The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like Arthrobotrys oligospora or Duddingtonia flagrans produce adhesive trapping networks to catch and immobilize nematodes. After penetration of the cuticle, hyphae grow and develop inside the worm and secrete large amounts of hydrolytic enzymes for digestion. In many microbial pathogenic interactions small-secreted proteins (SSPs) are used to manipulate the host. The genome of D. flagrans encodes more than 100 of such putative SSPs one of which is the cysteine-rich protein CyrA. We have chosen this gene for further analysis because it is only found in NTF and appeared to be upregulated during the interaction. We show that the cyrA gene was transcriptionally induced in trap cells, and the protein accumulated at the inner rim of the hyphal ring before Caenorhabditis elegans capture. After worm penetration, the protein appeared at the fungal infection bulb, where it is likely to be secreted with the help of the exocyst complex. A cyrA-deletion strain was less virulent, and the time from worm capture to paralysis was extended. Heterologous expression of CyrA in C. elegans reduced its lifespan. CyrA accumulated in C. elegans in coelomocytes where the protein possibly is inactivated. This is the first example that SSPs may be important in predatory microbial interactions.

Combined use of ivermectin, dimethyl sulfoxide, mineral oil and nematophagous fungi to control Rhabditis spp.

Rhabditis spp., is a nematode known to cause otitis externa, an infection difficult to control, in cattle reared within tropical regions. The objective of this study was to assess the combined use of ivermectin 1%, dimethyl sulfoxide 1% and mineral oil 100% containing nematophagous fungi of both Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) species to control in vitro Rhabditis spp. Thus, 12 experimental groups were designed with eight replicates each: G1 (nematodes + AC001); G2 (nematodes + NF34); G3 (nematodes + ivermectin 1%/positive control); G4 (nematodes + dimethyl sulfoxide 1%/positive control); G5 (nematodes + mineral oil 100%/positive control); G6 (nematodes + AC001 + ivermectin 1%); G7 (nematodes + NF34 + ivermectin 1%); G8 (nematodes + AC001 + mineral oil 100%); G9 (nematodes + NF34 + mineral oil 100%); G10 (nematodes + AC001 + dimethyl sulfoxide 1%); G11 (nematode + NF34 + dimethyl sulfoxide 1%); G12 (nematode + distilled water/negative control). The results demonstrated that all experimentally treated groups differed statistically (p < 0.01) from the control group. In the present study, the use of dimethyl sulfoxide 1% and mineral oil 100% in conjunction with conidia fungi portrayed noteworthy outcomes, which represents a future premise for the combined use of nematophagous fungi within these vehicles in both controlling Rhabditis spp.

Effect of temperature, pH, physical and chemical factors on germination rate of the chlamydospores of the nematophagous fungus Duddingtonia flagrans.

This study mainly investigated the effects of environmental factors on the germination/dormancy, sporulation and resistance of Duddingtonia flagrans chlamydospores. Results showed that the germination temperature of chlamydospores was >10°C and ≤35°C. After the chlamydospores were treated at -20, -40 and -80°C for 12-24 h, they still had the ability to germinate. The chlamydospores germinated at pH 3-13 but did not germinate at pH 1-2 and pH 14. The chlamydospores could tolerate ultraviolet rays for 720 min, but visible light irradiation for 24 h significantly reduced their germination rate. The chlamydospores did not germinate under anaerobic conditions. After the chlamydospores were cultured on water agar (WA) containing 5, 10 and 20% NaCl, their germination rate was significantly inhibited. Once NaCl was removed, the chlamydospores almost completely recovered their germination ability. Among the nine kinds of additives used in the study, 0.3% arginine significantly promoted spore germination (P < 0.05) but 1% trehalose and 1% glycerine significantly inhibited spore germination during incubation from 24 h to 48 h (P < 0.05). This work indicated that D. flagrans chlamydospores are highly resistant to environmental variations and so could be used for biocontrol of animal parasites.

Rhabditis spp., in the Espírito Santo, State of Brazil and evaluation of biological control.

