ELSEVIER Veterinary Parasitology 72 (1997) 470-492

vet v( Dlff

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

Michael Larsen ab'*, Peter Nansen ", JOrn GrOnvold ab, Jens Wolstrup ~b, Svend Aage Henriksen ~''~

~' Danish Ceno'e for E.werinwntal Parasitolob~,, Department of Veterinary Microbiolol, o,, Royal Veterinaly and Agricultural Universio,, 13 Biilowsvej, DK-1870 Frederiksberg C, Denmark

I'Department of Ecolo~3, and Molecular Biolo©,, Royal Veterina O, and Agricultural Universio', 13 Biilowsvej, DK-1870 Frederiksbel~ C, Denmark

¢National Veterinaly Laboratoyy, 27 Biilowsvej, DK-1790 Copenhagen V, Denmark

Abstract

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. © 1997 Elsevier Science B.V.

Keywords: Biological control; Nematodes; Livestock; Nematode-destroying fungi; Duddingto- nia flagrans

I. Introduct ion

Biological control (BC), involving nematode-des t roy ing fungi, has the potent ia l of becoming an impor tan t par t of a future integrated control strategy against

*Corresponding author: Tel.: +45 35 282794; fax: +45 35 282774; e-mail: mila@kvl.dk

0304-4017/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S0304-4017(97)00112-X

4SO M. L a r ~ o t ~'t al. I ~ ' l( ' / fnarr l>ura~itolo,~,,y 72 (1997) 470 492

parasitic nematodes in grazing livestock. Besides using BC as a supplement or adjunct in the attempt to control widespread anthelmintic resistance within nema- tode populations of livestock, the need for non-chemical alternative measures already exists with regard to control of parasitic nematodes of livestock on organic farms. Board regulations in Denmark exclude preventive use of anthelmintics arid when used for treatment of sick animals, an cxtended withdrawal period is required. Following an increase in the number of organic farms, one might also expect increased problems duc to parasites.

This brief presentation is not intended as an cxtcnsivc review of nematode-de- stroying fungi as potential biological control agents, but rather an outline of the present status and prospects for BC in a Danish context.

2. Biological control

The concept of microfungi as biological control agents against nematodes is not new. Linford et al. in Hawaii m the late 1930s experimented with control of parasitic nematodes oi1 pineapples (see Caswell and Apt, 1989). Inspired by those experiments, a group of scientists at the Pasteur Institute in France, in the late 1930s and early 1940s performed pioneering experiments with nematode-destroying fungi against animal parasitic nematodes both in in-vitro tests on agar media as well as in trials with a small number of animals (see review by Peloille, 1981).

Types of microfungi investigated as potential biological control agents are the so-called 'nematode-destroying' or nematophagous fungi. These microfungi occupy different niches in the soil/rhizosphere where they feed on a variety of free-living soil nematodes either as the main food source (obligate parasitic fungi) or as a supplement, living usually as saprophytes on dead organic matter.

A large proportion of the nematode-destroying fungi known today belong to the so-called 'imperfect' fungi or Deuteromycetes. These fungi, which are represented in all major fungal classes, have been divided into two major groups: Predacious fungi trap nematodes on the growing hyphae either by producing sophisticated traps (sticky knobs, branches, three-dimensional nets, constricting or non-constrict- ing rings) or, for the most simple forms, by producing sticky material directly on hyphae. The other group, the endoparasitic fungi, produce spores which either stick to and penetrate through the cuticle of the nematode or produce small spores of unusual shape, which then lodge in the oesophagus of the feeding nematode. After germinating, the fungi grow and engulf the contents of the nematode. The species of predacious fungus which has received most attention recently is Dud- dingtonia (=Arthrobotlys)flagrans, a fungus forming three-dimensional sticky nets and large numbers of thick-walled restingspores, called chlamydospores. It is likely that this latter feature is the basis for the survival of D. flagrans in its passage through the digestive tract of cattle and sheep.

In a control strategy using nematode-destroying fungi as BC agents against gastro-intestinal parasitic nematodes in grazing livestock, it is the aim to achieve a significant reduction in the larval population on herbage, a reduction which will consequently result in a significant reduction of the worm burden ira animals. The

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number of worms establishing in the animals should be reduced to such a level that not only clinical disease, but also signs of subclinical effects due to the parasites should be prevented. At the same time the reduced number of infective larvae on pasture should be sufficient to stimulate development of natural immunity in young animals.

