3 numbers were detected. The best results with the immunoenzymatic assay were obtained using IgG antibodies against the L1OES antigens (specificity = 89%; sensitivity = 100%; positive predictive value = 100%; negative predictive value = 57%). The IgG seroprevalence against L1OES was 78%. Thus, the analysis of IgG antibodies against antigens collected from L1 O. ovis larvae would seem to be a noninvasive method for reliably diagnosing oestrosis in naturally infested Iberian ibex. Nevertheless, additional immunological and methodological advances are still required because false positive and false negative results still represent a non-negligible part of the results of the ELISA tests.

The main goal in this research was to determine the beneficial effect of incorporating biological procedures in parasite control programs for equids in zoological parks. Two trials were developed for Equus quagga , E asinus , and E africanus asinus . The first trial (September 2010 to August 2011) consisted of chemotherapy only (ivermectin plus praziquantel), and the second trial (September 2011 to September 2012) consisted of administration of chemotherapy and chlamydospores of the nematophagous fungus Arthrobotrys ( Duddingtonia ) flagrans . The effect of these measures was evaluated by the estimation of the reduction in the fecal egg counts (FECR). In the first trial, 100% FECR values were achieved 15 days after treatment in all the animals. The egg reappearance period (ERP) was 2-3 months for the equids, and all of them were passing strongyle eggs in the feces at 2-4 months after their deworming. In the second experiment, the FECR values were 100% in the three species. ERPs of 3 months in the European donkeys, 4 months in the Africans, and 6 months in the zebras were recorded. All the equids had positive results for the coprological flotation test 4-8 months after anthelmintic administration. This preliminary study demonstrates the incorporation of chlamydospores of nematophagous fungus, as A flagrans appears highly promising for reduction of the infective stages of the strongyles affecting captive animals, but the experimental design precludes true determination of whether the treatment is fully efficacious.

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.

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.

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 (

Control of horse parasites often omits application of measures to eradicate the free-living stages in pastures and frequently relies on chemotherapy only. Selective therapy was used for Spanish Sport horses grazing either in the same pasture (continuous) or in rotated meadows. In each group, equines exceeding a cutoff value of 300 strongyle eggs per gram of feces received ivermectin or moxidectin. Efficacy of the treatment was assessed by estimating reduction of fecal egg counts and the number of horses shedding parasite eggs (PHR). Coprocultures revealed presence of the cyathostomins Cyathostomum and Gyalocephalus spp. In all treated groups, a 100% value for both reduction of fecal egg counts and PHR against cyathostomins was obtained, and PHR values ranged from 100% to 12%. The longest strongyle egg reappearance period was observed in horses undergoing rotation grazing and receiving ivermectin (9 weeks), compared with a 6-week period recorded for the other treated equines. Our results seem to point that the efficacy of selective therapy in equine herds could be reduced if the horses with fecal egg counts below the threshold value (thus not receiving chemotherapy) remain grazing in the same pastures with the treated ones. It is strongly suggested that interested parties consider performing periodic fecal analyses to monitor fecal egg counts, together with the percentage of horses passing eggs in feces, to improve the effect of this procedure.

Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs.

The efficacy of topical ivermectin (IVM) on foals naturally infected by parasitic nematodes was evaluated. Two dosages of IVM were applied pour-on (F-Nor0.5; 0.5 mg/kg body weight [BW] and F-Nor1; 1 mg/kg BW) and results compared with the oral administration (F-Eq0.2; 0.2 mg/kg BW of IVM). The efficacy was measured by estimating the reduction in the fecal egg counts (fecal egg count reduction) and in the numbers of horses shedding parasite eggs (positive horse reduction). Several biochemical and enzymatic serum parameters were measured in the groups F-Eq0.2 and F-Nor1. Before the deworming of the horses, eggs of Parascaris equorum, Cyathostomum, Gyalocephalus spp, and Oxyuris equi were identified. In all the treated groups, the excretion of ascarid eggs ended 4 days after the treatment. The orally administered IVM suppressed the egg output of strongyles and pinworms 4 days after the treatment, whereas for the F-Nor1 group this occurred 8 days after the treatment. Eggs of strongyles were detected in the F-Nor0.5 group throughout the study. The levels of blood urea nitrogen, creatinine, total proteins, albumin, globulins, and lactate dehydrogenase (LDH) reduced significantly after the administration of IVM, but values not within the normal range were only achieved for LDH. A significant positive correlation between the fecal egg output of cyathostomins and the LDH was investigated. Clinically, no adverse reactions in the horses receiving the topical IVM were observed. It was concluded that the pour-on administration of 1 mg/kg BW IVM provides similar results to the oral administration, and offers a very useful tool to control infestation by the intestinal nematodes affecting wild grazing horses.

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 1 year 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.

Three major protein complexes (51, 29, and 15 kDa, named P1 to P3, respectively) were resolved by gel filtration of the excretory/secretory antigens collected from a mixture of horse cyathostomin third-stage larvae (L3s). The potential application for the detection of infected horses was assessed with an enzyme-linked immunosorbent assay (ELISA) by the comparison of the serological and copromicroscopical results. The value of the area under the receiver operating characteristic (ROC) curve was higher than 0.9 when the three peaks were used. Elevated values (>90%) for the sensitivity, specificity, and the positive-likelihood ratio were also observed for all the antigen complexes. A significant increment in the IgG antibody levels 4 weeks prior to the observation of eggs in the feces of weanlings naturally infected was recorded. Our results indicate that the evaluation of chemotherapy is possible by using immunoenzymatic probes and fast protein liquid chromatography (FPLC)-purified antigens. Data collected in the present investigation indicate that FPLC isolation offers a very helpful one-step method for collecting antigens with diagnostic potential to be employed in immunoenzymatic probes.

