Hindawi Publishing Corporation

BioMed Research International

Volume 2015, Article ID 474879, 5 pages
http://dx.doi.org/10.1155/2015/474879

Research Article
Efficacy of Clonostachys rosea and Duddingtonia flagrans in
Reducing the Haemonchus contortus Infective Larvae

Manoel Eduardo da Silva,1,2 Fabio Ribeiro Braga,3

Pedro Mendoza de Gives,4 Miguel Angel Mercado Uriostegui,4 Manuela Reyes,5
Filippe Elias de Freitas Soares,6 Lorendane Millena de Carvalho,1
Francielle Bosi Rodrigues,6 and Jackson Victor de Araújo1
1
Departamento de Veterinária, Universidade Federal de Viçosa, 36570000 Viçosa, MG, Brazil
2
URECO/EPAMIG, 35650000 Pitangui, MG, Brazil
3
Universidade Vila Velha, Vila Velha, ES, Brazil
4
Área de Helmintologia, CENID-Parasitologia, INIFAP, Jiutepec, MOR, Mexico
5
Departamento de Microbiologı́a Ambiental y Biotecnologı́a, UAC, CAM, Mexico
6
Programa de Pós Graduação em Ciências Farmacêuticas (PPGF-UVV), Universidade Vila Velha, 29102920 Vila Velha, ES, Brazil

Correspondence should be addressed to Fabio Ribeiro Braga; fabioribeirobraga@hotmail.com

Received 29 January 2015; Accepted 10 April 2015

Academic Editor: José L. Campos

Copyright © 2015 Manoel Eduardo da Silva et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing
progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory
activity of fungal isolates of Duddingtonia flagrans and Clonostachys rosea species and its association on infective larvae (L3 ) of H.
contortus in microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an
aqueous suspension with 618 H. contortus L3 approximately. Group 1 was used as control and only received the infective larvae.
Groups 2 and 3 received D. flagrans chlamydospores and C. rosea conidia at doses of 5 × 106 . Group 4 received the combination of 5
× 106 D. flagrans chlamydospores + 5 × 106 C. rosea conidia. D. flagrans and C. rosea showed nematicidal effectiveness reducing by
91.5 and 88.9%, respectively, the population of H. contortus L3 . However, when used in combination efficiency decreased to 74.5%
predation of H. contortus L3 . These results demonstrate the need for further studies to determine the existence of additive effects,
synergistic or antagonistic, between these species.

1. Introduction measures of control is in most of the cases not based on

the adequate recommendations, that is, the general animal
Endoparasitic nematodiasis are considered as one of the state, body weight, diagnosis of parasitic burden, and other
main factors seriously affecting the livestock industry, since important factors that should be considered to establish a
they cause an important reduction not only in the animal successful control as quantity and quality of food, selection
weight but also their main products; that is, meat and of gastrointestinal parasitic genetically resistant animals, and
milk are also diminished [1]. Other problems derived from the presence of parasites resistance to chemical drugs [4].
nematode parasitic infections are the continuous expenses A number of alternatives of control different than the use
in chemical treatments and eventually the death of young of chemical drugs are being proposed, although the most
animals [2]. The use of chemical drugs is so far the unique promising measure of control is the use of natural nema-
method of anthelmintic control that is established around tode antagonists like nematophagous fungi [5, 6]. Allied to
the world [3]. Unfortunately the use of drugs and other chemical control, one of the most promising and sustainable
2 BioMed Research International

