Efficacy of the nematode-trapping fungus
Duddingtonia flagrans against infections by
Haemonchus and Trichostrongylus species in

lambs at pasture

R.A. Rocha1*, J.V. Araújo2 and A.F.T. Amarante1

1UNESP, São Paulo State University, Instituto de Biociências,
Departamento de Parasitologia, Caixa Postal 510, CEP 18618-000,

Botucatu-SP, Brazil: 2Departamento de Veterinária, Universidade Federal
de Viçosa, CEP 36570-000, Viçosa, MG, Brazil

Abstract

The efficacy of the nematode-trapping fungus Duddingtonia flagrans against
infections by trichostrongyle nematodes in sheep was assessed throughout 6
months. Twenty Ile de France lambs were divided into two groups (control and
treated groups), which were kept in separate pastures. Animals of the treated
group were fed with D. flagrans twice a week (Tuesdays and Fridays). Pellets
were prepared with the fungus mycelia in liquid culture medium and contained
approximately 20% fungus. They were mixed with the animals’ diet at a
concentration of 1 g pellet per 10 kg live weight. Faecal egg counts (FEC), packed
cell volume (PCV), total serum protein and the animals’ body weight were
determined fortnightly from 7 October 2005 to 24 March 2006. Comparison of
such parameters between groups showed no significant differences (P . 0.05),
except on 10 February 2006, when the control group presented a higher mean
FEC than the treated group (P , 0.05). Feeding sheep with pellets containing
D. flagrans had no benefit to the prophylaxis of nematode infections under the
experimental conditions used in the present study.

Introduction

Despite the successful development of anthelmintics in
the latter part of the twentieth century, helminth parasites
of domestic ruminants still continue to be the most
important cause of infectious disease problems in grazing
livestock systems worldwide (Waller, 2006). The outbreak
of nematode populations resistant to anthelmintics and
the scarce release of new drugs demanded alternative
strategies for the prophylaxis of parasitic gastroenteritis.

The use of nematophagous fungi has the potential to
become an important, integrated element in sustainable
strategies to control parasitic nematodes in livestock.
Effective biological control of parasitic nematodes
consists in the preventive reduction of infective stages
on pasture. Thus, a good candidate for control must

survive the passage through the host’s gastrointestinal
tract and subsequently be eliminated with the parasite
eggs in fresh faeces, where it must be able to germinate,
grow, trap and destroy the parasite larvae (Larsen, 1999).
This is the case of the thick-walled spores of Duddingtonia
flagrans, which caused significant larval reduction in faecal
cultures and on pastures when administered to small
ruminants as a daily feed supplement or when incorpor-
ated into a mineral/molasses feed block (Chandrawathani
et al., 2003; Fontenot et al., 2003).

The fungus D. flagrans has been the most used
nematophagous fungus in experiments regarding the
prophylaxis of parasitic gastroenteritis in small rumi-
nants, and good results have been obtained in several
countries, including Australia (Faedo et al., 1998; Larsen
et al., 1998; Waller et al., 2001), Spain (Gómez-Rincón et al.,
2006) and the United States (Terrill et al., 2004). In Brazil,
few field studies have used D. flagrans fungus; therefore,
the aim of the present experiment was to assess the

*Author for correspondence
E-mail: rrabdallah@hotmail.com

Journal of Helminthology (2007) 81, 387–392 doi: 10.1017/S0022149X07853697



efficacy of D. flagrans isolated in Brazil on the prophylaxis
of Haemonchus spp. and Trichostrongylus spp. infections
in sheep.

Material and methods

The present study was carried out in the city of
Botucatu, São Paulo State, Brazil, from October 2005 to
March 2006. During this period, mean monthly maximum
and minimum temperatures ranged from 28.68C to 19.58C
in January and from 27.18C to 16.78C in November,
respectively (fig. 1).

Twenty female Ile de France lambs, approximately 3
months old, were used. Before the beginning of the
experiment, faecal samples were collected from each
animal for faecal egg counts (FEC). The FEC values were
determined using a modified McMaster technique, in
which each nematode egg counted represented 100
eggs/g of faeces. Based on these values, animals were
ranked in pairs. Then, a lamb of each pair was randomly
allocated to one of the two experimental groups: one
group was fed with the fungus (treated group) and the
other group did not receive the fungus (control).

An area of 0.6 ha was divided into two paddocks (0.3 ha
each) presenting similar pasture and topography. Control
and treated groups were kept separately (each group
in one of the paddocks) in continuous grazing from
7 October 2005 to 24 March 2006. Pastures consisted of
Cynodon dactylon and Brachiaria decumbens and were kept
without animals for 18 months prior to the experiment.

The nematode-trapping fungus D. flagrans (AC 001
isolate) was isolated from soil in Viçosa, Minas Gerais
State, Brazil, according to the soil-spreading method of
Duddington (1955), modified by Santos et al. (1991). The
isolate was replicated periodically and kept in test tubes
containing 2% cornmeal agar (CMA 2%), at 48C in the dark.
Mycelia mass was obtained by inoculating culture discs
(approximately 5 mm diameter) containing 2% CMA. The
culture was incubated in 250 ml Erlenmeyer flasks
containing 150 ml liquid GPY culture medium (glucose,
sodium peptone and yeast extract, pH 6.5, kept under
120 rpm agitation), kept in the dark at 258C, for 7 days.

