Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping.

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.

Direct microscopy of bacillus endospore germination in soil microcosms.

Antagonistic endospore-forming Bacillus spp. offer a large potential as seed inoculants for control of soil-borne pathogens. In the soil, however, inoculated Bacillus endospores may remain dormant without germination, and plant protection can therefore be inefficient and unpredictable. A method based on direct fluorescence microscopy in soil microcosms was used to determine whether low-cost organic additives incorporated into seed coating material could stimulate endospore germination. Complex organic additives supported a high level of endospore germination of the fungal antagonist Paenibacillus polymyxa CM5-5. Skim milk is a low-cost additive that may be incorporated into seed coating material for efficient induction of Bacillus endospore germination in soil.

Absence of obvious short-term impact of the nematode-trapping fungus Duddingtonia flagrans on survival and growth of the earthworm Aporrectodea longa.

The nematode-trapping fungus Duddingtonia flagrans may be used in biological control of parasitic nematode larvae in faeces of domestic host animals after feeding the hosts with fungal chlamydospores. In this experiment a possible undesirable fungal impact on earthworms, of the species Aporrectodea longa, was investigated. As earthworms eat animal faeces, D. flagrans may come into contact with earthworms both in their alimentary tract and on their body surface. However during the experimental period of 20 days, when earthworms were living in soil and eating cattle faeces that were heavily infested with viable chlamydospores of D. flagrans there were no indications of internal or external mycosis among the earthworms.

The efficacy of two isolates of the nematode-trapping fungus Duddingtonia flagrans against Dictyocaulus viviparus larvae in faeces.

A series of experiments was carried out to examine the effects of two different isolates of the nematode-trapping fungus Duddingtonia flagrans to reduce the number of free-living larvae of the bovine lungworm, Dictyocaulus viviparus. A laboratory dose-titration assay showed that isolates CI3 and Troll A of D. flagrans significantly reduced (P < 0.05 to P < 0.001) the number of infective D. viviparus larvae in cultures at dose-levels of 6250 and 12,500 chlamydospores/g of faeces. The larval reduction capacity was significantly higher for Troll A compared to CI3 when lungworm larvae were mixed in faecal cultures with eggs of Cooperia oncophora or Ostertagia ostertagi and treated with 6250 chlamydospores/g of faeces. Both fungal isolates showed a stronger effect on gastrointestinal larvae than on lungworm larvae. Two plot trials conducted in 1996 and 1997 involved deposition of artificial faecal pats containing free-living stages of D. viviparus and C. oncophora on grass plots. Herbage around the pats was collected at regular intervals and infective larvae recovered, counted and identified. These experiments showed that both D. flagrans isolates reduced the number of gastrointestinal as well as lungworm larvae in faecal pats. During both plot trials, the transmission of C. oncophora larvae, but not D. viviparus, from faecal pats to the surrounding herbage was clearly affected by climatic conditions. After collection of faecal pats from the grass plots one month after deposition, the wet and dry weight of pats as well as organic matter content were determined. No differences were found between the fungus-treated and non-treated control pats. This indicated that the rate of degradation of faeces was not affected by the addition of the fungus.

Effect of Duddingtonia flagrans against Ostertagia ostertagi in cattle grazing at different stocking rates.

The efficacy of an isolate of the nematophagous fungus Duddingtonia flagrans against gastrointestinal nematodes of cattle was examined at 2 dose levels on 2 permanent pastures, with high and low stocking rates, respectively. Thirty calves, experimentally infected with Ostertagia ostertagi, were divided into 3 comparable groups and allocated to 3 similar paddocks in each of the 2 trials. Two of the 3 groups received fungal material once per day during the initial 2 months, either at high dose (10(6) fungal spores/kg body weight) or low dose (5 x 10(5) or 2.5 x 10(5) fungal spores/kg body weight). The third group remained as an untreated control group. Faecal, blood, and herbage samples were collected and animals were weighed every month from May to September. The pasture grazed at a high stocking rate had a large number of overwintering infective larvae, while the pasture grazed at a low stocking rate had a low overwintering herbage larval infectivity. The results showed that, at a high stocking rate, the recovery of infective larvae on pasture was diminished and calves were prevented from clinical ostertagiosis by using the D. flagrans Troll A-isolate. At low stocking rate, the parasite burden seemed not to be very heavy, and a conclusive effect of the fungi at the dose-level used could not be detected.