The objectives of this study were to describe occurrences of Rhabditis spp. causing parasitic otitis in dairy cattle of Gir breed in the state of Espírito Santo, southeastern Brazil, and to evaluate the biological control of this nematode using the nematophagous fungi Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34). After nematode detection and collection, three groups were formed: two groups that were treated, respectively, with the fungal isolates; and a control group, without fungus. The treatments were as follows: (a) Petri dishes containing the culture medium 2% water agar (WA) + 250 nematodes + AC001; (b) Petri dishes containing 2% WA + 250 nematodes + NF34; and (c) Petri dishes containing only 2% WA + 250 nematodes. After seven days at 27 °C the treatments with fungi were able to capture and destroy the nematodes, with percentages of 82.0% (AC001) and 39.0% (NF34) in relation to the control group. The results demonstrate the occurrence of Rhabditis spp. after animals physical examination and that there was efficacy of the in vitro predatory activity of both fungal isolates. Thus, these results are important because they can assist in future in vivo control of this nematode in cattle.

Rhabditis spp, in the Espírito Santo, State of Brazil and evaluation of biological control / Rhabditis spp, no Espírito Santo, estado do Brasil e avaliação do controle biológico

Abstract The objectives of this study were to describe occurrences of Rhabditis spp. causing parasitic otitis in dairy cattle of Gir breed in the state of Espírito Santo, southeastern Brazil, and to evaluate the biological control of this nematode using the nematophagous fungi Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34). After nematode detection and collection, three groups were formed: two groups that were treated, respectively, with the fungal isolates; and a control group, without fungus. The treatments were as follows: (a) Petri dishes containing the culture medium 2% water agar (WA) + 250 nematodes + AC001; (b) Petri dishes containing 2% WA + 250 nematodes + NF34; and (c) Petri dishes containing only 2% WA + 250 nematodes. After seven days at 27 °C the treatments with fungi were able to capture and destroy the nematodes, with percentages of 82.0% (AC001) and 39.0% (NF34) in relation to the control group. The results demonstrate the occurrence of Rhabditis spp. after animals physical examination and that there was efficacy of the in vitro predatory activity of both fungal isolates. Thus, these results are important because they can assist in future in vivo control of this nematode in cattle.

Resumo Os objetivos neste estudo foram descrever ocorrências do nematódeo Rhabditis spp., causando otite parasitária em bovinos leiteiros da raça Gir no estado do Espírito Santo, sudeste do Brasil, e avaliar o controle biológico desse nematódeo utilizando os fungos nematófagos Duddingtonia flagrans (AC001) e Monacrosporium thaumasium (NF34). Após a detecção e coleta dos nematódeos, três grupos foram formados: dois grupos que foram tratados com os isolados fúngicos, respectivamente; e um grupo controle, sem fungos. Os tratamentos foram os seguintes: (a) placas de Petri contendo o meio de cultura 2% ágar de água (WA) + 250 nematoides + AC001; (b) placas de Petri contendo 2% de WA + 250 nematoides + NF34; e (c) placas de contendo apenas 2% de nematódeos WA + 250. Após sete dias a 27 °C os tratamentos com fungos foram capazes de capturar e destruir os nematódeos, com porcentagens de 82,0% (AC001) e 39,0% (NF34) em relação ao grupo controle. Os resultados demonstram a ocorrência de Rhabditis spp., no Estado do Espírito Santo e a eficácia da atividade predatória in vitro dos isolados fúngicos utilizados. Assim, esses resultados são importantes, pois podem auxiliar no controle alternativo in vivo de Rhabditis spp. em bovinos com otite parasitária.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

The nematophagous fungus Duddingtonia flagrans reduces the gastrointestinal parasitic nematode larvae population in faeces of orally treated calves maintained under tropical conditions-Dose/response assessment.

