Introduction
Gastrointestinal nematode infections in dairy herds compromise the health and welfare of animals, leading to productive and economic losses (Geurden et al., Reference Geurden, Chartier, Fanke, Regalbono, Traversa and von Samson-Himmelstjerna2015; Ravinet et al., Reference Ravinet, Chartier, Bareille, Lehebel, Ponnau, Brisseau and Chauvin2016). Parasitism can negatively affect growing rate and puberty in heifers (Lacau-Mengido et al., Reference Lacau-Mengido, Mejía, Díaz-Torga, Gonzalez Iglesias, Formía, Libertun and Becú-Villalobos2000), in addition to promoting losses in milk production (Antonello et al., Reference Antonello, Cezar, Sangioni and Vogel2010). From an epidemiological point of view, only 5% of gastrointestinal nematodes are on the animals, while the remainder are on the pasture in the form of immature stages (Sagüés et al., Reference Sagüés, Fusé, Fernández, Iglesias, Moreno and Saumell2011). The biological cycle of these parasites occurs in pastures; when contaminated, the pasture is the main vehicle for the transmission of gastrointestinal nematodes to animals (Araújo et al., Reference Araújo, Assis, Campos and Mota2006).
Control of worms is a major challenge in livestock systems; to control these infections, anthelmintic drugs have been used for almost 40 years (Geurden et al., Reference Geurden, Chartier, Fanke, Regalbono, Traversa and von Samson-Himmelstjerna2015). Nowadays, a new challenge facing livestock systems is parasitic resistance to available drugs, a fact reported by Baiak et al. (Reference Baiak, Lehnen and Rocha2018). The authors demonstrated through a meta-analysis, involving results from several countries, that the indiscriminate use of medications (overdose) in cattle resulted in the ineffectiveness of the different chemical groups of anthelmintics (Baiak et al., Reference Baiak, Lehnen and Rocha2018). On the other hand, in agroecological systems, the main difficulty faced is restrictions on the use of anthelmintic drugs. The sanitary management used in these systems is usually carried out with homoeopathics, herbal medicines and factors inherent to the system management (Almeida, Reference Almeida2013); however, new control alternatives are being sought to minimize infection by gastrointestinal nematodes.
Biological control through nematophagous fungus is a promising alternative to reduce the number of infective larvae (L3) in livestock farming (Peña et al., Reference Peña, Miller, Fontenot, Gillespie and Larsen2002). Among the groups of nematophagous fungus (endoparasites, ovicides and predators), the group of predators is most commonly used in biological control. This group of fungi have the ability to slow the development of larvae in the environment without negative impacts on the host (Braga & Araújo, Reference Braga and Araújo2014). Among the most widely used species in this group, the following stand out: Arthrobotrys robusta and Duddingtonia flagrans. These species survive passage through the gastrointestinal tract of ruminants and germinate in the faecal pads, where they prey on the larvae (Campos et al., Reference Campos, Araújo and Guimarães2008). Furthermore, they are easy to grow in the laboratory and demonstrate the potential to be developed for commercial formulations (Mota et al., Reference Mota, Campos and Araújo2003).
Previous studies show promising results in the application of biological control in laboratory tests and field conditions (Jobim et al., Reference Jobim, Santurio and De La Rue2008; Silva et al., Reference Silva, Araújo, Braga, Soares and Rodrigues2013). In this way, the fungus contributes to the prophylactic control of nematodes, reducing the number of L3 in pasture and minimizing the risk of reinfection (Knox et al., Reference Knox, Josh and Anderson2002; Mota et al., Reference Mota, Campos and Araújo2003). However, in field conditions, the environment is influenced by several factors (rain, solar radiation, air temperature and relative humidity) that can affect the dynamics of nematophagous fungus (Hasanzadeh et al., Reference Hasanzadeh, Mohammadifar, Sahebany and Etebarian2012; Saumell et al., Reference Saumell, Fernández, Fusé, Rodríguez, Sagüés and Iglesias2015). Thus, the objective of this work was to evaluate the predatory activity of the nematophagous fungus D. flagrans on infective larvae of gastrointestinal nematodes from dairy heifers in pasture areas and in vitro, after gastrointestinal transit from faeces, soil and pasture.
