Acta vet. scand. 1997, 38, 175-179.

Nematode-Trapping Fungi in Biological Control of viviparus

By Sv. Aa. Henriksen 1, M Larsen 2, J. Grenvold3, P Nansen 2 and J. Wolstrup 4

1Danish Veterinary Laboratory, Copenhagen, 2Danish Centre for Experimental Parasitology, Department ofVe• terinary Microbiology, and 3Section of Zoology, and 4Section ofMicrobiology, Department ofEcology and Mol• ecular Biology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.

Henriksen, Sv. Aa., M. Larsen, J. Grenvold, P. Nansen and J. Wolstrup: • trapping fungi in biological control ofDictyocaulus viviparus. Acta vet. scand. 1997, 38, 175-179. - Larvae of the Dictyocaulus viviparus were cultured in experimental units of200 g cattle faeces placed in semi-transparent trays in the labora• tory. In each of4 experimental series using this experimental unit, chlamydospores (chi) of the nematode-trapping Duddingtonia jlagrans were admixed to half ofthe fa• ecal cultures in a concentration of 50.000 chl/g. In all 4 series there was a significant re• duction in the development and subsequent release of infective lungworm larvae from faecal cultures containing chlamydospores. The average reduction in larval release, cau• sed by fungal spores, was 86%.

parasites; lungworm; cattle.

Introduction The nematode-trapping fungus Duddingtonia laboratory (Nansen et al. 1988). The results jlagrans is able to trap migrating in showed that slow-moving nematodes, such as three-dimensional adhesive mycelial nets D. viviparus larvae, have poor trap-inducing (Cooke 1969). D. jlagrans only produces traps potentials. But if traps were induced by other when it is induced to do so, e.g. by physical nematodes, D. viviparus larvae were easily contact with migrating nematodes. trapped in the pre-formed A. oligospora nets. In field experiments, a D. jlagrans strain has The cattle lungworm D. viviparus is ovovivi• been able to control free-living stages of a range parous and excretes larvae in fae.ces. Under ofparasitic gastrointestinal nematodes ofcattle , normal summer temperatures in Denmark the ) and pig when fungal spores were fed to infective third stage larvae (L3 is reached the host (Grenvold et al. 1993, Wol• within one to 2 weeks in the cow pats. In con• strup et al. 1994, Larsen et al. 1995, 1996, Nan• trast to gastrointestinal nematodes, it has been sen et al. 1995, 1996). But, sofar, biological indicated that the coprophilous fungi control of the cattle lungworrn Dictyocaulus spp. may play an important role in transporting viviparus by D. jlagrans has not been tested the slow-moving infective D. viviparus larvae neither in the laboratory nor in the field. from faeces to herbage from where they can be Earlier on, another nematode-trapping fungus, taken up by susceptible grazing calves (Jer• Arthrobotrys o/igospora, has been tested aga• gensen et al. 1982). inst D. viviparus larvae in agar cultures in the In relation to a possible biological control effe.ct

Acta vet scand. vol. 38 no. 2, 1997 176 Sv. Aa. Henriksen et al.

Nematode-destroying fungi From a culture of D. jlagrans (strain Cl3), thick-walled chlamydospores (chi) were har• vested in tap water. The chlamydospore-su• spension was adjusted to approximately 106 chi/ml.

Experimental design The present study included 4 test series (I-IV) Figure I. Schematical cross-section of the experi• carried out from October to December 1995. mental design. A: Faecal sub-sample containing The following design was used for all series: infective larvae of Dictyocaulus viviparus and with From a thoroughly mixed batch of fresh faeces or without admixture of the nematode-trapping fun• gus Duddingtonia jlagrans. B: Transparent tray. containing lungworm larvae, 6 sub-samples of 200 g were made. Ten ml chlamydospore-su• spension were mixed into each of 3 ( experi• of D. jlagrans on D. viviparus, the following mental) faecal sub-samples, resulting in a fun• questions become pertinent: gal concentration of approximately 50,000 I) Does the presence of Pilobolus spp. myce• chl/g. The 3 other (control) faecal sub-samples liwn allow the nematode-trapping fungus D. received 10 ml of tap water. Each faecal sub• jlagrans to grow in cattle faeces?, and ifit does, sample was placed in individual semi-transpa• 2) can the nematode-trapping fungus subsequ• rent polystyrene trays (38x28xl2 cm) and for• ently be stimulated by the slow-moving D. vivi• med as a small cow pat (diam.: 20 cm; height: I

