Chlamydospore dormancy and predatory activity of nematophagous fungus Duddingtonia flagrans.

The chlamydospores of Duddingtonia flagrans are an essential survival and reproductive structure and also an effective ingredient for the biocontrol of parasitic nematodes in livestock. In this study, entering and exiting dormancy conditions and predatory activity of the fungal chlamydospores were conducted. During this fungal growth process, the cultivation time is negatively correlated with spore germination rates. After the spores were processed by vacuum drying for 168 h, their germination rate dropped to 0.94%. In contrast, the percentage of living spores remained 54.82%, suggesting that the spores entered structural dormancy in the arid environment. Meanwhile, the efficacies of the spore against Haemonchus contortus larvae were 93.05% (0 h), 92.19% (16 h), 92.77% (96 h), and 86.45% (168 h), respectively. After dormant spores were stored at 4°C, -20°C, and 28°C (RH90 ~ 95%) for 7 days, their germination rate began to increase significantly (p < 0.05). For in vitro predation assay under the condition of 28°C (RH90 ~ 95%), the predation rate was significantly higher on the 7th day after incubation than that on the 3rd day (p < 0.05). During the period when spores were stored at room temperature for 8 months, their germination rate decreased in the first 5 months and then increased slowly to reach a peak in the 7th month. However, the reduction rate of H. contortus L3 in feces captured by spores remained above 71% for the first 7 months. These results will help us increase the end products yield and the quality of biological control of parasitic nematodes in livestock.

Parkia platycephala lectin enhances the antibiotic activity against multi-resistant bacterial strains and inhibits the development of Haemonchus contortus.

Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2 mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31 mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MIC = 50 mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and H. contortus.

In vitro predatory activity of Arthrobotrys oligospora and after passing through gastrointestinal tract of small ruminants on infective larvae of trichostrongylides.

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.

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.

Larvicidal activity of Bacillus circulans against the gastrointestinal nematode Haemonchus contortus in sheep.

Efficient control of gastrointestinal parasites is necessary in sheep breeding. However, the available chemically based anthelmintics are becoming less effective due to the development of parasite resistance. An alternative to this problem is biological control. In the present study, we tested the larvicidal effect of Bacillus circulans by administering a spore suspension (2 × 109 colony forming units/ml) orally to lambs naturally infected with Haemonchus contortus. The number of faecal larvae was quantified daily and a significant reduction (~87%, P< 0.05) of larval development was observed after administration of B. circulans. Using a transformed B. circulans with green fluorescent protein, we were able to detect B. circulans in the faeces at 4 h post-administration and 72 h after cessation of its administration. These results suggest the use of B. circulans as a promising biological alternative for parasite control.

Duddingtonia flagrans chlamydospores in nutritional pellets: effect of storage time and conditions on the trapping ability against Haemonchus contortus larvae.

The study evaluated the effect of storage time and conditions of nutritional pellets (NP) containing Duddingtonia flagrans chlamydospores on its in vitro trapping ability against Haemonchus contortus L3 larvae. The treated batch (200 NP) contained 4 × 106 chlamydospores of the FTH0-8 strain, whereas the control batch (200 NP) was produced without spores. Both NP batches were exposed to four experimental storage conditions: (T1) shelves (indoors); (T2) refrigeration (4°C); (T3) outdoors under a roof; and (T4) 100% outdoors. Each group comprised 48 NP with spores and 48 NP without spores (control). The ability of D. flagrans spores to trap H. contortus L3 larvae was evaluated for 8 weeks for each storage condition. For that purpose, six randomly selected NP with spores were compared to their respective control NP. Each NP was individually crushed. The crushed material (1 g) was placed on the surface of a 2% water agar plate with 200 H. contortus L3 larvae. Plates were sealed and were incubated at room temperature for 8 days. The whole content of every plate was transferred to a Baermann apparatus to recover the remaining larvae. There was a clear larval reduction in the NP with spores, compared to the respective control NP in the four storage conditions (P< 0.05). The mean reductions ( ± SEM) of the storage conditions were 67 ± 4.9 (T2), 77 ± 6.1 (T1), 81.5 ± 3.8 (T4) and 82.1 ± 2.5 (T3). Larval reductions were similar at all times and were not affected by storage conditions or storage time (R 20.05). The long-term shelf-life of the chlamydospores in the NP suggests that this spore dosage technology is a viable option.