Growth rate and trapping efficacy of nematode-trapping fungi under constant and fluctuating temperatures.

The effect of temperature on radial growth and predatory activity of different isolates of nematode-trapping fungi was assessed. Four isolates of Duddingtonia flagrans and one isolate of Arthrobotrys oligospora were inoculated on petri dishes containing either cornmeal agar (CMA) or faecal agar and then incubated for 14 days under three different constant and fluctuating temperature regimes. The radial growth was similar on the two substrates at each temperature regime. All fungal isolates showed a higher growth rate at a constant 20 degrees C. At 10 degrees and 15 degrees C, all D. flagrans isolates showed very similar patterns of radial growth at both constant and fluctuating temperatures. At 20 degrees C, they grew significantly faster at constant than at fluctuating temperatures. A. oligospora grew significantly faster than all D. flagrans isolates except when incubated at a fluctuating 20 degrees C. Spores of each fungal isolate were added to faecal cultures containing eggs of Cooperia oncophora at a concentration of 6250 spores/g faeces. The cultures were incubated for 14 days at the same temperature regimes described above. Control faeces (without fungal material) were also cultured. More larvae were recovered from the fungus-treated cultures incubated at a constant 10 degrees or 15 degrees C than from those incubated at the respective fluctuating temperatures, except for one D. flagrans isolate. Incubation at 20 degrees C showed the opposite effect. The general reduction observed in the number of nematode larvae due to fungal trapping was 18-25% and 48-80% for a constant and fluctuating 10 degrees C, 70-96% and 93-95% for a constant and fluctuating 15 degrees C, and 63-98% and 0-25% for a constant and fluctuating 20 degrees C, respectively.

Dung-derived biological agents associated with reduced numbers of infective larvae of equine strongyles in faecal cultures.

Two sets of dung-derived organisms from soil routinely fertilized with manure (MA) and soil chemically fertilized (CH) were cultured separately in the laboratory. Baermannized organisms from these cultures were added to 20 g of faeces from strongyle-infected horses to form three treatment groups: (i) no soil organisms; (ii) low inoculum of soil organisms containing all organisms present in a suspension of approximately 100 adult female free-living nematodes; and (iii) high inoculum containing those soil organisms present with approximately 1000 adult female free-living nematodes. Three studies were conducted using MA cultures and faeces containing 50 stronglye epg, CH cultures and faeces containing 1500 strongyle epg, and a mixture of soil organisms from the two cultures (MC) and faeces containing 600 strongyle epg. Within each study, five control cultures and 15 each of low and high inoculum cultures were prepared and incubated at 24 degrees C and 95% humidity in a climate chamber for 15 days. Parasitic and free-living nematodes were then recovered by the Baermann technique and counted. The numbers of third stage larvae were significantly lower in the high inoculum group compared to controls. The percent reductions in the number of third stage larvae for the low and high inoculum groups were 63.6% and 90.9%, 85.1% and 97.1%, 84.5% and 98.4% for MA, CH, and MC studies, respectively, indicating that mortality increased with the number of soil organisms added to cultures. Examination of the source cultures detected the presence of two species of nematophagous fungi and three genera of free-living nematodes reported to be predacious.

Nematode-trapping fungi in biological control of Dictyocaulus viviparus.

Larvae of the cattle lungworm Dictyocaulus viviparus were cultured in experimental units of 200 g cattle faeces placed in semi-transparent trays in the laboratory. In each of 4 experimental series using this experimental unit, chlamydospores (chl) of the nematode-trapping fungus Duddingtonia flagrans were admixed to half of the faecal cultures in a concentration of 50.000 chl/g. In all 4 series there was a significant reduction in the development and subsequent release of infective lungworm larvae from faecal cultures containing chlamydospores. The average reduction in larval release, caused by fungal spores, was 86%.

