Wendeo Ferreira da Silveira

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.

In vitro association of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) to control horse cyathostomin (Nematoda: Strongylidae)

Abstract In vitro effects of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) were evaluated against eggs and third-stage infective larvae (L3) of horse cyathostomin (Nematoda: Strongylidae). The following percentage reductions compared with the control group were observed after a 20-day exposure period: AC001, 61.6%; NF34, 66.1%; VC1, 73.2%; group AC001 + VC1, 86.8%; NF34 + VC1, 77.3%; AC001 + NF34, 92.4%. The results showed that the fungal isolates (VC1, AC001 and NF34), acting alone or in conjunction, were efficient in controlling horse cyathostomin under in vitro conditions.

Interaction of the nematophagous fungus Pochonia chlamydosporia and Parascaris equorum eggs in different culture media

Research involving the use of nematophagous fungi in the biological control of parasites of interest to veterinarians has occurred over recent years, with promising results. This article reports the infection of Parascaris equorum eggs by the fungus Pochonia chlamydosporia (isolates VC1 and VC4). Six groups were formed for each isolate, with six different culture media: 2% water–agar (2% WA); agar–chitin (AC); YPSSA (yeast extract, K2HPO4, MgSO4·7H2O, soluble starch); AELA extract (starch + water + agar); 2% corn‐meal–agar (2% CMA); and 2% potato dextrose–agar (2% PDA). A total of 1000 eggs of P. equorum were transferred to each plate containing isolates grown for a period of 7 days (treatment group). Also, 1000 eggs were added to each plate without fungus (controlgroup). The plates were kept in an environmental chamber at 25 °C in the dark for 21 days. After, we analyzed the effects on ovicidal activity: effect 1 (accession shell); effect 2 (penetration hyphae); and effect 3 (destruction of the eggs). No differences were observed in the destruction of eggs between the two isolates. The decreasing effectiveness of the different culture media was: PDA (38.9%); CMA (38.3%); WA (36.7%); YPSSA (36.45%); and AC (32.5%). The highest percentage egg destruction was observed when the strains were grown in culture medium AELA (44.9%); this was the best medium.

First report of interaction of nematophagous fungi on Libyostrongylus douglassii (Nematoda: Trichostrongylidae)

Libyostrongylus douglassii is a gastrointestinal nematode parasite of ostriches that can cause up to 50% mortality in young birds. The objective of this study was to compare the predatory capacity of two isolates of the predatory fungi Duddingtonia flagrans (AC001 and CG722 isolates) and one of Arthrobotrys cladodes (CG719) on infective larvae (L3) of L. douglassii under laboratory conditions, in 2% water-agar medium. The results showed that the fungi tested were effective in preying upon the L3 of L. douglassii (P < 0.05), compared with the control group. However, there was no difference in predatory capacity between the fungi tested (P > 0.05) during the seven days of experimental testing. In comparison with the control, without fungus, there were significant decreases (P < 0.05) of 85.2% (AC001), 81.2% (CG722) and 89.2% (CG719) in the average numbers of L3 of L. douglassii recovered from treatments with the isolates tested. In the present study, the three isolates of the predatory fungi D. flagrans (AC001 and CG722) and A. cladodes (CG719) were efficient at in vitro destruction of the L3 of L. douglassii.

Proteolytic action of the crude extract of Duddingtonia flagrans on cyathostomins (Nematoda: Cyathostominae) in coprocultures.