3. Present status

As a result of renewed interest and intensified research in biological control during the last 10 years, a convincing amount of evidence on the potential for this principle has been gathered. Most of this work has been carried out in Europe (Denmark, UK and France), Australia but recently also in United States and Latin America (Brazil and Mexico). Both in some of the recently published articles as well as in earlier work, there appears to be some discrepancy as to the definition of what constitutes good BC candidates and how these are evaluated. It can be expected that many of the predacious fungi known today would be effective against free-living larvae of animal parasitic nematodes when tested simply on an agar media in petri dishes, or in faecal cultures under laboratory conditions. Unfortu- nately, such in-vitro results do not provide conclusive information regarding the capacity of the fungi to survive and trap/kill larvae in dung after passage through an animal.

With regard to the effect of nematophagous fungi against cattle parasitic nematodes, numerous articles have clearly demonstrated that it is possible both to reduce the number of infective larvae on herbage and subsequently the number of nematodes establishing in the grazing animals (e.g. Gronvold et al., 1993; Wolstrup et al., 1994; Larsen et al., 1995a, Nansen et al., 1995).

Recent results from field trials with horses, pigs and sheep are presented briefly. There have been a number of positive reports on the effect of nematode-trapping fungi against strongyles in horses. In-vitro studies on fungal-cyathostome interac- tions in faecal samples using Arthrobotrys oligospora (Charles et al., 1995) and dose-titration with D. flagrans and ,4. oligospora, in faecal cultures, (Bird and Herd, 1995) have shown that the fungi will develop and be efficient in the dung environment. In an in-vivo dose titration trial (Larsen et al., 1995b), it was shown that D. flagrans could survive gut passage and subsequently kill strongyle larvae in horse faeces. On the basis of that result, a field experiment was conducted to evaluate the potential of D. flagrans against free-living stages of horse strongyles (Larsen et al., 1996). In late spring, two groups of yearlings, with mixed natural infections of both cyathostomes and large strongyles, were turned out to contami- nate two clean pastures. One group of yearlings received D. flagrans spores daily mixed with a feed supplement, while the other group served as a non-treated control group fed a similar amount of supplement only. During the contamination period, faecal strongyle egg counts and numbers of infective strongyle larvae which developed in faecal cultures were determined. To evaluate pasture infectivity, grass samples were collected at 2-week intervals. After the contamination period, the yearlings were removed and two groups of tracer foals were allowed to graze the

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two pastures for 4 weeks, subsequently housed for approximately 3 months and then killed for worm burden determination. The number of larvae developing in faecal cultures from yearlings fed fungal material was significantly lower than that found in cultures from the control animals. Also, the herbage infectivity (number of infective larvae per kg dry grass) was reduced on the pasture grazed by horses fed fungal material. The number of large strongyle larvae (Strongylus vulgaris and S. edentatus) recovered from the tracer animals was significantly lower in foals from the fungus pasture. Cyathostome larvae found in the mucosa of the ventral and dorsal colon and from the caecum were significantly reduced, as well as the number of strongyles found in the gut contents of the dorsal colon.

A design similar to that used in the horse trial was used in an experiment with free-roaming pigs (Nansen et al., 1996). Two groups of pigs, experimentally infected with Oesophagostomum dentatum and Hyostrongylus rubidus, were allowed to con- taminate two pastures of equal size. During a 2-month period one group of pigs received daily doses of D. flagrans in the fodder. The number of infective larvae on the grass was lower on the pasture where pigs were treated with fungal material daily. The positive fungal control of grass infectivity was also reflected in the worm burdens of parasite-naive tracer pigs that were later introduced to the pasture plot. Reductions of 87% and 70% were observed for O. dentatum and H. rubidus, respectively, in the worm burden of tracers pigs from the pasture where pigs previously were fed fungal material on a daily basis compared to the worm burden of tracer pigs on the non-fungal treatment control pasture.

The survival of nematophagous fungi through the gut of housed sheep and subsequent fungal activity in the excreted faeces have been reported by several authors (Griiner et al., 1985; Larsen et al., 1994; Waller et al., 1994). The effect of daily adding the fungus D. flagrans to a feed supplement, given to animals during the first 3 months of a grazing season was tested in a recent field trial with sheep grazing on pastures naturally infected with Nematodirus spp. and Ostertagia spp. The design of this field trial was almost identical to the above-mentioned horse and pig trials. Groups of fungus-treated and non-treated control animals were allowed to graze the pastures for 5 months after which parasite-naive tracer lambs were turned out to graze the same pastures for 3 weeks. The tracers were subsequently housed and sacrificed to determine worm burdens.

