Biological Control 182 (2023) 105219

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Biological control of gastrointestinal nematodes in horses fed with grass in

association with nematophagus fungi Duddingtonia flagrans and
Pochonia chlamydosporia
Tábata Alves do Carmo a, *, Mateus Oliveira Mena a, Isabela de Almeida Cipriano a,
Giordani Mascoli de Favare a, Gabriel Jabismar Guelpa a, Sara da Costa Pinto a,
Alessandro Francisco Talamini do Amarante b, Jackson Víctor de Araújo c,
Ricardo Velludo Gomes de Soutello a
a
Universidade Estadual Paulista (UNESP), School of Agrarian and Technological Sciences (FCAT), Dracena, SP 17900-000, Brazil
b
Institute of Biosciences – UNESP, Botucatu, SP, Brazil
c
Federal University of Viçosa - UFV, Avenue Peter Henry Rolfs, Viçosa, Minas Gerais 36570-900, Brazil

H I G H L I G H T S

• D. flagrans and P. chlamydosporia fungus as alternative for the biological control.

• Reduction of EPG after oral administration of fungi.
• Animals treated with fungi showed a greater reduction of L3 in the pasture.
• Fungal association helps to reduce the use of macrocyclic lactones.

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

Keywords: Biological control with nematophagous fungi can be used to reduce populations of intestinal parasites, since
Anthelmintic these are their natural antagonists, minimizing the use of chemical anthelmintics. Therefore, the objective of the
Helminthiasis present work was to evaluate the combined effect of two nematophagous fungi Duddingtonia flagrans and
Macrocyclic lactones
Pochonia chlamydosporia in the biological control of gastrointestinal helminthoses in horses kept on pasture.
Nematodes
Thirty six mares were divided into three groups, one control and two trated, for twelve months. As for the group
of animals treated with fungi, inwhich animals in which the animals received a dose of 1 g of formulation
containing 105 chlamydospores of D. flagrans and 105 chlamydospores of P. chlamydosporia per gram of the
commercial product for each 10 kg of body weight, per day. The group Abamectin, with the animals being
treated every 3 months with the anthelmintic 1% abamectin, at a dose of 0.2 mg/kg, administered orally. The
proceeding of all treatments was evaluated based on the count of eggs per gram of faeces (EPG) with samples of
faeces collected every 28 days and preparation of fecal cultures to identify the different genera of infective
larvae, count of infective larvae retrieved from the pasture and microbiological analysis of the faeces. The data
were submitted for analysis of variance and means were compared by Tukey’s test (5%). The overall average of
EPG in the control group was 635 (±61.7); as for the fungi group, 385 (±60.4) and in the abamectin group 313
(±67.3), showing a statistically significant difference in the group treated with fungi and abamectin in com­
parison to the control group, with the control group having the highest averages, while the two other treated
groups were overall similar. In the count of infective larvae retrieved from the pasture, the general averages
presented in the control group was 2267 (±789.4), the group treated with fungi 1100 (±384.8) and the group
abamectin 3115 (±1038.4), writing down that the three groups differed significantly from each other, given that
the group being treated with fungi presented the lowest overall average. The fecal cultures showed a predom­
inance of small strongyles in the three groups. It is concluded that the use of Duddingtonia flagrans and Pochonia

* Corresponding author.
E-mail address: tabata.alves@unesp.br (T. Alves do Carmo).