Sodium alginate pellets were prepared with D. flagrans
mycelia in liquid culture medium, as described by Walker
& Connick (1983) and modified by Lackey et al. (1993).
Pellets contained 20% fungus and were administered to
the animals of the treated group (1 g pellet for each 10 kg
body weight) mixed in 1.2 kg of supplement feed (Ração
Noelw, Cafenoel, São Manoẽl Brazil) twice a week
(Tuesdays and Fridays). Animals of the control group
received a similar amount of sodium alginate pellets
prepared without fungus and mixed in the same quantity
of supplement feed.

All animals were vaccinated against clostridial infec-
tions (Clostridium chauvoei, C. septicum, C. perfringens,
C. novyi, C. sordellii and C. tetani, Sintoxan Polivalentew

(Merial, Paulinia, Brazil) at the beginning of the
experiment. Lambs had free access to mineralized salt
(Nutruminw, Nutrumin, Botucatu, Brazil) and drinking
water throughout the experimental period.

High FEC were observed at the end of the first month
(27 October 2005); thus, all animals were treated with
anthelmintic 20 days after entering the experimental
pasture. Therefore, animals with high FEC contaminated
the pastures in the first weeks of the study, before of the
treatment. To prevent deaths, salvage anthelmintic treat-
ments were provided individually to animals presenting
FEC higher than 4000 (Tembely et al., 1998; Amarante
et al., 1999) and/or packed cell volume lower than 21%
(Amarante et al., 1999). An association of levamisole
phosphate at 10 mg/kg (Ripercolw, Fort Dodge, Campi-
nas, Brazil) with albendazole at 10 mg/kg (Valbazenw 10
Cobalto, Pfizer, Guarulnos, Brazil) was used in the
treatments. All animals were treated with trichlorfon
(100 mg/kg; Neguvonw, Bayer, Belford Roxo, Brazil) on 1
February 2006, to control Oestrus ovis parasitism.

Measurements

All experimental samples were collected fortnightly.
Faecal samples were collected directly from the rectum of
animals for faecal cultures and FEC determination.
Composite faecal cultures (Roberts & O’Sullivan, 1950)
were prepared separately for each group, and the
infective larvae obtained were identified according to
Keith (1953). On the same day as faeces collection,
blood samples were taken from the jugular vein using
vacutainer tubes containing EDTA. Packed cell volume
(PCV) was determined using micro-haematocrit centrifu-
gation. Total serum protein concentration was estimated
using an optical refractometer (Model SPR-N, Atago,
Japan).

To determine the number of infective larvae (L3) per
kilogram of dry matter (L3/kg DM), grass samples were
collected manually from both paddocks, close to the
soil, approximately every 3.5 m. The collector followed a
W-track on the paddock (Taylor, 1939). Samples were
processed in the laboratory according to Amarante &
Barbosa (1995), and the infective larvae obtained were
identified according to Keith (1953).

Statistical analysis

The data obtained in the determination of FEC, PCV
and total serum proteins were subjected to one-way

Fig. 1. (A) Mean minimum ( ) and maximum ( ) temperatures,
(B) total rainfall ( ) and mean relative humidity ( ) in Botucatu,

São Paulo State, Brazil, from October 2005 to March 2006.

388 R.A. Rocha et al.



analysis of variance using the Minitab program (release 11;
State College, Pennsylvania, USA). Differences between
means were considered significant whenP, 0.05. The FEC
values were analysed using logarithmic transformation
2 log10(x þ 1); however, in the figures, they were presented
as arithmetic means. The numbers of larvae on herbage of
both paddocks were classified as inferior or superior to
300 L3/kg DM and compared using the chi-square test.

Results

Groups showed similar mean FEC (fig. 2) throughout
the experiment (P . 0.05), except on 10 February 2006,
when mean FEC of the treated group was significantly
lower than that of the control group (P , 0.05).

To prevent deaths, individual anthelmintic treatments
were administered to some animals. On 2 December 2005,
three animals of the control group and two animals of the

treated group required salvage anthelmintic treatment.
On 16 December 2005 and on 27 January 2006, one lamb
of each group was treated. Later, on 24 March 2006, two
lambs of the treated group received anthelmintic
treatment. Even after several anthelmintic treatments,
one highly susceptible lamb of the control group died on
20 March 2006. On the other hand, six animals of the
control group and eight of the treated group did not
require individual treatments.

Only Haemonchus spp. and Trichostrongylus spp.
infective larvae were identified in faecal cultures of both
control and treated groups (fig. 3). On average, faecal
cultures of the treated group showed 74.2% Haemonchus
spp. and 25.9% Trichostrongylus spp.; faecal cultures of the
control group presented 69.7% Haemonchus spp. and 24%
Trichostrongylus spp.

Comparison of mean PCV, total serum protein and
weight gain showed no significant differences between
groups (figs 4 and 5).