The reduced number of larvae recovered from faecal cultures of the fungus- treated group in the initial half of the experiment, demonstrated a high nematode- destroying potential of the fungus. Significantly (P < 0.05) reduced worm burdens (Ostertagia spp. + Trichostrongylus spp.) of the tracer lambs on 'fungus'-pastures subsequently confirmed this. The total average worm burden was reduced by approximately 90% in tracer lambs grazing pasture previously grazed by fungus- treated animals compared with the number of worms from tracers on the pasture of the untreated control animals.

The effect against Nematodirus spp. larvae in the sheep field trial mentioned above and a very high larval reduction potential recorded in a laboratory trial where D. flagrans was tested against Dictyocaulus viviparus (Henriksen et al.,

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1997), emphasized the wide spectrum of parasitic nematode species potentially controlled by this fungus.

4. Future for biological control

Thus, a major question is, how should microfungi be integrated into future control strategies? An integrated management strategy, based upon epidemiologi- cal knowledge of the parasite(s) involved, could be composed of two or more of the following elements: biological control agents, grazing management, anthelmintics (minimal treatment) and perhaps vaccines, if available.

Another important factor to consider is diet which has been shown to influence not only the pharmacokinetics of anthelmintics (Ali and Hennessy, 1993; Hennessy, 1993; Hennessy et al., 1994), but also establishment of nematodes (Petkevi6ius et al., 1995, 1996).

A major question requiring attention now and in the future is how well will BC candidates perform against free-living stages of different nematode species from different host species under climatic conditions which differ from those prevailing where the fungus originated. One aspect of this question concerns the feasibility of using the same species/isolates for a whole range of epidemiological and climatic conditions or will there then be a necessity of isolating indigenous fungi to overcome potential climatic problems, as well as restrictions or objections to importation and distribution of 'foreign' biological material.

Under intensive farming systems or on small-scale farms in certain parts of the world (cattle, pigs, sheep/goats or poultry) it may be possible to use fungi by adding them to the fodder, feedblocks or drinking water. As mentioned earlier, mixing of the fungus into the fodder seems to be feasible, although a specific formulation still needs to be developed. Recent reports indicate that nematode-de- stroying fungi are able to survive integration into a feed-block matrix and can survive and be active over an extended period of time.

Under extensive farming systems with cattle and sheep, clearly a different technology is needed. Here, the BC principle must be adapted to bolus systems/slow release devices (SRD) to be given to the young animals prior to the period(s) when the risk for heavy transmission of infective larvae is high. This area warrants future research.

Another aspect to consider in an integrated control strategy is the potentially negative or inhibiting effects certain anthelmintics might have on nematode-de- stroying fungi. Therefore, timing between and/or possible combinations in use of fungi and anthelmintics will need closer examination.

Having isolated and tested some very promising BC candidates, there are still questions requiring solutions. Considering the complex epidemiological picture due to the different species of nematodes involved in grazing livestock, do we then have the most effective and broad spectrum BC candidate(s)? By studying why spores from only a limited number of species of nematode-destroying fungi have been able to survive passage through the GI tract of ruminants in sufficiently high

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numbers we might get some indicat ions as to where to look for potent ia l ly be t te r candidates . Also, there is a need to get more insight into the ecology of these fungi to be able to measure any potent ia l impact on the env i ronment as well as effects of o ther biotic factors on the activity and survival of the nema tode -des t roy ing fungi when used in in tegra ted control strategies.

In this br ie f p resen ta t ion , only fungi which a t tack the free-l iving larval stages of animal paras i t ic n e m a t o d e s have been addressed , however, o the r organisms, includ- ing fungi, which are able to a t tack and kill the eggs of paras i tes having long lasting egg stages in the env i ronment (e.g. Ascaris, Trichuris, Ascaridia spp.), should be examined for possible use in future BC strategies, a imed specifically at these paras i tes or as par t of a BC p a c k a g e / c o c k t a i l covering a wider range of parasi tes .