This research assessed the dose/response to Duddingtonia flagrans chlamydospores (Df-C) administered to calves naturally infected with gastrointestinal nematodes (GINs), and its effect in reducing the infective larvae (L3) population in faeces in a farm in the Mexican tropics. Forty zebu calves, between 6 and 12 months of age, were randomly distributed into four groups of 10 calves. One control and three groups treated with different oral doses of Df-C based on their body weight (BW) were established as follows: group 1 (control); group 2, 0.250 × 106Df-C per kg/BW; group 3, 0.5 × 106Df-C per kg/BW and group 4, 1 × 106Df-C per kg/BW. The fungal doses were administered daily for 10 days. Every group was confined to individual pens, and they received a nutritional regime based on Buffel grass, concentrated supplement and water ad libitum. Every third day, starting one week before treatments, faeces were taken from the rectum of each animal to determine the number of eggs per g of faeces (epg) through the McMaster technique. Four coprocultures of 20 g each from each individual faecal sample were prepared and incubated for 14 days. The efficacy of the treatments was based on the mean of the GIN L3 recovered from coprocultures of the different groups. Data were analysed using a completely randomized design through an ANOVA analysis, followed by a Duncan multiple range test. The efficacy of treatments was expressed as the larval reduction rate. High variation in the epg in the different groups along the experiment was recorded. The reduction in the GIN L3 population was observed from the 4 to 11 day post-treatment in the three assessed doses. Results in group 2 (lowest fungal dose), showed 88.5, 57.6, 55.9 and 30% (58% overall reductions) in the GIN L3 in the faeces of animals 4, 7, 9 and 11 days post-treatment, respectively. In group 3 (medium fungal dose), 95.8, 80.4, 63.4 and 52.7% GIN L3 reductions (73% overall reduction) were recorded, respectively. At the highest Df-C dose used (1 × 106 per kg/BW), the results were 88.9, 78.0, 59.3 and 67.3% (73.5% overall reduction), respectively (p < 0.05). The species of identified nematodes through L3 morphometric and molecular taxonomy were Cooperia spp. and H. contortus. From the three Df-C assessed doses, the medium dose (0.5 × 106Df-C per kg/BW) was sufficient to substantially reduce the GIN L3 in zebu calves maintained under conditions in the Mexican tropics.

Predatory effect of Duddingtonia flagrans on infective larvae of gastro-intestinal parasites under sunny and shaded conditions.

Duddingtonia flagrans is a natural strain of Nematophagous-Fungi isolated around the world. It has demonstrated efficacy and ease of use in laboratory as well as in field conditions. The fungus contributes to the prophylactic control of the worms by reducing the number of L3 on pasture. The aims of this study were to test and analyze the predatory effect of D. flagrans under sunny and shaded conditions on the L3 in the faeces, and to verify the reduction of translation to pasture during summer and winter seasons. Faecal Mass Units (FMUs) were assigned to two treated groups (groups treated with D. flagrans chlamydospores, TG) and two untreated groups (without D. flagrans chlamydospores, UG), in summer and winter, under sunny and shaded conditions. FMUs and herbage samples were taken for parasitological workup. Predatory activity of D. flagrans was evident under both conditions for the summer experiment but was not manifest for the winter experiment. In summer, an interaction between sunny and shaded conditions and predatory activity of D. flagrans was found. Environmental conditions on predatory activity should be considered when designing strategies for the implementation of D. flagrans in grazing systems to smooth the infectivity curve of L3.

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.

Field evaluation of Duddingtonia flagrans IAH 1297 for the reduction of worm burden in grazing animals: Tracer studies in sheep.

