Nematophagous fungi from Mexico with activity against the sheep ...

Artículo Original
Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 101-108
Nematophagous fungi from Mexico with activity
against the sheep nematode Haemonchus contortus
ACEVEDO-RAMÍREZ P.M.C. 1 , QUIROZ-ROMERO H. 1 , VALERO-COSS R.O. 2 ,
MENDOZA-DE GIVES P. 2 and GÓMEZ J.L 3
1
Departamento de Parasitología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de
México UNAM.
2
Laboratorio de Helmintología, CENID-Parasitología Veterinaria, Instituto Nacional de Investigaciones Forestales,
Agrícolas y Pecuarias, Jiutepec, Morelos.
3
Departamento de Bioquímica, Facultad de Medicina, UNAM.
ABSTRACT
The use of nematophagous fungi (NF) is a biological control alternative for gastrointestinal parasitic
nematodes (GIN) in ruminants. The aim to the present work was to isolate and to identify nematophagous
fungi with potential predatory activity against Haemonchus contortus larvae (L 3
) in some regions of
Mexico. Two hundred and fifty-nine samples from the soil, roots and ovine, caprine, bovine and porcine
feces were collected from diverse zones of the country. Samples were transferred into agar plates,
sprinkling technique was carried out and H. contortus L 3
were used to determine in vitro nematophagous
fungi predatory effect. Plates were incubated at room temperature and were revised under a stereoscopic
microscope at 7, 14 and 28 days. The taxonomic identification revealed the presence of seven species of
nematophagous fungi: Arthrobotrys oligospora, A. musiformis, A. kirghizica, A. brochopaga, A. conoides,
A. superba and Monacrosporium gephyropagum. This is the first report on the presence of A. kirghizica and
M. gephyropagum in Mexico. All species had predatory effect against H. contortus L 3
however, A. conoides
and M. gephyropagum showed the highest predatory activity with 93% and 83%, respectively.
Key words: Nematophagous fungi, Arthobotrys, Monacrosporium, Haemonchus contortus.
RESUMEN
El propósito del presente trabajo fue aislar e identificar hongos nematófagos con potencial actividad
contra larvas 3
de Haemonchus contortus en algunas regiones de México. Para ello se recolectaron 259
muestras de suelos, raíces y excrementos de ovinos, caprinos, bovinos y porcinos en diferentes localidades
del país. Estas muestras se transfirieron a placas Petri en delgadas capas y larvas 3
de H. contortus se usaron
para determinar “in vitro” efectos nematófagos predatorios de hongos. Las placas Petri se incubaron a
temperatura ambiente y se revisaron bajo microscopio estereoscópico a los 7, 14 y 28 días. La taxonomía
Recibido: 24 de Febrero de 2011. Aprobado: 08 de Mayo de 2001.
Correspondencia: Av. Universidad 3000, Coyoacan, C.P. 04510, México D. F., México.
Phone, fax : 52(55) 5622-5898.
E-mail: hquiroz@unam.mx, pedromdegives@yahoo.com, perlacevedoram@hotmail.com
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P. M. C. ACEVEDO et al.
reveló la presencia de 7 especies de hongos nematófagos Arthrobotrys oligospora, A. musiformis, A.
kirghizica, A. brochopaga, A. conoides, A. superba y Monacrosporium gephyropagum. Este es el primer
informe de A. kirghizica y M. gephyropagum en México. Todas las especies tuvieron efecto predatorio
contra l 3
de H. contortus, sin embargo, A. conides y M. gephyropagum mostraron la mayor actividad
predatoria con un 93% y 83% respectivamente.
Palabras clave: Nematophagus fungi, Arthrobotrys, Monacrosporium, Haemonchus contortus.
INTRODUCTION
Gastrointestinal nematode (GIN) control is
based on the use of a therapeutic chemical strategy;
nevertheless, the periodical and irrational use
of anthelmintics, subtherapeutic doses administration
and short periods of applications, as well as
the scarce alternation of different family of drugs,
have resulted in rapid appearance of parasites resistant
to chemicals. Haemonchus is no the exception,
there are resistant strains to bencimidazoles, imidazathiazoles
and macrocyclic lactones, specially
to ivermectin (Campos et al,1992; 2001; Torres et
al, 2003; Encalada-Mena et al, 2008). Thus, the use
of nematophagous fungi is a biological control alternative
for this parasite.