In conclusion, research efforts covering aspects of using fungi as biological control agents against paras i t ic nema todes in l ivestock should be intensified. It should be recognized that imp lemen ta t ion of this concept as a pract ical control op t ion cannot be quickly achieved. It is most likely that a m in imum of 2 - 3 years of research would be requ i red before an active p ro to type could be widely tes ted under app l ied condi t ions . As par t of an in tegra ted control p rog ra m for gastro- in- test inal n e m a t o d e s in a wide spec t rum of host animals, nema tode -des t roy ing fungi could pe rhaps be commerc ia l ly avai lable in approx imate ly 3 - 5 years.

References

Ali, D.N., Hennessy, D.R., 1993. The effect of feed intake on the rate of flow of digesta and the disposition and activity of oxfendazole in sheep. Int. J. Parasitol. 23, 477-484.

Bird, J., Herd, R.P., 1995. In vitro assessment of two species of nematophagous fungi (Arthrobotrys oligospora and Arthrobotrys flagrans) to control the development of infective cyathostome larvae from naturally infected horses. Vet. Parasitol. 56, 181 187.

Caswell, E.P., Apt, W.J., 1989. Pineapple nematode research in Hawaii: past, present, and future. J. Nematol. 21, 147-157.

Charles, T.P., Rodrigues, M.L.A., Santos, C.P., 1995. Reducao do nfimero de larvas de Cyathostominae em fezes de eqfiinos tratadas com conidios de Arthrobotrys oligospora. Arq. Bras. Med. Vet. Zootec. 47, 87-89.

Griiner, L., Peloille, M., Sauv6, C., Cortet, J., 1985. Parasitologie animale. - - Survie et conservation de I'activite predatrice vis-a-vis de nematodes trichostrongylides apres ingestion par des Ovins de trois hyphomyctes predateurs. C.R. Acad. Sci. III 300, 525-528.

Gr0nvold, J., Wolstrup, J., Larsen, M., Henriksen, S.A., Nansen, P., 1993. Biological control of Ostertagia ostertagi by feeding selected nematode-trapping fungi to calves. J, Helminthol. 67, 31-36.

Hennessy, D.R., 1993. Pharmacokinetic disposition of benzimidazole drugs in the ruminant gastrointestinal tract. Parasitol. Today 9, 329-333.

Hennessy, D.R., Ali, D.N., Tremain, S., 1994. The partition and fate of oxfendazole in soluble and particulate digesta material in the gastrointestinal tract of sheep. Int. J. Parasitol. 24, 327-333.

Henriksen, Sv.Aa., Larsen, M., Gronvold, J., Nansen, P., Wolstrup, J., 1997. Nematode-trapping fungi in biological control of Dictyocaulus viviparus. Acta. Vet. Scand. 38, 175-179.

Larsen, M., Faedo, M., Waller, P.J., 1994. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: survey for the presence of fungi in fresh faeces of grazing livestock in Australia. Vet. Parasitol. 53, 275 281.

Larsen, M., Nansen, P., Wolstrup, J., Gr0nvold, J., Henriksen, S.A., Zorn, A., 1995a. Biological control of trichostrongyles in calves by the fungus Duddingtonia flagrans ted to animals under natural grazing conditions. Vet. Parasitol. 60, 321-330.

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Larsen, M., Nansen, P., Henriksen, S.A., Wolstrup, J., Gr0nvold, J., Zorn, A., Wed¢, E., 1995b. Predacious activity of the nematode-trapping fungus Duddingtonia flagrans against cyathostome larvae in faeces after passage through the gastrointestinal tract of horses. Vet. Parasitol. 60, 315-320.

Larsen, M., Nansen, P., Grondahl, C., Thamsborg, S.M., Gronvold, J., Wolstrup, J., Heriksen, S.A., Monrad, J., 1996. The capacity of the fungus Duddingtoniaflagrans to prevent strongyle infections in foals on pasture. Parasitology 113, 1-6.

Nansen, P., Larsen, M., Gronvold, J., Wolstrup, J., Zorn, A., Henriksen, S.Aa., 1995. Prevention of clinical trichostrongylidosis in calves by strategic feeding with the predacious fungus Duddingtonia flagrans. Parasitol. Res. 81,371-374.

Nansen, P.. Larsen, M., Roepstorff, A., Grcnvold, J., Wolstrup, J., Henriksen, S.A., 1996. Control of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor-reared pigs through daily feeding with the microfungus Duddingtoniaflagrans. Parasitol. Res. 82, 580-584.

Peloille, M., 1981. Les hyphomyc&es pr6dateurs de n6matodes: ph6nom~ne de pr6dation; 6cologie; utilisation en lutte biologique. Agronomie 1, 331-337.