https://doi.org/10.1016/j.biocontrol.2023.105219
Received 8 September 2022; Received in revised form 15 March 2023; Accepted 18 March 2023
Available online 23 March 2023
1049-9644/© 2023 Elsevier Inc. All rights reserved.
T. Alves do Carmo et al.
chlamydosporia fungi is efficient in the control of gastrointestinal nematodes in horses kept on pasture, promoting
a reduction in pasture infestation by infective larvae and so the degree of helminthoses.
1. Introduction
Brazil has about 6 million horses, which allows the country to occupy
the 3rd position in the ranking of countries with the largest equine herds
in the world, only behind the United States and Mexico (Anualpec,
2021). Most of the horse breeding in Brazil is carried out under an
extensive regime, in which the animals are kept in pastures contami­
nated with infective nematode larvae, a factor that constantly favors
helminthoses (Braga, 2009).
Gastrointestinal helminth infections stands out due to the conse­
quent losses caused such as loss performance loss in racing animals,
gastric and intestinal colic, in addition to diarrhea in foals (Lagaggio
et al., 2007).
The main method to control helminths, in most farming systems, is
the usage of chemical anthelmintics (Martinho, 1997). The main
chemical groups used to control helminthoses in horses are benzimid­
azoles and macrocyclic lactones (Pérez-Álvarez et al., 2013). The dif­
ferences between these classes are the modes of action for each one
(Martinho, 1997).
The impasse of using commercial anthelmintics has been their
misuse, indiscriminately, not associated with auxiliary control strategies
(Molento, 2005). Because of this, the current world situation is parasitic
resistance to many classes of commercial anthelmintics available on the
market (Nielsen et al., 2014). Consequently, the spread of parasiticide
resistant nematode populations has become a serious threat to animal
health and production in several countries.
Therefore, it is required to assign new forms of parasite control,
promoting the reduction in numbers of infective larvae in the pastures,
thus causing a decrease in reinfection rates of animals and consequently
reduction the use of chemical anthelmintics (Araújo et al., 2004a, b;
Braga et al., 2009).
Biological control with nematophagous fungi can be used to reduce
parasite populations, as these are their natural antagonists. The use of
nematophagous fungi in biological control is also beneficial due to the
synergism in chemical control, which achieves greater action over the
infective forms present in the faeces, as well as the adult helminths
present in the gastrointestinal tract (Araújo & Ribeiro, 2003; Braga et al.,
2014).
Duddingtonia fagrans is widely used out as the ideal biocontrol agent,
proven to be efective in controlling domestic animals gastrointestinal
parasites (Buzatti et al., 2017; Vilela et al., 2018, 2020; Luns et al., 2018;
Rodrigues et al., 2018). This fungus produces chlamydospores, which
are highly resistant structures that when ingested resist digestion and
pass through the gastrointestinal tract of animals (Larsen et al., 1992).
They are eliminated in the faeces and are capable of germinating and
destroying the infecting larvae, thus interrupting the life cycle of the
parasite in the environment (Araújo et al., 2021; Baiak et al. 2021).
Pochonia chlamydosporia parasitises eggs and female nematodes
(Dallemole-Giaretta et al., 2013; Vieira et al., 2019) and belongs to the
group to the nematophagous fungal group known as“ovicidal fungi”
(Araújo et al., 2021).
Thus, the aim of this study was to evaluate the combined action of
Duddingtonia flagrans and Pochonia chlamysdoporia fungi as an alterna­
tive in the biological control of nematodes in horses kept on naturally
infected pasture, compared to conventional treatment with
anthelmintic.
2
Biological Control 182 (2023) 105219
2. Methods
2.1. Region and development
The study was carried out at Córrego Seco Farm, located in the city of
Castilho in the state of São Paulo, in Brazil (latitude 20◦ 52′ 09.0′′ south
51◦ 29′ 22.9′′ west longitude), in partnership with the Faculty of Agri­