The aim of these studies was to determine the reduction in pasture infectivity likely to be achieved by the supplementation of grazing sheep with BioWorma®, a product containing the chlamydospores of the nematophagous fungus Duddingtonia flagrans strain IAH 1297. Four placebo-controlled trials were conducted between 2009 and 2013 in sheep in different climatic regions of New South Wales and Queensland, Australia and across several seasons. The effectiveness of BioWorma was assessed by total worm counts in tracer sheep placed in paddocks grazed by parasitised sheep which were fed a daily supplement with and without BioWorma under group-feeding conditions. Further proof of concept was obtained by assessing the worm burdens and weight gains of the parasitised sheep, as well as the number of anthelmintic ("salvage") treatments required when faecal egg counts exceeded a threshold level. Significant reductions ranging from 57 to 84% (P < 0.05) in worm burdens of the tracer sheep placed in the paddock grazed by BioWorma treated sheep were obtained in all four trials, compared to the Control group. In two of the studies the treatment effect was greater at the end of the trial, indicating that pasture infectivity in the Control paddocks had risen considerably. The main nematodes encountered were Haemonchus spp., Trichostrongylus spp., and Teladorsagia spp. (including multi-resistant strains) and significant reductions were demonstrated for each of these species. Given the results of the four trials it can be concluded that supplementation of pastured sheep with BioWorma was effective in reducing the numbers of parasitic nematode larvae ingested by tracer sheep. It is considered that these levels of reduced pasture larvae would result in productivity increases in grazing sheep and reduce the requirement for intervention with anthelmintic chemicals. Therefore, use of BioWorma will provide an alternative means for control of gastrointestinal nematode (GIN) parasites on pasture.

Duddingtonia flagrans formulated in rice bran in the control of Oesophagostomum spp. intestinal parasite of swine.

Three experimental assays with Duddingtonia flagrans (isolated AC001) were carried out. The growth of the genus Duddingtonia present in formulation of rice bran, its predatory capability on Oesophagostomum spp. infective larvae (L3) in petri dishes (assay 1), its action in faecal cultures with eggs of that parasite (assay 2) and isolate's capability of predation after passing through gastrointestinal tract of swine (assay 3) was evaluated. At assay 3, feces were collected at time intervals of 12, 24, 36, 48, and 60 h after feed animals with the formulation. Assays 1 and 2 showed a statistical difference (p < 0.01) by the F test when comparing the treated group with the control group. At the both assays, was observed in the treated group a reduction percentage of 74.18% and 88.38%, respectively. In assay 3, there was a statistical difference between the treated group and the control group at all collection times (p < 0.01). Regarding the collection periods, there was no statistical difference over time in the treatment group (p > 0.05). The results demonstrate that the fungal isolate AC001 formulated in rice bran can prey on L3 of Oesophagostomum spp., in vitro and after passing through the gastrointestinal tract, without loss of viability. This isolate may be an alternative in the control of Oesophagostomum spp. in swine.

Viability of Strongyloides venezuelensis eggs and larvae in vermiculite containing the fungus Duddingtonia flagrans.

Strongyloidiasis is the most clinically important disease among the infections caused by geohelminths, seeing that this parasite can cause autoinfection. The use of nematophagous fungi like Duddingtonia flagrans, that have predation action on eggs and infecciososas forms of helminths, emerges as an alternative method for environmental control. For this reason, analyzing the viability of larvae and eggs of Strongyloides venezuelensis and the action of Duddingtonia flagrans AC001 in vermiculite, as well as the action of the nematophagous fungi in different growth stages, is important to elaborate and define the best culture conditions that favor the activity of the fungus. Two different growth conditions were applied: both eggs and AC001 fungi were added at the same time to the vermiculite (assay A) and the addition of eggs after the growth of the AC001 fungi in the vermiculite (assay B). To recover the L3 larvae, the Baermann-Moraes method was applied, followed by the counting of L3 dead and alive. At last, it was observed that the vermiculite enriched with organic material is an adequate culture medium not only for the growth of the S. venezuelensis but also for the growth of the D. flagrans fungus, being therefore, a satisfactory culture medium for tests of viability and predatory action of this fungus. It was also observed that the activity of the AC001 fungus is greater when it is growing concomitantly with the eggs, in other words, when it is in the adaptation phase.

