Analysis of the effect of soil saprophytic fungi on the eggs of Baylisascaris procyonis.

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.

Formulating Parasiticidal Fungi in Dried Edible Gelatins to Reduce the Risk of Infection by Trichuris sp. among Continuous Grazing Bison.

Control of infection by gastrointestinal nematodes remains a big problem in ruminants under continuous grazing. For the purpose of decreasing the risk of infection by Trichuris sp. in captive bison (Bison bison) always maintained in the same plot, dried gelatins having ≥106 chlamydospores of both Mucor circinelloides and Duddingtonia flagrans were given to them for one week, and at the end, fecal samples (FF) collected each week for four weeks were analyzed immediately. Feces taken one week prior to gelatin administration served as controls (CF). Eggs of Trichuris sp. were sorted into non-viable and viable, then classified into viable undeveloped (VU), viable with cellular development (VCD), or viable infective (VI). Ovistatic and ovicidal effects were determined throughout the study. In FF, viability of Trichuris eggs decreased between 9% (first week) and 57% (fourth week), egg development was delayed during the first two weeks, and VI percentages were significantly lower than in CF (p = 0.001). It is concluded that the preparation of gelatins with chlamydospores of parasiticidal fungi and their subsequent dehydration offer an edible formulation that is ready to use, stress-free to supply, and easy to store, as well as being well-accepted by ruminants and highly efficient to reduce the risk of Trichuris sp. infection among animals under continuous grazing regimes.

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 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.

A New Comestible Formulation of Parasiticide Fungi to Reduce the Risk of Soil-Transmitted Helminth Infections in a Canine Shelter.

Dogs cared for in a shelter are dewormed every three-four months, but they all become infected one-two months later by the soil-transmitted helminths (STHs) Toxocara canis, Toxascaris leonina, Trichuris vulpis, and Ancylostoma caninum. For the purpose of reducing their risk of infection by decreasing the survival of helminths' infective stages in soil, chlamydospores of two parasiticide fungi, Mucor circinelloides (ovicide) and Duddingtonia flagrans (larvicide) were formulated as handmade edible gelatins and given three days per week for 17 months to 18 dogs (DRF, dogs receiving fungi); a second group was maintained without fungi (CD, control dogs). All individuals were dewormed at months 0, 3, 7, 10 and 13, and it was observed that the levels of helminths egg-output were reduced by 96-98% fourteen days after each treatment. Fecal egg counts of STHs were similar in both groups until the 6th-8th months, and then remained significantly lower in DRF than in CD (42-100% ascarids; 30-100% trichurids and ancylostomatids). According to the results, and considering that gelatin treats have always been fully accepted, it is concluded that this new formulation offers an efficient solution to decrease the risk of infection among dogs maintained in shelters, and is therefore recommended.

Control of Strongyles in First-Season Grazing Ewe Lambs by Integrating Deworming and Thrice-Weekly Administration of Parasiticidal Fungal Spores.

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 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 <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 Control of Zoonotic Soil-Transmitted Helminthoses Using Saprophytic Fungi.

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.

Integrating the control of helminths in dairy cattle: Deworming, rotational grazing and nutritional pellets with parasiticide fungi.

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.

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.

A combined effort to avoid strongyle infection in horses in an oceanic climate region: rotational grazing and parasiticidal fungi.

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.

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

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.

Abstract: 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.

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 treatment on the dynamics of circulating hypodermin C in cattle naturally infested with Hypoderma lineatum (Diptera: Oestridae).

An antigen capture ELISA, using a murine monoclonal antibody recognising recombinant hypodermin C (rHyC), was used to evaluate the influence of early treatment with eprinomectin (Eprinex) or fenthion (Spotton) on the kinetics of circulating hypodermin C in calves naturally infested with Hypoderma lineatum. No viable larvae were collected from treated animals, whereas a variable number of warbles were found in control animals. Treatment provoked a decrease in circulating HyC levels that was significant 9 days post-treatment (p.t.). Circulating antigen levels in the treated cattle remained detectable for approximately 99 days p.t. In contrast, control animals had no detectable antigen at 64 days p.t., 42 days earlier than in the treated animals. These results suggest that larvae were either gradually killed, resulting in slow release of antigen or they were encapsulated, leading to the slow liberation of antigen. Kinetics of circulating HyC did not differ among the two insecticide treatments. Antibodies persisted, in all groups, throughout the 120-day study. These results suggest that the antigen capture ELISA will be useful as a technique for detecting successful treatment of cattle grub infestations and for the detection of new infestations in previously infested cattle.

Reliability of an ELISA test for diagnosing oestrosis in Iberian ibex.

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.

Trematodes enhance the development of the nematode-trapping fungus Arthrobotrys (Duddingtonia) flagrans.

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.

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.

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.