VETERINARSKI ARHIV 69 (2),
69-78, 1999

ISSN 1331-8055 Published in
Croatia

Passage of nematode-trapping fungi
through
the gastrointestinal tract of calves

Jackson Victor de Araújo*, Marcelo
Antônio Stephano,
and Weverton Marcos Sampaio

Departamento de Veterinária,
Universidade Federal de Viçosa, Viçosa-MG, Brazil

* Contact address:
Prof. Dr. Jackson
Victor de Araújo,
Departamento de Veterinária, Universidade Federal de
Viçosa, CEP 36571-000, Viçosa-MG, Brazil,
Phone: 55 031 8991464, Fax: 55 031
8992314, E-mail: [email-masked]

Araújo, J. V., M. A. Stephano, W. M.
Sampaio: Passage of nematode-trapping fungi through the gastrointestinal tract
of calves. Vet. arhiv 69, 69-78, 1999.

ABSTRACT

In vivo experiments were
performed to test six isolates of nematode-trapping fungi: two of
Arthrobotrys robusta, one of A. conoides, one of Monacrosporium
ellypsosporum, one of M. thaumasium and one of M. sinense for
passage through the gastrointestinal tract of calves without losing the ability
to entrap infective Haemonchus placei larvae. Following administration of
conidia and mycelium of fungi isolates to the calves, two isolates of A.
robusta, one isolate of A. conoides and one isolate of M.
thaumasium met that goal when harvested from faecal samples and was later
able to entrap the infective H. placei larvae. There was no evidence that
the other isolates (M. ellypsosporum and M. sinense) passed
through the gastrointestinal tract of the calves. Isolation from the faeces
occurred from between 15 and 110 h following oral administration of fungi, with
more isolation, mainly at the 15th hour.

Key words: biological control,
nematode-trapping fungi, Arthrobotrys, Monacrosporium, nematodes,
calves

Introduction

The fungi antagonistic to nematodes
consist of a wide variety of organisms, including nematode-trapping or
predacious fungi, endoparasitic fungi, parasites of nematode eggs and cysts, and
those producing nematotoxic metabolites. It is remarkable that fungi belonging
to highly divergent orders and families occur in each of the above groups
(MANKAU, 1980).

The predacious group, which includes
the genus Arthrobotrys and Monacrosporium, produces an extensive
system of hyphae in the environment. Along the hyphae there are organs able to
capture living nematodes (BARRON, 1977).

The fungi inhabit a wide variety of
substrates including soil, moulds, animal dung, decaying plant material, root
plants and others. The most practical use of nematophagous fungi in the control
of animal parasitic nematodes is by means of the oral administration of fungal
material. After this material passes through the gastrointestinal tract of
animals and is eliminated, together with the faeces, into the environment,
faecal material is colonised by these fungi and a close contact between the
recently hatched larvae and the fungi takes place, promoting the production of
traps and further capture and death of nematodes. However, most
nematode-trapping fungi are extremely susceptible to destruction by the severe
conditions met in the gastrointestinal tract.

The present study was designed to
select nematophagous fungi of the genera Arthrobotrys and
Monacrosporium which are able to pass through the gastrointestinal tract
of calves without loss of viability to prey on nematodes.

Materials and methods

Six isolates of nematode-trapping
fungi, two isolates of Arthrobotrys robusta (A and B), one isolate of
A. conoides (C), one isolate of Monacrosporium ellypsosporum (D),
one isolate of M. thaumasium (E) and one of M. sinense (F) were
obtained from Brazilian soil and kept in small flasks containing 2%
Potato-Dextrose-Agar (2% PDA) at 4 °C.

Free-living nematodes (Panagrellus
spp) originating from Brazilian soil were cultivated at room temperature in
Petri dishes containing dampened and mashed oat flakes. The nematodes were
extracted from surrounding material using the Baermann funnel.

Two Holstein calves, 100 kg of live
weight each and free of helminth infection, were infected with 1000 infective
Haemonchus placei larvae per kg of body mass. After 30 days of parasitic
infection, and for a period of one month thereafter, daily faecal samples from
the animals were obtained and, along with sterile charcoal, were kept at 26 °C
for two weeks. Larvae were recovered in a Baermann apparatus, with water at 42
°C and washed three times with 0.9% NaCl physiological solution, and were then
stored at 4 °C for one to two months. In order to obtain H. placei and
Panagrellus spp free from bacteria and fungi, the nematodes were washed
ten times in distilled water by centrifugation at 1000 rpm for five minutes. The
nematodes were stored at 4 °C for one week in a solution containing 0.05% of
streptomycin sulfate, 0.01% of chloramphenicol and 0.05% of amphotericin B.
Washing twice with distilled water was repeated. Before their use, nematodes
were maintained at 25 °C in the light for six hours and their viability was
checked by stereoscopic microscope.