Predatory activity of Butlerius nematodes and nematophagous fungi against Haemonchus contortus infective larvae / Atividade predatória de nematóides Butlerius e fungos nematófagos contra larvas infectantes de Haemonchus contortus

Abstract The purpose of this study was to evaluate the predatory activity of the nematode Butlerius spp. and fungal isolates of Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis and Trichoderma esau against H. contortus infective larvae (L3) in grass pots. Forty-eight plastic gardening pots containing 140 g of sterile soil were used. Panicum spp. grass seeds (200 mg) were sown into each pot and individually watered with 10 mL of tap water. Twelve days after seeding, the pots were randomly divided into 6 groups (n=8). Two thousand H. contortus infective larvae (L3) were added to each group. Additionally, the following treatments were established: Group 1 – 2000 Butlerius spp. larvae; group 2 – A. musiformis (1x107 conidia); group 3 – T. esau (1x107 conidia); group 4 – C. rosea (1x107 conidia), group 5 – D. flagrans (1x107conidia) and Group 6 – no biological controller (control group). The larval population of H. contortus exposed to Butlerius spp. was reduced by 61.9%. Population reductions of 90.4, 66.7, 61.9 and 85.7% were recorded in the pots containing A. musiformis, T. esau, C. rosea and D. flagrans, respectively. The results of this study indicate that the predatory nematode Butlerius spp. and the assessed fungi display an important predatory activity can be considered suitable potential biological control agents.

Resumo O objetivo deste estudo foi avaliar a atividade predatória do nematoide Butlerius spp. e isolados fúngicos de Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis e Trichoderma esau contra larvas infectantes (L3) de Haemonchus contortus em vasos plantados com Panicum spp. Foram utilizados quarenta e oito potes plásticos de jardinagem contendo 140 g de solo estéril, 200 mg de sementes de Panicum spp.. Cultivar colonião, foi semeado em cada vaso e, diariamente, molhados com 10 mL de água da torneira. Doze dias após, os vasos foram divididos em 6 grupos (n = 8), e duas mil L3 de H. contortus foram adicionadas a cada vaso. Foram estabelecidos os seguintes tratamentos: Grupo 1 - 2.000 larvas de Butlerius spp.; Grupo 2 - A. musiformis (1x107 conídios); grupo 3 - T. esau (1x107 conídios); grupo 4 - C. rosea (1x107 conídios); grupo 5 - D. flagrans (1x107conidia); e Grupo 6 – somente L3 de H. contortus que serviu como controle negativo. A população de L3 de H. contortus expostas a Butlerius spp. foi reduzida em 61,9%. Redução populacional de 90,4, 66,7, 61,9 e 85,7% foram observadas nos vasos contendo A. musiformis, T. esau, C. rosea e D. flagrans, respectivamente. Os resultados deste estudo indicaram que o nematoide Butlerius spp. e os fungos avaliados exibiram importante atividade predatória e podem ser considerados como agentes de controle biológico.

Predatory activity of Butlerius nematodes and nematophagous fungi against Haemonchus contortus infective larvae.

The purpose of this study was to evaluate the predatory activity of the nematode Butlerius spp. and fungal isolates of Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis and Trichoderma esau against H. contortus infective larvae (L3) in grass pots. Forty-eight plastic gardening pots containing 140 g of sterile soil were used. Panicum spp. grass seeds (200 mg) were sown into each pot and individually watered with 10 mL of tap water. Twelve days after seeding, the pots were randomly divided into 6 groups (n=8). Two thousand H. contortus infective larvae (L3) were added to each group. Additionally, the following treatments were established: Group 1 - 2000 Butlerius spp. larvae; group 2 - A. musiformis (1x107 conidia); group 3 - T. esau (1x107 conidia); group 4 - C. rosea (1x107 conidia), group 5 - D. flagrans (1x107conidia) and Group 6 - no biological controller (control group). The larval population of H. contortus exposed to Butlerius spp. was reduced by 61.9%. Population reductions of 90.4, 66.7, 61.9 and 85.7% were recorded in the pots containing A. musiformis, T. esau, C. rosea and D. flagrans, respectively. The results of this study indicate that the predatory nematode Butlerius spp. and the assessed fungi display an important predatory activity can be considered suitable potential biological control agents.

In vitro and in vivo studies of the native isolates of nematophagous fungi from China against the larvae of trichostrongylides.

