Combined use of chemical and biological compounds to control hookworm.

The aim of this study was to evaluate the combined use of different chemical (albendazole, ivermectin, glycerine and Vaseline) and biological (Monacrosporium thaumasium) compounds in the control of Ancylostoma caninum. Infective larvae of A. caninum were obtained from coprocultures of positive faeces from naturally infected dogs. We used 1% ivermectin, 1% albendazole, 100% glycerine, 100% Vaseline and an isolate of the nematophagous fungus M. thaumasium (NF34), alone or in combinations. Next, an experimental test was set up with 16 groups in microtubes, with a 24-h interaction. The groups (G1 to G15) that contained any chemical or biological compound (NF34) and/or their combined use (chemical + biological) showed a difference in relation to the control group, except G5 - Vaseline 100% without combinations. It was concluded that, even on an experimental basis, the combined use of anthelmintic drugs with biological control was efficient; however, more studies must be carried out in order to elucidate the synergistic action between chemical and biological compounds to be used in the effective control of hookworms in the future.

An isolate of the nematophagous fungus Monacrosporium thaumasium for the control of cattle trichostrongyles in south-eastern Brazil.

A mycelial formulation in sodium alginate pellets of the nematophagous fungus Monacrosporium thaumasium (isolate NF34A) was assessed in the biological control of beef cattle trichostrongyles in tropical Brazil. Two groups of ten male Nellore calves aged 6 months, a fungus-treated group and a control group, were fed on a pasture of Brachiaria decumbens naturally infected with larvae of cattle trichostrongyles. The fungus-treated group received doses of sodium alginate mycelial pellets orally (1 g pellets (0.2 g fungus)/10 kg live weight) twice a week for 12 months. At the end of the study there was a significant reduction (P< 0.01) in the number of eggs per gram of faeces and coprocultures of the fungus-treated group--47.8% and 50.2%, respectively--in relation to the control group. There was a 47.3% reduction in herbage samples, collected up to 0-20 cm from faecal pats, between the fungus-treated and control groups, and a 58% reduction when the sampling distance was 20-40 cm from faecal pats (P< 0.01). The treatment with sodium alginate pellets containing the nematode-trapping fungus M. thaumasium reduced trichostrongyles in tropical south-eastern Brazil and could be an effective tool for the biological control of this parasitic nematode in beef cattle. However, in such a tropical climate with low rainfall the fungal viability can be reduced.

Parasitism by Paratanaisia bragai (Digenea, Eucotylidae) in commom waxbill (Estrilda astrild) / Parasitismo por Paratanaisia bragai em bico-de-lacre (Estrilda astrild)

O trematodeo digenético Paratanaisia bragai é descrito como parasito dos túbulos coletores renais e ureteres de aves. Esse helminto é usualmente pouco patogênico para a maioria dos hospedeiros; porém, em altas cargas parasitárias e em animais mais suscetíveis, pode causar sérios danos que resultam em apatia, perda de peso, desidratação e até mesmo a morte. O presente trabalho objetivou relatar pela primeira vez o trematoide P. bragai parasitando os rins de um passeriforme da espécie Estrilda astrild, conhecido popularmente como bico-de-lacre, de distribuição cosmopolita. A partir da análise histopatológica dos rins da ave, observou-se a presença dos vermes adultos e de ovos, grande dilatação dos ductos coletores renais, destruição e achatamento das células epiteliais de revestimento e, ao redor dos parasitos, observou-se moderada reação inflamatória. Os autores sugerem que a infecção pode ter sido acidental, uma vez que os moluscos terrestres que servem de hospedeiros intermediários para P. bragai não fazem parte da dieta natural descrita para os bico-de-lacre. Os resultados encontrados no presente estudo confirmaram um bico-de-lacre como possível hospedeiro de P. bragai, acrescentando essa espécie de ave e possivelmente todo o grupo dos passeriformes na epidemiologia da infecção por esse trematoide.(AU)

Pochonia chlamydosporia in the biological control of Fasciola hepatica in cattle in Southeastern Brazil.

Biological control with the use of nematophagous fungi has been described very successfully by many authors and presents itself as a complementary control method, acting on the free-living forms of helminths. The efficacy of a formulation containing the fungus Pochonia chlamydosporia in controlling Fasciola hepatica eggs in faeces was evaluated in an experimental field assay. Two bovine groups (six animals each) were used: A (control) and B (treated with fungus). At 30 days after deworming, the animals were separated into two similar paddocks with flooded areas and were given pellets containing 25 % mycelial mass (group B) or no fungus (group A) at a dose of 1 g/10 kg body weight, twice a week, during 18 months. Faecal samples were harvested fortnightly in the animals of groups A and B and they were submitted at examination of quantitative sedimentation. The mean count of F. hepatica eggs per grams of faeces was significantly higher in group A (1.19) compared with those from group B (0.82) (P < 0.01). After 18 months, animals from group B had gained 42.33 kg above (17.82 % more by weight) (P < 0.01), compared with the control group (A). Every month, faecal samples from paddocks A and B were collected and they were incubated. P. chlamydosporia was identified only in sample source of the paddock B. It can be concluded that the application of this fungical formulation with P. chlamydosporia 25 % mycelial mass was effective in reducing the availability of eggs in the environment and reinfections in calves in natural conditions.

