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This study aimed to evaluate the efficiency of the powdered formulation with the nematophagous fungi Duddingtonia flagrans against gastrointestinal nematodes of sheep under three feeding systems in...

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.

Background/aims The expression of transcription factor Zbtb1 is essential for the maintenance and development of various blood cells in the hematopoietic system. In the current study, we found that the total number of thymocytes in PLZF deficient mice was reduced compared with thymocytes in wild-type mice, and the number of early T-cell progenitors decreased. However, the decrease of thymocytes in PLZF deficient mice was not cell intrinsic. This study adds new information regarding the regulation of the PLZF gene in the development and self-renewal of T cells. Methods The thymus was isolated from newborn mice, and the two lobes of each thymus were physically separated. Each host received a thymus, two lobes, each placed at one end of the kidney, as described in the literature. Tail vein blood was periodically collected from some of the recipients and analyzed for the presence of peripheral blood T cells. Results In PLZF-EGFP reporter mice and neonatal thymus transplantation to the kidney, we found that PLZF was highly expressed in DN1 (Lineage-CD44+CD25-) cells of thymic grafts of Rag2/γc-/- recipient mice. We found that the proportion of PLZF wild-type and mutant-derived cells in the thymocytes of recipient mice after bone marrow transplantation is approximately equal to the competitive bone marrow chimeric mouse model, and all mice contain a normal thymus. Conclusion The development of T cells suggests that the effect of the PLZF gene on T cell differentiation and development is not cell intrinsic. However, in the neonatal mouse thymic transplant model in the Rag2/γc-/- recipient mouse, deletion of the PLZF gene results in a significant decrease in the proportion of DN1 cells from the donor in the thymic graft.

This study aimed to determine the effects of Duddingtonia flagrans contained in sodium alginate pellets on trichostrongylide larvae under different storage conditions and durations. The in vitro pr...

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC50 ) of each tested drug to inhibit the germination of fungal spores. EC50 ranged between 0·7 and 47·2 μg ml-1 for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5-4057·8 μg ml-1 ). EC50 of tested fungicides was 0·6-2·3 μg ml-1 for carbendazim, 55·9-247·4 μg ml-1 for metalaxyl, 24·4-45·2 μg ml-1 for difenoconazole, and 555·9-1438·3 μg ml-1 for pentachloronitrobenzene (PCNB). EC50 of two antifungal drugs was 0·03-3·4 μg ml-1 for amphotericin B and 0·3-10·9 μg ml-1 for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.

We isolated and identified nematophagous fungus Arthrobotrys musiformis from materials derived from cattle and sheep. We also molecularly characterised the native fungal isolates and evaluated the ...

In vitro predatory activity of 157 native isolates of Arthrobotrys oligospora from China on larvae of trichostrongylides (Trichostrongylus colubriformis and Haemonchus contortus) in feces of sheep were assessed. The results showed that 135 of tested isolates of A. oligospora reduced the development of trichostrongylide larvae in feces by 90-99.99%, 11 isolates by 80-89.46% and 11 isolates by 14.58-78.82%. To understand their capacity of passing through gastrointestinal tract of sheep, 50 native isolates of A. oligospora were selected and assessed in sheep. Among these isolates, 16 isolates significantly reduced the number of larvae developing in the feces (P 0.05); their percentage reduction of L3 ranged from 42.87% to 99.51% and the isolates tested were harvested in 5 g sub-samples of from sheep in each treatment group, indicating that these isolates had the capacity of preying larvae of trichostrongylides after the passage through gastrointestinal tract of sheep. The remaining isolates of A. oligospora were not able to survive after passage through gastrointestinal tract of sheep. In the following, the 16 isolates that presented more or less viability after sheep gastrointestinal passage were selected and assessed in goats. The results showed that the 11 isolates out of them could be able to pass through the digestive tract of goats without loss of ability of preying larvae of trichostrongylides in feces and their efficacies ranged from 53.88% to 94.28%, and that the isolates tested were harvested in 5 g sub-samples of feces from goats in each treatment group. In the current study, these isolates which demonstrated outstanding properties in vitro and could survive in the passage through the alimentary tract of sheep and goat should be potential candidates as a possible feed additive.

The objective of this study is to select a native isolate of Arthrobotrys thaumasia (Monacrosporium thaumasium), a nematophagous fungus that shows potential for use in biocontrol programmes for ruminants. First, we looked for native isolates of A. thaumasia and characterised them using light microscopy and molecular markers. Then, we determined the effect of temperature, pH and nutrition on the growth rate and trap formation of a representative isolate. Of the 1532 samples of different types related to sheep and cattle, 11 isolates of A. thaumasia were isolated, and their occurrence frequency in the samples was 0.71% (11/1532). We sequenced the rDNA internal transcribed spacer of isolate NBS005, submitted it to GenBank (ID: KX640093) and then sequenced it using BLAST. The NBS005 could not grow at 37.5°C but could grow from 11°C to 35°C, and it exhibited its optimum growth at 30°C on 1% corn meal agar (CMA). Over 4 days, the fungus did not grow in the pH interval from 1 to 3 or from 13 to 14 but di...

