Small Ruminant Research 223 (2023) 106980

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Small Ruminant Research

journal homepage: www.elsevier.com/locate/smallrumres

Evaluation of new formulations of nematophagous fungi Duddingtonia

flagrans to control gastrointestinal nematodes in post-weaning lambs in
Colombia Andean region
Jaime Andrés Cubides-Cárdenas a, 1, Jimmy Jolman Vargas Duarte b, Henry Grajales Lombana c, 2,
Elizabeth Céspedes-Gutiérrez d, 3, Martha Isabel Gómez-Álvarez d, 4,
Diego Francisco Cortés-Rojas d, *, 5
a
Corporación colombiana de investigación agropecuaria - AGROSAVIA, Sede Tibaitatá, Mosquera, Colombia
b
Universidad Nacional de Colombia, Instituto de genética, Colombia
c
Universidad Nacional de Colombia, Facultad de Medicina Veterinaria y de Zootecnia, Grupo de Investigación: Gestión Tecnologica e Innovación en Sistemas Pecuarios,
Colombia
d
Corporación Colombiana de Investigación Agropecuaria - AGROSAVIA, Sede Central, Mosquera, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: Gastrointestinal nematodes (GIN) can reduce or limit sheep production. Nematophagous fungi are natural en­
Biological control emies of GIN and can be used as a biological control tool by reducing the use of anthelmintic drugs. This study,
Bioproducts formulations conducted in the Colombian Andean region (Mosquera, Cundinamarca) under tropical conditions, aimed to
Integrated pest management
evaluate the effectiveness of novel granular formulations (E3, E16) based on Duddingtonia flagrans (native strain)
Anthelmintics resistance
against GIN in a controlled field trial. Four different groups of post-weaning lambs (n = 9) naturally infected with
GIN were allocated in separate paddocks and received daily fungi formulations identified as E3, E16, and fungus
dried biomass (DB) at a dosage of 1 × 106 chlamydospores/kg of body weight for 18 weeks, excepting control
group. Parasitological criteria such as the number of eggs per gram of feces (EPG) and larvae pasture count (L3/
Kg DM) recovered were monitored. The estimated EPG reduction of the developed formulations was 62.7 %, 80.2
%, and 62.3 % for E3, E16, and DB, respectively. There was a significant reduction between the treated groups
and the control group (p < 0.05). Regarding EPG, no significant differences arose between E3 and E16 formu­
lations. The formulation E16 reduced pasture larvae count up to 89 %. The effect of formulations persisted for at
least 42 days after the last fungus administration. Therefore, using these formulations would provide an alter­
native option to control GIN parasites on pasture and in animals.

1. Introduction The control of gastrointestinal nematodes is based on conventional

Gastrointestinal nematode diseases in cattle and sheep cause losses in
the livestock industry related to the reduction of weight gain and milk
production (Mendoza de Gives et al., 2022). Particularly in tropical re­
gions such as Colombia, the environmental conditions of humidity and
temperature are optimal for the development and survival of parasites
(Márquez et al., 2008).
anthelmintic drugs (Medina et al., 2014; Mendoza de Gives et al., 2022),
widely adopted due to their easy administration, high efficacy, a wide
margin of safety, and broad spectrum. However, the inappropriate use of
these anthelmintics and the lack of knowledge about integrated control
by producers caused problems such as (1) resistance of parasites to
synthetic molecules reducing the control capacity of current products,
(2) bioaccumulation of antiparasitic drugs in products derived from the

* Corresponding author.
E-mail address: dfcortes@agrosavia.co (D.F. Cortés-Rojas).
1
Orcid: 0000-0002-4286-2018
2
Orcid: 0000-0003-0827-6899
3
Orcid: 0000-0002-8587-2152
4
Orcid: 0000-0003-4919-2747
5
Orcid: 0000-0002-5618-6284

https://doi.org/10.1016/j.smallrumres.2023.106980
Received 8 February 2023; Received in revised form 8 April 2023; Accepted 11 April 2023
Available online 13 April 2023
0921-4488/© 2023 Elsevier B.V. All rights reserved.
J.A. Cubides-Cárdenas et al. Small Ruminant Research 223 (2023) 106980

