Alexandre de Oliveira Tavela

Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil.

The viability of a fungal formulation using the nematode-trapping fungus Duddingtonia flagrans was assessed for the biological control of horse cyathostomin. Two groups (fungus-treated and control without fungus treatment), consisting of eight crossbred mares (3-18 years of age) were fed on Cynodon sp. pasture naturally infected with equine cyathostome larvae. Each animal of the treated group received oral doses of sodium alginate mycelial pellets (1g/(10 kg live weight week)), during 6 months. Significant reduction (p<0.01) in the number of eggs per gram of feces and coprocultures was found for animals of the fungus-treated group compared with the control group. There was difference (p<0.01) of 78.5% reduction in herbage samples collected up to (0-20 cm) between the fungus-treated group and the control group, during the experimental period (May-October). Difference of 82.5% (p<0.01) was found between the fungus-treated group and the control group in the sampling distance (20-40 cm) from fecal pats. During the last 3 months of the experimental period (August, September and October), fungus-treated mares had significant weight gain (p<0.01) compared with the control group, an increment of 38 kg. The treatment with sodium alginate pellets containing the nematode-trapping fungus D. flagrans reduced cyathostomin in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in horses.

Observação in vitro da ação dos isolados fúngicos Duddingtonia flagrans, Monacrosporium thaumasium e Verticillium chlamydosporium sobre ovos de Ascaris lumbricoides (Lineu, 1758)

The in vitro action of the nematophagous fungi Duddingtonia flagrans, Monacrosporium thaumasium and Verticillium chlamydosporium on eggs of Ascaris lumbricoides was observed. After 7, 10 and 14 days of interaction, the fungus showing most promise for use in biologically control over Ascaris lumbricoides was Verticillium chlamydosporium (26-30%). The other fungi did not present satisfactory results.

In vitro evaluation of the effect of the nematophagous fungi Duddingtonia flagrans, Monacrosporium sinense, and Pochonia chlamydosporia on Ascaris suum eggs

The in vitro effect of four isolates of the nematophagous fungi Duddingtonia flagrans (AC 001), Monacrosporium sinense (SF 53), and Pochonia chlamydosporia (VC 1 and VC 4) on eggs of Ascaris suum was evaluated. One hundred thousand A. suum eggs were plated on 2% water–agar with the grown isolates and control without fungus. After 7, 14, and 21 days, 100 eggs were removed and classified according to the following parameters: type 1, lytic effect without morphological damage to eggshell; type 2, lytic effect with morphological alteration of embryo and eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, besides hyphal penetration and internal egg colonization. P. chlamydosporia showed ovicidal activity (p < 0.01), mainly of the type 3 effect, on A. suum eggs in the studied intervals of 13.3% (isolate VC 1) and 17.3% (isolate VC 4), 13.9% (VC 1) and 17.7% (VC 4), and 19% (VC 1) and 20% (VC4), respectively, at 7, 14, and 21 days. The other fungi showed no type 3 effect. P. chlamydosporia is a potential biological control agent of A. suum eggs.

Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first-stage larvae.

Angiostrongylus vasorum is a nematode that parasitizes domestic dogs and wild canids. We compared the predatory capacity of isolates from the predatory fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34), Monacrosporium sinense (SF53) and Arthrobotrys robusta (I31) on first-stage larvae (L1) of A. vasorum under laboratory conditions. L1 A. vasorum were plated on 2% water-agar (WA) Petri dishes marked into 4 mm diameter fields with the four grown isolates and a control without fungus. Plates of treated groups contained each 1000 L1 A. vasorum and 1000 conidia of the fungal isolates AC001, NF34, SF53 and I31 on 2% WA. Plates of the control group (without fungus) contained only 1000 L1 A. vasorum on 2% WA. Ten random fields (4 mm diameter) were examined per plate of treated and control groups, every 24 h for 7 days. Nematophagous fungi were not observed in the control group during the experiment. There was no variation in the predatory capacity among the tested fungal isolates (P>0.05) during the 7 days of the experiment. There was a significant reduction (P < 0.05) of 80.3%, 74.5%, 74.2% and 71.8% in the means of A. vasorum L1 recovered from treatments with isolates AC001, NF34, SF53 and I31, respectively, compared to the control without fungi. In this study, the four isolates of predatory fungi were efficient in the in vitro capture and destruction of A. vasorum L1, confirming previous work on the efficiency of nematophagous fungi in the control of nematode parasites of dogs and as a possible alternative method of biological control.