Comparison between the action of nematode predatory fungi Duddingtonia flagrans and Monacrosporium thaumasium in the biological control of bovine gastrointestinal nematodiasis in tropical southeastern Brazil.

Sodium alginate pellets of the nematode predatory fungi Duddingtonia flagrans and Monacrosporium thaumasium were evaluated in the biological control of bovine gastrointestinal nematodiasis. Three groups (A-C) of ten six month old male Nelore bulls were kept in paddocks of Brachiaria decumbens for 12 months. Each animal of group A received 1g/10 kg of body weight (b.w.) of pellets of D. flagrans (0.2 g of fungus/10 kg b.w.) and of group B, 1g/10 kg of b.w. of pellets of M. thaumasium (0.2 g of fungus/10 kg b.w.), twice a week, for 12 months. Animals of the group control received no fungus. The monthly averages of egg count per gram of feces of the animals of groups A and B were 56.67% and 47.8% smaller, than the animals of group C (p<0.05), respectively. Treatment of bulls with pellets containing the nematophagous fungi D. flagrans and M. thaumasium can be used as an alternative treatment of bovine gastrointestinal nematodiasis, however, D. flagrans was more efficient than M. thaumasium for the biological control in the environmental conditions of the present study.

First report of austrodiplostomum compactum and ithyoclinostomum dimorphum in trahira (hoplias malabaricus) from the middle course of the Rio Doce, Minas Gerais, Brazil / Primeiro relato de austrodiplostomum compactum e ithyoclinostomum dimorphum em traíras (hoplias malabaricus) provenientes do médio curso do rio doce, Minas Gerais, Brasil

Austrodiplostomum compactum and Ithyoclinostomum dimorphum are two trematodes commonly found in trahira, but these parasites were never reported in trahiras from Rio Doce. Thus, the aim of this study is to describe the occurrence of A. compactum and I. dimorphum metacercariae in trahira from the middle course of the Rio Doce and to record the presence of eggs in I. dimorphum metacercariae. The parasites were identified and analyzed using methods described previously. There were found 10 A. compactum metacercariae in the aqueous humor of eyes in four of the trahiras and 12 I. dimorphum metacercariae encysted in the peritoneal cavity in five of the trahiras. Maceration of the I. dimorphum metacercariae revealed the presence of eggs. These results demonstrate the broad distribution of these parasites and the first report of these parasites in trahira from Rio Doce.

Austrodiplostomum compactum e Ithyoclinostomum dimorphum são dois trematódeos comumente encontrados em traíras, contudo, esses parasitas nunca foram relatados em traíras do Rio Doce. O objetivo do estudo é descrever a ocorrência de metacercárias de A. compactum e I. dimorphum, e presença de ovos em metacercárias de I. dimorphum em traíras provenientes do médio curso do Rio Doce. Os parasitas foram analisados e identificados utilizando métodos descritos anteriormente. Foram encontradas 10 metacercárias de A. compactum no humor aquoso dos olhos em quatro traíras e 12 metacercárias de I. dimorphum encistadas na cavidade peritoneal de cinco traíras. A maceração de metacercárias de I. dimorphum revelou a presença de ovos nestes parasitos. Esses resultados demonstram a ampla distribuição desses trematódeos e este é o primeiro relato destes parasitos em traíra do Rio Doce.

Biological control of trichostrongyles in beef cattle by the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil.

The efficacy of a fungal formulation based on the nematophagous fungus Duddingtonia flagrans was assessed in the control of cattle trichostrongyles. Twenty male Nellore calves, six-month-old, divided in two groups (fungus-treated and control without fungus) were fed on a pasture of Brachiaria decumbens naturally infected with larvae of bovine trichostrongyles. Animals of the treated group received doses of sodium alginate mycelial pellets orally (1 g/10 kg live weight, twice a week), for 12 months. Feces samples were collected for egg count (eggs per gram of feces-EPG) and coprocultures during 12 months. There was a significant reduction in EPG (56.7%) and infective larvae (L3) in coprocultures (60.5%) for animals of the treated group in relation to the control group at the end of the study. There was a significant reduction of L3 (64.5%) in herbage samples collected up to 0-20 cm from fecal pats and 73.2% in distant samples (20-40 cm) between the fungus-treated group and the control group. The treatment with sodium alginate pellets containing the nematode trapping fungus D. flagrans reduced trichostrongylid in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in beef cattle.