livestock system such as meat and milk, (3) change in the beneficial 2.2. D. flagrans formulations
microbial flora present in manure and animals (4) increase in the fre­
quency of application of the products increase the cost of parasite con­ The strain of D. flagrans: AGROSAVIA: BGMA: H4–0001 was origi­
trol (Cubides et al., 2015; Gilleard et al., 2021; Medina et al., 2014). nally isolated from soil samples from the region of Cota, Cundinamarca –
The fungus Duddingtonia flagrans has a well-stablished nem­ Colombia (4.8099◦ N, 74.1018◦ W). Its use is authorized by the contract
atophagous capacity and high production of chlamydospores which are for access to genetic resources No. 168 of 2017. The biomass for the
resistance structures characterized by a thick wall protecting them in formulations was produced by following the solid-state fermentation
different environments of the ruminant gastrointestinal tract methodology described by Castillo-Saldarriaga et al. (2020). The
(Céspedes-Gutierrez et al., 2021; Ojeda-Robertos et al., 2009). biomass was dried at 35 ◦ C ± 2 ◦ C to reduce the moisture to 10–15 %,
Currently, there are two products based on D. flagrans for biological and subsequently, the coating formulations (Table 2) were sprayed on
control of gastrointestinal nematodes in ruminants one of them was the air-fluidized biomass using the Wurster fluid bed coating technique
developed in Australia (Bioworma®) (Healey et al., 2018); and the other (Glatt® Uni-glatt 8512).
one was created in Brazil (Bioverm®) (Braga et al., 2020). However,
those products are not registered in Colombia. 2.3. Formulation characterization
A Colombian native strain of the fungus D. flagrans was isolated and
the optimal conditions for biphasic fermentation to obtain a high The formulations were analyzed at microbiological and physico­
number of chlamydospores were developed (Castillo-Saldarriaga et al., chemical level, as follows.
2020). The design of delivery systems that protect fungus chlamydo­
spores through the gastrointestinal tract of animals is of utmost impor­ 2.3.1. Microbiological characterization
tance for obtaining a high-efficacy bio-product considering previous Solid formulation (5 g) was diluted in 50 mL of a Tween 80 solution
reports in which only 10 % of the administered chlamydospores were (0.1 % v/v), aliquots of this suspension were employed to evaluate
recovered in some cases (Ojeda-Robertos et al., 2009). In this work two chlamydospore concentration, viability and nematode predatory ability.
formulations based on D. flagrans were developed using a coating pro­
cess and adding excipients to maintain the integrity of the fungus after ■ Chlamydospores concentration (chlamydospores/mL): chlamydospores
passing through the gastrointestinal tract of the animals. The objective were counted by optical microscopy employing a Neubauer chamber
of this work was to evaluate the impact of the compositions developed to as described by (Céspedes Gutiérrez et al., 2021).
control gastrointestinal nematodes in post-weaning wool sheep under ■ Viability (CFU/g): was analyzed by plate count in Petri dishes with
Colombian high mountain conditions. Viability and in vitro nematode YMA Agar plus Triton X-100 (1 g/L) incubated for 5 days at 28 ◦ C ±
predatory ability were monitored during the production process 0.5 ◦ C following the methodology described by (Céspedes Gutiérrez
knowing in advance that these are the most important variables related et al., 2021).
to the expected effect (Céspedes Gutiérrez et al., 2021). ■ Nematode predatory ability (%): was performed using the Baermann
funnel technique following the methodologies described by (Men­
2. Materials and methods doza de Gives, 2011; Rodríguez-Martínez et al., 2018) using the
biological model of the nematode Panagrellus redivivus (Braga et al.,
2.1. Location 2012). The larvae recovered were counted, and the percentage of
nematode predatory ability was calculated compared to control
The field trial was conducted at the CIDTEO (Centro de Inves­ (Rodríguez-Martínez et al., 2018).
tigación, Desarrollo Tecnológico y Extension Ovina) of the Universidad ■ Contaminant concentration: to determine the presence and concen­
Nacional de Colombia, located in the agricultural center Marengo, tration of bacterial and yeast-like contaminants, samples were
Mosquera, Colombia at 2510 m above sea level (4◦ 40′ 57′′ N 74◦ 12′ 50′′ inoculated in Petri dishes with Nutritive Agar (24 h) and east malt
W). The weather conditions (maximum temperature, minimum tem­ extract agar (YMA) (48 h) correspondingly and incubated (at 26 ◦ C
perature and precipitation) during the experimental period (every 14 ± 0.5 ◦ C). Potato extract agar (PDA) was used to determine fungal
days) are presented in Table 1. contaminants. The incubation time was 7 days at 28 ◦ C ± 0.5 ◦ C.
The technological development of the formulations based on chla­ Results were expressed as colony-forming units (CFU).
mydospores of the nematophagous fungus D. flagrans was carried out at
the Bioproducts department of AGROSAVIA. The diagnostic coprology 2.3.2. Physicochemical characterization
techniques were carried out in the animal health laboratory - helmin­
thology, at the Tibaitatá Research Center Mosquera, Colombia. ■ Specific gravity: Briefly, 5 g of each formulation were weighed and
placed in a 100 mL beaker, 25 mL of distilled water were added. The
mixture was left overnight for moistening and transferred to a 100
mL volumetric flask, and the volume was completed with distilled
water. Equation 1 was employed to calculate the specific gravity
Table 1 (Materials testing FM 5–472, 2001).
Weather conditions during the experimental period in Mosquera, Cundinamarca
– Colombia.
Experimental Temperature Máx Temperature Mín Precipitation Table 2
day (ºC) (ºC) (mm) Composition of the two coating formulations applied over the fungal biomass of
0 16.6 5 4 Duddingtonia flagrans (% weight/weight).
14 20.6 9.6 0
Excipient E3 E16
28 21.4 5.2 1
42 18.6 4 0.8 Vegetal oil 50.5 52.6
56 21 7.2 13.3 Eudragit® S100 3.1 26.3
70 19.8 8 0 Tween® 80 0 15.8
84 20.6 4.8 0 Arabic gum 3.1 0
98 20.4 5 13 Kaolin 28.3 0
112 22 5.4 2 Soy protein isolate 15.0 0
126 20.8 4.6 0 Oleic acid 0 5.3

