J. Grønvold

In vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants.

Laboratory experiments were designed to select nematophagous fungi that were able to survive in vitro conditions simulating passage through the gastro-intestinal tract of cattle. All of the tests were conducted at 39 degrees C. In a primary stress selection step in diluted rumen fluid, 21 isolates were obtained. Each of the primary stress selected isolates was tested in synthetic saliva, rumen fluid simulating the activity in the rumen, rumen fluid followed by pepsin-hydrochloric acid treatment simulating the additional effect of ruminal and abomasal activity, pepsin-hydrochloric acid solution simulating conditions in the abomasum and finally in a trypsin solution as an example of enzyme activity in the gut. The effect of the rumen fluid alone, or rumen fluid followed by pepsin-hydrochloric acid treatment, were responsible for the reduction in surviving fungal isolates. Only six of thirteen isolates belonging to the genus Arthrobotrys survived while seven of eight isolates of the genus Duddingtonia survived. Fourteen isolates were tested for their predatory capacity in a dung pat bioassay. Fungi of the genera Arthrobotrys and Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by approximately 75% and 96% respectively compared to the number of larvae that developed from fungus-free control pats.

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.

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%.

In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes

The experiment was designed to test the survival and performance of stress selected nematophagous fungi after passage through the gastro-intestinal tract of cattle. Ruminating calves were fed daily with a fixed amount of fungal material grown on barley grains. The excreted dung was collected on days four and five after the start of the feeding experiment. Barley grains were washed out of the excreted dung and incoculated on water-agar plates. After incubation for one week, nine of ten fungal isolates were re-isolated from these plates. The predatory capacity of the fungi in the excreted faeces was tested in a dung pat bioassay and a faecal culture system. In the dung pat bioassay, two fungi of the genus Arthrobotrys and six of the genus Duddingtonia reduced the development of Ostertagia ostertagi third stage larvae by 85% (61%-93%), compared to the number of larvae developed from fungus-free control pats. In seven out of these eight isolates, the reduction of larvae in the faecal cultures was 92% (76%-99%).

Interactions between the predacious fungus arthrobotrys oligospora and third-stage larvae of a series of animal-parasitic nematodes

Interactions between the predacious hyphomycete Arthrobotrys oligospora and third-stage larvae of nine animal-parasitic nematodes were tested in vitro. The trap-inducing capabilities of the ruminant trichostrongylus Cooperia oncophora, C. curticei, Haemonchus contortus and Ostertagia ostertagi and of equine cyathostomes were almost comparable to those of free-living soil nematodes, and significantly higher than those of the porcine Oesophagostomum dentatum and Oe. quadrispinulatum and of the murine Nematospiroides dubius. The trap-forming potential of Dictyocaulus viviparus was poor. All animal-parasitic nematodes were rapidly captured when fungal traps had been pre-induced in high numbers. The possible influence of predacious fungi on animal-parasitic nematode populations under natural conditions in the field is discussed.

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 of Ostertagia ostertagi by feeding selected nematode-trapping fungi to calves.

Three nematode-trapping fungi, one Arthrobotrys oligospora and two Duddingtonia flagrans isolates, were fed to Ostertagia ostertagi-infected calves to test their ability to destroy larvae of this parasite in faeces and consequently to reduce the transmission of infective larvae to herbage. The fungi had previously been selected for their capability to pass the alimentary tract of cattle without losing growth and nematode-trapping potentials. Dung was collected from three calves each fed one of the three fungi and placed as 1-kg cow pats on a parasite-free grass plot together with control cow pats from a calf that was not given fungi. The cow pats contained comparable concentrations of parasite eggs. The two D. flagrans isolates were highly effective in that they reduced herbage larval infectivity by 74-85%. In contrast, A. oligospora did not show any effect in the present experiment. Field experiments will demonstrate if D. flagrans represents a potential organism for biological control of bovine gastrointestinal nematodes under practical agricultural management 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.

Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus

Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus. Experiments were performed to evaluate the potential role of A. oligospora in destroying third-stage larvae of Cooperia spp. on agar plates and in cattle faeces. The fungus had a growth rate optimum at 23 degrees C and pH 6. Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C. Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp. was captured within three days of the experiment. The trapping efficiency in faeces was shown to depend upon the inoculation level. At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed. The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.

EFFECT OF THE NEMATODE-TRAPPING FUNGUS DUDDINGTONIA FLAGRANS ON THE FREE-LIVING STAGES OF HORSE PARASITIC NEMATODES : A PLOT 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.