Biological control of gastro-intestinal nematodes--facts, future, or fiction?

The potential of using fungi to prevent nematodosis caused by parasites with free-living larval stages is well documented today. In this respect Duddingtonia flagrans, a net-trapping, nematode-destroying fungus, appears to be the most promising candidate. Laboratory experiments and in-vivo studies, where fungal spores have survived passage through the gastro-intestinal tract of cattle and horses, plus field studies with cattle, horses and pigs, demonstrate significant reduction in the number of infective larvae that develop in the faecal environment. In field trials this reduction subsequently leads to reduced infectivity of herbage and also reduced worm burdens in grazing animals. A status of the present situation, primarily based upon work performed in Denmark within the last 6-8 years, plus an outlook for practical implementation of an integrated control strategy including the use of nematode-destroying fungi in the future is discussed.

Effect of the nematode-trapping fungus Duddingtonia flagrans on the free-living stages of horse parasitic nematodes: a pilot study.

A plot experiment was conducted to investigate the ability of the nematode-trapping fungus Duddingtonia flagrans to reduce the transmission of infective horse strongyle larvae from deposited dung onto surrounding herbage. At three different times during the summer 1995, three groups of horses, naturally infected with large and small strongyles, were fed different doses of D. flagrans spores, while a fourth group of animals served as non-fungal controls. Faeces from all four groups of horses were deposited as artificial dung pats on a parasite-free pasture. Every second week for 8 weeks after dung deposition, a subsample of the herbage surrounding each dung pat was collected and the number of larvae on the grass determined. Also, the larval reduction capacity of the fungus was evaluated by faecal cultures set up from all groups of horses. The faecal cultures showed that a sufficient number of spores of D. flagrans survived passage through the horses alimentary tract to significantly reduce the number of developing larvae. A lower reduction of larval numbers was observed when a different batch of fungal material was used at the beginning of the season. Dry climatic conditions affected the transmission of infective larvae in all groups, resulting in low numbers of larvae on the herbage. During the rainy periods a significant reduction in the number of larvae recovered was observed around all fungal containing pats. There were no significant differences between the number of fungal spores and the level of reduction caused by the fungus.

Induction of traps by Ostertagia ostertagi larvae, chlamydospore production and growth rate in the nematode-trapping fungus Duddingtonia flagrans.

Biological control of parasitic nematodes of domestic animals can be achieved by feeding host animals chlamydospores of the nematode-trapping fungus Duddingtonia flagrans. In the host faeces, D. flagrans develop traps that may catch nematode larvae. In experiments on agar, D. flagrans had a growth rate between 15 and 60 mm/week at temperatures between 20 and 30 degrees C. The presence of nematodes induces the fungus to produce traps. The rate of trap formation in D. flagrans has an optimum at 30 degrees C, producing 700-800 traps/cm2/2 days, when induced by 20 nematodes/cm2 on agar. Approaching 10 and 35 degrees C the ability to produce traps is gradually reduced. The response of chlamydospore production on agar to changes in temperature is the same as that for trap formation. On agar, at 10, 20 and 30 degrees C D. flagrans loses its trap inducibility after 2-3 weeks. During the ageing process, increasing numbers of chlamydospores are produced up to a certain limit. The time for reaching maximum chlamydospore concentration coincided with the time for loss of induction potential. The implications of these results in relation to biological control in faeces are discussed.

Biological control. Aspects of biological control--with special reference to arthropods, protozoans and helminths of domesticated animals.