Materials and methods
This study was approved by the Ethics Committee on Animal use at the Universidade Federal do Paraná under protocol number 083/2018, and was performed in accordance with the ethics of animal experimentation. This study was conducted between April and July of 2019. To meet our objectives two experiments were performed: (1) evaluation of the predatory activity of the nematophagous fungus in vitro; (2) evaluation of the predatory activity of the nematophagous fungus in a pasture area.
Evaluation of the predatory activity of the nematophagous fungus in vitro
The first experiment was carried out in order to confirm the passage of the fungus through the gastrointestinal system and its presence in faeces for further testing in a pasture area. The experiment was performed between May and July of 2019 at the Laboratório de Parasitologia Animal da Universidade Estadual de Ponta Grossa, Paraná, Brazil. Ten Jersey heifers were used in this study, with an average age of (mean ± SD) 11.7 ± 4.3 months, and an average weight of 236 ± 81 kg. The animals were divided into two homogeneous groups (five animals per group) according to the number of gastrointestinal nematode eggs per gram of faeces (EPG, fig. 1). An EPG test was performed according to the methodology of Gordon & Whitlock (Reference Gordon and Whitlock1939), modified by Ueno & Gonçalves (Reference Ueno and Gonçalves1998).
Fig. 1. Average values ± standard deviation (SD) of the EPG values from animals used in experiments 1 and 2. Average values followed by the same letter did not differ (P < 0.05).
Our treatments consisted of faeces obtained from animals treated with fungus (treated group) and faeces from animals that did not receive fungus (control group). For this purpose, we adopted the methodology proposed by Silva et al. (Reference Silva, Araújo, Braga, Soares and Rodrigues2013), in which the treated group received 150 g of pellets (single dose) containing a mycelial mass of the fungus D. flagrans (0.2 g of mycelium) mixed with 500 g of wheat bran, while the control group received 150 g of pellets (single dose) without the mycelial mass of D. flagrans fungus mixed with 500 g of wheat bran. The fungus isolate of D. flagrans (AC001) used was produced and provided by the Laboratório de Controle Biológico de Fitonematóides (Instituto de Biotecnologia Aplicada à Agropecuária – BIOAGRO) of the Department of Phytopathology at the Universidade Federal de Viçosa (UFV), Minas Gerais, Brazil.
These isolates of the nematophagous fungus D. flagrans (AC001) were obtained from Brazilian agricultural soil in the municipality of Viçosa, in the Zona da Mata region of the state of Minas Gerais. They were collected using the soil-sprinkling method of Duddington (Reference Duddington1955). To produce fungal mycelia of D. flagrans (AC001), culture discs approximately 4 mm in diameter, in 2% water agar (2% WA), were transferred to 250-ml Erlenmeyer flasks containing 150 ml of the liquid medium GPY (glucose, sodium peptone and yeast extract). The flasks were continually stirred at 120 rpm in the dark and at the temperature of 26°C for 10 days. After this period, the mycelia were removed, filtered and weighed on an analytical scale. All the procedures followed the methodology of Braga et al. (Reference Braga, Araújo, Silva, Araujo, Carvalho and Tavela2009).
For the in vitro analyses, 12 h after pellet administration (with and without fungus), faeces samples were collected directly from the rectal ampoule of the animals (n = 10; 5 by group). The time of 12 h was chosen based on Silva et al. (Reference Silva, Araújo, Braga, Soares and Rodrigues2013) and Braga et al. (Reference Braga, Ferraz, da Silva and de Araújo2020); these authors reported that D. flagrans activity did not differ among different time periods (range: 12–72 h). The experimental design to evaluate the in vitro predatory activity of the fungus D. flagrans was adapted from Mauad (Reference Mauad2008), pooled for each group (treated and control) and used a 1-g faeces sample from each animal. At the end of this step, 2 g of each pooled sample was removed and placed in a 9-cm Petri dish, containing solid Agar and antibiotic (Chemitril 0.05 g/l). In total, we used four Petri dishes, two for each group. A suspension of approximately 3000 larvae (L3) of Haemonchus spp. (41.1%) and Cooperia spp. (58.9%), obtained through coprocultures according to the technique proposed by Roberts & O'Sullivan (Reference Roberts and O'Sullivan1950), was added to the Petri dishes.