methods of control is biological control, which has shown Experimental Sheep Farm (INIFAP) in Hueytamalco Munici-
a good effectiveness either in laboratory trials or in pasture pality in the state of Puebla, Mexico. A H. contortus egg donor
experiments which is an important activity reducing the lamb was orally infected with an aqueous suspension contain-
free-living stages of mayor gastrointestinal helminths. This ing 350 larvae/kg of body weight. Twenty days after infection
method can be considered as a useful tool of control which the presence of H. contortus eggs in faecal samples was
reduces the economic losses caused by parasites and can detected through the McMaster technique. Faecal cultures
promote a higher animal productivity [7, 8]. were prepared by mixing faeces with polystyrene particles in
The species Clonostachys rosea belonging to the order plastic bowls. Water was added to the faecal cultures and the
Hypocreales (family: Bionectriacea) has being recorded as a mixture was homogenized to keep the cultures hydrated and
parasite of other microfungi and also as a nematode parasite, oxygenated to promote better larval hatching [20]. Faecal cul-
apart from being a saprophyte fungus [9]. This species tures were covered with paper foil and incubated for 7 days at
colonizes plants and it has attracted interest by workers as a room temperature (25–30∘ C). Infective larvae were extracted
potential biological control agent, since it produces volatile from faeces using the Baermann funnel technique [21]. The
organic compounds toxic for some living organisms [10, 11]. larvae were washed several times in density gradients of 50%
Duddingtonia flagrans is an “old friend” of scientists who sucrose solution and rinsed and suspended in sterile water.
envision biological control as an alternative to the treatment
of herd animals by chemical means and also in the future
2.3. Assay. The pots were conformed as follows: thirty-six
of prevention of high levels of contamination by nematodes
plastic (gardening) pots with a capacity of 100 grams (75
in public areas and squares used by humans [3, 12]. Again it
× 55 mm) were used. Seventy grams of soil acquired from
must be remembered here that the ability to pass through the
commercial nurseries was autoclaved at 1.2 atm and 115∘ C
gastrointestinal tract of domestic animals makes this species
for 15 minutes and added to the plastic pots. Every pot with
of fungus an eternal ally in combating nematodes present in
soil was added with 0.06 grams of a commercial mixture of
the environment [13].
different genera/species grass seeds; about 70 graminaceous
This fungal species is said to be the “most promising.” Its
seeds were added to the soil surface in order to obtain a
main focus of activity culminates in the predation of infective
homogeneous grass growing. Six mL of top water was added
helminth forms (in this case, nematode larvae), by means of
to every pot every day during 18 days of the experiment using
simple adhesive hyphae.
a Pasteur pipette to maintain the pots humidity.
The fungus D. flagrans belonging to the order Monil-
After germination the pots were randomly divided into
iales (family: Moniliaceae) is one of the most promising
four (4) experimental groups with 8 replicates each. All
microorganisms for biological control of ruminant parasitic
groups were added with 10 mL average of an acqueos sus-
nematodes [14]. This species is harmless to plants, animals,
pension with 618 H. contortus L3 approximately. Group 1 was
and human being [15, 16] and it is possible to isolate it mainly
used as control and only received the infective larvae. Groups
from soil samples and animal faeces [17]. This microorganism
2 and 3 received D. flagrans chlamydospores and C. rosea
possesses dual food habits, being saprobius in absence of
conidia at doses of 5 × 106 , respectively. Group 4 received the
nematodes and becoming either predatory or parasite of
nematodes in the presence of them [18]. combination of 5 × 106 D. flagrans chlamydospores + 5 × 106
Taking into account the representation of the world C. rosea conidia.
livestock and its socioeconomics, it is important to evaluate, The pots were maintained under a shading rate hedging
improve, and provide the use of technologies that can con- protected screen and after 12 days of interaction between
tribute to the development of this segment of agrobusiness. nematophagous fungi and parasitic nematodes, the whole
The aim of this study was to evaluate the predatory activity of nematode population from total soil and grass of every single
fungal isolates of the species C. rosea and D. flagrans and their pot was recovered using the Baermann Funnel method [21].
association on H. contortus infective larvae (L3 ) in microplots
with graminaceous forage plant. 2.4. Location. The experiment was conducted in CENID-
PAVET-INIFAP-MEXICO, located at 18∘ 53󸀠 04󸀠󸀠 N latitude
2. Material and Methods and 98∘ 51󸀠 34󸀠󸀠 W and 1615.16 meters above sea level, in the
municipality of Jiutepec, Morelos, Mexico.
2.1. Fungi. The C. rosea Yucatan strain, belonging to the CICY
collection, was used. This strain was maintained in 2% water- 2.5. Weather Data. Meteorological data including maximum
agar (2% WA) at the Department of Helminthology (CENID- and minimum temperatures, relative humidity, and rain-
PAVET-INIFAP-MEXICO). The D. flagrans fungal strain fall during the experiment were obtained from the closest
(FTHO-8) belonging to the Fungal Collection of CENID- experimental weather station (National Institute for Water
Parasitologı́a Veterinaria, INIFAP-Mexico, was used. This Technology), Jiutepec, Morelos, Mexico.
strain was originally obtained from a sheep faecal sample
from a farm at Fierro del Toro Village, Huitzilac Municipality,
Morelos State, Mexico [19]. 2.6. Statistical Analysis. Data were analysed using an ANOVA
test and the Tukey test was used as a complementary tech-
2.2. Nematodes. A H. contortus isolate was originally ob- nique for discriminating the mean different from the others.
tained from a naturally infected sheep from “Las Margaritas” Data were analysed using the software BioEstat 5.3.
BioMed Research International 3