Biological control of cyathostomin (Nematoda: Cyathostominae) with nematophagous fungus Monacrosporium thaumasium in tropical southeastern Brazil

Horses are hosts to a wide variety of helminthes; the most important are the cyathostomin, or small strongyles. The viability of a fungal formulation (pellets) using the nematode-trapping fungus Monacrosporium thaumasium was assessed in biological control of horse cyathostomin. Two groups (fungus-treated and control) consisted of six mares in each group, crossbred (ages of 2.5 and 3.5 years), were placed in pastures of Cynodon sp. naturally infected with horse cyathostomin larvae. In the treated group, each animal received 1g/10 kg body weight (0.2g/10 kg live weight of fungus) of pellets of sodium alginate matrix containing the fungus M. thaumasium orally, twice a week for 6 months. In the control group, animals received (1g/10 kg body weight) of pellets without fungus. The egg count per gram of feces showed difference (p<0.01) in the animals treated with the fungus in relation to the control animals during all months of the experiment. The EPG percentage decrease were 87.5%, 89.7%, 68.3%, 58.7%, 52.5% and 35.2% during June, July, August, September, October and November, respectively. In faecal cultures, there was difference (p<0.05) among animals treated with fungus was found in relation to the control animals during all the experiment month, with percentage reduction of 67.5%, 61.4% and 31.8% in September, October and November, respectively. Difference (p<0.01) was observed in the recovery of infective larvae from pastures that were collected up to 20 cm from the dung pats in pastures in the group treated with the fungus in relation to the control group with a reduction of 60.9% and between 0-20 and 0-40 cm from the faecal pat reduction (p<0.01) was about 56% in the group treated with the fungus M. thaumasium in relation to the control group pasture. There was no difference (p>0.05) between the average weight gains in both animal groups. The treatment of horses with pellets containing the nematophagous fungus M. thaumasium can be effective in controlling cyathostomin in the tropical region of southeastern Brazil.

Predatory activity of nematophagous fungi on infective larvae of Ancylostoma sp.: evaluation in vitro and after passing through the gastrointestinal tract of dogs

The predatory capacity of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34a), M. appendiculatum (CGI), M. sinense (SF53), Arthrobotrys conoides (I-40), A. cladodes (CG719) and A. robusta (I-31) on infective Ancylostoma sp. larvae (L3) was evaluated. Compared with the control without fungi there was a significant reduction (P < 0.05) of 87.02%, 82.74%, 47.93%, 60.49%, 76.89%, 71.33% and 86.02% in the mean number of Ancylostoma sp. (L3) recovered from treatments with the isolates AC001, NF34a, CGI, SF53, I-40, CG719 and I-31, respectively. Isolates AC001, I-31 and NF34a were more effective in capturing L3 during the in vitro assay. Isolates were then in vivo evaluated for the capacity to remain viable after passing through the gastrointestinal tract of dogs, while still maintaining their predatory activity against L3. Fungal isolates survived the passage and showed efficient predation 48 h after fungal administration to the dogs (P < 0.05). After this time, only the isolate NF34a remained effective up to 96 h after administration (P < 0.05). Monacrosporium thaumasium, D. flagrans and A. robusta are potential biological control agents of Ancylostoma sp. in dogs.