pams L3-larvae to produce traps that can effe• cm) with a smooth surface (Fig. I). ctively control the cattle lungworm? The trays were stacked randomly and the upper tray was supplied with a lid. The set-up was pla• ced in a room with a window, which allowed Materials and methods daylight to hit the faecal sub-samples. Light is Experimental animals necessary for Pilobolus to produce fruit bodies Two parasite-naive calves, housed in an experi• and therefore this arrangement allowed us to re• mental unit, were each infected (3 larvae per kg gister if Pilobolus was present in the faeces. bodyweight) with a Danish strain of D. vivi• The room temperature was approximately parus. After the end of the prepatent period 20°C and the incubation period was 8 days. fresh faeces containing lungworm larvae were Daily each cow pat was gently sprayed with tap sampled per rectum. water leaving a thin film of moisture on the faecal surface. Pilobolus fungi At the end of the incubation period, the surface No particular measures were taken to ensure of each pat and the inside of each tray were rin• that the calves excreted Pilobolus spp. spores sed off gently with approximately 50 ml of tap with their faeces, since it is known that calves water. The water from each sample was poured fed on stable usually excrete Pilobolus spores into a test tube. Following centrifugation for I 0 more or less constantly all year round, because min at 1500 rpm, the supernatant was discar• their food (hay, straw etc.) naturally contains ded, leaving a remnant of I 0 ml. From each spores ( Grenvold & Jergensen 1985). remnant 2 sub-samples of0.5 ml were transfer-

Acta vet. scand. vol. 38 no. 2, 1997 Nematode-trappingfungi in Dictyocaulus viviparus 177

Table l. The number of infective Dictyocaulus viviparus larvae released from cow pats with(+) and without (-) admixture of the nematode-trapping fungus Duddingtonia jlagrans, in each of the 4 series (I-IV) of experi- ments.

Ful\gal material Reduction Series Statistics + (%)

65 494 105 1160 95 286 Mean of serie I 88 647 86 •

799 3150 JI 1520 1743 720 5256 Mean of serie II 1013 3383 70 *

138 3360 III 54 4758 90 5454 Mean of serie III 94 4524 98 •

216 2247 TV 320 1950 360 3712 Mean of serie IV 299 2636 89 •

Total mean 86%

Asteriks indicate significant differences in larval count between the 2 groups ofcow pats using a the Mann-Whit• ney U-test. *: p<0.05; **:p<0.01; ***:p<0.001. red to slides for counting D. viviparus larvae. lower than counts from the control cow pats in Subsequently, the total number of infective particular in series I, III and IV On average, the lungworm larvae, released from each cow pat in addition of the nematode-trapping fungus 8 days, was calculated. resulted in a 86% (range:70%-98%) reduced release of infe.ctive lungworm larvae from the Statistics faeces. Statistical analyses were made by the Mann• Pi/oho/us spp. fungi were observed in all trays, Whitney U-test (Siegel 1956). The level of sig• but no quantification was attempted. nificance was 5%. Discussion Results This paper shows that there was a significant Table I shows that larval counts from the expe• reduction in the number of infective lungworm rimental cow pats, which had received chla• larvae (D. viviparus) released from the cow pats mydospores of D. jlagrans, were significantly containing the nematode-trapping fungus (D.