Mycelium grown of the fungi was
performed in liquid medium of KADO and HESKET (1970) after 7 days of incubation
at 25 °C in the dark. A part of the liquid medium was supplemented with 0.05 mg
of L-Phenilalanine and 0.05 mg of L-Valine per ml of medium in order to induce
the trap formation in the isolates according to NORDBRING-HERTZ (1973). Conidia
of the isolates were obtained from the stock (2% PDA) and cultures were
transferred to Petri dishes containing calf faecal agar made with 50 g macerated
faeces, filtered through a 61 µm sieve. This filtrate was adjusted to 500 ml,
added to 2% agar-agar and then autoclaved for 15 min. at 120 °C. To these dishes
was added 1 ml suspension with 1000 Panagrellus over a period of 2 days.
On the 5th day the dishes became completely overgrown by the various
fungal isolates. Two ml of distilled water was added to each Petri dish, conidia
removed with a delicate brush and the isolated fungal material stored in 50 ml
Erlenmeyer flasks.

Twenty-four, six- to eight-month-old
housed male Holstein × Zebu calves were randomly separated into six (four
animals in the test of each isolate) and four groups of one animal each: groups
1, 2, 3 and 4.

Group 1 - Animals received
20×106 conidia; orally.

Group 2 - Animals received 100 g
mycelium with trap, orally.

Group 3 - Each animal received 100 g
mycelium without trap, orally.

Group 4 - Each calf received 100 ml
water.

Five days before and five days
following the administration, each animal was fed daily an autoclaved special
ration of 1 kg of crushed corn and 3 kg of Pennisetum purpureum grass to
control other nematodes and fungi.

Faecal samples were collected from each
animal at 15, 18, 21, 24, 48, 72, 96 and 110 h after administration. Two grammes
of faeces were removed from these samples and individually added to 9-cm Petri
dishes containing 2% water agar at 25 °C, in the dark. The dishes were baited
with 1000 infective H. placei larvae. The amount of conidiophore growth
was observed daily. A count was performed at 24-h intervals from the time when
the material was placed on the Petri dishes, during a period of 10 days for the
Arthrobotrys isolates and during a period of 15 days for the
Monacrosporium isolates. These assays were repeated three times.

Results and discussion

Figures 1, 2, 3 and 4 show the number
of conidiophores in the faecal samples after administration of the A (A.
robusta), B (A. robusta), C (A. conoides) and E (M.
thaumasium) isolates, respectively. No fungi were found in the other faecal
samples belonging to the calves after administration of the D (M.
ellypsosporum) and F (M. sinense) isolates. The A and C isolates
presented a greater production of conidiophores than the B and E isolates. In
all the isolates that passed through the gastrointestinal tract of the calves in
group 1 (animals received 20×106 conidia, orally) the growth curve
was higher than in the other groups. There was no fungal growth in the dishes
containing faeces of the calves used as control (group 4). Ten days after
administration of the infective H. placei larvae on isolates that passed
through the gastrointestinal tract of the calves, no infective larvae free from
fungi trapping were observed.

Fig. 1. Amount of the conidiophores
of A (Arthrobotrys robusta) isolate, which appeared on 2% wateragar after
the administration conidia (G1); mycelium with trap (G2) and mycelium without
trap (G3)

Fig. 2. Amount of the conidiophores
of B (Arthrobotrys robusta) isolate, which appeared on 2% wateragar after
the administration conidia (G1); mycelium with trap (G2) and mycelium without
trap (G3)

Fig. 3. Amount of the conidiophores
of C (Arthrobotrys conoides) isolate, which appeared on 2% wateragar
after the administration conidia (G1); mycelium with trap (G2) and mycelium
without trap (G3)

Fig. 4. Amount of the conidiophores
of E (Monacrosporium thaumasium) isolate, which appeared on 2% wateragar
after the administration
conidia (G1) and mycelium without trap (G3)

According to LARSEN et al. (1991) the
major problem in the use of nematophagous fungi as bio-control agents is the
deposition of the fungal material in dung pats where the entrapment of the
parasite larvae should take place. The most obvious possibility would be to add
nematophagous fungi to the alimentary tract without loss of viability. The
present study was designed to select trapping fungi of the genus Arthrobotrys
and Monacrosporium, according to the above explanation. In this
experiment, the A, B, C and E isolates were successful in passing through the
gastrointestinal tract of calves, subsequently demonstrating its viability to
prey on infective H. placei larvae. The D and F isolates did not perform
as well, which could be due to their possible destruction while passing through
the gastrointestinal tract of the calves.

These results demonstrate the
importance of screening nematode-trapping fungi according to their ability to
pass through the gastrointestinal tract before administering them in
bio-control. HASHMI and CONNAN (1989) were first to mention this passage after
performing oral administration of A. oligospora conidia on calves.
Further, LARSEN et al. (1992) achieved this goal using isolates of A.
oligospora, A. superba and Duddingtonia flagrans, as well as
GRONVOLD et al. (1993), WOLSTRUP et al. (1994), NANSEN et al. (1995) using D.
flagrans and ARAÚJO et al. (1996) using A. robusta.