To screen potential nematophagous fungi candidates for the biological control of parasitic nematodes in livestock, in vitro and in vivo studies of the native isolates of nematophagous fungi against the larvae of trichostrongylides were conducted. The in vitro predatory activity of 16 native nematophagous fungal isolates on the larvae of trichostrongylides in sheep feces was assessed. In the ten isolates of Duddingtonia flagrans, the reduction percentage for the infective larvae (L3) of Trichostrongylus colubriformis ranged from 57.21 to 99.83%, and that of Haemonchus contortus ranged from 62.12 to 99.88%. The analysis of the same assay on five isolates of Arthrobotrys superba and one isolate of A. cookedickinson (Monacrosporium cystosporum) showed comparable results with those for D. flagrans. To determine the excretion time of fungal isolates in feces after oral administration, D. flagrans (SDH035) were studied in vivo in sheep and rabbits. Results showed that the tested fungal isolates existed in sheep feces from 12 to 72 h after fungal treatment, and the fungal excretion in rabbit feces occurred at 4 h, reached a peak at 10 h, and declined gradually 18 h after oral administration. All the native fungal isolates were assessed after passing through the gastrointestinal tract of sheep. Treatment with isolates of D. flagrans significantly reduced the number of developing larvae in the feces, and the efficacies ranged from 55.15 to 98.82%. One out of the five isolates of A. superba and A. cookedickinson (BS002) survived after passing through the gastrointestinal tract, and the L3 reduction rates were 83.79 and 81.33%, respectively. Results of the present study provide information about the in vitro predatory activity of nematophagous fungi from China on the L3 of trichostrongylides and their ability to pass through the gastrointestinal tract before administering them for biocontrol.

Consumption of nutritional pellets with Duddingtonia flagrans fungal chlamydospores reduces infective nematode larvae of Haemonchus contortus in faeces of Saint Croix lambs.

Two groups of six Haemonchus contortus infected Saint Croix lambs each received different diets for 11 weeks: control group, commercial food, molasses and lucerne hay; and treated group, nutritional pellets (NPs) containing Duddingtonia flagrans at 2 × 106 chlamydospores/kg body weight (BW), sorghum and lucerne hay. Mean BW gain (BWG), body condition score (BCS) and packed cell volume (PCV) and also eggs/g of faeces (EPG) and recovered L3 were compared using a repeated measures across time model. Groups had similar BWG (control 139.7 ± 0.035 g/day and treated 167.7 ± 0.041 g/day), BCS (control 3.6 ± 0.39 and treated 3.4 ± 0.46) and PCV (control 32.5 ± 1.68% and treated 30.0 ± 1.68%). The mean EPG of the control group was 1215 ± 1040 and in the treated group it was 2097.91 ± 2050. No reduction in larval population was observed during weeks 2 and 3. The greatest larval population reduction in the faeces of treated lambs was observed during the first week (70.5%) and from weeks 6 to 11, with a mean value close to 70% (P < 0.05). In general, both experimental groups showed a similar feed conversion. It was concluded that both diets resulted in similar lamb growth, PCV, BCS and H. contortus EPG. However, NP consumption significantly reduced the H. contortus L3 population in lamb faeces.

Coadministration of nematophagous fungi for biological control over gastrointestinal helminths in sheep in the semiarid region of northeastern Brazil.

This study aimed to evaluate coadministration of Duddingtonia flagrans and Monacrosporium thaumasium in a sodium alginate matrix for controlling gastrointestinal helminths in young and adult sheep in the semiarid region of northeastern Brazil. An area of 1ha was divided into two paddocks, in which two experimental groups (fungus and control) were formed, each consisting of six adult females and ten young males. In each group, two subgroups were formed in accordance with the animal category (adult or young). In the fungus group, each animal received 3g of pellets containing 0.6g of fungal mycelium, with 0.3g of D. flagrans and 0.3g of M. thaumasium for each 10 kg of body weight, in their feed twice a week, for six months. In the control group, each animal received 3g of pellets without fungus for each 10 kg of body weight, in their feed twice a week, for six months, serving as a witness group. Reductions in numbers of eggs per gram of feces of 76% among the adult sheep in the fungus group and 83% among the young sheep in the fungus group were observed, in comparison with their respective control subgroups. The groups that received these fungi needed less salvage deworming and presented better packed cell volume percentages, better weight gain and lower levels of L3/kg dry matter in their paddock than the control groups. Thus, it was concluded that coadministration of D. flagrans and M. thaumasium was effective in controlling gastrointestinal helminths of adults and young sheep in the semiarid region of northeastern Brazil.