Similarly to faecal cultures, pastures presented only
Haemonchus spp. andTrichostrongylus spp. infective larvae,
generally at equivalent percentages on both paddocks
(fig. 6). However, the paddock grazed by control animals
presented higher numbers of Trichostrongylus spp. L3/kg
DM on 4 November 2005, 16 December 2005, 13 January
2005 and 24 February 2006, and of Haemonchus spp. larvae
on 18 November 2005 and 2 December 2005. The opposite
occurred on 10 February 2006 and 24 March 2006, when a
higher number of larvae was found in the paddock grazed
by the treated group (fig. 6).

Except for those obtained in the first grass collection,
the total number of larvae was higher than 300 L3/kg DM
on seven occasions in the paddock grazed by the control
group and on four occasions in the paddock grazed by the
treated group. Such occurrences were not statistically
different (x 2 ¼ 1.51; P . 0.05).

Fig. 2. Arithmetic mean fecal egg counts (FEC) of lambs fed with
the fungus Duddingtonia flagrans (A) or not fed (B). The arrow

indicates the treatment with albendazole þ levamisole.

Fig. 3. Percentage of Haemonchus spp. ( ) and Trichostrongylus spp. ( ) infective larvae identified in faecal cultures of lambs fed with the
fungus Duddingtonia flagrans (treated) or not fed this fungus (control).

Efficacy of D. flagrans against nematode infection in sheep 389



Discussion

The treatment scheme using D. flagrans, tested in the
present study, had no evident benefit to the prophylaxis of
infections caused by Haemonchus spp. and Trichostrongylus

spp. in sheep. Similar results were obtained in The
Netherlands, where administration of D. flagrans had no
effects on the weight gain, FEC or worm burden of lambs
treated daily with 50,000 spores of the fungus (Eysker
et al., 2006). In Brazil, Graminha et al. (2005) studied the
efficacy of Arthrobotrys musiformis fungus in naturally
infected sheep and did not detect differences between the
FEC of the control group and that of treated group;
however, they observed that A. musiformis reduced the
number of Trichostrongylus colubriformis by more than 50%
in tracer lambs.

Unlike the present study, Gómez-Rincón et al. (2006), in
Spain, reported that daily dosing grazing ewes with
D. flagrans clearly reduced autumn and spring pasture
contamination. Chandrawathani et al. (2003), in Malaysia,
also observed efficacy in daily treatment of sheep with
D. flagrans. Fontenot et al. (2003), in the United States,
reported L3 reduction on pasture and in faecal cultures
after daily administration of D. flagrans; however, FEC,
PCV or animal weight did not differ between fungus-fed
and control groups. Terrill et al. (2004) also reported a
reduction in larvae in the faeces of goats predominantly
infected by Haemonchus contortus. These authors observed
that daily supply of the fungus (D. flagrans) provided
more consistent larval reduction than intermittent feeding
(every 2 or 3 days).

In the studies cited above, good results were obtained
when animals had daily access to the fungus. The
frequency of the treatments used in the present study
(only twice a week) was probably insufficient to reduce
pasture contamination. However, promising results have
been obtained in Brazil with the administration of
Monacrosporium thaumasium fungus to cattle twice a
week; treated animals presented a lower degree of
infection than those of the control group (Alves et al.,
2003; Araújo et al., 2004).

The dosage used (1 g/10 kg live weight) may also
have been insufficient. Araújo et al. (2006) treated goats
with a higher dose (20 g/animal) of the same fungus
and observed a 59.3% reduction in the production of
H. contortus larvae in faecal cultures.

Replicates of the experimental paddocks were not used
in the present study, which may have influenced the
results, although the two paddocks used were contiguous
and pastures were alike. This was only an initial study

Fig. 4. (A) Mean packed cell volume (PCV) and (B) total serum
protein of lambs fed with the fungus Duddingtonia flagrans (A) or

not fed this fungus (B). Bars: standard deviation.

Fig. 5. Mean body weight (kg) of lambs fed with the fungus
Duddingtonia flagrans (A) or not fed this fungus (B). Bars:

standard deviation.

Fig. 6. Number of Haemonchus spp. ( ) and Trichostrongylus spp. ( ) infective larvae per kilogram of dry matter (L3/kg DM) on herbage
of the paddocks grazed by lambs fed with the fungus Duddingtonia flagrans (treated) or not fed this fungus (control).

390 R.A. Rocha et al.



carried out with sheep in Brazil; further studies aimed at
increasing the efficacy of nematophagous fungi in the
prophylaxis of sheep parasitic gastroenteritis are needed.
Other fungal species may be more adapted to the
Brazilian environmental conditions and should be tested
for the biological control of gastrointestinal nematodes.

The results obtained also reinforced the importance of
identifying animals in a flock that are highly susceptible
to parasites. This problem was observed in the present
study: some highly susceptible individuals were present
in both groups. Susceptible animals are responsible for a
great part of pasture contamination and, once identified,
they must be removed from the flock.

Acknowledgements

The authors thank CAPES for the grant to R.A.R. and
the National Council for Scientific and Technological
Development (CNPq) for the grants to A.F.T.A and J.V.A.

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