cultural and Technological Sciences (FCAT), UNESP - Dracena Campus,
from November 2020 to November 2021.
2.2. Experimental delimitation
Thirty-six adult female Quarter Horse mares were used, identified by
numbers or names. The animals were split into 3 groups with twelve
animals each, in a completely randomized delimitation. Each group
stayed in separate paddocks from the other groups, being similar, and
with naturally helminth infested pastures, formed by Paspalum atratum.
All animals received an anthelmintic treatment with 1% abamectin,
orally, 28 days before the beginning of the experiment, to achieve ho­
mogeneity in their parasite load. They were also weighed on an elec­
tronic scale at the experiment start and every 28 days for weight
monitoring. The groups were distributed as follows: Control group: the
animals were treated daily with equine mashed concentrate manufac­
tured by Premix®, with 500 g provided to each animal. Fungi treated
group: the animals were treated using BiovermPlus® which is manu­
factured by the Cinergis company, using 1 g/10 kg of body weight,
containing 105 chlamydospores of the fungi D. flagrans and
P. chlamydosporia, administered daily, in association with 500 g of
Premix® mashed concentrate for horses (Fig. 1). And Abamectin treated
group: treated every 3 months with an abamectin based anthelmintic –
Animax Gel® 10 g, at a dosage of 2 g for every 100 kg of body weight,
presented in paste provided orally, in association with 500 g of Premix®
mashed concentrate for horses, daily. The administration of the
anthelmintic abamectin was preconized to individuals who, regardless
of group, had an EPG value above 1000, for the sake of the health of the
animals used in this experiment.
This study was certified by the Animal Ethics Committee (CEUA) of
the Faculty of Agricultural and Technological Sciences - UNESP – pro­
tocol number 05/2020.R1.
2.3. Fungal formula
A formulation composed of 105 chlamydospores per gram of the
Duddingtonia flagrans fungus and 105 chlamydospores of the Pochonia
chlamydosporia BiovermPlus® fungus was evaluated. The product used
was in the form of fine-grained powder, packed in crystal-colored
polypropylene bags, hermetically sealed and kept under room temper­
ature, guarded from direct sunlight, being supplied by the company
Cinergis Saúde e Nutrição Animal Ltda.
2.4. Faeces collection and coproparasitological analysis
Faecal samples were collected on D0 and then every 28 days, directly
from the rectal ampulla of each animal. The samples were packed in
plastic bags, with identification of the animals and kept in a thermal box
with chemical ice, until their arrival at the laboratory. Subsequently,
they were refrigerated until the time of the analysis, which would take
place within a maximum window of up to 24 h. For the egg count tests
per gram of faeces (EPG), the McMaster chamber was used, according to
the modified Gordon and Whitlock (1939) technique. This technique
consists of diluting 4 g of faeces, followed by homogenization, in 56 ml
T. Alves do Carmo et al.
of hypersaturated saline solution (NaCl), so that the eggs are suspended.
Then the suspension is filtered and placed in McMaster chambers. Thus,
the egg count was performed and the result multiplied by 50, expressing
the final number in eggs per gram of faeces.
Cultivation of infective larvae in faeces and extraction of larvae were
performed by the method of Roberts and O’Sullivan (1950). This tech­
nique consists of placing 50–60 g of faeces in disposable plastic cups,
moistening and covering the samples with perforated aluminum foil and
then placing them in a BOD oven for 14 days at a temperature of
26–28 ◦ C and relative humidity of 70–80%. After 14 days, the cup with
faeces are to be filled with water and inverted over a Petri dish, which
will also be filled with warm water. Subsequently, it will be placed in a
test tube after 24 h, covered and stored in a refrigerator at 4–5 ◦ C until
count and larval identification according to Madeira de Carvalho (2001)
of the different genera and/or species, whenever possible, based on the
100 copy count. The analysis were conducted out at the Laboratory of