Samples for coprocultures were collected from the heifers before the beginning of the experiment. The amount of L3 present in the coprocultures was estimated by counting larvae in ten aliquots of 10 μl. The Petri dishes were sealed with plastic film and incubated in an oven (25°C) for 14 days. After this period, non-predated larvae (L3) were recovered by the Baermann method (see Staniland, Reference Staniland1954) and identified using the methodology proposed by Keith (Reference Keith1952). The effectiveness of the fungus D. flagrans in preying on infective larvae (L3) was evaluated through the percentage of larvae reduction (PR, %) according to equation (1):
where PR is the percentage of larvae reduction, X is the number of larvae in the treated group and Y is the number of larvae in the control group.
Evaluation of the predatory activity of the nematophagous fungus in a pasture area
Experimental area and climate pattern
The second experiment was carried out at the Estação de Pesquisa Agroecológica – CPRA at the Instituto de Desenvolvimento Rural do Paraná, Paraná state, in Southern Brazil (25°26′41″S, 49°11′33″W). This is an agroecological farm, where deworming of animals with anthelmintic drugs is not permitted; sanitary management is usually carried out with the use of homeopathic herbal medicines and factors inherent to management. The climate in this region is characterized as humid maritime temperate (Cfb) according to the Köppen's classification. The region has mild summers with an average annual temperature between 18 and 20°C (Alvares et al., Reference Alvares, Stape, Sentelhas, Moraes Gonçalves and Sparovek2013).
At the farm, animals are raised permanently on pasture, mainly composed of plant species of Paspulum spp., Arachis spp., Lolium spp. and Brachiaria spp. The farm is divided into paddocks, under the Voisin's Rotational Grazing system (Voisin, Reference Voisin2001), in which mineral salt and water are offered ad libitum. In this system, the pasture area is divided into paddocks, and the herd enters a new paddock every day so that the plants have enough time to recover. Voisin's system is ecologically useful, sustainable and offers a key solution for the problems faced by climate change, since this system improves the structure and productivity of the soil due to biocenosis; reduces greenhouse gas emissions from the animals; decreases fertilizer and pesticide use; and improves the health of animals (Pinheiro Machado, Reference Pinheiro Machado2010). Before the animals enter a new paddock, they receive supplementation (wheat bran) at the trough. The experimental area consisted of a paddock (900 m²), with a 60-day pasture rest period set before the start of the tests.
Animals and definition of treatments
The definitions and characteristics of the animals and treatments used have already been described in the methodology of the first experiment.
Definition of the experimental unit
Twelve hours after pellet administration (with and without fungus), animals were taken to the experimental area, where they remained for 7 h (7:00 to 14:00). Four observers were positioned, one on each corner of the paddock, to help to record the time that each animal defecated. All observers remained at a safe distance (approximately 5 m) so as not to interfere in the animal's behaviour, entering the paddock only to fix the pole. All faecal pads were identified with poles with colours representing each treatment (treated and control). After the animals left the paddock, 20 faecal pads (treated, n = 10 and control, n = 10) were selected at random and the diameter (cm) was measured. During the observational period (7:00 to 14:00), the time that each animal defecated was recorded so that the passage time corresponding to the time between intake of the pellets and defecation in the experimental areas could be calculated.
Each faecal pad was delimited to identify the experimental unit (n = 20) and divided into four equal parts. To demarcate the collection areas, transects that exceeded 10 cm from the largest radius of the faeces were used (fig. 2). During the collections, a minimum distance of 100 cm and a maximum distance of 350 cm between the faecal pads were respected; in this way interaction between faecal pads was not possible. This methodology was adopted because it is known that approximately 90% of infective larvae (L3) do not migrate more than 10 cm away from faecal pads (Skinner & Todd, Reference Skinner and Todd1980).
Fig. 2. Schematic representation of the collection areas.