Efficacy of Clonostachys rosea and Duddingtonia flagrans in Reducing the Haemonchus contortus Infective Larvae.

The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory activity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L3) of H. contortus in microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an aqueous suspension with 618 H. contortus L3 approximately. Group 1 was used as control and only received the infective larvae. Groups 2 and 3 received D. flagrans chlamydospores and C. rosea conidia at doses of 5 × 10(6). Group 4 received the combination of 5 × 10(6) D. flagrans chlamydospores + 5 × 10(6) C. rosea conidia. D. flagrans and C. rosea showed nematicidal effectiveness reducing by 91.5 and 88.9%, respectively, the population of H. contortus L3. However, when used in combination efficiency decreased to 74.5% predation of H. contortus L3. These results demonstrate the need for further studies to determine the existence of additive effects, synergistic or antagonistic, between these species.

Nematocidal activity of extracellular enzymes produced by the nematophagous fungus Duddingtonia flagrans on cyathostomin infective larvae.

Duddingtonia flagrans produces chitinases, however, optimization of the production of these enzymes still needs to be explored, and its nematocidal activity should still be the subject of studies. The objective of the present study was to optimize chitinase production, and evaluate the nematocidal activity of extracellular enzymes produced by the nematophagous fungus D. flagrans on cyathostomin infective larvae. An isolate from D. flagrans (AC001) was used in this study. For the production of enzymes (protease and chitinase), two different culture media were inoculated with AC001 conidia. Both enzymes were purified. The statistical Plackett-Burman factorial design was used to investigate some variables and their effect on the production of chitinases by D. flagrans. After that, the design central composite (CCD) was used in order to determine the optimum levels and investigate the interactions of these variables previously observed. Only two variables (moisture and incubation time), in the evaluated levels, had a significant effect (p<0.05) on chitinase production. The conditions of maximum chitinase activity were calculated, with the following values: incubation time 2 days, and moisture 511%. The protease and chitinase derived from D. flagrans, individually or together (after 24h), led to a significant reduction (p<0.01) in the number of intact cyathostomin L3, when compared to the control, with following reduction percentage values: 19.4% (protease), 15.5% (chitinase), and 20.5% (protease+chitinase). Significant differences were observed (p<0.05) between the group treated with proteases in relation to the group treated with proteases+chitinases. In this study, the assay with the cyathostomins showed that chitinase had a nematocidal effect, suggesting that this enzyme acts on the "fungus versus nematodes" infection process. It is known that nematode eggs are rich in chitin, and in this case, we could think of a greater employability for this chitinase.

Duddingtonia flagrans in the control of gastrointestinal nematodes of horses.

Horses can harbor a large amount of parasites that may cause serious clinical signs even death. The aim of this study was to evaluate the predatory activity of the fungus Duddingtonia flagrans against infective larvae (L3) of gastrointestinal nematodes of horses in fecal culture. The experimental design was completely randomized with three treated groups (G1, G2 and G3) and one control (CG), using eight animals/group. The treated animals received G1: 1.5 × 10(5); G2: 3 × 10(5) and G3: 6 × 10(5) chlamydospores of D. flagrans/kg body weight during 21 days. The fungi preparation was given at every other three-day interval. Faecal samples were collected during 30 days, on the same interval, to perform the fecal egg counts (EPG) and fecal culture for each horse. All groups demonstrated similar results for the EPG (P > 0.05) counts. D. flagrans significantly reduced (P < 0.05) the number of infective larvae after 72 h-interval between treatments. The G2 and G3 promoted higher results (P < 0.05) of L3 reduction compared to the CG. The biological control with the predacious fungi D. flagrans is still a promising free-living parasite regulator alternative to be use in livestock.