Parasitology and Animal Health of the Faculty of Animal Science
UNESP/Dracena Campus - SP.
2.5. Infectious larvae recovered from pasture
As for the larvae retrieval from pastures, samples were collected in
the 3 paddocks of the 3 groups in a “Z” pattern from 5 alternate points,
according to Taylor (1939) and Raynaud and Gruner (1982). The sam­
ples were weighed and 200 g of pasture were reserved for each paddock
of the groups, which were later placed in an oven at 65 ◦ C for 72 h to
obtain the dry matter used to recover the infective larvae (L3). The
sediment was examined under an optical microscope and counting and
identification of larvae was performed according to the criteria estab­
lished by Madeira de Carvalho (2001).
2.6. Microbiological analysis of faeces
The microbiological analyzes of the faeces were carried out through
the faeces obtained in the collections every 28 days, which were
collected and stored as previously described in the EPG technique.
Subsequently, they were carried out at the Laboratory of Parasitology,
Faculty of Animal Science UNESP/ Dracena – SP, in duplicates from
pools of each group in which the plate counting method was used (Silva
et al., 1997; Hajdenwurcel, 1998). For the evaluation of total bacteria, 5
g of the faeces composite samples from each group were diluted in 45 ml
of sterile saline solution, followed by serial decimal dilutions of 105, and
0.1 ml of aliquots will be transferred to Petri dishes containing
Fig. 1. Monthly rainfall (mm), average temperature (◦ C) and relative humidity (%) from November 2020 to November 2021, in Castilho, São Paulo, Brazil.
3
Biological Control 182 (2023) 105219
approximately 15 ml of nutritive agar culture medium, and incubated in
the BOD oven. Kept at a temperature of 27 ◦ C +/- 1 ◦ C and humidity
55% for 48 h. For the quantification of bacteria, and using the CFU
method (colony forming unit) which, according to Franco and Landgraf
(1996), is the most used in analysis laboratories. Distinct groups of
microorganisms can be enumerated according to the medium of culture
and/or the incubation conditions used, consisting of identifying and
counting the viable colonies present on the plates.
2.7. Climate data
Information on precipitation, relative humidity and average hu­
midity, minimum and maximum temperatures were obtained from re­
cords from the Castilho city weather station, in state the São Paulo,
found 5 km from the experiment site, a partnership between Embrapa
Solos and the Virálcool plant – Unit II (sugar and ethanol company) and
the UDOP (União dos Produtores de Bioenergia).
3. Results
3.1. Eggs per gram of faeces
At the beginning of the experiment, the three groups had similar
averages in the count of eggs per gram of faeces, which reflects the
homogeneity among the groups (Table 1). In the first quarter, in
November, December and January, the three groups did not present a
statistically significant difference (p < 0.05). After the third month of
treatment, in February, it was seen that only the abamectin group
showed a significant difference in relation to the control group, and the
group treated with fungi. In March, the control groups, and the group
treated with fungus and treated with abamectin, presented, k being
possible to see that the treated groups differed from the control group. In
July, the three groups differed significantly from each other, the highest
average (643 EPG) in the control group, an average of 475 EPG in the
group treated with fungi and 296 EPG in the abamectin treated group. In
September, it was seen that only the abamectin group showed a statis­
tically significant difference (p < 0.05), with EPG 0. In the last two
months of the study (October and November) the group treated with
fungi differed significantly from the control group, which, in turn, did
not differ from the abamectin group. When analyzing the general av­
erages of EPG among groups, it was seen that in the control group (6 3 5)
it was higher than the fungus group (3 8 5) and in the abamectin group
(3 1 3), showing a statistically significant difference (p < 0.05) between
T. Alves do Carmo et al. Biological Control 182 (2023) 105219