Although in Mexico, some nematophagous fungi
species have been identified (Lappe and Ulloa,
1982; Llerandi-Juarez and Mendoza de Gives,
1998); it remains as fundamental to perform studies
that allow to know adittional genera and species of
nematophagous fungi with potential against GIN of
ruminants. The aim of this work was to isolate and
to identify nematophagous fungi from diverse substrates
of several localities in Mexico with predatory
activity against H. contortus third stage larvae.
MATERIAL AND METHODS
Area of study. Material collection (soil, roots,
and ruminant and pig feces) was carried out from
September 2008 to June 2009 in several federal
entities of Mexico (Morelos, Puebla, Veracruz,
Estado de México, Guanajuato, Sinaloa, Hidalgo
and Distrito Federal). Samples were collected and
placed into polyethylene bags for shipment and
process at the laboratory.
Sampling of different substrates for the isolation
of nematophagous fungi. Samples consisting
of soil, roots and ovine, bovine and caprine feces
were taken from the soil surface and from a depth
of 5 cm. Samples were collected from different localities
of Mexico (Table 1). The collected material
was placed in separate sterile plastic bags, with its
respective identification. They were sent to the laboratory
of Helmintology at CENID-PAVET and to
the Department of Parasitology at the Facultad de
Table 1. Sampling collection site for predatory nematophagous fungi search
Study areas
Jardines del Cenid-Pavet, Jiutepec, Morelos
Rancho Experimental Las Margaritas, Hueytamalco, Puebla
Centro de Enseñanza, Investigación y Extensión en Ganadería Tropical
(CEIEGT), Martínez de la Torre, Veracruz
Centro de Enseñanza, Investigación y Extensión en Producción Agro-
Silvo-Pastoril (CEiepasp), Chapa de Mota, Edo. de México
Centro de Enseñanza, Práctica e Investigación en Producción y Salud
Animal (CEPIPSA); facultad de Estudios Superiores Zaragoza; Tlapan
San Miguel de Allende, Guanajuato
Facultad de Veterinaria de la Universidad Autónoma de Sinaloa, Culiacán,
Sinaloa
Ixmiquilpan, Hidalgo
Climate (García 1987)
Semiwarm
Warm tropical rain
Warm tropical rain
Template cool and log summer
Template cool and long summer
Dry, warm summer
Dry, cool summer
Dry, semiwarm
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NEMATOPHAGOUS FUNGI AGAINST HAEMONCHUS CONTORTUS
Medicina Veterinaria y Zootecnia de la Universidad
Nacional Autónoma de México for further analysis.
Samples were stored at 4ºC until their processing
(Su et al, 2007).
Nematophagous fungi isolation. The soil
sprinkling on water-agar plates technique described
by Barron, 1977 was used to isolate nematophagous
fungi. Chloramphenicol (500 mg/L) was added to
the culture media to avoid bacteria growth. The assay
was done by duplicate. Seven days after seeding,
an aqueous suspension with an undetermined
number of H. contortus larvae was added, they
were incubated at room temperature for another
7 days (Wyborn et al, 1969). For identification of
fungal structure (rings, nets, conidia) as well as the
detection of trapped nematodes, a stereoscopic microscope
was used for observation at 7, 14 and 28
days of incubation.
Taxonomic identification of nematophagous
fungi. Fungi identification was carried out by
observation of morphometric characteristics under
a light microscope. The taxonomic identification
codes published by Cooke and Godfrey (1964), Van
Oorschot (1985) and Rubner (1996) were used.
Sheep faecal cultures to obtain Haemonchus
contortus infective larvae. For the obtainment of
H. contortus larvae, fecal samples from a sheep,
previously infected with H. contortus L 3,
were used.
Feces were collected and a coproculture prepared
during 10 days at room temperature. Afterwards,
larvae were collected using Baermann fondle were
stored at 4ºC until their use (Hendrix, 1999).
Evaluation of the in vitro trapping capability
of nematophagous fungi against Haemonchus
contortus third stage larvae. Each fungus species
was grown into Petri dishes of 6 cm in diameter
with agar-water medium. Plates were incubated
for 3 weeks at room temperature (25-28°C). After
this period, one hundred H. contortus infective
larvae were placed on the surface of each plate
and incubated for 5 days at room temperature.