The objective of this study was to examine the action of the crude extract of Duddingtonia flagrans (isolates AC001 and CG722) on infective larvae (L3) of cyathostomins in coprocultures and to confirm its proteolytic activity by means of a zymogram. The following groups were formed in coprocultures: Group 1: 10 mL of crude extract of D. flagrans (AC001); group 2: 10 mL of crude extract of AC001 with 10 mM of Ca2+; group 3: 10 mL of crude extract of D. flagrans (CG722); group 4: 10 mL of crude extract of CG722 with 10 mM of Ca2+; and group 5: control group (distilled water). The third-stage larvae (L3) were obtained after eight days. The crude extract of D. flagrans was effective in reducing the number of L3, with the following percentage reductions: group 1, 49.5%; group 2, 52.5%; group 3, 36.8%; and group 4, 57.7%; in relation to the control group (p > 0.05). The proteolytic activity of the crude extract was confirmed through the zymogram. The results from this study confirmed that the crude extract of the fungus D. flagrans could be used for controlling cyathostomin L3, and suggested that at least one protease of approximately 38 kDa was present.

Nematophagous fungi combinations reduce free-living stages of sheep gastrointestinal nematodes in the field

Gastrointestinal nematodes (GIN) can reduce or limit sheep production. Currently there is a clear deficiency in the action of drugs for the control of these parasites. Nematophagous fungi are natural enemies of GIN. Fungal combinations have potential for reducing GIN populations. The aim of this study was to evaluate the efficiency combinations of nematophagous fungi in sodium alginate matrix pellets for the biological control agents of gastrointestinal sheep nematode parasites in the field. The nematophagous fungi (0.2mg of fungus per kg of body weight), Arthrobotrys conoides, A. robusta, Duddingtonia flagrans, and Monacrosporium thaumasium were used. The treated groups were administered mycelium combinations in the following combinations: group 1 (D. flagrans+A. robusta); group 2 (M. thaumasium+A. conoides). The control group did not receive any fungal pellets. We used three groups with eight Santa Inês sheep each. Each animal was treated with approximately 1g of pellet per 10kg of live weight. During the experimental period, we evaluated: number of eggs per gram of feces (EPG), infective larvae (L3) per kg of dry matter, larvae recovered from coprocultures, packed cell volume, total plasma protein concentration of sheep, and environmental conditions. Group 2 EPG (M. thaumasium+A. conoides) differed from the control group in September and October. The number of L3/kg of dry matter recovered from animals of groups 1 and 2 at distances of 0-20 and 20-40cm from the fecal pats was lower than the control group. The packed cell volume and total plasma proteins of treated animals were similar to those of the control group. The combination of treatment groups (D. flagrans+A. robusta and M. thaumasium+A. conoides) reduced the number of L3/kg of pasture. Therefore, treatment of nematophagous fungal combinations have the potential to manage free-living stages of GIN in sheep.

Biological control on gastrointestinal nematodes in cattle with association of nematophagous fungi

ABSTRACT This study aimed to evaluate the efficacy of the association of the nematophagous fungi (Duddingtonia flagrans-AC001); (Pochonia chlamydosporia-VC4) and (Arthrobotrys robusta-I31) in a pelletised formulation of a sodium alginate matrix. The viability and activity of pellet germination and fungal activity (after encapsulation) were evaluated using in vitro and in vivo tests. Next, 12 heads of cattle, Dutch mestizo x zebu, with an average age of 12 months were dewormed with an anthelmintic. Next, 20 days after treatment with the anthelmintic, the animals were randomly divided into two groups of six animals each, and placed in two paddocks with 7.0 ha each of Brachiaria decumbens with historical grazing by animals naturally infected by gastrointestinal nematode parasites. At first, each animal was treated with 2 g of pellets per 10 kg of animal, containing the associated fungi (AC001 + VC4 + I31) administered twice a week in conjunction with commercial feed. Each animal in the control group received 2 g of pellets without mycelia added to the feed. The percentage reduction of infective larvae in the in vitro test was 94% (p < .01). In the in vivo test, the treated animals with fungal association had lower egg counts per gram of faeces (p < .01) compared to the control group animals – a reduction of 91.8%. The reduction in the number of infective larvae recovered from the pasture was 27.5% at a distance of 0–20 cm from the stool bulk, and 26.7% at a distance of 20–40 cm. This association (AC001 + VC4 + I31) of nematophagous fungi was effective in nematode control.