Table 1 recommended months (February, May, August and November). In

Mean, mean standard error of egg count per gram of stool (EPG) of the control general, the animals in this group showed a significant difference in
group, fungi group and abamectin group, from November 2020 to November number of applications, totaling 58, followed by the control group with
2021, in Castilho, São Paulo, Brazil. 26 applications, and the group treated with fungi, with 12 applications
Month EPG in the experimental period.
Control Fungi Abamectin

Nov 108 (±42,3) 104 (±45,2) 117 (±46,6) 3.2. Fecal cultures
Dec 447 (±117,5) 321 (±92,0) 427 (±199,8)
Jan 641 (±144,8) 779 (±160,9) 581 (±124,9)
Table 3 presents the percentage values of small and large strongyles
Feb 791 (±125,8) a 700 (±191,8) a 704 (±150,6) a *
Mar 634 (±100,6) a 200 (±70,4) b 179 (±67,2) b
recovered from the fecal cultures. Initially, the three groups showed a
Apr 1075 (±147,3) 500 (±124,0) 742 (±136,3) prevalence of 100% of small strongyles (cyathostomins), remaining in
May 747 (±112,1) a 600 (±168,8) a 279 (±78,2) b * this way from November to July. In both treated groups, there was a
Jun 706 (±76,4) a 433 (±151,6) a 20 (±16,8) b 100% prevalence of small strongyles throughout the experimental
Jul 643 (±84,4) a 475 (±292,1) b 296 (±109,8) c
period. Whereas, for large strongyles, the recovered amounts were
Aug 590 (±71,3) a 133 (±52,0) b 246 (±68,9) ab *
Sept 637 (±100,4) a 346 (±104,3) a 0b extremely low, being found after eight months of study, only in the
Oct 467 (±73,4) a 183 (±88,4) b 208 (±149,5) ab control group, with a gradual increase, being in the months of August
Nov 764 (±96,0) a 233 (±109,3) b 496 (±200,1) ab (1%), September (3%), October (5%) and November (17%) of preva­
Average 635 (±61,7) a 385 (±60,4) b 313 (±67,3) b
lence. Therefore, the presence of large strongyles was not found in the
Arithmetic means with different letters on the same line differ significantly by two treated groups.
Tukey’s test (P < 0.05).
(*) months of abamectin application in the abamectin group. 3.3. Infectious larvae retrieved from the pasture