Data collection
Pasture, faecal pad and soil collections occurred at CPRA in April of 2019 with an interval of 7 days, totalling four assessments. The weekly collection was always performed in the morning at the same time (beginning 8:00; ending 11:00) and by the same person. During this period the paddock remained free of animals. For the recovery of infective larvae (L3) from the pasture, samples were collected within 10 cm of a faecal pad at two heights: 50% of the upper pasture (corresponding to the aerial part of the pasture) and 50% of the lower pasture (corresponding to the pasture stalk). To evaluate the infective larvae in the faecal pad, all the matter of a part was collected, as well as the soil sample under the faecal pad to a depth of 10 cm. All samples were placed in thermal boxes and sent to the Laboratório de Parasitologia Animal at the Universidade Estadual de Ponta Grossa, where the laboratory analyses were performed.
Laboratory analysis – recovery of infective larvae
For recovery of infective larvae (L3) from pasture, samples were placed in a sedimentation calyx. These samples were wrapped in gauze and fixed in the upper part of the sedimentation calyx, where they remained submerged in mineral water for 24 h. At the end of this stage, the pasture samples were removed from the sedimentation calyx and conditioned in a forced ventilation oven (55°C) for 72 h to determine the dry matter. Meanwhile, the sedimentation calyx supernatant was discarded and the sediment (1.5 ml) transferred to a graduated conical tube with a lid. The sediment was examined under a microscope and the infective larvae were quantified according to the methodology proposed by Keith (Reference Keith1952); the results were expressed in number of L3 per kg of dry matter (L3/kg DM).
For recovery of infective larvae (L3) from the faecal pad and soil, the samples were conditioned on a tissue, inside a sieve and placed in a sedimentation calyx. Mineral water was added to the sedimentation calyx to cover the samples, where they remained for 24 h. Sediment collection as well as quantification and identification of larvae were carried out in the same way as described for pasture. After the larvae were recovered, both samples (faecal pad and soil) were transferred to paper packages of known weight and conditioned in a forced ventilation oven (55°C) for 72 h to determine the dry matter. The larvae genera were identified according to their characteristics described by Keith (Reference Keith1952).
Statistical analysis
In order to confirm there was no significant difference in eggs per gram (EPG) between the groups (treated and control), the data of the EPG test were submitted to a simple generalized linear model with Poisson distribution, logarithmic bonding function, and 95% confidence interval. The experimental design of the in vitro biological control was composed of two treatments (faecal pad with fungus and faecal pad without fungus) and four experimental units (two Petri dishes from the treated group and two Petri dishes from the control group). For analysis, a simple generalized linear model with Poisson distribution, logarithmic bonding function and 95% confidence interval was used.
The experimental design of the pasture area was composed of two treatments (faecal pad with fungus and faecal pad without fungus), four replications (parts of faecal pad) and 20 experimental units (ten faecal pads from treated group and ten faecal pads from control group). For analysis a mixed model (generalized linear model – GLM) with Poisson distribution was used and a 95% confidence interval. Animals, diameter of faecal pad (DF) and EPG were defined as random effects following the model
where Yij is the non-predated infective larvae (L3/Kg DM); αijk is the fixed effect (i corresponds to group (treated and control), j corresponds to collections, k corresponds to passage time); Ai is the random effect of animals; βij is the random effect (i corresponds to DF, j corresponds to EPG); and eij is the residual effect.
The analyses were performed separately, and for each site of collection (pasture (upper and lower), faecal pad and soil) a GLM model was obtained. The model was adjusted by statistical package lme4 (Bates et al., Reference Bates, Mächler, Bolker and Walker2015). The confidence intervals were estimated using Type II Wald chi-square tests and the fit of the model was given by a likelihood test. The normality of the random facts was given by quartile plot means with 95% confidence interval. In order to evaluate fungus predatory activity in the faeces collections, the data were submitted to confirmatory analysis by a paired samples Wilcoxon test at the confidence level of 95%.
The environment data were used for a descriptive analysis of the climate during the experimental period. All analyses (descriptive, influence and confirmatory) were performed using statistical software R (R Core Team, 2017).