Coadministration of sodium alginate pellets containing the fungi Duddingtonia flagrans and Monacrosporium thaumasium on cyathostomin infective larvae after passing through the gastrointestinal tract of horses

The predatory nematophagous fungi have been used as an alternative control of gastrointestinal nematodes of domestic animals in natural and laboratory conditions. However, it is unclear if the association of some of these species could bring some kind of advantage, from a biological standpoint. In this context, this study consisted of two tests in vitro: in assay A, the assessment of the viability of the association of pellets in sodium alginate matrix containing the fungus Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) and its predatory activity on infective larvae (L3) of cyathostomin after passing through the gastrointestinal tract of horses and assay B, assessment of the cyathostomin L3 reduction percentage in coprocultures. Twelve crossbred horses, females, with a mean weight of 356 kg and previously dewormed were divided in three groups with four animals each: group 1, each animal received 50 g of pellets containing mycelial mass of the fungus D. flagrans and 50 g of pellets of the fungus M. thaumasium, associated and in a single oral dose; group 2, 100 g of pellets containing D. flagrans and 100 g of pellets containing M. thaumasium, associated and in a single oral dose; group 3, control. Faecal samples were collected from animals in the treated and control groups at time intervals of 12, 24, 36, 48, 60 and 72 h after the administration of treatments and placed in Petri dishes containing 2% water-agar (assay A) and cups for coprocultures (assay B). Subsequently, 1000 cyathostomin L3 were added to each Petri dish (assay A) and 1000 cyathostomin eggs were added to each coproculture (assay B) of fungi-treated and control groups. At the end of 15 days, there was observed that the two associations of pellets containing the fungi tested showed predatory activity after passing through the gastrointestinal tract of horses (assay A). In assay B, all the intervals studied showed reduction rate in the number of L3 recovered from coprocultures exceeding 80%. However, no difference (p>0.01) was seen in recovery of not predated L3 between the fungi-treated groups in the time intervals studied. The results obtained showed that the associations of pellets (50 or 100 g of each fungal isolate) were viable after passage through the gastrointestinal tract in horses and could be used in natural conditions.

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

Abstract The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 106 conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K2HPO4 (5.0 g/l), MgSO4 (0.10 g/l), ZnSO4 (0.0050 g/l), FeSO4 (0.001 g/l) e CuSO4 (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.

Control of Strongyloides westeri by nematophagous fungi after passage through the gastrointestinal tract of donkeys

Strongyloides westeri is the most prevalent nematode among equines aged up to four months and causes gastrointestinal disorders. The objective of this study was to observe the control of infective S. westeri larvae (L3) by the nematophagous fungi Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34) after passage through the gastrointestinal tract of female donkeys. Twelve dewormed female donkeys that were kept in stables were used. Two treatment groups each comprising four animals received orally 100 g of pellets made of sodium alginate matrix containing a mycelial mass of either D. flagrans (AC001) or M. thaumasium (NF34). The control group consisted of four animals that received pellets without fungus. Feces samples were then collected from the animal groups at different times (after 12, 24, 48 and 72 hours). These feces were placed in Petri dishes containing 2% water-agar medium and 1000 L3 of S. westeri. AC001 and NF34 isolates showed the ability to destroy the L3, after gastrointestinal transit, thus demonstrating their viability and predatory activity.

Pochonia chlamydosporia fungal activity in a solid medium and its crude extract against eggs of Ascaridia galli.

The present study aimed to evaluate the ovicidal activity (type 3 effect) of VC1 and VC4 isolates of Pochonia chlamydosporia in a solid medium and the action of a crude extract of P. chlamydosporia against eggs of Ascaridia galli. To evaluate ovicidal activity in culture medium, 1000 A. galli eggs were plated on Petri dishes containing 2% water-agar with grown fungal isolates (VC1 or VC4) and without fungus (control group) and were examined at 1, 3 and 5 days post-inoculation (assay A). Then, to test the action of crude extracts of P. chlamydosporia (VC1 or VC4), 500 eggs of A. galli were plated on Petri dishes of 4.5 cm diameter with 5 ml of fungal filtrate from each tested isolate. The control group consisted of 500 eggs of A. galli with 10 ml of distilled water on each Petri dish (assay B). Fungal isolates were effective (P < 0.01) at destroying these eggs, showing a type 3 effect at the studied intervals. On the other hand, the crude extract of isolates (VC1 or VC4) reduced the number of A. galli eggs in the treated group compared with the control group by 64.1% and 56.5%, respectively. The results of the present study show that P. chlamydosporia is effective at destroying eggs of A. galli and could therefore be used in the biological control of nematodes.