the groups. treated in relation to the control group, but not differing As shown in Table 4, the three groups started the study with similar
from each other. averages of infective larvae recovered from the pasture (L3). In the
As previously described, in addition to the anthelmintic treatment in second month (December), the three groups showed a significant dif­
the abamectin group every three months, the administration of aba­ ference (p < 0.05) among themselves. In March, there was a significant
mectin was recommended to all animals taking part in the experiment difference between the control and fungal treated groups, and the aba­
that presented EPG count values equal to or greater than 1000. There­ mectin group did not differ from these. Thus, throughout the study
fore, Table 2 shows the number of animals and the months which the period, the group treated with fungi showed lower mean values of
applications were carried out. Thus, it was possible to see that after the infective larvae recovered in pastures than the control group and aba­
beginning of the experiment, in the control group, every month there mectin group. The overall averages presented in the control group was
were animals in need of applications of abamectin, which ranged from 1 2267, the group treated with fungi 1100 and the group abamectin 3115,
to 5 animals per month. In the fungus group, unlike the other groups, the showing that the three groups differed significantly (p < 0.05) from each
animals took longer to present the need for abamectin application, since other, with the group treated with fungi having a lower average of
only in the third month (February) there were applications, ranging infective larvae recovered from pastures.
from 1 to 2 animals, and on 4 occasions during the experimental period
none of the animals belonging to this group required an anthelmintic 3.4. Faecal microbiology
treatment. In the group treated with abamectin, as well as the control
group, there were animals that needed applications one month after the Table 5 presents the average values of CFU/ml among the three
beginning of the experiment, ranging from 1 to 4 animals, and only on 3 groups. It was observed that the initial averages were similar in the three
occasions there were no need for abamectin application. However, in the groups, in the first two months (November and December), not differing
abamectin group, 10 applications were needed in addition to the significantly among them. The group treated with fungi obtained higher
monthly average values of CFU/ml than the other groups, during most of
the entire experimental period, except for the months of April and May,
Table 2 after the applications of abamectin in some animals belonging to the
Number of horses treated individually with abamectin in the control group, group (Table 2). The general average of CFU/ml presented by the groups
fungi group and in the abamectin group, from November 2020 to November
2021, in Castilho, São Paulo, Brazil.
Table 3
Month Number of Treated Animals Percentage values of infective larvae of small and large strongyles recovered
Control Fungi Abamectin from coprocultures with feces of animals belonging to the control group, fungi
Nov 0 0 0 group and abamectin group from November 2020 to November 2021, in Cas­
Dec 2 0 1 tilho, São Paulo.
Jan 2 1 2
Small strongyles Large strongyles
Feb 2 2 12*
Mar 1 0 0 Month Control Fungi Abamectin Control Fungi Abamectin
Apr 5 2 4
Nov 100% 100% 100% 0% 0% 0%
May 2 1 12*
Dec 100% 100% 100% 0% 0% 0%
Jun 2 2 0
Jan 100% 100% 100% 0% 0% 0%
Jul 2 2 2
Feb 100% 100% 100% 0% 0% 0%
Aug 1 0 12*
Mar 100% 100% 100% 0% 0% 0%
Sept 3 1 0
Apr 100% 100% 100% 0% 0% 0%
Oct 1 0 1
May 100% 100% 100% 0% 0% 0%
Nov 3 1 12*
Jun 100% 100% 100% 0% 0% 0%
Total 26 12 58
Jul 100% 100% 100% 0% 0% 0%
Individual treatment was performed whenever the animal had a count greater Aug 99% 100% 100% 1% 0% 0%
than 1000 EPG. Sept 97% 100% 100% 3% 0% 0%
Oct 95% 100% 100% 5% 0% 0%
(*) applications of abamectin in all animals of the abamectin group every three
Nov 83% 100% 100% 17% 0% 0%
months.