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.

Comparative analysis of destruction of the infective forms of Trichuris trichiura and Haemonchus contortus by nematophagous fungi Pochonia chlamydosporia; Duddingtonia flagrans and Monacrosporium thaumasium by scanning electron microscopy.

The present study aimed to demonstrate by scanning electron microscopy (SEM) the in vitro predatory activity of nematophagous fungi Pochonia chlamydosporia (VC1 and VC4 isolates) Duddingtonia flagrans (AC001 isolate) and Monacrosporium thaumasium (NF34a isolate) on eggs of Trichuristrichiura and infective larvae (L3) of Haemonchus contortus. The work was divided into two experimental tests (A and B). In tests A and B, the predatory activity of nematophagous fungi P. chlamydosporia, D. flagrans and M. thaumasium on eggs of T. trichiura and H. contortus L3 was observed. After 6h, in test A, isolates P. chlamydosporia (VC1 and VC4) had a role in destroying eggs of T.trichiura. For fungi D. flagrans and M. thaumasium the ovicidal activity on T. trichiura eggs was not observed. Test B showed that D. flagrans (AC001) and M. thaumasium (NF34a) were capable of predating H. contortus L3, but no predation by the fungus P. chlamydosporia was seen. These fungi can offer potential for the biological control of nematodes.

Ovicidal activity of different concentrations of Pochonia chlamydosporia chlamydospores on Taenia taeniaeformis eggs.

Three concentrations of chlamydospores of the nematophagous fungus Pochonia chlamydosporia (1000, 10,000 and 20,000 per Petri dish) were evaluated in vitro on Taenia taeniaeformis eggs. Chlamydospores at each concentration were cultured in two different media: 2% water-agar (2%WA) and 2% corn-meal-agar (2%CMA). Taenia taeniaeformis eggs were plated in each chlamydospore concentration in 2%WA and 2%CMA (treated groups) and without fungus (control group). Eggs were removed from each Petri dish at intervals of 7, 14 and 21 days and classified according to ovicidal activity (type 1, type 2 and type 3 effects). Plates containing 2%CMA showed the highest percentages for type 3 effect (81.3%) on the 21st day of observation. A difference (P < 0.01) between the media 2%WA and 2%CMA for type 1 effect was observed only at a concentration of 1000 chlamydospores on the 7th day. There were differences (P < 0.01) between 2%WA and 2%CMA on the 14th and 21st days, at the concentration of 20,000 chlamydospores, for type 1 and type 3 effects. Regression curves for type 3 effect in 2%WA and 2%CMA at the tested concentrations showed higher ovicidal activity with increasing chlamydospore concentrations. Results indicate that, at concentrations of 1000, 10,000 and 20,000 per Petri dish, chlamydospores of P. chlamydosporia effectively destroyed T. taeniaeformis eggs and can be considered a potential biological control agent for this cestode.

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.

Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins.

Protease production from Duddingtonia flagrans (isolate AC001) was optimized and the larvicidal activity of the enzymatic extract was evaluated on infective horse cyathostomin larvae (L3). Duddingtonia flagrans was grown in liquid medium with eight different variables: glucose, casein, bibasic potassium phosphate (K2HPO4), magnesium sulphate (MgSO4), zinc sulphate (ZnSO4), ferrous sulphate (FeSO4), copper sulphate (CuSO4) and temperature. The Plackett-Burman analysis showed a significant influence of MgSO4, CuSO4 and casein (P < 0.05) on protease production by D. flagrans in liquid medium. Central composite design indicated that the highest proteolytic activity was 39.56 U/ml as a function of the concentrations of casein (18.409 g/l), MgSO4 (0.10 g/l) and CuSO4 (0.50 mg/l). A significant difference (P < 0.01) was found for the larval number between the treated and control groups at the end of the experiment. A reduction of 95.46% in the number of free-living larvae was found in the treated group compared with the control. The results of this study suggest that protease production by D. flagrans (AC001) in liquid medium was optimized by MgSO4, CuSO4 and casein, showing that the optimized enzymatic extract exerted larvicidal activity on cyathostomins and therefore may contribute to large-scale industrial production.

In vitro ovicidal activity of the nematophagous fungi Duddingtonia flagrans, Monacrosporium thaumasium and Pochonia chlamydosporia on Trichuris vulpis eggs.

The in vitro effect of four isolates of the nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34a) and Pochonia chlamydosporia (VC1 and VC4) on the eggs of Trichuris vulpis was evaluated. One thousand eggs of T. vulpis were plated on Petri dishes with 2% water-agar with the fungal isolates grown and without fungus as control. After 7, 14 and 21 days 100 eggs were removed from each plate 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 demonstrated ovicidal activity (p<0.05) on the eggs of T. vulpis in the studied intervals presenting type 3 effect of 29.5% (VC1) and 36.5% (VC4), 59.5% (VC1) and 2.5% (VC4), 94.8% (VC1) and 2.95% (VC4) at 7, 14 and 21 days, respectively. The other fungi showed no type 3 effect. P. chlamydosporia should be a potential biological control agent of T. vulpis eggs.