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

A total of 1502 samples, including feces of sheep (793) and cattle (348), pasture soil (118), dung compost (147) and barn soil (96), were examined between October 2012 and August 2014 to discover potential strains of nematophagous fungi for the biological control of livestock-parasitic nematodes. These samples were collected from 87 sites located in 48 counties of 20 provinces (autonomous regions/municipalities) of China. Fungi were identified down to a species level. Four hundred and seventy-seven isolates, which were distributed in 8 genera and 28 taxa, were identified as nematophagous fungi. Nematode-trapping fungi included 17 species and one unidentified species of Arthrobotrys, two of Dactylella, Drechslerella dactyloides, and Duddingtonia flagrans. Five identified species and two unidentified species of endoparasitic fungi were isolated. The predominant species from all regions were Arthrobotrys oligospora, followed by Arthrobotrys musiformis, Arthrobotrys (Monacrosporium) thaumasiun, and Arthrobotrys (Monacrosporium) microscaphoides. Species with adhesive networks were the most frequently isolated. Among the endoparasitic fungi, Podocrella harposporifera (Harposporium anguillulae) was the most common species, followed by Harposporium lilliputanum and Harposporium arcuatum. Based on Shannon diversity index, the diversity levels of nematophagous fungi were relatively higher in samples associated with cattle, barn soil, and subtropical monsoon climate zone. Three species isolated from this study, namely, Duddingtonia flagrans, Arthrobotrys salina (Monacrosporium salinum), and Arthrobotrys oligospora var. sarmatica, are newly recorded in China, and 20 species (including one unidentified species) are newly recorded in sheep and cattle barn soils worldwide.

The nematophagous fungus Duddingtonia flagrans has been investigated as a biological agent for the control of gastrointestinal nematodes infecting domestic animals in other countries. However, D. flagrans has not been detected in China. In this study 1,135 samples were examined from 2012 to 2014; 4 D. flagrans isolates (SDH 035, SDH 091, SFH 089, SFG 170) were obtained from the feces of domestic animals and dung compost. The 4 isolates were then characterized morphologically. The SDH 035 strain was characterized by sequencing the ITS1-5.8S rDNA-ITS2 region. A BLAST search showed that the SDH 035 strain (GenBank KP257593) was 100% identical to Arthrobotrys flagrans (AF106520) and was identified as D. flagrans. The morphological plasticity of the isolated strain and the interaction of this strain with the nematode targets were observed by subjecting the infected trichostrongylide L3 to scanning electron microscopy. At 6 and 8 hr after trichostrongylide L(3) was added, hyphal ramifications were observed and L(3) were captured, respectively. Scanning electron micrographs were obtained at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hr, where 0 is the time when trichostrongylide L(3) were first captured by the fungus. The details of the capture process by the fungus are also described. Chlamydospores were observed in the body of L(3) in the late stage of digestion. A sticky substance and bacteria could be observed in contact areas between predation structures and nematode cuticle.

Nematophagous fungi are considered to have the best potential as biological agents for the control of gastrointestinal nematodes in domestic animals. However, relatively few studies have been conducted with the genus Monacrosporium, especially with strains native to China. In the present study, we isolated and identified nematophagous fungi from fresh sheep feces. A pure fungal strain was molecularly characterized, and its nematophagous activity was evaluated. The morphological plasticity of the isolated strain, as well as its interaction with the nematode targets, was observed by scanning electron microscopy of the infected Trichostrongylus colubriformis L3 and the free-living nematode Caenorhabditis elegans. Three isolated fungal strains from the 30 fresh fecal samples of sheep from Inner Mongolia, China exhibited predatory activity; however, only a single strain was successfully purified (SF 0459). The SF 0459 strain was characterized by morphological analysis of its conidia and sequencing of its ITS1-5.8S rDNA-ITS2 region. This strain was identified to be Monacrosporium salinum (GenBank ID: KP036623). Nematophagous fungus helper bacteria were found at the interaction points between fungi and nematodes. The percentage of live T. colubriformis L3 was reduced by 83.79-88.69% based on the in vitro assay.

With the development of anthelmintic resistance of parastic nematodes, it is necessary to isolate and study nematophagous fungi to screen out the native isolates for their potential in the biocontrol of domestic animal nematodosis. This study aimed to isolate theArthrobotrys sinense(Monacrosporium sinense) of nematophagous fungus, to characterize representative molecular isolates using scanning electron microscope (SEM), and to determine the effect of the temperature and pH values on radial growth of the isolate. Five isolates were isolated from 1532 samples of different types, and their occurrence frequencies were 0.32% of the total samples. They were identified asA. sinenseby means of morphology and the sequence of the 5.8S, 18S, and 28S rDNA, as well as internal transcribed spacers 1 and 2. The isolate NBS003 could grow from 11°C to 35°C and had optimal growth at 30°C. The isolate could grow at pH 4 to 11, and its optimal value was obtained at pH 9. SEM results showed that 6 h after their addition, the second stage larvae (L2) and the third stage infective larvae (L3) ofHaemonchus contortuswere captured. L2 and L3 were penetrated by the fungus at 18 and 24 h post-capture, respectively. L2 and L3 were completely digested at 84 and 90 h post-capture, respectively. The NBS003 of theA. sinenseshould have a certain potential to be used for capturing the free-living stage of nematodes in sheep.

