Assessing the efficacy of the ovicidal fungus Mucor circinelloides in reducing coccidia parasitism in peacocks.

The biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15-t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism.

Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community.

Parasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds' gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors-iron permease, iron receptors, ADP-ribosylation factors, and GTPase-and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

First insights on the susceptibility of native coccidicidal fungi Mucor circinelloides and Mucor lusitanicus to different avian antiparasitic drugs.

BACKGROUND: The combined application of predatory fungi and antiparasitic drugs is a sustainable approach for the integrated control of animal gastrointestinal (GI) parasites. However, literature addressing the possible interference of antiparasitic drugs on the performance of these fungi is still scarce. This research aimed to assess the in vitro susceptibility of six native coccidicidal fungi isolates of the species Mucor circinelloides and one Mucor lusitanicus isolate to several antiparasitic drugs commonly used to treat GI parasites' infections in birds, namely anthelminthics such as Albendazole, Fenbendazole, Levamisole and Ivermectin, and anticoccidials such as Lasalocid, Amprolium and Toltrazuril (drug concentrations of 0.0078-4 µg/mL), using 96-well microplates filled with RPMI 1640 medium, and also on Sabouraud Agar (SA). RESULTS: This research revealed that the exposition of all Mucor isolates to the tested anthelminthic and anticoccidial drug concentrations did not inhibit their growth. Fungal growth was recorded in RPMI medium, after 48 h of drug exposure, as well as on SA medium after exposure to the maximum drug concentration. CONCLUSIONS: Preliminary findings from this research suggest the potential compatibility of these Mucor isolates with antiparasitic drugs for the integrated control of avian intestinal parasites. However, further in vitro and in vivo studies are needed to confirm this hypothesis.

Influence of seasonality and biological activity on infection by helminths in Cantabrian bear.

This study aimed to investigate the variations of parasites in the feces of brown bears Ursus arctos inhabiting the Cantabrian Mountains (NW Spain). A total of 248 bear fecal samples were collected throughout one year, spanning from August 2018 to September 2019, at an approximate frequency of 20 samples per month. The results were analyzed in relation to both the season and the biological activity of the brown bears, i.e., hibernation, mating and hyperphagia. Among the examined samples, eggs of Dicrocoelium dendriticum (32.2%; 95% Confidence Interval: 26.4-38.1), Baylisascaris sp. (44.8%; 38.5-50.9), ancylostomatids (probably belonging to Uncinaria spp.) (16.5%; 11.9-21.1) and Trichuris sp. (1.2%; 0-2.6) were observed. Significant seasonal differences were noted for Baylisascaris and ancylostomatids (χ2 = 21.02, P = 0.001 and χ2 = 34.41, P = 0.001, respectively). Furthermore, the presence of helminth eggs was correlated with the activity phase of the brown bears. Dicrocoelium attained the highest prevalence during the mating phase, while Baylisascaris and ancylostomatids were more frequent during hyperphagia. Notably, the highest egg-output counts for Dicrocoelium and Baylisascaris sp. were recorded during the mating phase and hibernation, respectively, whereas ancylostomatids eggs peaked during hyperphagia. Additionally, variations in egg-output counts were significant for all helminths concerning the season, with the exception of Trichuris sp., and for Dicrocoelium and Baylisascaris sp. According to bear activity. It is concluded that infection by gastrointestinal helminths depends on the season and the biological activity of the bears from the Cantabrian Mountains, and their health status could result influenced.

First Record of Flavocillium subprimulinum (Cordycipitaceae, Hypocreales) in Mexico: Morphological and Molecular Characterisation, Nematocidal Activity of Its Liquid Culture Filtrates against Haemonchus contortus and Protease Activity.

This is the first record of the fungus Flavocillium subprimulinum in Mexico. The isolate was taxonomically characterised and cultured in potato dextrose broth (PDB), Czapek-Dox broth (CzDoxB), and sweet potato dextrose broth (SPDB) to obtain its filtrates (FLCF). The nematocidal activity (NA) of three FLCF concentrations was assessed against Haemonchus contortus L3. Protease activity (PA) was assessed with SDS-PAGE, followed by a zymogram. The NA of the FLCF reached 94.43% in PDB and 95.82% in CzDoxB, respectively, at 100 mg/mL. Lower mortality (64%) was found in SPDB at 100 mg/mL. SDS-PAGE showed bands (in PBS) of ~25, ~40, and ~55 kDa. The zymogram showed protein bands (PBs) with PA in the media, including PBs of ~14, ~40, and ~55 kDa. This study establishes the basis for exploring the potential use of this fungus against H. contortus, which is considered the most pathogenic parasite affecting lambs.

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.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts.

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

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.

Arthrobotrys musiformis (Orbiliales) Kills Haemonchus contortus Infective Larvae (Trichostronylidae) through Its Predatory Activity and Its Fungal Culture Filtrates.

Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek−Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.

Implementation of Mini-FLOTAC in Routine Diagnosis of Coccidia and Helminth Infections in Domestic and Exotic Birds.