4
T. Alves do Carmo et al. Biological Control 182 (2023) 105219

Table 4 and in the months where temperatures were lower, there was a lack of
Mean and standard error of the mean number of infective larvae per kilogram of rain period.
dry matter (Kg.DM) recovered from the pasture the control group, of the fungi
group and of the abamectin group, in the period of November of 2020 to 4. Discussion
November 2021, in Castilho, São Paulo.
Month L3/KG.DM The abamectin anthelmintic application conduct scheme, in all ani­
Control Fungi Abamectin mals, before starting the experiment is reflected in the average EPG
Nov 120 (±34,6) 153 (±0) 135 (±0) average values presented by the three groups in the first phase of the
Dec 238 (±8,8) c 355 (±12,4) b 540 (±11,8) a study. The decrease in EPG values mean that in the group treated with
Jan 7444 (±704,1) 2163 (±969,0) 9905 (±149,5)
fungi, from the fourth month of study, a reduction of infective larvae
Feb 7672 (±235,2) 2325 (±80,67) 7672 (±4181,8)
Mar 3430 (±231,2) a 718 (±209,23) b 2833 (±1031,6) ab recovered from the pastures was seen. As in studies carried out by Larsen
Apr 1818 (±194,5) ab 423 (±151,8) b 2032 (±1736,4) a et al. (1996), where they used the fungus D. flagrans in foals kept on
May 1686 (±123,6) a 84 (±54,7) b 3810 (±1933,4) a pasture, a significant difference in the EPG averages only after 5 months
Jun 6265 (±495,5) 4837 (±3659,9) 8801 (±1878,1) of study was seen. Braga et al. (2009), in their 6-month study evaluating
Jul 271 (±27,8) b 260 (±44,37) b 486 (±37,6) a
Aug 407 (±3,7) a 251 (±14,4) b 470 (±15,27) a
the use of D. flagrans in mares kept on pasture, also saw a significant
Sept 304 (±9,8) b 0c 625 (±92,5) a difference in EPG averages from the third month onwards, as shown in
Oct 919 (±138,6) 436 (±7,13) 675 (±33,3) this study.
Nov 922 (±33,2) a 295 (±53,7) b 510 (±9,8) a Biological controlling agents have a different method of action from
Average 2267b 1100 a 3115c
chemical treatment, which in turn acts directly to reduce EPG values. A
Arithmetic means with different letters on the same line differ significantly by EPG reduction is expected in the first months of treatment, since the
Tukey’s test (P < 0.05). result is a consequence of the reduction in the infestation of infective
larvae in the pasture, which will become smaller throughout the treat­
ment period.
Table 5
It is not possible to say which of the fungi showed greater predatory
Mean and standard error of the mean of colony forming units (CFU), of the
activity, but it is likely that D. flagrans influenced the predatory activity
control group, fungi group and abamectin group, from November 2020 to
November 2021.
of L3, and P. chlamydosporia influenced the EPG reduction in the group
treated with fungi when compared to the control group. According to
Month CFU/ml
Mauchline et al. (2003). the fungus P. chlamydosporia has been impli­
Control Fungi Abamectin
cated as a predator of several helminth genera, and the ability to para­
Nov 203 (±40,0) 295 (±5,0) 209 (±9,0) sitize helminth eggs through their structures, when in contact with
Dec 243 (±52,5) 280 (±10,0) 162 (±11,0)
Jan 478 (±22,0) a 319 (±23,0) b 219 (±21,0) b *
them. Given the above, it is possible to tell that the use of fungi requires
Feb 22 (±0,50) b * 339 (±0) a * 111 (±74,5) ab * a treatment with less need for anthelmintic applications, while Campos
Mar 15 (±0,5) b 27 (±0) a 3 (±0,5) c et al. (2008) states that one of the favorable points for the use of nem­
Apr 190 (±0,5) a * 9 (±0,5) b * 37 (±27,5) b * atophagous fungi in the control of gastrointestinal helminths is the
May 160 (±20,0) a * 8 (±0,5) b 1 (±0) b
reduction of commercial anthelmintics usage.
Jun 150 (±25,0) 400 (±104,5) * 288 (±8,0)
Jul 7 (±0,5) 25 (±10,5) 7 (±0,5) * Small strongyles (cyathostomins) prevalence seen in the three groups
Aug 3 (±0,5) b * 600 (±0) a 2 (±0,5) b * of the present study, especially in the treated groups, according to
Sept 300 (±0) a 300 (±10,0) a * 38 (±0,5) b Kaplan and Nielsen (2010) is related to the high adaptability of these
Oct 12 (±7,0) 10 (±0) 3 (±1,0) nematodes to new molecules and types of treatments. According to
Nov 38 (±16,0) 82 (±16,0) 15 (±0) *
Average 147b 207 a 84c
Barbosa et al. (2001); Pereira and Vianna (2006), cyathostomins are
found with more prevalence and with greater parasitic intensity in
Arithmetic means with different letters on the same line differ significantly by horses in Brazil, standing for 80–100% of the total parasite load. They
Tukey’s test (P < 0.05).
are commonly found in field-raised animals (Reinemeyer and Nielsen,
(*) indicates the months in which some animals received applications of
2012), as well as the animals in the present study, which were kept on
abamectin.
naturally infected pastures.
The exclusive presence of small strongyles in the group treated with
were 147 in the control group, 207 in the group treated with fungi and
fungi shows their predatory activity on the larvae of large strongyles,
84 in the abamectin group. Therefore, the three groups differed signif­
which were observed in the control group only, thus comprising the
icantly (p < 0.05) from each other, with the abamectin group having the
presence of Strongilus vulgaris, Andersen et al. (2013), which is
lowest CFU/ml general average, due to the greater number of abamectin
considered the most pathogenic nematode of large strongyles, due to its
applications in this group, followed by the control group that also suf­
extensive mesenteric arterial system migration, the presence of these
fered influence of treatments with abamectin, with more frequent ap­
larvae in the arterial system causes arteritis and thrombosis, with risks of
plications in comparison to the fungi treated group, which presented the
intestinal involvement.
highest CFU/ml averages, due to the lower number of applications of
The control group had the presence of animals that spent an
abamectin in this group.
extended period without anthelmintic treatment. And according to au­
thors, long intervals in the use of anthelmintic treatments may favor the
3.5. Climate data reappearance of large strongyles, considered a high pathogenic parasite
(Austin, 1994; Andersen et al., 2013). Therefore, the identification of
The climatic data from Castilho – SP, obtained during the experi­ large strongyles from the eighth month of the experiment, only in the
mental period, are represented in Fig. 1, showing the highest tempera­ control group, is related to this fact, which for Larsen et al. (1996), large
ture, rainfall, and relative humidity averages in the first months of the strongyles have a relatively long prepatent period of 6 to 7 months, thus
study, from November to March. No rainfall was recorded in the months decreasing the rate of reinfection. In a study carried out by Vera et al.
of April, May, June and July (dry period). There was a rain period return (2020), helminths of the species S. vulgaris, considered sensitive to
in August, but with low rainfall until the last month of the study macrocyclic lactones, were found in one of the properties studied by the
(November). Therefore, it was observed that in the months with higher author, being the same where the present study was carried out.
temperatures (first months) of the year, rainfall rates were the highest, Currently, resistance to anthelmintics in equine gastrointestinal