Petkevi~ius, S,, Bj¢rn, H., Roepstorff, A., Nansen, P., Bach Knudsen, K.E., Barnes, E.H., Jensen, K., 1995. The effect of two types of diet on populations of Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs. Parasitology 111, 395-402.

Petkevi~ius, S., Bach Knudsen, K.E., Nansen, P., Roepstorff, A., Skjcth, F., Jensen, K., 1996. The impact of diets varying in carbohydrates resistant to endogenous enzymes and lignin on populations of Ascaris suum and Oesophagostornum dentatum in pigs. Parasitology (in press).

Waller, P.J., Larsen, M., Faedo, M., Hennessy, D.R., 1994. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies. Vet. Parasitol. 51, 289-299.

Wolstrup, J., Gronvold, J., Henriksen, Sv.Aa., Nansen, P., Larsen, M., Bcgh, H.O., Ilsoe, B., 1994. An attempt to implement the nematode-trapping fungus Duddingtonia flagrans in biological control of free-living stages of trichostrongyles in first year grazing calves. J. Helminthol. 68, 175-180.

Discussion: Biological control of gastrointestinal nematodes - - facts, future or fiction? (Larsen)

(Eysker m The Netherlands) Abou t the Nematodirus results, it is surprising that you saw differences, it will be very interesting to see the follow-up next spring, but when you look at the three different species, they behave a little bit differently according to my experience. I don ' t have experience with N. battus, but we did do some tracer studies in the '70 's which indicated that you will get N. spathiger on pasture in late summer and N. fillicolis follows; N. battus reaches a peak in spring and this seems to be quite specific for this species.

Tape Problem: Larsen response to Eysker lost as well as question to him regarding effect of nematophagus fungi on nematode eggs.

(Larsen - - Denmark) W h e n the eggs were taken directly f rom the female they were invaded by the fungus within 10 to 20 days. It became problematic when we washed eggs out of the faeces. Those eggs, aged and hardened in the faeces, showed no infection by the fungus within the 20-day period. In the study I previously ment ioned, where one of the Master students was looking at survival and did recovery trials with eggs of different pig parasites, he noticed that Ascaris

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eggs could disappear or die within a few weeks from the time he deposited the samples and he often noticed hyphae of fungi inside the eggs. Such fungi could be purely opportunistic, invading otherwise damaged eggs. So, we don't know if they (the observed fungi) are able to cope with the hardened shell of say Ascaris spp. or Trichuris spp. eggs. That's something we are trying to include in the program, investigating the effect of fungi on eggs at different developmental stages. We have at present some 15 to 20 different isolates that we are planning to investigate.

(Coles - - UK) Could I ask you two unrelated questions. The first one is on weight gain on the graphs that you put up on the screen and the second one is if you culture certain fungi that attack nematodes in the laboratory without the presence of nematodes, do they lose their ability to trap and kill nematodes? Have you seen this problem or have you got a way of getting over it?

(Larsen Q Denmark) To your last question, we have not seen fungi lose their trapping ability after short term cultivation, but I'm aware of culture collections where you have problems with old senile cultures, that have been cultivated for years, and that of course is always a problem when you're trying to keep an isolate in culture. You have to store and keep them fresh, e.g. frozen in liquid nitrogen to be sure that you have the same isolate and preserve the activity you wanted. Now, I'm not quite sure what was your question on weight gains?

(Coles - - UK) Did those gains shown in the graph include gains for cattle?

(Larsen - - Denmark) They did, but it is not always that we see such marked differences. If you look at the number of field experiments performed since 1992, it adds up to four or five involving cattle. Of course we need an extended number of experiments to document a consistency on weight gains.

(Fox - - UK) How do naturally occurring faecal spore numbers compare with spore numbers in the faeces of animals you have fed the fungi, too? What is the difference in order of magnitude?

(Larsen Q Denmark) We have never actually measured the actual number of fungal spores of nematode-destroying fungi naturally occurring in the soil, poten- tially invading the faeces, with those passed through an animal, but I 'm convinced that such a number might come out lower than that occurring in the excreted faeces when we feed animals fungal spores. If the animals are exposed to a million units per kilogram of liveweight per day, their faeces should be well-seeded with the fungal spores. We made small dung pats in laboratory studies, and when we looked at the succession of fungi on those pats (inoculated with a very large amount of fungal material) there appeared to be a difference between what developed on the control pats and the fungus-treated pats. So, this indicated that there is competition going on between fungi in the pats and if the inoculum is high

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enough, the nematode-trapping fungus might get an advantage, at least in the short term (initial weeks).