Results
In experiment 1 (in vitro) there was a difference (P < 0.05) in the number of non-predated infective larvae (L3) between the groups (treated and control). There was a difference (P < 0.05) in L3 recovered between the groups (fig. 3). The highest number of L3 was obtained in the control group (454), while in the treated group, only 52 infective larvae were non-predated. After gastrointestinal transit of dairy heifers, a percentage reduction of 88% in vitro was obtained.
Fig. 3. Average values of infective larvae (L3) of Cooperia spp. and Haemonchus spp. recovered. Average values followed by the same letter did not differ (P < 0.05).
Monthly average temperature during the experimental period is shown in table 1. The average maximum temperature throughout the experimental period was 23.6°C, the average minimum temperature was 15.2°C and the average precipitation was 0.38 mm.
Table 1. Average values ± standard deviation (SD) during experimental period for air temperature (AT, °C), relative humidity (RH, %) and rainfall (RF, mm).
The predominance of gastrointestinal nematode genera recovered during the experimental period from pasture (upper and lower), faecal pad and soil by group is shown in table 2. Regarding genera, there was a predominance of Cooperia spp. and Haemonchus spp. in both groups.
Table 2. Recovery of infective larvae by pasture samples (upper and lower), faecal pad and soil at the four collections by groups.
In experiment 2, there was no influence (P = 0.87) of the passage time (treated group range: 473 min to 835 min) on the fungus predatory activity. There was a difference (P < 0.05) in the amount of non-predated larvae in the faecal pad (range: 0.00–86,294) and upper pasture (range: 0.00–37,419) between treatments. In addition, the collections influenced (P < 0.05) the number of non-predated infective larvae in the evaluated sites. The third and fourth collections showed the highest mean values of L3/kg DM (table 3).
Table 3. Average values and coefficient of variation (CV, %) of non-predated infective larvae (L3/kg DM) from pasture (upper and lower), faecal pad and soil at the four collections by treated and control groups.
Averages followed by P < 0.05 in the line had the influence of treatment by the generalized linear model.
When analysing the number of non-predated infective larvae (L3) in the faecal pads from the pasture area, there was a difference (P < 0.05) between collections 3 and 4 for both groups (treated and control); in the treated group a reduction of 65% was obtained, while in the control group there was an increase of 217% in the number of L3.
Discussion
This is the first study to report the predatory activity of the fungus D. flagrans in faecal pads divided into parts and evaluated over time. The nematophagous fungus D. flagrans stands out due to its large production of chlamydospores that are highly resistant to adverse conditions (Silveira et al., Reference Silveira, Oliveira, Braga, Carvalho, Domingues and Silva2017). Its predatory activity has been widely studied in vitro (Braga et al., Reference Braga, Araújo, Soares, Araujo, Ferreira, Frassy and Queiroz2011; Silva et al., Reference Silva, Araújo, Braga, Soares and Rodrigues2013; Silveira et al., Reference Silveira, Oliveira, Braga, Carvalho, Domingues and Silva2017) since research in controlled environments (in vitro) is important to find fungi with the greatest potential for biological control. However, our study demonstrates that even after gastrointestinal transit, the fungus D. Flagrans is an efficient biological control not only in vitro, but also for pasture areas. Regarding the genera of larvae recovered from the samples, there was a predominance of groups Cooperia spp. and Haemonchus spp. This fact has already been reported in other studies (Assis et al., Reference Assis, Luns, Araújo, Braga, Assis and Marcelino2013; Fernandes et al., Reference Fernandes, Aguiar, Costa Silva, Senna, Mello and Oliveira2017), demonstrating that in tropical regions, these genera are common in cattle. However, the fungus D. flagrans is not selective for specific genera of parasites, which demonstrates that the genera of the parasite does not affect the predatory activity of the fungus (Araújo et al., Reference Araújo, Assis, Campos and Mota2006).