2
J.A. Cubides-Cárdenas et al.
Ws
GS =
(Ws + Wbw − Wbws
Where, Ws is the weight of the solid formulation, Wbw is the weight of
the volumetric flask with water, and Wbws is the weight of the volu­
metric flask with the stable formulation and water.
■ pH: 1 g of each formulation was diluted in 10 mL of distilled water
and homogenized with an immersion blender (KitchenAid®
KHB1231ER). A calibrated pH-meter (Consort®, C1010, Turnhout,
Belgium) was employed for analysis.
■ Moisture content: 0,5 g of each formulation were placed in the
moisture analyzer at 105 ◦ C (KERN, MLS50-3, Southampton, United
Kingdom) until constant weight.
■ Particle size: was analyzed by sieving. 200 g of each formulation were
placed in a column of sieves with a nominal opening of 4750 µm,
2000 µm, and 1000 µm. Sieves were shake for 10 min (Tyler 12) and
the amount retained in each sieve was weighed. Results were
expressed as percentages.
2.4. In vivo tests
The animals selected for the experiment were lambs of approxi­
mately 4 months old post weaning of the Hampshire and wool Creole
breeds, located in a pasture with a low load of gastrointestinal nema­
todes established by pre-trial grazing. For the evaluation, a mixed model
was used with a completely randomized design supported by a structure
of repeated measures over time. The animals were distributed in four
groups of nine lambs (n = 9) each. Group 1 was administered the
formulation (E3), group 2 was administered the formulation (E16),
group 3 was administered the dried fungus biomass (DB) and group 4
was the control treatment, to which no formulation or anthelmintic drug
was administered. The formulations were administered orally, five days
Fig. 1. Rotational grazing scheme for animals (n = 9) through the paddocks of Pennisetum clandestinum grass for each experimental group to evaluate the effect of
Duddingtonia flagrans formulations. *The occupation period for each paddock was 21 days. A cycle (63 days) was considered as the rotation through three exclusive
paddocks of each experimental group.
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Small Ruminant Research 223 (2023) 106980
a week, based on the work published by Paraud et al. (2005). The
amount of granular formulation to achieve the dose of 1 × 106 chla­
mydospores/Kg of body weight for each animal was mixed with a
commercial concentrate composed of 20 % protein 3 % fat, 9 % crude
fiber, and 10 % ashes. A rotational grazing scheme was carried out in a
monoculture meadow of Pennisetum clandestinum. Pasture was divided
by fencing and 3 paddocks were assigned to each experimental group
(Fig. 1), the occupation period for each paddock was 21 days giving 42
days of meadow rest. Each paddock was used exclusively by only one of
the treatment groups. A cycle (63 days) was considered as the rotation
through three exclusive paddocks of each experimental group. The
experiment lasted 18 weeks, therefore two cycles were completed dur­
ing the experiment. The response variables monitored were egg count
per gram of feces (EPG) weekly and pasture larvae count every 21 days.
EPG was evaluated according to Gordon and Whitlock (1934) tech­
nique; fecal samples were taken directly from the rectal ampulla of each
animal. For pasture larvae count samples from each paddock were
collected according to Minguetto et al. (2021) and processed according
to Molento et al. (2016), results were expressed as the number of larvae
per kilogram in dry basis (L3/ Kg db).
Any animal presenting an EPG higher than 4000 was dewormed
using Levamisole (Coopersol®, MSD).
The genera of gastrointestinal nematodes of the different experi­
mental groups were monitored weekly (100 larvae from each experi­
mental group) by means of stool cultures (Van Wyk and Mayhew 2013).
2.5. Post-cessation persistence effect
Once the administration of the formulations finished, the evaluation
of the response variables EPG and pasture larvae count (L3/kg dry basis
[db]) in the treated and control groups continued for two up to 42 days.
Two samplings were carried out at 21 and 42 days, the results were
compared with the last two reports of the in vivo test in which the
J.A. Cubides-Cárdenas et al. Small Ruminant Research 223 (2023) 106980