(Hertzberg - - Switzerland) I have two questions. The first is, is there any chance for these spores to start to develop as soon as inside the rumen or inside the intestinal tract? And the second is, if you give one of your recommended doses of spores to cattle, for how long will you see sufficient reduction of larval develop- ment in the faeces?

( L a r s e n m D e n m a r k ) To your first question, I 'm sure there is no germination going on inside the cattle. The fungus need oxygen to germinate and grow. To my mind going through the GI tract is just a passive transport through the animals. Now of course we have to perform studies to document that no pathological changes are taking place in the animals, but as I said, I don't expect any activity in that respect at all. In answer to your second question, when you feed a single dose of fungus to the animal, the spores are excreted from 8-12 hours after feeding, but the peak concentration will be approximately 12-24 hours after feeding and then it tails off, depending on the initial fungal dose. If you give a very high dose, spores disappear over maybe 2 or 3 days. We've seen this in a horse trial where a single dose of up to 107 units per kg liveweight was given on day one and subsequently the number of larvae in faecal cultures was significantly reduced for the next 2 days, but in the days thereafter, the number of larvae increased again.

(Klei - - USA) Relative to the horse system, it seems like this would be a very useful tool in that horse people aren't as concerned about costs and control, number 1, and number 2, the handling of animals is easier and daily feeding is more common. In that system there is a need for a therapeutic treatment of young animals for Parascaris. What effect does treatment and reduction of worm burdens periodically have on efficacy of fungus? I know you said before that worm burdens might have an effect on how many nets are produced. In a recent study by Peter Waller and the Australian group, the efficacy required or that they predict is necessary for fungal control as determined by modeling, is much higher than they predict for a vaccine, at least the way I read that review. Maybe you could comment on that?

( L a r s e n m D e n m a r k ) To your last comment, I agree. They said that you need a 90% reduction over 3 months to get a good effect with fungi. But to me this indicates that we have to develop an integrated control strategy where you also use grazing management and perhaps vaccines, that don't have to be highly effective. But, in the right combination with the other measures you might get a very good effect. With regard to the number of parasites in the faeces that would stimulate the activity of the fungus, I think it doesn't matter whether you have a very low or high level of egg counts because of the often massive invasion of these different types of soil nematodes. They would themselves stimulate activity of the fungus,

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that do not distinguish between parasitic larvae and free-living soil nematodes. So I'm sure you still have good activity, but it might be a good idea when you experience a low egg count in the beginning of the season to start with a higher dose of fungi and then perhaps adjust this later in the season, cutting the dose down a bit.

(Armour - - UK) I enjoyed your paper very much. In the west of Scotland, particularly on higher ground of old permanent pastures, one sees a mat at the surface where there's a much higher humidity right at the interface of the soil and the grass. There was a theory back in the '50's that you seldom saw clinical problems or recognizable parasite problems on these old pastures. An old col- league of mine, who died a few years ago, Angus Dunn, followed the theory that this was due to the increase in the numbers of nematode-trapping fungi in the mat of these old pastures. I was never sure whether that was true or whether it was the fact that the stocking of animals was very much lower on these relatively poor pastures. It could be that what you've just shown was in fact a confirmation of what Angus suggested, although not proven then, that the area in the mat was particu- larly suitable for the development of these nematode-trapping fungi.

(Larsen - - Denmark) I think what you are saying is a possibility. When we look at what has been going on within plant parasitic nematode control, researchers have been talking about suppressive soils, being soils where they found different nema- tode antagonists, including nematophagous fungi, killing the nematodes. So, it could be that over the years you reach a balance or a point, with an increased population of nematode-destroying fungi or other enemies of nematodes, thereby keeping the parasitic nematode larvae at a very low level in this natural control situation. That is something we might get an opportunity to look at with the increased interest and money going into research on ecological, organic farming systems in Denmark at present.

(Guerino - - USA) Can you tell us what is known about the mode of action of these fungi?