Predatory capability of the nematophagous fungus Arthrobotrys robusta preserved in silica gel on infecting larvae of Haemonchus contortus.

Biological control of gastrointestinal nematodiasis in ruminants is an alternative to reduce the number of infective larvae. The fungal isolates predatory activity preservation is a basic requirement for the success of this control type. The aim of this work is to evaluate the predatory capacity of the fungus Arthrobotrys robusta (isolate I-31), preserved on silica gel on infective larvae of Haemonchus contortus under laboratory conditions on 2 % water agar (2 % WA). In this essay, A. robusta storage on silica gel showed successful predatory activity on H. contortus L3 larvae (p < 0.01) compared to the control group. Nematophagous fungi were not observed in the control group during the experiment. There was a significant reduction (p < 0.01) of 73.84 % in the means of H. contortus (L3) recovered from treatment with isolate I-31 compared to the control without fungi. Results indicate that A. robusta (I-31) could survive stored on silica gel for at least 7 years and keep its predatory activity on H. contortus (L3).

Study of a Mexican isolate of Arthrobotrys musiformis (Orbiliales): Predatory behavior and nematocidal activity of liquid culture filtrates against Haemonchus contortus (Trichostrongylidae), protein profile and myco-constituent groups.

A Mexican isolate of the nematophagous fungus Arthrobotrys musiformis was obtained from a soil sample from the Chapultepec ecological reserve zone, in Cuernavaca, Morelos, Mexico. This isolate demonstrated an important predatory activity (74.9%) against the parasitic nematode Haemonchus contortus (L3) and its fungal liquid culture filtrates (LCF) grown in two media showed the following highest nematocidal activities (NA): In Czapek-DoxBroth (CzDoxB) 80.66% and potato-dextrose broth (PDB) 49.84%. Additionally, two major compounds derived from carboxylic acids and two derivates from alkane group were identified by GC-MS. These compounds have been associated to many biological activities. On the other hand, the protein profile analysis by SDS-electrophoresis followed by a zymogram revealed a 10 kDa protein with protease activity. This study provides important information for future experiments focused to explore the potential use of this protein as well as the identified bioactive compounds presents in the LCF as potential candidates against sheep haemonchosis.

Bacillus spp. toxicity against Haemonchus contortus larvae in sheep fecal cultures.

The gastrointestinal nematode Haemonchus contortus is a major productivity constraint in sheep. In this study, the nematicidal effects of Bacillus circulans, Bacillus cereus, Bacillus thuringiensis var. israelensis, Bt. var. osvaldocruzi, Bt. var. morrisoni, and Bt. var. kurstaki were assessed in free-living larval stages of H. contortus. A spore-crystal suspension containing approximately 2×10(8)UFC/mL of each strain was added to sheep feces that were naturally infected with H. contortus eggs, and the presence of larvae was then evaluated. We observed a significant (p>0.05) reduction in larval development when using B. circulans, B. thuringiensis var. israelensis, Bt. var. osvaldocruzi and Bt. var. kurstaki, and these effects were proportional with the amount of bacteria added to the feces. However, no effect was observed when Bt. var. morrisoni or B. cereus was added. These observations suggest that these bacteria might be effective as nematicides and may allow for the development of integrated biological control of zooparasitic nematodes.

A Nematode Crude Extract Acts as an Elicitor of the Nematocidal Activity of Nematophagous Fungi Liquid Culture Filtrates Against Haemonchus contortus (Nematoda: Trichostrongylidae).

AIM: This study was designed to investigate if culturing nematophagous fungi (NF) in the presence of a Haemonchus contortus larva crude extract (HcCE) enhances the nematocidal activity of nematophagous fungi liquid culture filtrates (NFCF). MATERIALS AND METHODS: Four NF Arthrobotrys oligospora, A. musiformis, Duddingtonia flagrans and Clonostachys rosea were cultured in flasks (n = 5) containing Czapek-Dox broth medium (CDB) in the presence or absence of HcCE. NFCF recovered by filtration of each fungus (200 mg/mL) were assessed on H. contortus infective larvae (L3) using 96-well micro-titer plates (n = 4). Additionally, CDB and water were considered negative controls, while Ivermectin acted as a positive control. After 48 h confrontation, ten 10-µL aliquots of each well were deposited on slides and observed under the microscope (40 ×). Dead and alive larvae in the aliquots were quantified, and a mortality rate (MR) was estimated. RESULTS: The MR of the different NFCF was greatly enhanced by the presence of HcCE. The four NF incubated in the absence of HcCE showed low mortality percentages from 8.2 to 25.8%; in contrast, when the assessed NF growth in the presence of HcCE showed a lethal activity ranging from 66.8 to 80.5%. Only C. rosea showed a moderate increase in the presence of the elicitor (42.7%). CONCLUSION: This study shows evidence about the HcCE enhances the production of nematocidal activity in NFCF. Future studies should be performed to elucidate the compounds responsible of the nematocidal activity that could have important implications in the control of sheep haemonchosis.