WALLER et al. (1994) demonstrated
through an in vivo experiment that the average time spent in the passage
of the conidia through the digestive system of bovines after oral administration
is about 24 h. In the present study, this time varied from 15 to 110 h after
oral administration of fungi in calves, with more isolation mainly at the
15th h.

It is very important to screen
nematophagous fungi in each location as they can be effective, or even be found,
in certain ecological niches. Future experiments will demonstrate whether our
fungi isolates can be used for the biological control of gastrointestinal
nematodes of grazing calves under natural conditions. The final challenge is to
develop an efficient, low cost means of scaling up production of fungal material
to satisfy industrial needs for the commercial exploitation of this technology.

References

ARAÚJO, J. V., A. P. NETO, M. H. F.
AZEVEDO (1996): Screening parasitic nematode-trapping fungi Arthrobotrys
for passage through the gastrointestinal tract of calves. Arq. Bras. Med.
Vet. Zootec. 48, 543-552.

BARRON, G. L. (1977): The
Nematode-Destroying Fungi. Canadian Biological Publications. Guelph.

GRONVOLD, J., J. WOLSTRUP, M. LARSEN,
S. A. HENRIKSEN, P. NANSEN (1993): Biological control of Ostertagia ostertagi
by feeding selected nematode-trapping fungi to calves. J. Helminthol.
67, 31-36.

HASHMI, H. A., R. M. CONNAN (1989):
Biological control of ruminant Trichostrongylids by Arthrobotrys
oligospora, predacious fungus. Parasitol. Today 5, 28-30.

KADO, C. I., M. G HESKET (1970):
Selective media for isolation of Agrobacterium, Corynebacterium,
Erwinia, Pseudomonas and Xanthomonas. Phytopathol. 60,
969-976.

LARSEN, M., J. WOLSTRUP, S. A.
HENRIKSEN, C. DACKMAN, J. GRONVOLD, P. NANSEN (1991): In vitro stress
selection of nematophagous fungi for biocontrol of parasitic nematodes in
ruminants. J. Helminthol. 65, 193-200.

LARSEN, M., J. WOLSTRUP, S. A.
HENRIKSEN, J. GRONVOLD, P. NANSEN (1992): In vivo passage through calves
of nematophagous fungi selected for biocontrol of parasitic nematodes. J.
Helminthol. 66, 137-141.

MANKAU, R. (1980): Biological control
of nematode pests by natural enemies. Ann. Rev. Phytopathol. 18, 415-440.

NANSEN, P., M. LARSEN, J. GRONVOLD, J.
WOLSTRUP, A. ZORN, S. A. HENRIKSEN (1995): Prevention of clinical
trichostrongylidosis in calves by strategic feeding with the predacious fungus
Duddingtonia flagrans. Parasitol. Res. 81, 371-374.

NORDBRING-HERTZ, B. (1973):
Peptide-induced morphogenesis in the predacious fungus Arthrobotrys
oligospora. Physiol. Plant. 29, 223-233.

WALLER, P. J., M. LARSEN, M. FAEDO, D.
R. HENNESSY (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., J. GRONVOLD, S. A.
HENRIKSEN, P. NANSEN, M. LARSEN, H. O. BOGH, B. ILSOE (1994): An attempt to
implement the nematode-trapping fungus Duddingtonia flagrans in
biological control of trichostrongyle infections of first year grazing calves.
J. Helminthol. 68, 175-180.

Received: 10 August 1998
Accepted: 5
May 1999

Araújo, J. V., M. A. Stephano, W. M.
Sampaio: Prolazenje nematofagnih gljivica kroz probavnu cijev teladi. Vet. arhiv
69, 69-78, 1999.

SAZETAK

Pokusima in vivo istrazeno je
sest izolata nematofagnih gljivica: dva od Arthrobotrys robusta, jedan od
A. conoides, jedan od Monacrosporium ellypsosporum, jedan od M.
thaumasium i jedan od M. sinense, na prolaz kroz probavnu cijev
teleta bez gubljenja sposobnosti da blokiraju invazione licinke oblica
Haemonchus placei. Nakon aplikacije izolata konidija i micelija gljivica
teladi, dva izolata od A. robusta, jedan od A. conoides i jedan od
M. thaumasium su postigli cilj da su nakon njihova prikupljanja iz
uzoraka balege bili sposobni blokirati invazione licinke Haemonchus
placei. Za druga dva izolata (M. ellypsosporum i M.
sinense) nije bilo znakova da su prosli kroz probavni trakt teladi.
Izolacija iz balege uspjela je izmedu 15 i 110 sati nakon oralne aplikacije
gljivica, s time da su novi izolati uspjevali najvise u 15. satu.

Kljucne rijeci: bioloska
kontrola, nematofagne gljivice, Arthrobotrys, Monacrosporium,
nematodi, oblici, telad

Back