(Larsen - - Denmark) The invasion of the nematode is partly a mechanical andenzymatic event. It has been shown that active enzymes degrade the cuticle, but also that penetration is assisted by a high mechanical pressure in the fungal cells, the fungus forming an infection bulb inside the nematode after penetration, effectively sealing off the point of entrance for other organisms (bacteria, etc.). So, you do not get competition from bacteria and other organisms inside the nematode victim at the same time, leaving the nematode-trapping fungus time to digest and redistribute the contents of the nematode into other parts of the fungal mycelium. Colleagues in Sweden have been looking into these mechanisms of trapping and digesting of nematodes, in particular focusing on the enzymes and what kinds could be active in the process, perhaps something that could be used in control.

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(Borgsteede - - The Netherlands) Michael, you know that the nematodes are a very flexible group. They have occupied almost every niche with us in nature and you have mentioned yourself that there are also nematodes which eat fungi. Do you think on the long-term that this system will be destroyed by the nematodes which eat the fungi?

(Larsen - - Denmark) I hope not, but I don't have an answer to that.

(Borgsteede - - The Netherlands) Have you seen an increase?

(Larsen - - Denmark) We haven't checked it, but we are planning to set-up a model system where we can introduce different groups of soil nematodes, if we can get them. And subsequently, if you introduce nematodes in different quantities, try to see what happens with regard to the balance in such a model system. But, so far we have not looked at it yet and nor am I aware of others who have done this. If any in the audience have information on this issue I would be happy to hear about it.

(Taylor - - USA) You gave these by bolus primarily or were they put in as part of a complete feed or how have they been added?

(Larsen - - Denmark) We gave the canes fungal material grown on barley grain and in the horse experiment we used material washed off the grain, then subse- quently mixed into a horse supplement mix. We have never tried a bolus system or lick stone but I know that they have been working on this aspect in Australia.

(Taylor - - USA) Do you have any data looking at palatability and feed consump- tion when these products are added? Is there any change in how much the animals will eat or is there any kind of rejection at all?

(Larsen - - Denmark) In the beginning we were a little bit concerned about that when we performed the horse field trial, but after 1 to 2 days where one to two horses needed adjustment to the supplement containing fungal spores, there was no apparent difference between the two groups. They both got approximately 0.5 kg of supplement with or without fungus and they ate it all.

(Vercruysse - - Belgium) Are there any commercial companies interested in this fungal approach to control? We know of people with vaccines, but companies don't appear interested in investing.

(Larsen - - Denmark) At the moment we have to comply with the Danish company we are working with and see what their plans are. It is up to the company to decide when or if they want to go to a third party, so in that respect our hands are tied.

(Donoghue - - USA) Can you speculate at all on whether or not when you feed

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x-number of spores per day, you can overwhelm beneficial soil nematodes of a pasture and what would the long-term effects of that be? In the United States a product like this is much more easily registered because it's a quote-unquote 'bio-control', but there may be long-term effects that are not observed in the short-term.

( L a r s e n - - Denmark) Yes, we are aware that there potentially could be unfore- seen problems, but we do not know yet if there will be any long-term effects or not. We haven't had either the time or the opportunity to perform long-term trials. As Peter Waller mentioned in his talk, if you consider the dung pat compared to the soil environment, the pat initially contains a lot of microorganisms such as coprophilic fungi, bacteria and others, but compared to soil it is a relatively n~iive media for fungi to develop and function in. It is a relatively large amount of material we introduce in the dung, but in the soil we know that it is very difficult for any new organism to get established and survive due to heavy competition in that environment. But, of course, studies need to be performed to answer these questions, which any authority that is asked to approve the use of such fungi would require an answer.

(Wallace - - USA) Have you thought about the practical application of scaling up for a country or the world and what's the cost of this? Have you thought about that, put some figures to it?

(Larsen - - Denmark) I haven't and it's luckily not my headache. The company has been doing some calculations and as many others, they preferably want to produce fungi in liquid cultures, but there are other techniques for large scale production of fungi available. One thing that is not an option is moving around and handing out large numbers of spores on barley, driving loads of material from farm to farm. If biological control is going to be a practical option, it has to be at a price level maybe a bit higher than the cheapest. I do not know exactly, but it has to be at a reasonable level to be able to compete with the price of anthelmintics. As I said, production is really not my headache, what we try to do is provide as much data as we can from a scientific point of view on the potential for exploitation of these fungi.

(Corwin - - USA) I know that last year Peter Nansen was going to attend or did attend a plant nematology meeting in the States and I was wondering what the intercourse was there as far as how far advanced they are with their use of nematophagus fungi and what suggestions they had for him?