Mini-FLOTAC (MF) has recently been proposed for the fecal quantification of gastrointestinal (GI) parasites in birds due to its higher sensitivity and precision in comparison with the McMaster method. The current research aimed to test the use of MF in routine diagnosis of coccidia and helminth infections in several domestic and exotic bird collections in Portugal. Between July 2020 and April 2021, a total of 142 fecal samples from organic layers, peacocks and ratites were collected in four Portuguese bird collections and processed using MF and fecal cultures to identify and calculate GI parasite shedding and prevalence. The McMaster method was also used to compare the shedding levels obtained for both quantitative techniques. MF's relative sensitivity and specificity were also assessed, using McMaster as the reference technique. The implementation of MF resulted in an average Eimeria spp. shedding higher in peacocks from bird collection 2 (502 OPG), followed by peacocks from collection 1 (107 OPG) and organic layers (24 OPG) and peacocks from collection 3 (9 OPG). Peacocks were also positive for Capillaria spp., Trichostrongylus tenuis and Strongyloides pavonis, whereas ostriches and emus were infected by L. douglassii. The MF protocol for exotic animals and the McMaster method did not differ significantly for each parasitic agent and bird species, and MF achieved relative sensitivities and specificities higher than 70% for Galliform Eimeria spp., peacock helminths and ratites' L. douglassii infections. Higher L. douglassii EPG values were identified using the MF protocol for exotic species (2 g of feces/38 mL of sucrose solution), followed by McMaster 2/28, MF 5/45 and MF 2/18. The use of MF allowed for obtaining different intestinal parasitic populations in several bird species and locations, and MF 2/38 is globally proposed as the most suitable protocol for bird fecal samples as an alternative to the McMaster method in the diagnosis of avian intestinal parasitic infections.

Gastrointestinal Parasitism in Miranda Donkeys: Epidemiology and Selective Control of Strongyles Infection in the Northeast of Portugal.

In Portugal, equine parasitism in pasture animals is characterized by high parasitic burden and high helminthic biodiversity; both factors are potentially pathogenic for their hosts. The decrease in the number of donkeys over the last years in Portugal, their importance in rural lowland and mountain ecosystems and pastures and the scarce information regarding their parasitism led to this research, which aimed to evaluate the parasitological status of a Miranda donkey breed population, a native breed mainly located in the northeast of Portugal. This study provides better knowledge of their gastrointestinal parasitism, particularly strongyles, and the assessment of a targeted selective treatment (TST) as an alternative control approach of their parasitism. A longitudinal observational study was developed during a period of five years in a population of 62 Miranda donkeys. At first, strategic deworming of these animals was performed every semester, but this was progressively replaced by a TST approach according to the levels of Eggs per Gram (EPG). This new deworming strategy was conducted in association with a regular parasitological monitoring of the animals every three months, being dewormed with ivermectin when egg shedding was higher than 500 EPG. Over the study period, a decrease of the annual prevalence rate of infection by gastrointestinal strongyles was observed, from 35.5% to 19.4%, as well as a negative binomial distribution of parasitic strongyles in donkeys submitted to selective anthelminthic control. The prevalence rate of infection was higher in females (39.5%), in individuals under four years (46.7%) and in those presenting a lower body condition (40.8%). The egg output was higher in animals younger than four years (589.3 EPG) than in those older than 10 years (533.6 EPG) (p < 0.05). However, no differences were observed according to sex during the study period. Results from this study allowed to note the influence of swampy pastures and of the weather changes in the epidemiology of strongylosis in Miranda donkeys. Moreover, it was possible to establish the annual epidemiological curve of strongyle egg shedding, with June being the month with the highest EPG, December having the lowest EPG and March and September showing intermediate numbers. Overall, a lower biodiversity of gastrointestinal parasites was observed. Cyathostomum sensu lato was the most prevalent genus and Strongylus vulgaris was the most observed large strongyle of the Strongylidae family. Trichostrongylus axei and Parascaris sp. were other nematodes with a minor frequency. The higher prevalence of strongyles at the beginning of the study showed a progressive decrease throughout the research period, and also for parasite biodiversity. Therefore, a targeted selective treatment seems to be a rational anthelminthic control approach in Miranda donkey strongyle infection and in other gastrointestinal parasites, since it reduces the antiparasitic treatments, the parasite' prevalence and the EPG level. However, a loss of parasite biodiversity was noted at the end of the study period, as Cyathostominae were the only isolated strongyles. This can be a challenging situation in the long run, taking the ability of these nematodes to adapt easily to any deworming program, meaning that fecal EPG monitoring should be kept as a rule to a rational parasite control program.

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.

Implementation of Biological Control to the Integrated Control of Strongyle Infection among Wild Captive Equids in a Zoological Park.

The integrated control of strongyles was assayed for a period of three years in wild equids (zebras, European donkeys, and African wild asses) captive in a zoo and infected by strongyles. During three years control of parasites consisted of deworming with ivermectin + praziquantel; equids also received every two days commercial nutritional pellets containing a blend of 104 - 105 spores of the fungi Mucor circinelloides + Duddingtonia flagrans per kg meal. Coprological analyses were done monthly to establish the counts of eggs of strongyles per gram of feces (EPG). The reductions in the fecal egg counts (FECR) and in the positive horses (PHR) were calculated fifteen days after deworming; the egg reappearance period (ERP) and the time elapsed from the previous deworming (TPD) were also recorded. Four anthelmintic treatments were administered during the assay, three times throughout the first 2 yrs, and another treatment during the last one. FECR values of 96-100% and 75-100% for the PHR were recorded. The ERP oscillated between eight and twenty-eight weeks, and the TPD ranged from four to eighteen months, increasing to the end of the trial. No side effects were observed in any of the equids. It is concluded that integrated control of strongyles among equids captive in a zoo can be developed by anthelmintic deworming together with the administration of pellets manufactured with spores of parasiticide fungi every two days.

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.

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.