formulations were evaluated. Table 3

Physical, microbiological and biological characterization of formulations.

2.6. Statistical analysis Dried biomass E3 E16

Concentration 3.09 1.64 2.93

The selection of the variance/covariance structure for each variable (chlamydospores/g) ± 0.73 × 107 a ± 0.66 × 107 a ± 0.41 × 107 a
was carried out by comparing the BIC, AIC, and AICC criteria. Its se­ Viability (UFC/g) 5.90 3.30 5.60
± 0.00 × 106 a ± 0.14 × 106 b ± 0.99 × 106 a
lection comes from combining the lowest values of the three criteria. The
In vitro nematode 82.58 ± 10.71a 92.03 ± 6.55b 93.58 ± 4.92b
data were transformed into a log(x + 1) before the analysis and sub­ predatory ability (%)
jected to analysis of variance (ANOVA) with repeated measures and Bacterial contaminants 2.95 4.50 5.00
Tukey’s test using a randomized design at a probability level of 5 %. (UFC/g) ± 0.49 × 104 a ± 3.54 × 103 b ± 7.00 × 103 b
Yeast contaminants < 10 1.00
Statistical analyzes were performed with the GLIMMIX procedure of the <10
(UFC/g) ± 0.00 × 103
SAS 9.4 statistical package (SAS Institute Inc., Cary, NC, USA). For in Fungal contaminants 6.20 4.85 1.40
vivo test of all the measured variables, the assumptions of normality and (UFC/g) ± 0.57 × 104 a ± 0.78 × 104 a ± 0.28 × 104 b
homoscedasticity of the residuals were evaluated, from these analyzes it pH (-) 5.72 ± 0.04a 6.97 ± 0.03b 6.91 ± 0.06b
was decided to transform larvae and EPG count to normalize data, using Moisture (%) 3.92 ± 0.44a 0.82 ± 0.16b 2.70 ± 0.95a
Specific gravity (-) 1.233 ± 0.020a 1.304 ± 0.026b 1.354 ± 0.030b
the logarithm in base 10 of the observed value plus 100, to change the
Particle size 92.5 73.7 51.3
magnitude of the variable and reduce the number of zeros in the results. 2000–4750 µm (%) 7.5 26.3 48.7
The treatment effectiveness based on the variables was carried out 1000–2000 µm (%)
through a repeated measure complete randomized design, accordingly
* Values with the same letter do not present statistically significant difference
to the following model: with respect to time zero (Tukey p < 0.05).
yij = μ + τi + θj + (τi ∗ θk ) + oij + eij
in the control group, in fact two animals (Hematocrit level below 20 %)
yij = evaluatedvariable of this group required anthelmintic treatment, one in experimental day
56 and another later in experimental day 84. No anthelmintic treatment
μ = generalmean was needed in the groups treated with nematophagous fungi. The sta­
tistical model showed statistically significant differences (p < 0.05)
τi = treatmenteffect between the control group and the treated groups. The cumulative
reduction for the formulations were 62.7 %, 62.3 % and 80.2 % for DB,
θk = samplingtimeeffect E3 and E16 respectively, no significant differences were observed be­
tween formulation E3 and E16.
(τi ∗ θk ) = treatmentinteractioninsamplingtimes
In Fig. 3, it is observed that pasture larvae count increased gradually
after each rotation, especially for the control group (without fungi).
oij = randomerroramongovine(replicate)
During the last rotation cycle, statistically significant differences
eij = randomerrorinanimals(repeatedmeasure) (p < 0.05) were found in the mean larvae pasture count (L3/Kg db); the
E16 group showed a more substantial reduction (89%).