Biological control describes situations in which a living antagonist (a predator, parasite, parasitoid or a pathogen) is distributed by man to lower pest (parasite) populations to acceptable sub-clinical densities or to keep the population at a non-harmful level. Ideally, biological control has no negative effects on the environment, whereas chemical control is not always so harmless. Laboratory and field observations have revealed many organisms, such as viruses, bacteria, fungi, protozoans, turbellarians, nematodes, earthworms, tardigrades, insects, copepods and mites as antagonists to parasitic arthropods, protozoans and helminths of domesticated animals. However, only very few of these antagonists have shown promising qualities as biological control agents within veterinary science. The lack of success should be linked to the lack of knowledge about complex natural biological systems and the antagonists that may be found there. This situation has restricted the interest of industry in developing biological products. In the future, however, industry may become more interested in biological control considering the increasing problems with parasite resistance to drugs in combination with the increasing cost of developing new chemical products, and because of increasing public concern about chemical residues in animal products and in the environment.

The capacity of the fungus Duddingtonia flagrans to prevent strongyle infections in foals on pasture.

A field trial was conducted to evaluate the potential of the nematode-destroying fungus Duddingtonia flagrans to control free-living stages of horse strongyles. In late Spring 2 groups of horses (yearlings) with mixed infections of strongyles were allowed to contaminate 2 equal-sized pastures. One of the groups (F) received a daily dose of D. flagrans mixed in a feed supplement, while the other (C) received a similar amount of supplement without fungus. During a 3-month contamination period strongyle egg counts in faeces and number of infective strongyle larvae harvested from faecal cultures were determined. Grass samples were collected fortnightly. After the contamination period the yearlings were removed and 2 groups of young tracer foals (TF and TC) grazed the fungus and control pastures respectively for 4 weeks, housed for another 15 weeks and then killed to determine their worm burdens. The number of larvae in cultures from group TF was significantly lower than that in TC and herbage infectivity was reduced to a very low level on the pasture grazed by horses fed fungi. The number of Strongylus vulgaris and Strongylus edentatus larvae was also significantly lowered in group TF. Cyathostome larvae recovered from the mucosa of the ventral and dorsal colon and from the caecum were significantly lowered in group TF foals. Also, the number of strongyles found in the gut contents of group TF foals were significantly reduced in the dorsal colon, but numbers of worms in the ventral colon and in the caecum were similar to those of the controls.

Control of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor-reared pigs by daily feeding with the microfungus Duddingtonia flagrans.

An investigation of pasture-reared pigs experimentally infected with Oesophagostomum dentatum and Hyostrongylus rubidus showed that daily doses in the feed with the microfungus Duddingtonia flagrans over a 2-month period led to lowered herbage larval infectivity of both species. This was further substantiated by low worm recoveries in initially parasite-naive tracer pigs that were later introduced to the pasture plot. The control setup comprised the release of similarly infected but nondosed pigs on a plot of the same area, followed by a group of tracer pigs. This paper discusses the potentials for using this biological control principle in the pig industry and emphasizes the research required, primarily regarding production technology and elaboration of feasible epidemiology-based dosing regimens, before such control can be implemented in practice.

Toward practical biological control of parasitic nematodes in domestic animals.

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.

Biological control of trichostrongyles in calves by the fungus Duddingtonia flagrans fed to animals under natural grazing conditions.

The present study was conducted in the 1993 grazing season with yearling calves exposed to a pasture with a natural mixed trichostrongyle larval infection. It was shown that daily feeding with the microfungus Duddingtonia flagrans during the first 2 months of the season led to a lowered herbage infectivity and a reduced acquisition of Ostertagia sp. and Cooperia sp. later in the season. In addition, the procedure delayed the onset of clinical disease. This was due to the nematode-destroying effects of the fungi in the dung excreted by the fungus-treated calves, as evidenced by results from a parallel in vitro assay on faecal larval cultures. The paper discusses future research needs before practical biological control can be implemented.

Predacious activity of the nematode-trapping fungus Duddingtonia flagrans against cyathostome larvae in faeces after passage through the gastrointestinal tract of horses.