Ten replicates were made for each fungus species;
besides, agar-water dishes without fungus were
placed and used as control. After 5 days, the surface
of the Petri dishes were scrapped with a spatula
and washed with distilled water and the liquid
was collected into assay tubes and refrigerated for
2 hours at 4ºC to allow larvae migrate to the tube
bottom. The volume of the tubes was adjusted to 2
mL where larvae were found. Ten aliquots of 20 µL
were taken and the recovered larvae were counted
with the aid of a stereoscopic microscope.
Data interpretation. Statistical analyses.
Recovered larvae were counted and a capture percentage
was estimated. A non-parametric U-Mann-
Witney test was carried out to identify trapping difference
between species.
RESULTS
Nematophagous fungi identification: From
Figure 1. Arthobotrys oligospora. A: conidia (40X). B. Trapped larva (40X)..
Rev. Ibero-Latinoam. Parasitol. (2011); 70 (1): 101-108
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P. M. C. ACEVEDO et al.
Figure 2. Arthobotrys misifprmis. A: conidia (40X). B. Three-dimensional networks (10X).
Figure 3. Arthobotrys kirghizica. A: conidia (40X). B. Three-dimensional networks (10X).
Figure 4. Arthobotrys brochopaga. A: conidia, constrictor rings (10X). B. Trapped Haemonchus contortus (40X).
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NEMATOPHAGOUS FUNGI AGAINST HAEMONCHUS CONTORTUS
Figure 5. Monacrosporium gephyropagum. A: conidia (40X). B: Scalariform net (40X).
the total of 259 samples, in 29 (11.2%) it was
possible to isolate nematophagous fungi belonging
to 7 species. All species showed predatory effect
against H. contortus infective larvae. In Table
2, the number of collected samples that resulted
positive to predatory nematophagous fungi and
corresponding species is summarized.
In Figures 1-7, the different nematophagous
fungi identified in different study areas of Mexico
are shown. The majority of the positive samples
for nematophagous fungi were from warm weather
zones. In the temperate regions, positive samples
were found only in the Distrito Federal. In the lat-
Figure 6. Arthrobotrys superba conidia (40X).
Figure 7. Arthrobotrys conoides A: conidia (40X). B: Trapped Haemonchus contortus in three-dimensional network
(10X).
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P. M. C. ACEVEDO et al.
Figure 8. Haemonchus contortus larvae 3 (L 3
) trapping percentage by nematophagous fungi. Different species of
nematophagous fungi were in Petri dishes with agar-water (2%), at room temperature and after twenty-one days, 100 H.
contortus L 3
were added. Seven days later, the dishes were washed and the surface scrapped. Free larvae were collected
and trapping percentage was calculated.
Study
areas
Table 2. Nematophagus fungi isolation in different study areas
Positives samples
/ Total samples
Species
Samples
1 1/10 Arthrobotrys
Bovine feces colected garden
oligospora (Figure 1)
2 6/47 A. musiformis (Figure 2) Soil from garden, decaying soil
from coffee, orange tree and mint
pot
3 6/33 A. oligospora
A. musiformis
A. kirghizica (Figure 3)
Monacrosporium sp
4 0/55
5 2/34 Arthrobotrys sp
A. brochopaga (Figure 4)
6 40/9 A. oligospora
Monacrosporium gephyropagum
(Figure 5)
A. superba (Figure 6)
A. conoide (Figure 7
7 4/25 No identified
Soil from garden, decaying soil
Decaying soil
Sheep feces colected from soil
Soil from garden
Sheep feces with soil
Soil from garden, compost
Soil from garden
Soil from garden
8 1/15 A. oligospora Soil from garden
Total 29/259 (11,2%) 7 different species
ter region, four different species were identified
and the highest quantity of samples with predatory
nematophagous fungi was recorded. A. oligospora
was the most frequent species observed, since it was
found in several localities, followed by A. musiformis.
A. brochopaga fungi (producer of constrictor
rings) and A. kirghizica were identified in samples
collected from Sinaloa and Veracruz, respectively.
A. superba, A. conoides and Monacrosporium gephyropagum
were recorded in the Distrito Federal.
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NEMATOPHAGOUS FUNGI AGAINST HAEMONCHUS CONTORTUS
Trapping capability of nematophagous fungi:
All the fungi species shown predatory effect
against H. contortus infective larvae. There was
statistical difference in trapping capability between
species (P < 0.05). All species were different to the
control; there was no statistical difference between
A. oligospora, A. kirghizica, A. brochopaga and A.
musiformis, but A. conoides and M. gephyropagum
statistically differed from all other species (P <
0.05) with 94% and 83% trapping activity respectively.