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 × 106/100 g 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.

During the 2007 and 2008 hunting seasons (April-October) the skin of 367 roe deer (Capreolus capreolus L.), hunted in different preserves from Galicia (Northwestern Spain), were examined for ticks (Acari: Ixodidae). The overall prevalence of infestation by ticks was 83.1%. The predominant species was Ixodes ricinus (83.1%), whereas a single Dermacentor marginatus specimen appeared in one roe deer. All developmental stages of I. ricinus were found parasitizing roe deer, the adults being the most frequent (82.2%), followed by nymphs (45.6%) and larvae (27.2%). The mean intensity of infestation by I. ricinus was 43.2 ± 49.85; most of them were adults (30.7 ± 31.64) and in a lesser extend nymphs (16.9 ± 24.74) and larvae (10.7 ± 29.90). Ixodes ricinus was present all over the study with percentages that oscillated between 100% in spring and 57.4% in autumn. CHAID algorithm showed the sex of roe deer as the most influential factor in tick prevalence, followed by the climatic area. The different developmental stages of I. ricinus were more frequent in males than in females, and the prevalence of adults and larvae were higher in roe deer from coastal areas than in those from mountainous and central areas, whereas nymphs were more frequent in mountainous areas. Host age and density were not determinants for tick infestation. Our results confirm that roe deer are important hosts for I. ricinus in northwestern Spain, serving as a vehicle for the geographic distribution of these ticks.

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.

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41–48 days post-infection. The mice vaccinated with ADAD only with PAL + Fh15 present a survival rate of 40–50% and those vaccinated with ADAD containing PAL + Qs + Fh15 had a survival rate of 50–62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL + Qs + Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.

Baylisascaris procyonis is a soil-transmitted helminth mainly found in raccoons (Procyon lotor) which can also affect other domestic and sylvatic animals, as well as humans, when the eggs released in the feces of parasitized raccoons are accidentally ingested. Three assays have been conducted to assess the effect of three saprophytic fungi, Mucor circinelloides, Paecilomyces lilacinus, and Verticillium sp., on the eggs of B. procyonis. Firstly, their ovicidal effect was in vitro ascertained by placing 1 mL with 2 × 10(6) spores of each fungus in Petri plates with water-agar (2 %) and simultaneously adding 200 eggs of Baylisascaris/plate. Two in vivo probes were carried out, by spraying the fungal spores (3 mL containing about 2 × 10(6) spores/mL) on the feces of raccoons and coatis (Nasua narica) passing eggs of B. procyonis in a zoological park; the other assay consisted of evaluating the activity of the fungi after adding sand to fecal samples from raccoons. An ovicidal type 3 activity characterized by morphological damage of the eggshell with hyphal penetration, internal egg colonization, and embryo alteration was observed for all the tested fungi. In the plate assays, viability of Baylisascaris eggs reduced significantly by 53-69 % with Mucor, 45-62 % with Paecilomyces, and 52-67 % with Verticillium. A similar ovicidal effect was detected in the feces with sand. These results demonstrate the usefulness of spraying spores of M. circinelloides, Pa. lilacinus, or Verticillium sp. on the feces of animals infected by Baylisascaris to decrease the numbers of viable eggs and, thus, the risk of infection.

The IgG antibody response to Calicophoron daubneyi (Digenea: Paramphistomidae) excretory/secretory antigens was evaluated in naturally infected cattle from Lugo (Galicia, NW Spain) by using an ELISA procedure. Two studies were conducted, first a survey in 524 cattle separated into three groups according to age, G-1 (0-2 years old), G-2 (3-5 years old) and G-3 (> 6 years old). In the second study, three groups of cattle were employed: G-I, naturally infected; G-T, naturally infected and treated with oxyclozanide plus levamisole (Nilzan Plus); G-C, cattle maintained in a farm where C. daubneyi has never diagnosed. Variations on egg-output and haematic parameters (erythrocytes, haematocrite, leukocytes and lymphocytes) were also analyzed. The ELISA procedure showed that 61.2% of the cattle in the first study had been exposed to the trematode, but only 10.1% passed eggs in the feces. Age-association with egg-output was shown but not with the IgG values. In the second experiment, the administration of the anthelmintic reduced significantly the IgG kinetic levels and the C. daubneyi-egg-output was suppressed during 12 weeks in the G-T group. The values of red cells, haematocrite, leukocytes and lymphocytes increased significantly in the treated cattle 5 weeks after chemotherapy; however, new reduction after week 5 was recorded, as results of the challenge of these cattle. This is the first investigation in which evaluation of the IgG humoral response against C. daubneyi in cattle has been carried out. We proved that a notable IgG response in naturally infected cattle is induced, and can be detected by using an ELISA procedure. The IgG antibodies did not increase after challenge infection. Our results proved an important percentage of cattle were exposed to this trematode in the area of study and suitable measures for preventing this relationship must be considered.