Table 1: Number and percentage of reduction of Haemonchus contortus L3 recovered by the method of Baermann 12 days after interaction
with fungal isolates Clonostachys rosea and Duddingtonia flagrans, and association of these microfungi.

Treatment Control C. rosea D. flagrans D. flagrans + C. rosea

H. contortus 118 (±114)a 13 (±19)b 10 (±15)b 30 (±19)b
% reduction L3 0 88.9% 91.5% 74.5%
∗
Different small letters in rows indicate the existence of a statistical difference (𝑝 < 0.01).

3. Results and Discussion in grazing cattle by administering fungal pellets obtaining

a reduction of 47.8 and 56.67% of the number of larvae,
The results are shown in Table 1, which shows the mean, respectively, and these results were lower than those found
standard deviation, and the H. contortus larvae reduction in our study, although conducted in animals grazing.
percentage after being recovered from pots after 12 days of In the present study, the nematophagous fungus C. rosea
interaction between nematophagous fungi and nematode lar- reduced by 76.9% the number of H. contortus L3 under envi-
vae. The average temperature value during the experimental ronmental conditions. Similar results were recently obtained
period was 16.1∘ C, ranging from 15 to 33∘ C; the environmental using two isolates, C. rosea (Yucatán strain) and Clonostachys
relative humidity was 71.03%, ranging from 15.2 to 108% and sp.
7 mm of rain. These environmental conditions are considered One Campeche strain C. rosea reduced 84.2 and 59.5% H.
as ideal for the development of ruminant parasitic larvae [22] contortus L3 , respectively. These fungi also showed 94.3 and
and nematophagous fungi [23]. 95.9% reduction on the phytonematode Meloidogyne sp. (J2),
The results obtained using D. flagrans in reducing the respectively [26].
nematode larvae population into animal faeces have gen- Baloyi et al. [27] evaluated the in vitro predatory activity
erated the development of studies focused on the produc- of C. rosea (in faeces and in water) against trichostrongylids
tion of chlamydospores which are resistant stages of some larvae and they reported 69.9 and 89.3%, reductions, respec-
microfungi for the control of ruminant parasitic nematodes tively, of the number of trichostrongylids larvae in bioassays
[13]. The results of the present work are promising and made in sheep feces and water. On the other hand, Rodriguez-
an important reduction in the larval population recovered Martı́nez [28] evaluated in vitro predatory activity of C. rosea
from treated pots has shown evidence that fungi produce against Rhabditis sp., Caenorhabditis elegans, Panagrellus
satisfactory results with the use of 5 × 106 conidia and redivivus, Butlerius sp., and H. contortus and they recorded
chlamydospores per pot; and although this is only a model 71.9, 94.7, 92.7, 100, and 87.7%, larvae reductions, respectively.
of study, it gives a clear image about the potential use of Their results have shown evidence about the potential of these
this biological system of control of infective larvae on the species in the control of parasites of veterinary importance.
contaminated grass. These results are similar to those described in the present
The recovery larvae mean values have shown statistical work performed in soil pots.
difference among the treated groups when compared with Dong et al. [10] showed that the metabolites obtained
control group (𝑝 < 0.001). However, no differences were of C. rosea were lethal 24 hours after treatment to 50%
found among the treated groups (𝑝 > 0.005) (Table 1). Such of C. elegans, P. redivivus, and B. xylophilus larvae; in our
results corroborate those published of De Almeida et al. [24] work we obtained better results of predation for free-living