The different times of passage of the fungus through the gastrointestinal system of animals did not influence the predatory action of the fungus evaluated in the pasture area. Under controlled conditions (in vitro), it is possible to observe the viability and capacity of the fungus to prey on infective larvae after 72 h of gastrointestinal transit (Silva et al., Reference Silva, Araújo, Braga, Soares and Rodrigues2013). However, with our results, we suggest that the fungus can remain viable after 12–19 h of gastrointestinal transit. For in vitro experiments, the faecal pads were collected 12 h after the fungus was offered; and collection of faecal pads in the pasture areas was carried out after 19 h (12 h after offered + 7 h animals remained at the paddock). This information is important since environmental conditions can influence the predatory action of the fungus in the faecal pad (Saumell et al., Reference Saumell, Fernández, Fusé, Rodríguez, Sagüés and Iglesias2015).
No infective larvae were collected in the soil and faecal pads in the first and second collections in either group (treated and control). This may be because of development of the larvae, which in favourable conditions (air temperature ≥25°C) takes around 7 days (Amarante, Reference Amarante2014). In our study, the average air temperature was 23.6°C, which may lead to later development of infective larvae. In the third collection, the increase in the number of infective larvae recovered may be related to the increase in air temperature. With this effect, it is suggested that the fungus started to act in the third collection (14 days after deposition of faecal pad) since they form their traps (three-dimensional network) quickly when they are stimulated by free-living nematodes or parasites. Generally, traps are formed in the first 4 h of contact of the fungus with the larvae (Nansen et al., Reference Nansen, Gronvold and Henriksen1986; Charles et al., Reference Charles, Santos and Alvim1993), with predation of the larvae occurring up to approximately 20 h (Nansen et al., Reference Nansen, Gronvold and Henriksen1986).
In the fourth faecal pad collection, there was an increase in non-predated infective larvae in the control group; whereas in the treated group, there was a reduction in larvae from collection 3 to collection 4. Previous studies also showed a reduction (range: 60–90%) in the number of infective larvae present in faecal pads of cattle that received D. flagrans (Saumell et al., Reference Saumell, Fernández, Fusé, Rodríguez, Sagüés and Iglesias2015; Bilotto et al., Reference Bilotto, Fusé, Sagües, Iglesias, Fernández and Zegbi2018; Healey et al., Reference Healey, Lawlor, Knox, Chambers and Lamb2018).
However, the infective larvae recovery in the soil and in the different heights of the pasture (upper and lower) increased in both groups (treated and control) from collection 3 to 4. This result suggests that the non-predated infective larvae in the faecal pad continued with their growth and development, resulting in a greater number of non-predated infective larvae in the final evaluation, as a consequence of the amount of eggs that were eliminated in the animals’ faeces. This occurred because the fungus D. flagrans has little growth beyond the faecal pad and in the surrounding soil (Faedo et al., Reference Faedo, Larsen, Dimander, Yeates, Höglund and Waller2002). In addition, the migration of infective larvae is controlled by environmental factors (e.g. solar radiation, air temperature and relative humidity), with relative humidity being the main factor that promotes migration, since the larvae need a film of water for their movement (Sciacca et al., Reference Sciacca, Ketschek, Forbes, Boston, Guerrero and Ashton2002). In uncontrolled conditions (pasture areas), the difficulties in studying biological control are greater than in laboratory conditions, due to the direct action of environmental factors. Thus, further research should be carried out to deepen knowledge of this biological control technique in pasture areas. Biological control has growth potential, mainly due to popular pressure for safer food, in addition to not requiring a grace period for the consumption of products of animal origin.
In conclusion, the fungus D. flagrans was demonstrated to withstand the gastrointestinal transit of animals by reducing the number of infective larvae in the in vitro test and in faecal pads in the field test. The results of this study demonstrate that biological control of gastrointestinal nematodes with nematophagous fungi is a promising alternative; however, due to the complex influence of the environment on the predatory activity of the fungus, there is a need for more research on different types of grazing over time. In this way, knowledge of the development characteristics of nematophagous fungi will enable better decision making in the use of fungi in the environment. This, in turn, will allow researchers to improve current knowledge in order to apply nematophagous fungi to livestock systems.
Acknowledgements
We acknowledge the support of the Universidade Estadual de Ponta Grossa, Estação de Pesquisa Agroecológica – CPRA, and the Laboratório de Controle Biológico de Fitonematóides do Departamento de Fitopatologia da Universidade Federal de Viçosa (UFV).
Conflicts of interest
None.