For the post-cessation persistence effect of nematophagous fungi, the The genera of gastrointestinal nematodes of the experimental groups
comparison of the results of the last two sampling times of the admin­ is presented in Table 4. No wide variations were observed among
istration of the formulation with the two sampling results of the post- groups, although in the case of the control group the Haemonchus genera
cessation months, was performed using the Wilcoxon test for related predominated.
groups.
3.3. Post-cessation persistence effect of formulations
3. Results
For the EPG variable, the results of the animals in the previous
3.1. Formulation characterization experimental groups increased considerably at 42 post cessation days
(Figs. 4 and 5), showing significant differences in the experimental
The formulations selected for this study, E3 and E16, as well as the groups with the Wilcoxon test. The counts for the E3 group increased
DB, were analyzed in terms of their physical- and microbiological from an average of 600 EPG to 2560 EPG. For the E16 group, the change
properties and in vitro predatory ability (Table 3). The concentration of was from 54.6 to 2042 EPG, and for the DB group, from 92.7 to 2645
chlamydospores in each formulation was higher than one × 107 chla­ EPG. These results indicate that after 42 days of cessation, the treated
mydospores/g; no significant differences were observed with the DB. groups achieve loads equal to the control group; therefore, the persis­
Regarding viability, values were lower than the concentration in all the tence time of the fungus is less than 42 experimental days.
formulations, which means that not all the chlamydospores produced by In the case of larvae in a pasture, there was an increase in the treated
the fungus can develop. The pH of the formulations dispersed in water groups after 21 days after the last administration of fungus in the groups
was very close to 7, this indicates that the added excipients do not alter (mean 775 and 232 L3/Kg db for E3 and E16, respectively).
the pH of the medium in which the formulation is diluted. There were no significant differences (p > 0.05) compared to the last
rotation and the rotation carried out 42 days after the previous fungi
3.2. In vivo test: EPG and pasture larvae count administration; the mean pasture larvae count was 1832 L3/Kg db for E3
and 2042 L3/Kg db for E16. These results indicate that the persistence
The EPG results during the field study in the different experimental period could be between 21 and 42 days because there are no significant
groups showedan evident effect of the use of nematophagous fungus as increases in larvae on the pasture before 21 days; however, after 42
biocontrol agent (Fig. 2). The mean EPG in the group that did not receive days, the EPG increased.
D. flagrans (control) is considerably higher than the other groups. An
increase of EPG in all groups was observed after a rainy period (Table 1)
in experimental day 48. This efffect began to decrease for the groups
treated with the nematophagous fungi formulations, but remained high

4
J.A. Cubides-Cárdenas et al. Small Ruminant Research 223 (2023) 106980

Fig. 2. Mean eggs per gram (EPG) over time of each experimental group receiving Duddingtonia flagrans formulations E3, E16, DB (dried biomass), and a group did
not receive fungi (without fungi).