that demonstrated high effectiveness of the specie D. flagrans nematodes same test being done in vivo. Other researches
against different genera of parasites affecting ruminants such in phytopathology highlight the relevance and efficiency of
as Haemonchus, Teladorsagia, and Trichostrongylus. the species C. rosea as entomopathogenic against Cicadellidae
The nematophagous C. rosea fungus was able to reduce hemiptera, causing, respectively, 82.5 and 45% mortality in
by 88.9% the number H. contortus L3 . D. flagrans was able to Oncometopia tucumana and Tapajosa rubromaginata at 14
reduce by 91.5% the number H. contortus L3 . The association days after incubation [29]. Vega et al. [30] showed that C.
of the micro fungi D. flagrans and C. rosea was able to reduce rosea and B. bassiana associated were able to reduce 82.5%
74.5% of H. contortus (L3 ) infective larvae. of the insects Hypothenemus hampei in coffee beans and
Higher in vitro reductions (94.21–99.61%) in the H. Carreño-Perez et al. [31] showed that C. rosea reduction 79%
contortus L3 population by different D. flagrans isolates were of diseases caused by plant pathogenic fungus Phytophthora
recently found by Priego-Cortes [25], although it is important cactorum isolated on apple fruits.
to remark that this research was performed under in vitro According to Ayers et al. [32] a group of compounds
conditions on water agar plates. In the same work the in extracted from C. candelabrum showed 90% of efficacy
vitro activity of D. flagrans isolates, FTHO-8, M3, and DFIPC, against H. contortus. These compounds were synthesized by
showed 99.2, 98.23, and 98.8% reductions against the root- Rama Rao [11], the macrolide clonostachydiol derived from
knot nematode Meloidogyne sp. Such results are encouraging C. cilindrospora that showed 80–90% of reduction of H.
in searching for a fungal candidate as a potential agent of contortus in artificially infected sheep when subcutaneously
agricultural pest control. administered at a dose of 2.5 mg/kg, corroborating with 21
Assis et al. [5] evaluated the predatory activity of D. our results, although we have worked with environmental
flagrans and M. thaumasium in the control of nematodiasis control.
4 BioMed Research International

The association of the micro fungi D. flagrans and C. parasitic diseases of ruminants,” Alternative and Integrative
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[6] P. Mendoza-de-Gives and J. F. J. Torres-Acosta, “Biotechnologi-
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In the present study, the association of Clonostachys and nematophagous fungus Duddingtonia flagrans on Amblyomma
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designs under laboratory conditions and in the field. C. its chitinase,” Biocontrol Science and Technology, vol. 23, no. 5,
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Conflict of Interests [13] A. Paz-Silva, I. Francisco, R. O. Valero-Coss et al., “Ability of the
fungus Duddingtonia flagrans to adapt to the cyathostomin egg-
The authors declare that they have no conflict of interests.
output by spreading chlamydospores,” Veterinary Parasitology,
vol. 179, no. 1–3, pp. 277–282, 2011.
Acknowledgments [14] N. F. Ojeda-Robertos, J. F. J. Torres-Acosta, A. J. Ayala-Burgos,
C. A. Sandoval-Castro, R. O. Valero-Coss, and P. Mendoza-de-
The authors also would like to thank CNPq, Capes, Fapes, and Gives, “Digestibility of Duddingtonia flagrans chlamydospores
Fapemig for financial support and grant concession. in ruminants: In vitro and in vivo studies,” BMC Veterinary
Research, vol. 5, article 46, 2009.
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