Activity in vitro of fungal conidia of Duddingtonia flagrans and Monacrosporium thaumasium on Haemonchus contortus infective larvae.

The objective of this work was to evaluate the predatory activity of the fungi Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34a) on Haemonchus contortus infective larvae (L3) in two experimental assays (A and B). In assay A, two treatments and one control were formed and kept for 7 days in Petri dishes with 2% water-agar. Each treatment consisted of 1000 H. contortus L3 and 1000 conidia of only one fungal isolate, and the control group consisted of 1000 L3, without fungus, with 10 repetitions per group. In assay B, 1000 conidia of one of the fungal isolates, AC001 or NF34a, were added to coprocultures made from 20 g of faeces collected from sheep naturally infected with H. contortus. At the end of the experiment, the Baermann method was used to count the non-predated larvae of all Petri dishes from treatment and control groups. In assay A, no difference was observed (P>0.05) between the groups treated with AC001 and NF34a fungi. A difference was observed (P < 0.05) between the treated and control groups. The L3 reduction percentages at the end of the experiment were 87.75 and 85.57%, respectively, for the fungal isolates compared to the control group. In assay B, the reduction percentages for conidia of these isolates were 85.82 and 87.32%, respectively. The results obtained show that D. flagrans (AC001) and M. thaumasium (NF34a) were effective in the in vitro control of sheep H. contortus L3 and could be used in the biological control of this nematode.

Bacterial profiling of Haemonchus contortus gut microbiome infecting Dohne Merino sheep in South Africa.

A metagenomic approach was used to study the gut microbiome of Haemonchus contortus field strains and that of its predilection site, the abomasum of Dohne Merino sheep. The abomasum contents and H. contortus were collected from 10 naturally infected Dohne Merino sheep. The H. contortus specimens were classified and sexually differentiated using morphometric characters and was further confirmed through molecular identification. We investigated differences and similarities between the bacterial composition of the adult male and female H. contortus gut microbiomes, which were both dominated by bacteria from the Escherichia, Shigella, Vibrio and Halomonas genera. Major abundance variations were identified between the shared adult male and female H. contortus microbiomes. The results also revealed that Succiniclasticum, Rikenellaceae RC9 gut group and Candidatus Saccharimonas were the predominant genera in the Dohne Merino abomasum. This study provides insight into the highly diverse bacterial composition of the H. contortus gut microbiome and the Dohne Merino abomasum which needs to be studied further to explore the complex interactions of different gastrointestinal nematode microbiomes with the host.

Digestibility of Duddingtonia flagrans chlamydospores in ruminants: in vitro and in vivo studies.