This study was undertaken to examine the potential of the nematode-trapping microfungus Duddingtonia flagrans to survive passage through the gastrointestinal tract of horses and subsequently to destroy free-living stages of cyathostomes in faecal cultures. Three different oral dose levels were tested, two horses being used for each level. Faeces were collected twice daily and the numbers of parasite eggs per gram of faeces were determined. The numbers of infective third stage larvae which developed in faecal cultures were determined after the cultures had been incubated for 2 weeks at 24 degrees C. Results showed a positive relationship between dose level and reduction in the number of infective larvae. Fungi were recovered in faeces at times which corresponded to high larval reduction.

Prevention of clinical trichostrongylidosis in calves by strategic feeding with the predacious fungus Duddingtonia flagrans.

The present investigation showed that strategic feeding of first-season calves with the microfungus Duddingtonia flagrans through the initial 3 months of the grazing season could prevent severe clinical trichostrongylidosis in the late summer. The successful prevention of disease was particularly noteworthy in view of the high stocking rate practiced on this permanent pasture, which was widely contaminated with a range of gastrointestinal nematodes. The results showed that larval populations of Ostertagia and Cooperia were significantly reduced on the pasture grazed by the fungus-treated calves. Numbers of Nematodirus seemed less affected. The present paper discusses the complexity of fungus-nematode interactions in dung pats under natural conditions.

An attempt to implement the nematode-trapping fungus Duddingtonia flagrans in biological control of trichostrongyle infections of first year grazing calves.

An attempt was made to control Ostertagia ostertagi by feeding the nematode-trapping fungus Duddingtonia flagrans (DSM 6703) to grazing calves. One group of calves (group E) was fed the fungal material in the first two months of the grazing season while another group was a non-treated control group (group C). Group E showed significantly lower faecal egg count in August and September. On four occasions in July and September, the herbage larval counts were significantly lower on the plot with the fungal-treated group than those recorded on the control plot. The average abomasal larval and adult worm counts were significantly reduced in August in group E and the average total worm count in the abomasum of group E was reduced by 87% in August compared to the non-treated group C. In October, the difference in average abomasal worm counts between group E and C was insignificant. Due to weight loss at the end of the grazing season, the control group showed a significantly lower average weight increase.

Biological control of nematode parasites in cattle with nematode-trapping fungi: a survey of Danish studies.

In Denmark two series of experiments have been performed to study the interactions between larvae of bovine gastrointestinal nematode parasites and nematode-trapping fungi. For practical reasons we were interested in the possibility of depositing nematode-trapping fungi in cattle faeces after passage through the gastrointestinal tract. In the first series, laboratory tests with the fungus Arthrobotrys oligospora showed that motile free-living larvae of a wide range of animal-parasitic nematodes and some soil-living nematodes effectively induce the formation of traps. Larvae of all parasitic nematodes are rapidly captured in these traps. The induction of nets was influenced by temperature, number of larvae, atmosphere, light, and media composition. Captured first- and second-stage larvae were quickly penetrated and killed while third stage larvae were killed slowly, perhaps because they are partially protected by an outer dead sheath. Laboratory and field studies showed that when A. oligospora material was directly mixed into dung a significant reduction in the number of infective parasite larvae in the dung and surrounding herbage occurs. This reduction was also reflected in the acquired worm burden of calves grazing on fungal treated pasture. However, the A. oligospora strain studied in the above mentioned experiments did not survive passage through the alimentary tract of cattle. This prompted us to start a second series of experiments to isolate fungi that could survive gut passage of cattle. Different soil and compost samples were screened by an in vitro stress selection technique. This simulated certain important stress factors which occur during passage through the alimentary tract of ruminants. Rumen exposure was found to be a major limiting factor, but some Arthrobotrys and Duddingtonia strains survived submersion in rumen fluid. In a subsequent in vivo experiment, some of these survivors were fed to calves, and it was hereby demonstrated that isolates of both genera, i.e. Arthrobotrys and Duddingtonia, were able to survive passage through calves and significantly reduce the number of developing preparasitic larvae in dung of fungal treated calves. In a controlled field experiment, isolates of Duddingtonia reduced the level of infective third-stage larvae in herbage by 74-85%.