Acta vet. scand. vol. 38 no. 2, 1997 178 Sv. Aa. Henriksen et al. jlagrans). On the average, a nematode-trapping Referen ces efficacy of 86% was observed. In previous ex• Cooke RC: Two nematode-trapping Hyphomycetes, Duddingtoniaflagrans gen.et comb.nov. and Mo• periments, on gastrointestinal trichostrongyles, nacrosporium mutabilis sp. nov. Transactions of similar high reductions in Ostertagia and Coo• the British Mycological Society. 1969, 53, 315- peria larval numbers were reached as a result of 319. feeding calves with 200x I 06 chlamydospores Granvold J. Jergensen RJ: PILOBOLUS Tode 1784. per per day (Nansen et al. 1995). Danmarks Veterinrer- og Jordbrugsbibliotek, Copenhagen. 1985, 124 pp. As Pilobolus spp. fungi were observed in all se• Grenvold J. Wolstrup J, Larsen M, Henriksen SA, ries, the present results indicate that Pi/obolus Nansen P: Biological control of Ostertagia oster• fungi and D. jlagrans can coexist in cattle fae• tagi by feeding selected nematode-trapping fungi ces. Moreover, the results indicate that D. vivi• to calves. J. Helminthol. 1993, 67, 31-36. Jergensen RJ: Studies on the lungworm Dictyocau• parus larvae can induce trap-production in D. lus viviparus (Bloch, 1782) and its epidemiology jlagrans in cattle faeces, as no other nematodes in young cattle with a description of an attempt to were observed in the faeces. However, future prevent parasitic bronchitis. Thesis. Danmarks experiments will have to clarify whether the Veterinrer- og Jordbrugsbibliotek, Copenhagen. present results were caused mainly by: 1) redu• 1981, 77 pp. Jargensen RJ, Renne H, Heisted C, Iskander AR: ced release oflungworm larvae caused by redu• Spread of infective Dictyocaulus viviparus larvae ced Pilobolus growth in competition with the in pasture and to grazing cattle: Experimental nematode-trapping fungus, or by 2) the nema• evidence of the role ofPilobolus fungi. Vet. Para• tode trapping activity of the latter alone. sitol. 1982, JO, 331-339. If the present laboratory observations reflect Larsen M, Nansen P. Wolstrup J, Grenvold J, Henrik• sen SA, Zorn A: Biological control of trichostron• the field situation reasonably, which is not ne• gyles in calves by the fungus Duddingtonia cessarily the case, dosing of grazing calves with flagra11s fed to animals under natural grazing D. jlagrans chlamydospores might reduce the conditions. Vet. Parasitol. 1995, 60, 321-330. herbage contamination, perhabs to a level that Larsen M. Nansen P. Grendahl C, Thamsborg SM, Grenvold J, Wolstrup J. Henriksen SA, Monrad J: will prevent clinical manifestations of lun• The capacity of the fungus Duddingtonia gworm infection. According to Jergensen flagrans to prevent strongyle infections in foals ( 198 1) strategic anthelmintic treatment of cal• on pasture. Parasitology. 1996, 113, 1-6. ves 6 and 8 weeks after turn-out onto contami• Nansen P. Gronvold J, Henriksen SAa, Wolstrup J.: nated pasture, was effective in controlling D. vi• Interactions berween the predacious fungus Art• hrobotrys oligospora and third-stage larvae of a viparus. Thus, given the efficacy of nematode series of animal-parasitic nematodes. Vet. Parasi• destroying fungi as described above, dosing tol. 1988, 26, 329-337. with fungi during the initial 2 months of the Nansen P. Larsen M, Grenvold J, Wolstrup J, Zorn A. grazing season may be sufficient for practical Henriksen SAa: Prevention of clinical tricho• control of bovine dictyocaulosis. strongylidosis in calves by strategic feeding with the predacious fungus Duddingtonia flagrans. Parasitol. Res. 1995, 81, 371-374. Acknowledgement Nansen P. Larsen M. RoepstorffA, Granvold J, Wol• The authors highly appreciate the technical assi• strup J. Henriksen SAa: Control of Oesophago• stance of Leif Eiersted, and the financial support of stomum dentatum and Hyostrongylus rubidus in Danish National Research Foundation and Chr. Han• outdoor-reared pigs through daily feeding with sen's Bio Systems. the micro fungus Duddingtonia flagrans. (sent to Parasitol. Res.).

Acta vet. scand. vol. 38 no. 2, 1997 Nematode-trappingfungi in Dictyocaulus viviparus 179

Siegel S: Non-parametric statistics for the behavioral Sammendrag sciences. McGraw-Hill, Kogakusha, Tokyo, Biologisk kontro/ af Dictyocaulus viviparns ved 1956, 312 pp. hjalp afrovsvampe. Wolstrnp J. Grenvold J. Henriksen SA, Nansen P. Lar• sen M, Begh HO, I/see 8: An attempt to imple• Larver af kvregets lungeorm, Dictyocaulus v1v1- ment the nematode-trapping fungus Duddingto• parus, blev dyrket i enheder af 200 g kva:gg0dning nia jlagrans in biological control of first year placeret i semi-transparente kasser i laboratoriet. I grazing calves. Journal of Helminthology. 1994, hver af 4 eksperimentelle serier, hvor ovennrevnte 68. 175-180. opstilling blev anvendt, blev chlamydosporer (chi) af den nematode-fangende svamp Duddingtonia jlagrans tilblandet halvdelen af opstillingeme i en koncentration pA 50.000 chVg. I alle 4 serier var der en signifikant reduktion i spredningen af infektive lungeormlarver fta kulturer tilblandet chlamydospo• rer. Den gennemsnitlige reduktion i spredningen, for• arsaget af chlamydosporer, var 86%.

(Received December 4, 1996; accepted Febrnary 27, 1997).

Reprints may be obtained from: Sv. Aa. Henriksen, The Danish Veterinary Laboratory, Biilowsvej 27, DK-1790 Copenhagen Y, Denmark. E-mail: [email protected], tel: +45 35 30 02 11 , fax : +45 30 01 20.

Acta vet. scand. vol. 38 no. 2, 1997