Ovicidal activity of Pochonia chlamydosporia and Paecilomyces lilacinus on Toxocara canis eggs.

An assessment was made of the ovicidal activity of egg-parasitizing fungi Pochonia chlamydosporia (isolates VC1 and VC4) and Paecilomyces lilacinus on Toxocara canis eggs in vitro. The fungal isolates were inoculated onto Petri dishes with 2% water-agar (2% WA) and stored at 25 degrees C for 10 days in an incubator, in the dark. The control group was comprised of Petri dishes without fungi, containing the 2%WA medium only. Later, 1000 embryonated eggs were placed on the surface of the plates with fungal isolates and also on the control plates, and were then incubated at 25 degrees C for 7, 14 and 21 days. At these intervals, the eggs were retrieved and underwent percentage assessment according to the following parameters: no changes; type 1 effect, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell; type 2 effect, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell; type 3 effect, lytic effect with morphological changes in embryo and eggshell, with hyphal penetration and internal egg colonization. All the fungal isolates showed ovicidal activity (type 3 effect) on T. canis eggs, with 13.8%, 20.5% and 20.3% of ovicidal activity using P. chlamydosporia isolate VC1 after 7, 14 and 21 days, whereas isolate VC4 showed 15.2%, 19.0% and 21.7% of ovicidal activity at the same time intervals. P. lilacinus showed ovicidal activity of 12.3%, 18.8% and 20.0% after 7, 14 and 21 days. P. chlamydosporia and P. lilacinus were effective in vitro on T. canis eggs and can be considered a potential candidate to biological controller of those nematodes.

Duddingtonia flagrans, Monacrosporium thaumasium and Pochonia chlamydosporia as possible biological control agents of Oxyuris equi and Austroxyuris finlaysoni.

The action of four fungal isolates of the species Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34a) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Oxyuris equi and Austroxyuris finlaysoni was evaluated in two assays (A and B). Eggs of O. equi (Test A) and A. finlaysoni (Test B) were plated on Petri dishes with 2% water-agar with grown fungal isolates and control without fungus. After 5, 10 and 15 days, 100 eggs were collected and classified according to the following parameters: type 1 effect, physiological and biochemical effect without morphological damage to the eggshell; type 2 effect, lytic effect with morphological alteration of the eggshell and embryo; and type 3 effect, lytic effect with morphological alteration of the eggshell and embryo, hyphal penetration and internal egg colonization. Pochonia chlamydosporia isolates VC1 and VC4 showed ovicidal activity for type 1, 2 and 3 effects on eggs of O. equi and eggs of A. finlaysoni. In vitro assays A and B showed that P. chlamydosporia had a negative influence on eggs of O. equi and A. finlaysoni and can be considered as a potential biological control agent of nematodes.

Biological control of Ancylostomosis in dogs using the nematode-trapping fungus Monacrosporium thaumasium in southeastern Brazil.

Parasitic nematodes Ancylostoma caninum and Ancylostoma braziliense affect dogs and cats and have great medical and veterinary importance for their high prevalence, zoonotic potential, cosmopolitan characteristic and soil contamination by eggs and larvae. In order to evaluate the efficiency of the nematophagous fungus Monacrosporium thaumasium (isolate NF34a) in the biological control of dog hookworm, 12 adult animals, average weight between 7 and 19 kg, were separated into groups and kept in 2 different kennels: control group (without fungus) and a group treated with 0.5 g of fungal mycelium per kilogram of body weight. The animals were treated and feces samples were collected for egg count (eggs per gram of feces-EPG) and coprocultures during six months, twice a week. Every 15 days soil samples were collected from each group and examined for infective larvae (L(3)) in the period between March and September 2008. From April onwards, EPG and coproculture recordings in the treated group were lower than the control group (p<0.05). Linear regression coefficients for the control group were -30.79 and -160.79 for coproculture and EPG means, respectively. The linear regression coefficients for the treated group were -5.64 and -67.64 for EPG and coproculture means, respectively. Larvae were detected in the soil throughout the experimental period. From June to the end of the experiment (September), means of L(3) recovered from the kennel soil of the control group were higher than the means of the kennel soil of the treated group (p>0.05). The regression coefficient was higher for the treated group (-5.36) than the control group (-1.14), confirming the action of M. thaumasium against larvae in the soil. M. thaumasium can be therefore considered as an alternative environmental control of Ancylostoma spp. in dogs.

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.