In Figure 8, trapping percentages of H. contortus
L 3
in different nematophagous fungi (NF)
species at 28 days of NF culture are shown.
DISCUSSION AND CONCLUSIONS
There is a variety of predatory nematophagous
fungi in Mexico. A. oligospora is a worldwide
frequent species and has a high incidence in several
entities in Mexico, followed by A. musiformis.
Previously, these fungi have been recorded in
Morelos and in this study they were also recorded
in Veracruz, Hidalgo and Federal District. Arthrobotrys
conoides was recorded in a locality near the
Ajusco, just as Lappe and Ulloa (1982) had already
identified it in nearby places; hence this is a distribution
zone for this fungus. This is the first record
about the presence of A. kirghizica and Monacrosporium
gephyropagum in Mexico. A. superba has
been previously recorded in the State of Morelos
(Llerandi-Juarez and Mendoza-de Gives, 1998);
nevertheless, this is the first record made on this
species in the Distrito Federal. All mentioned fungi
species showed in vitro predatory activity against
H. contortus infective stage.
In comparison to other countries, in Germany, 9
species of Arthrobotrys, 2 species of Monacrosporium,
1 species of Duddingtonia, were identified;
the large number of predatory nematophagous fungi
isolated might be due to the fluorescence technique
that they used (Saxena and Lysek, 1993). A. conoides,
A. musiformis, A. oligospora and A. superba
have been identified in Germany, Scotland and in
this study, also A. gephyropagum and A. kirghizica
and Drechslerella brochopaga (Sexena, 2008) were
similarly recorded in Scotland and Mexico with the
difference that in Scotland A. gephyropaga and Dr.
brochopaga were most frequent and in this study it
was A. oligospora.
In the province of Yunan, China, found 17 different
species from 660 fecal samples (Su et al,
2007), most of them belonged to A. oligospora and
A. musiformis, which coincides that these species
were the most frequently recorded.
Kelly et al, (2009) collected 12 species of
nematophagous fungi in 120 samples from Irish
pastures. From these, Monacrosporium cianopaga,
Duddingtonia flagrans and D. coniospora were
detected in fresh feces samples. In contrast, in
the present study only four positive samples to A.
oligospora and A. brochopaga were isolated from
bovine feces, and A. kirghizica was isolated from
ovine feces; nevertheless, it worth to mention that
samples were collected from the soil; therefore,
the presence of fungi on feces could have been a
secondary contamination and not necessarily that
they had already been on them, hence having
passed through the digestive tract of the animals.
No predatory nematophagous fungi were identified
in ovine, caprine, bovine and porcine feces
samples, probably because they are pen confined
animals and it coincides with the recorded by Larsen
et al, (1994) who only observed a scarce number
of fungi from ruminant feces.
Predatory nematophagous fungi positive samples
correspond, in their majority, to soil collected
from the garden as decomposed matter or under
ornamental or aromatic plants. By the number of
positive samples, it can be concluded that nematophagous
fungi are more widely distributed in
warm humid zones, its presence decreases in temperate
zones, although this must be taken with
caution.
Gonzalez-Cruz et al, (1998) recorded a trapping
percentage of 93.1% of M. gephyropagum against
Strongyloides papillosus, in contrast with the
present study where trapping activity was 84% for H.
contortus L 3
. Several species of Arthrobotrys genus
have demonstrated to have around 80% trapping
activity against H. contortus infective larvae; A.
oligospora was the most active (Waller and Faedo,
1993), which contrasted with the present study,
since A. conoides resulted with a higher trapping
percentage, followed by M. gephyropagum.
It is concluded that in Mexico there are
nematophagous fungi with predatory effect
against H. contortus L 3,
from which A. conoides
and M. gephyropagum had a high trapping
percentage.
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P. M. C. ACEVEDO et al.
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Acknowledgements: Special thanks to CONACYT for the
scholarship granted to Perla Maria del Carmen Acevedo-
Ramirez, to Dr. Enrique Liebano for to provide H. contortus
L 3
and to Ana Arana Contreras, Esau Villarreal Olvera,
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collaboration in the collection and handling of samples.
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