veterinary parasitology

ELSEVIER Veterinary Parasitology 60 (1995) 315-320

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

tract of horses

Michael Larsen a, * , Peter Nansen a Svend Aage Henriksen a,b Jens Wolstrup a,c, J0m Gr~nvold a,d, Anders Zorn a, Erik Wed~ e

a Danish Centre for Experimental Parasitology, Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, 13 Biilowsvej, DK-1870 Frederiksberg C, Denmark

b National Veterinary Laboratory, 27 B~lowsvej, DK-1790 Copenhagen V, Denmark c Microbiology Section, Department of Ecology and Molecular Biology,

Royal Veterinary and Agricultural University, 21 Rolighedsvej, DK-1958 Frederiksberg C, Denmark d Section of Zoology, Department of Ecology and Molecular Biology,

Royal Veterinary and Agricultural University, 13 B~lowsvej, DK-1870 Frederiksberg C, Denmark Statens Seruminstitut, Animal Department, 71 Frederiksborgvej, DK-3450 Allered, Denmark

Accepted 11 January 1995

Abstract

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°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.

Keywords: Horse; Cyathostominae-general; Duddingtonia flagrans; Fungi; Control methods-Nematoda

* Corresponding author.

0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0304-4017(95)00792-X

316 M. Larsen et a l . / Veterinary Parasitology 60 (1995) 315-320

1. Introduction

Various nematode-trapping fungi have been isolated from horse dung (Zopf, 1888; Duddington, 1951a,b; Juniper, 1957; Larsen et al., 1994), but the potential of such nematode-trapping fungi to control free-living stages of parasitic nematodes in horses has been given virtually no attention except for a few limited studies (Descazeaux and Capelle, 1939; Soprunov, 1958; Bird and Herd, 1994).

There is a growing awareness of the role of cyathostomes in parasite-associated equine diarrhoea (Love, 1992) and surveys seem to document that anthelmintic resis- tance in cyathostomes against benzimidazoles has become a widespread phenomenon (Bjcrn et al., 1991; Boersema et al., 1991). Therefore, attention has turned to alternative management and treatment practices to sustain parasite control (Herd, 1993). Apart from different grazing management practices, increased pasture hygiene has been suggested as an additional option (Herd and Gabel, 1990). Biological control of free-living stages of horse strongyles by using nematode-trapping microfungi might be another practical option which in the future could alleviate the present heavy reliance on intensive anthelmintic treatments.

The aim of the present experiment was to test the ability of spores, especially the thick-walled chlamydospores, of the nematode-trapping fungus Duddingtonia flagrans, to survive passage through the gastrointestinal tract of horses and subsequently to evaluate the potential of the fungus to destroy cyathostome larvae in faecal cultures.

2. Materials and methods

A Danish isolate of D. flagrans (Larsen et al., 1991) was grown for 6 weeks on barley grains at 24°C, the cultures being prepared according to the procedure of Gr~nvold et al. (1993a). Fungal material was washed off the grains by thorough shaking and mixing with tap water. The material was poured sequentially through a kitchen sieve, a layer of fine gauze, and finally a 38 /zm nylon net. Fungal material in the resulting suspension contained less than 5% conidia, the rest were chlamydospores. The concentration of fungal units in the suspension was determined using a haemocytometer.

Six horses were used in this experiment, two for each level of fungal material tested. The horses, weighing from 520 to 650 kg, harboured a natural infection with cyathos- tomes and excreted eggs at a moderate to high level during the experiment. The animals were confined to individual boxes during the 5 days the experiment lasted.

From each horse all faeces excreted was collected daily at 07:00 h and 15:00 h, respectively. At 07:00 h on Day 2 of the experiment each horse received a single oral dose of fungal suspension mixed with wheat bran. The dose levels tested on each pair of horses were 105, 10 6 and 107 fungal units per kg bodyweight, respectively. At each sampling time all faeces were thoroughly mixed and triplicate 4 g aliquots were used to determine the numbers of eggs per gram (EPG). In addition, triplicate subsamples were used to establish faecal cultures, modifying the procedure described by Henriksen and Korsholm (1983). Each faecal culture consisted of 25 g of faeces mixed with 10 g of vermiculite and 10 ml of tap water. After incubation for 2 weeks at 24°C the cultures