Fig. 3. Mean pasture larvae count (L3 larvae/Kg dry basis) during the two cycles of paddock rotation for experimental groups receiving Duddingtonia flagrans
formulations: E3, E16, D.B. (dried biomass) and the group that did not receive fungi (without fungi).

coating with excipients that avoid the action of enzymes or the effect of
Table 4
pH fluctuation in the ruminal digestive tract such as Eudragit® S100.
Gastrointestinal nematodes genera of the experimental groups.
This polymer is intended for intestinal delivery and dissolves at pH
Nematode genera E3 E16 Dried biomass Control values above 7. Previous in vitro studies (data not presented) showed
Haemonchus 56 48 52 60 that this substance was compatible with the fungi chlamydospores, and
Teladorsagia 33 25 26 22 its physicochemical properties are adequate for the coating formulation
Trichostrongylus 5 10 14 9
process employed in this work. The higher amount of gastro-resistant
Cooperia 3 8 6 5
Nematodirus 2 7 2 2 polymer could be related to the higher efficacy of formulation E16.
Oesophagostomum 1 - - 2 The composition of the formulations developed for this study contains
Chabertia - 2 - - substances that prevent this type of damage. Vegetable oils could also
protect chlamydospores from enzymes in the rumen, ensuring a higher
number of viable chlamydospores come through the animal’s fecal
4. Discussion
matter to exert their effect. The pH of the formulation compositions
developed was monitored considering that this characteristic can
4.1. Formulation characterization
negatively affect the cell membrane of microorganisms and the devel­
opment of traps to capture nematodes (Hasanzadeh et al., 2012); usu­
Considering the mechanism of action of the nematophagous fungus
ally, neutral pH favors the integrity of fungus (Samaniego-Gaxiola,
D. flagrans, which implies the oral administration and the passage
2007). Accordingly, Grønvold et al. (1999) reported that pH values close
through the ruminant gastrointestinal tract, so protection of fungus
to 7 favor the nematophagous capacity of the fungus.
chlamydospores will help ensure that there are more chlamydospores in
Another strategy to avoid the degradation of chlamydospores in the
the feces. One strategy to protect the chlamydospores of the fungus is the