5
T. Alves do Carmo et al.
nematodes has become a worldwide phenomenon (Lester et al., 2014;
Cernea et al., 2015). cyathostomins in all groups. According to Chapman
et al., (1996), this distribution is already expected, since small stron­
gyles are more resistant to anthelmintic medications available on the
market.
The use of D. flagrans and P. chlamydosporia fungi reduced the
contamination of infective larvae (L3) recovered in the pasture, acting
directly in the environment, from the second month of the experiment.
In a similar study, carried out by Baudena et al. (2000), also lasted 12
months and saw that D. flagrans promoted a significant reduction in the
number of L3 in the second month of use.
The high average temperatures associated with increased rainfall, in
addition to promoting greater larval migration to the pasture, it is also
possible that they have increased fungal growth and, so, increased their
predatory activity. Buzatti et al., 2017 states that the fungus Dudding­
tonia flagrans achieves its best growth at temperatures between 25 ◦ C
and 30 ◦ C, with 30 ◦ C being the best temperature for development, and
environmental variables can also directly affect the development of the
fungus and pre-growth stages. parasites of equine gastrointestinal
nematodes. Being similar the climatic conditions presented in the pre­
sent study, being favorable to the development of the fungi. Therefore,
the fungi D. flagrans and P. chlamydosporia showed their activity in the
biological control of equine gastrointestinal nematodes, even under
lower temperatures and precipitation. Thus, even with the challenge in
pastures, the group treated with fungi showed a lower degree of infes­
tation of infective larvae compared to the other groups, due to the action
of fungi. Because, they supply a reduction in the number of infective
larvae in the environment, and so exposing the animals to a lower
parasitic load of helminths (Fausto, 2020).
It was seen in the present study that the use of abamectin anthel­
mintic interferes with the development of bacteria present in equine
faeces. Omansen et al. (2015) states that avermectins inhibited the
growth of M.ulcerans and showed dose-dependent deaths in culture and
bioluminescence assays. Researchers confirmed that ivermectin showed
bactericidal activity, acting against a range of mycobacterial organisms,
including S. aureuse M. tuberculosis isolates. (Lim et al., 2013; Ashraf
et al., 2018). Because, after applications in animals, a reduction in the
averages of CFU/ml was observed, mainly in the abamectin group,
which presented the lowest average values of CFU, and the highest
number of animals with applications, Floate et al., (2001) state that this
is due to the high concentrations of macrocyclic lactones excreted in the
faeces, mainly in the first weeks after treatment, as they affect the
populations of coprophagous fauna that maintained contact with these
faeces and there are vulnerable species even in minimal concentrations
of these drugs.
Everything shows that the fungi did not interfere with the develop­
ment of microorganisms present in the faeces, since the group treated
with fungi there was only a reduction in the CFU/ml values followed by
the necessary applications of abamectin in some animals of the group,
Knox et al. (2002) proved that D. flagrans did not have a negative
environmental impact when used in a pasture production system. The
authors saw that the number of free-living nematodes and micro­
arthropods were not reduced (P greater than 0.05) by the fungus, nor
was there any effect (P greater than 0.05) on the presence of other
predatory fungi.
The use of D. flagrans and P. chlamydosporia fungi did not interfere in
the development of the microbial population present in the faeces as
shown in the abamectin group, where the development of microorgan­
isms was influenced by the applications of abamectin needed in the
studied period. Therefore, the disturbances that macrocyclic lactones
can cause in non-target invertebrates and on the participation of their
associates in faeces and soil degradation are unpredictable, and can
negatively influence the biodiversity and sustainability of agricultural
ecosystems (Kolar et al., 2006).
6
Biological Control 182 (2023) 105219
5. Conclusion
The use of Duddingtonia flagrans and Pochonia chlamydosporia fungi is
efficient in the control of gastrointestinal nematodes in horses kept on
pasture, promoting a reduction in pasture infestation by infective larvae
and so the degree of helminthiasis. Therefore, it can be considered as an
alternative to minimize the use of anthelmintic drugs in parasite control
programs for horses.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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