M. Larsen et al. / Veterinary Parasitology 60 (1995) 315-320 317

were harvested using a modified Baermann technique, and the number of third stage parasite larvae was determined. The effect of the fungus was expressed as the percentage of eggs that developed into infective larvae in the actual sample for each day (morning and afternoon). Before extracting larvae, a check for the presence of nematode-trapping fungi was performed by taking a blot of each culture on water agar plates (2% Difco bacto agar) containing an antibiotic (0.02% (w/v) chlortetracycline). A quantitative estimate of fungal units in the faecal cultures was not attempted.

3. Results

Oral dosing of cyathostome-infected horses with the nematode-trapping fungus D. flagrans showed that the fungus was able to survive passage through the gastrointestinal tract of the horse and subsequently to grow and reduce the number of infective larvae in faecal cultures.

No larval reduction in cultures could be observed when the horses were given 105 fungal units kg-1 bodyweight, despite the presence of the fungus in cultures over a period from 24 to 48 h after the single dose was given (data not shown). At dose levels of 106 and 107 fungal units kg- 1 bodyweight (Figs. I(A) and I(B)) a clear reduction in the number of larvae was seen 32 h after dosing. At 106 fungal units kg -~, the percentage of developed larvae based on the EPG was 7.9% and 10.8%, respectively, for the two horses. The level of development was 0.1% and 1.7% for the two horses at 10 7

fungal units kg -~. For 107 fungal units kg -~ the larval reducing effect was already pronounced by 24 h after dosing with 0.4% and 7.5% larvae developing, respectively. The large deviation recorded in the afternoon on Day 1 for Horse B (Fig. I(A)) might be due to the unexpectedly low EPG of 147, especially in the context of an average EPG during the rest of the week of approximately 700 (minimum, 520; maximum, 933).

The fungus was positively identified in faecal cultures from 8 and 24 h after dosing with 106 and 107 units kg -1, respectively. The fungus persisted in the cultures for 56 and 80 h after dosing, the latter for the highest dose level.

4. Discussion

This study clearly demonstrated the ability of the fungus D. flagrans to survive passage through the gastrointestinal tract of horses and subsequently to reduce the number of infective larvae which develop in the excreted faeces.

Successful laboratory and field experiments have been performed in attempts to control free-living larvae of trichostrongyles in cattle and sheep by means of nematode- trapping fungi (Grcnvold et al., 1993a,b; Waller and Larsen, 1993; Waller et al., 1994; Wolstrup et al., 1994). This experiment strongly indicates that a similar concept might also be successful for controlling free-living stages of horse parasitic nematodes.

As the problem of anthelmintic resistance increases, particularly among horse cy- athostomes, the need will arise for new approaches to combat these parasites. Biological control of free-living larval stages might provide us with a practical and flexible

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M. Larsen et al. / Veterinary Parasitology 60 (1995) 315-320 319

approach to be considered for both intensive and extensive grazing practices. At present, however, further studies on, for example, dose levels and methods of application, are required. Furthermore, it is important to examine the versatility of these fungi with regard to controlling free-living larvae of species of parasitic nematodes of the horse under different climatic conditions.

Acknowledgements

This work was supported by Chr. Hansen's BioSystems. Margrethe Pearman, Lillian Sztuk and John Jorgensen are thanked for their valuable technical assistance during the experiment.

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Fig. 1. The percentage of eggs that developed to infective cyathostome larvae in faeces of two horses treated with chlamydospores as a single dose in the morning on Day 2. Each bar represents the mean of three faecal cultures. Two times standard deviation is shown for each sampling time. Faeces from each of the two horses were collected in the morning (07:00 h) and the afternoon (15:00 h). Arrows indicate the time of oral dosing with fungal material. Bars marked 'Fungi in cultures' indicate the period through which the fungus could be positively identified in faecal cultures. Cross-hatched bars, Horse A: stippled bars, Horse B; *, only afternoon samples; * *, not determined; $, oral dosing with fungus; m, morning sampling (faeces collected at 07:00 h); a, afternoon sampling (faeces collected at 15:00 h).

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