BACKGROUND: The use of Duddingtonia flagrans as a tool for the biological control of gastrointestinal nematodes (GIN) is a promising alternative to anthelmintics. The chlamydospores of D. flagrans are orally dosed and their thick cell wall gives them the capacity to resist digestion and pass through the gastrointestinal tract (GIT). Chlamydospores reaching the faeces are able to germinate and trap nematode larvae. The efficacy of this control method is based on reducing the numbers of infective larvae leaving the faeces. Techniques have recently been developed for quantifying the numbers of chlamydospores in faeces. As the number of non-digested spores could be relevant in the design and optimization of dosing programmes for the control of GIN infective larvae, the aim of the present study was to estimate the loss of D. flagrans chlamydospores during their passage through the ruminant gastrointestinal tract using in vitro and in vivo techniques. RESULTS: After in vitro rumen digestion, chlamydospore recovery was not different from the quantity originally incubated (undigested spores) (P > 0.05). In vitro rumen+abomasum digestion caused nearly 36% loss of the chlamydospores originally incubated (P < 0.05). Germination of chlamydospores classified as viable was 24.3%. Chlamydospores classified as non-viable did not germinate. Rumen digestion resulted in more spore germination (R1 = 35.7% and R2 = 53.3%) compared to no digestion (time 0 h = 8.7%). Subsequent abomasal digestion reduced germination (R1+A = 25%) or stopped it (R2+A = 0%). In vivo apparent chlamydospore digestibility in sheep showed a loss of 89.7% of the chlamydospores (P < 0.05). CONCLUSIONS: The loss of chlamydospores was evident under in vitro and in vivo conditions. Negligible amounts of spores were lost during the in vitro rumen digestion. However, in vitro rumen+abomasum digestion resulted in a chlamydospore loss of approximately 36%. In vivo passage through the sheep GIT resulted in a total loss of 89.7% of the orally administered spores.

Resistance of different fungal structures of Duddingtonia flagrans to the digestive process and predatory ability on larvae of Haemonchus contortus and Strongyloides papillosus in goat feces.

The dynamics of the passage of conidia, chlamydospores, and mycelia of the fungus Duddingtonia flagrans through the digestive tracts of goats was evaluated. Four groups with five goats each were formed. In the group conidia, each animal received 1 x 10(6) D. flagrans conidia per kilogram of live weight. In the group chlamydospore, each animal received 1 x 10(6) chlamydospores per kilogram of live weight. In the group mycelia, each animal received 1 g of mycelium mass per kilogram of live weight. In the control group, the animals received no fungal structure. Feces were obtained 3 h before and 12, 24, 30, 36, 42, 48, 60, 72, 84, and 96 h after the inoculation. The feces were placed in Petri dishes containing water-agar. The Petri dishes were examined to detect the fungus and trapped nematodes. A second trial evaluated the effect of the fungal structures on the number of gastrointestinal larvae of Haemonchus contortus and Strongyloides papillosus harvested from the fecal cultures of the goats. The feces were obtained from the goats in the 12-24, 24-30, 30-36, 42-48, 60-72, 72-84, and 84-96 intervals after the inoculation. D. flagrans survived the digestive process of the goats and maintained its predatory activity, being observed from 12 to 96 h before inoculation in the animals that received chlamydospores and conidia.

Microbiome analysis as a platform R&D tool for parasitic nematode disease management.

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host's health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite's microbiome for drug development and treatment purposes.

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae.

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 x 10(6), (T3) 2.5 x 10(6) and (T4) 5 x 10(6). Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0-6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (> 10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.

Interaction between the nematophagous fungus Duddingtonia flagrans and infective larvae of Haemonchus contortus (Nematoda: Trichostrongyloidea).

The interaction between Duddingtonia flagrans and infective larvae of Haemonchus contortus was studied in vitro under optical and scanning electron microscopy. Trap formation by the fungus started 9 hours after inoculation and first larvae were found 11 hours after larval inoculation on colonies grown on the surface of dialysis membranes. Scanning electron micrographs were taken 12, 24, 36 and 48 h after larval predation. Details of predation structures and fungus-larvae interaction are described. A mucilaginous substance occurred at the points of adherence of traps to nematode cuticle. Bacteria were also found at some points of interaction between fungus and larval cuticle. Cuticle penetration by fungus hyphae occurred only 48 h after predation.

In Vitro Lethal Activity of the Nematophagous Fungus Clonostachys rosea (Ascomycota: Hypocreales) against Nematodes of Five Different Taxa.

This study was aimed to evaluate the in vitro lethal activity of the nematophagous fungi Clonostachys rosea against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained C. rosea cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: Haemonchus contortus, Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus, and Butlerius sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: H. contortus (L3) 87.7%; C. elegans 94.7%; Rhabditis sp. 71.9%; P. redivivus 92.7%; and Butlerius sp. 100% (p ≤ 0.05). The activity showed by C. rosea against the H. contortus can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.

Isolation, identification and characterization of the nematophagous fungus Arthrobotrys (Monacrosporium) sinense from China.

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 the Arthrobotrys 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 as A. sinense by 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) of Haemonchus contortus were 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 the A. sinense should have a certain potential to be used for capturing the free-living stage of nematodes in sheep.