(Larsen m Denmark) I'm afraid that the advances in control of plant nematodes are few and limited. Again, I think that this is due to the special circumstances in the soil in which such antagonists have to be efficient. They have been labouring with nematophagus fungi and other antagonists for a number of years and to a certain extent they have made some, but limited progress, one being the discovery

M. Larsen et al. / Veterinary Parasitology 72 (1997) 479-492 491

of, and attempts to understand the phenomenon of suppressive soils. They would like to develop this further and you could say this represents a success story, but otherwise, I am not aware of any new, major breakthrough; rather, they are a bit disappointed with the prospect of biological control of plant parasitic nematodes with fungi. Comparing animal parasitic nematodes with plant parasitic forms, I think there are great differences and that attempts to use biological control against larvae of the first group are going to be a different ball game from attempts to control the latter.

(Waller - - Australia) Michael has mentioned that we have been working on this and certainly Michael has been one of our very close collaborators. He spent a year with my group back in 1992 and we've certainly enjoyed this very close collabora- tion, but unfortunately the research environment that we work in these days with the guarding of intellectual property, everybody has to look for commercial partners. I think that Michael mentioned that the intellectual shutters have been drawn to some extent on our close collaboration which is a pity for science as far as I'm concerned. We're particularly interested in other forms of deployment. At present we are investigating this in a number of environments in grazing sheep, because under our system, supplementary feeding is not a common situation for sheep and we suggest that there would be large areas of the world where this would also be the case. Supplementary feed-blocks are certainly a possibility particularly for livestock in the tropics and subtropics where nightly housing is very common. Administration of fungi through very cheap forms of supplementary feed-blocks is a realistic option. Another option is the incorporation of these fungal spores in control release devices as Michael mentioned and we have done some early work on that and it's looked quite promising. These spores are really very resistant to all manner of different treatments. They can survive tableting pressures which are required for the manufacture of control release devices and they can survive very nicely at body temperature for extended periods of time, under laboratory condi- tions. We haven't investigated in situ survival, but certainly shelf-life survival is very good as well. There are a lot of pluses going for this technology and I think the last hurdles are the most difficult and this is coming up with deployment options such as feed-blocks, supplementary feed additives, or the control release devices. I'm optimistic that this approach will be of great benefit to biological control, and as Michael has very clearly demonstrated, applies to all livestock species. It applies to all nematode parasites with exception of those he indicated and also to all livestock systems as well, from the intensive management that you find in Japan to the situations that you might find for example in Australia.

( P r i c h a r d - - C a n a d a ) You've shown significant reductions in larval counts in the faeces from the animals that have been treated with the fungi. Do you know if there's any effect of the spores on the adult or immature worms in the animals themselves? Has this been studied?

( L a r s e n - - D e n m a r k ) No, it hasn't.

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(Prichard - - Canada) Is it conceivable that those reductions are due to some effect on the parasites rather than on the free-living stages?

(Larsen ~ Denmark) I 'm not aware of that, but we know that diet can affect establishment of worms, and maybe the amount of fungal material fed to the animals could influence the digestion process in an unknown manner, thereby also having some secondary effect on the worm population in the animals. We have not been looking into such an aspect, but since dosing animals with the fungal product should not have any negative influence on the digestion of the animals, this aspect should be checked out. If the fungus does not negatively affect digestion, but merely as a side effect the adult worm population, besides the positive larval reducing effect performed in the deposited faeces, then it could just be seen as a positive side effect.

(Waller m Australia) If I could just add to that. Work that Michael, Des (Hennessy) and I did on this aspect in Australia, included some fungus dose titration work. We can say currently that there was absolutely no anthelmintic effect of dosing spores to animals. The egg counts remained exactly the same.

(Larsen m Denmark) Yes, that is true, but there could be, as suggested, other effects than just the anthelmintic or chemical effects.

(Williams - - USA) Michael, I have a comment that's not directly related to fungi; it relates to predacious nematodes. In past ecological research, I occasionally noted rather large free-living nematodes in the act of ingesting or sucking out body contents of smaller nematodes. The victims were usually other small larval free-liv- ing nematodes, but in a few cases, infective larvae of cattle nematodes were destroyed in this manner. I was wondering if you or anybody else has ever observed this?

(Larsen - - Denmark) I 've never seen it, but Fm sure there must be a lot of predacious nematodes described, so something must be going on out there.