5
J.A. Cubides-Cárdenas et al.
Fig. 4. Eggs per gram (EPG) excretion after 21 and 42 days after cessation of Duddingtonia flagrans administration (formulations E3, E16, DB [dried biomass]) and
without fungi administration).
Fig. 5. Pasture larvae count (L3/Kg dry basis.) after 21 and 42 days after
cessation of Duddingtonia flagrans administration (formulations E3, E16, DB
[dried biomass]) and without fungi administration).
gastrointestinal tract of ruminants is to bypass the rumination process or
decrease the residence time. In this sense, the physical properties of the
administered substances, such as particle size and shape and specific
gravity, play an essential role (Pell et al., 2000). The specific gravity of
the formulations developed in the present study was among the ranges
that prevent retention in the ruminal cavity. Therefore, it is expected
that the fungus chlamydospores do not experience a high residence time
in the rumen due to the bypass phenomenon (Seyama et al., 2017).
According to a study published by Kaske and Engelhardt (1990), plastic
particles were orally administered to sheep. It was observed that the
specific gravity and its particle size influenced between 28 % and 59 %
of the variations in the retention time on the gastrointestinal segments.
In addition, Seyama et al. (2017) used plastic spheres to study how
specific gravity and particle diameter affected dairy cows’ excretion rate
and the rumination process. The results showed that particles with a
particular gravity between 1.19 and 1.41 and diameters between 6.35
and 7.94 mm are ideal for the design of post-ruminal release systems.
King and Moore (1957) also found that particles with a specific gravity
greater than 1.20 and less than 1.77 have a lower tendency to be
retained in the rumen, ideally being in a range between 1.20 and 1.40.
Despite the above, it should be considered that rumination time also
depends on the type of animal and the diet it consumes. For example, in
the case of forage digestion, a prolonged rumination process is desirable.
The dosage of chlamydospores of D. flagrans reported in the litera­
ture for sheep and cattle is 3 × 104 to 5 × 106 chlamydospores/Kg/day
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Small Ruminant Research 223 (2023) 106980
(Healey et al., 2018; Mendoza-de Gives et al., 2018). Considering that
the chlamydospore concentration of formulations E3 and E16 is high
(approximately 1 × 107 chlamydospores/g db), only a small amount of
these formulations would be required in order to achieve the recom­
mended dose (1 × 106 chlamydospores/Kg body weight).
4.2. Efficacy of the new formulations
The new formulations developed are a vital differential factor to
consider for the survival of the fungus through the GIT and on its
accumulated efficacy measured as the percentage of reduction of EPG or
pasture larvae count (L3/Kg MS). The results obtained in this work
suggest that the formulation composition and its physicochemical
characteristics could directly impact the nematode control and excretion
over time.
The in vivo efficacy studies described in literature employing
D. flagrans must be analyzed carefully since it will depend on several
variables such as the indicator being evaluated, the fungus strain, the
evaluation method, the animal species, the dose of chlamydospores
administrated to animals, the simultaneous implementation of other
strategies, the grazing system, and the environmental conditions
employed, among others (Canhão-Dias et al., 2020; Chavarría-Joya
et al., 2022).
The reduction of EPG obtained at the end of the assay with the
formulation E16 (up to 98,6%) was higher than the results found in
lambs by Vilela et al. (2016), Liu et al. (2020) and Silva et al. (2009) In
the study by Aguilar-Marcelino et al. (2016), a reduction range between
30.5% and 78.7% was obtained, using twice the dose of our experiment.
Healey et al. (2018) did not find significant differences in the reduction
after 15 weeks of fungi administration in a similar number of lambs to
those used in the present study due to the impact of salvage drenches but
did find significant differences in tracer lambs. Other authors, such as
Liu et al. (2020), reported a 92% reduction of EPG in goats after 120
days. However, the animals were kept in confinement conditions, which
reduces the possibility of re-infection from the meadow.
A recent assay used an aqueous suspension of D. flagrans without any
formulation excipient. After 17 days of the administration, a reduction
of only 62% was obtained, demonstrating the importance of the
formulation to improve its efficacy or reduce the administration period
(Chavarría-Joya et al., 2022).
Regarding the EPG, it is essential to highlight that the trapping ef­
ficiency also depends on the EPG/chlamydospore ratio in the feces
(Federica Sagües et al., 2021) since larvae in feces stimulate traps for­
mation and, therefore, the efficacy of D. flagrans. Our results showed
that the dose of 1 × 106 Kg of live weight for five days a week was
adequate for natural infections of sheep during the trial.
The efficacy measured as pasture larvae count could be considered
J.A. Cubides-Cárdenas et al.
the most relevant indicator of activity by D. flagrans. A reduction of
larvae counts by the fungi is subsequently reflected in EPG reduction in