It is important to isolate potential candidates from the local isolates of nematophagous fungi and to investigate interaction between the fungal strains and gastrointestinal nematodes for the biological control of parasitic nematodes in livestock. In the present study, we assessed the in vitro predatory activity and the viability of isolates of Arthrobotrys thaumasia ( Monacrosporium thaumasium) after passage through the gastrointestinal tract of sheep. The predatory process of a representative isolate selected against the larvae of trichostrongylids was prepared with a scanning electron microscope (SEM). In vitro experiments tested the ability of 9 native isolates of A. thaumasia to prey on larvae of feces of sheep infected with natural mixed nematodes ( Haemonchus contortus, Trichostongylus colubriformis, Marshallagia mongolica). These isolates of A. thaumasia decreased infectivity of third stage infective larvae (L3) by 75.54-99.97%; 7 isolates decreased infectivity by more than 90%. In vivo experiments also demonstrated significant reductions of L3 numbers in the feces treated with the 9 isolates after passing through the gastrointestinal tract of sheep, and these decreases ranged from 51.68 to 88.16%. The isolates tested were re-isolated in 5-g sub-samples of feces from sheep in each treatment group, indicating that these isolates had the capacity to prey upon larvae of trichostrongylids after the passage through gastrointestinal tract. SEM shows that at 6 hr after the larvae were added, including the second stage larvae (L2) and L3 of trichostrongylids, the isolate NBS 005 caught them; at 8 hr after being caught L2 was penetrated by the fungus while penetration of L3 occurred at 12 hr; at 78 hr post-capture L2 was completely destroyed by the fungus while complete digestion of L3 occurred at 84 hr.

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.

A total of 1502 samples, including feces of sheep (793) and cattle (348), pasture soil (118), dung compost (147) and barn soil (96), were examined between October 2012 and August 2014 to discover potential strains of nematophagous fungi for the biological control of livestock-parasitic nematodes. These samples were collected from 87 sites located in 48 counties of 20 provinces (autonomous regions/municipalities) of China. Fungi were identified down to a species level. Four hundred and seventy-seven isolates, which were distributed in 8 genera and 28 taxa, were identified as nematophagous fungi. Nematode-trapping fungi included 17 species and one unidentified species of Arthrobotrys, two of Dactylella, Drechslerella dactyloides, and Duddingtonia flagrans. Five identified species and two unidentified species of endoparasitic fungi were isolated. The predominant species from all regions were Arthrobotrys oligospora, followed by Arthrobotrys musiformis, Arthrobotrys (Monacrosporium) thaumasiun, and Arthrobotrys (Monacrosporium) microscaphoides. Species with adhesive networks were the most frequently isolated. Among the endoparasitic fungi, Podocrella harposporifera (Harposporium anguillulae) was the most common species, followed by Harposporium lilliputanum and Harposporium arcuatum. Based on Shannon diversity index, the diversity levels of nematophagous fungi were relatively higher in samples associated with cattle, barn soil, and subtropical monsoon climate zone. Three species isolated from this study, namely, Duddingtonia flagrans, Arthrobotrys salina (Monacrosporium salinum), and Arthrobotrys oligospora var. sarmatica, are newly recorded in China, and 20 species (including one unidentified species) are newly recorded in sheep and cattle barn soils worldwide.

Nematophagous fungi are considered to have the best potential as biological agents for the control of gastrointestinal nematodes in domestic animals. However, relatively few studies have been conducted with the genus Monacrosporium, especially with strains native to China. In the present study, we isolated and identified nematophagous fungi from fresh sheep feces. A pure fungal strain was molecularly characterized, and its nematophagous activity was evaluated. The morphological plasticity of the isolated strain, as well as its interaction with the nematode targets, was observed by scanning electron microscopy of the infected Trichostrongylus colubriformis L3 and the free-living nematode Caenorhabditis elegans. Three isolated fungal strains from the 30 fresh fecal samples of sheep from Inner Mongolia, China exhibited predatory activity; however, only a single strain was successfully purified (SF 0459). The SF 0459 strain was characterized by morphological analysis of its conidia and sequencing of its ITS1-5.8S rDNA-ITS2 region. This strain was identified to be Monacrosporium salinum (GenBank ID: KP036623). Nematophagous fungus helper bacteria were found at the interaction points between fungi and nematodes. The percentage of live T. colubriformis L3 was reduced by 83.79-88.69% based on the in vitro assay.