animals. Larvae presence in the pasture is not influenced by animal
genetics, immune system, feeding, or physiological stage of the animals
(Hoste et al., 2016; Naeem et al., 2021; Nisbet et al., 2016); after all, it
can be influenced by the quantification methodology as well as by
environmental variables (Molento et al., 2016; Healey et al., 2018).
The ability of D. flagrans to reduce pasture larvae counts has been
proven by several authors; the formulation E16 developed had a mean
reduction of pasture larvae count of 74%, ranging from 55% to 95.6% in
the last rotation, is higher than the reported in the literature. Eysker
et al. (2006); Fontenot et al. (2003); Hernández Hernández and de Gives
Mendoza (2004). da Silveira et al. (2017) showed a synergism of
D. flagrans with Monacrosporium thaumasium to reduce pasture larvae
count.
Another strategy to reduce pasture larvae counts is the association of
a nematophagous fungus with an anthelmintic drug; Vilela et al. (2016)
observed a reduction of pasture larvae count more than 40% using
D. flagrans and levamisole.
Our study could be the first step in integrated control of gastroin­
testinal nematodes in sheep in Colombia, implementing three strategies:
restricted rotational grazing, nutritional supplementation, and nem­
atophagous fungi. In the case of specialized dairy, authors such as Voinot
et al. (2020) had already proposed an integrated control by imple­
menting both rotational grazing, nematophagous fungi, and anthel­
mintic treatments; with this approach, they managed to reduce EPG
loads by 95% in just two months. In the study, Voinot et al. (2020) re­
ported, that the use of anthelmintic drugs, rotational grazing, and the
nematophagous fungi D. flagrans and Mucor circinelloides could be
implemented in a two-year assay with encouraging results. This high­
lights the role of nematophagous fungi as part of an integrated parasite
management program".
4.3. Persistence effect of formulations
To the best of our knowledge, this is the first post-cessation report of
D. flagrans formulations after a continuous supplementation scheme.
Studies reported with the registered product Bioworma® show that the
fungus does not spread widely from the fecal matter, but penetrates
superficially into the soil by a mechanical translocation of chlamydo­
spores (Bampidis et al., 2020).
Our results indicate that the effect of the developed formulations (E3
and E16) could persist for 42 days after the cessation of fungus admin­
istration. After this period, there is a significant increase in the EPG of
the animals. This behavior could be related to the low nematode pop­
ulation that remains in the pasture after a high excretion of chlamydo­
spores; however, without any anthelmintic treatment, this population
increased progressively until day 42. Consequently, the EPG of the an­
imals in the treated groups increased. It should be noted that in the E16
group, the average increase in the EPG was mainly associated with one
animal in the whole group.
A recent study demonstrates the limited ability of D. flagrans to
control larvae after 20 cm of distance from fecal pads (Minguetto et al.,
2021). Therefore, the persistence effect could not be related to the
constant colonization of the fungus in the soil, instead, it could be
associated with nematode-load reduction by fungus in the pasture which
delays the meadow reinfestation.
Finally, the persistence effect of D. flagrans can be influenced by
several environmental factors such as solar radiation, air temperature,
and humidity. Baiak et al. (2021) showed the increase of pasture larvae
count after 21 days of follow-up and it was related to a significant in­
crease in air temperature. The relationship between humidity and sur­
vival of the larvae has been clearly established; higher humidity
promotes larvae survival and dissemination in the pasture (Rossanigo
and Gruner, 1995).
The formulations developed in E3 and E16 presented suitable
7
Small Ruminant Research 223 (2023) 106980
physical, chemical, and microbiological parameters to be administrated
to animals; the efficacy in terms of EPG was higher for E16 than either
the control or the DB. The effect of the formulations remains up to 42
days after the last administration in a schedule of 18 weeks.
Funding
This work was supported by the corporación colombiana de inves­
tigación agropecuaria AGROSAVIA project ID 1000449 and Ministerio
de Agricultura y Desarrollo Rural de Colombia (MADR) [Ministry of
Agriculture and Rural Development].
Declaration of Competing Interest
All authors have participated in (a) conception and design, or anal­
ysis and interpretation of the data; (b) drafting the article or revising it
critically for important intellectual content; and (c) approval of the final
version. This manuscript has not been submitted to, nor is under review
at, another journal or other publishing venue. The authors have no
affiliation with any organization with a direct or indirect financial in­
terest in the subject matter discussed in the manuscript.
Acknowledgments
The authors would like to thank the CIDTEO – Universidad Nacional
de Colombia, to John Sebastian Martinez Lopez, Juan Carlos Barrios
Murcia and Tatiana Aldana for their support in executing the activities
carried out in the current study.
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