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. 2020 Oct 27;45(1):204–210. doi: 10.1007/s12639-020-01297-3

Antagonistic effects of native strains of the soil fungus Paecilomyces against gastrointestinal nematode and protozoan parasites of pigs in Panama

Génesis Cruz 1, Lenin De León 1, Ariadna Bethancourt 1, Nivia Ríos 1, Rachel Krause 2,✉, Nidia Sandoval 1
PMCID: PMC7921268  PMID: 33746405

Abstract

A variety of gastrointestinal parasites naturally infect domestic pigs in Panama which may also occur as zoonotic infections in humans. Anthelmintic drug treatment, including mass drug administration, can lead to drug resistance, reflecting a need for alternatives. The objectives of this exploratory and observational study were: (1) to isolate and cultivate natives species of Paecilomyces from natural soils in Panama, and (2) to evaluate isolated strains for their capacity to parasitize endemic gastrointestinal nematode and protozoan parasites recovered from naturally infected domestic pigs by observing cultures for spore adhesion and hyphae penetration phases. Using microcultivation and inoculation techniques, four strains of Paecilomyces were isolated from three locations in Panama, out of which three successfully adhered to and penetrated free-living stages (eggs, cysts and oocysts) of Balantidium suis, coccidia, Trichuris suis and hookworm. To our knowledge, this is the first published report of a nematophagous fungus such as Paecilomyces successfully infecting this range of gastrointestinal parasites, particularly protozoan parasites.

Keywords: Paecilomyces, Nematophagous fungi, Pigs, Antagonistic effects, Gastrointestinal nematodes, Protozoan parasites, Panama

Introduction

Incomplete treatment of gastrointestinal parasites with anthelmintics, particularly in the context of preventative chemotherapy and mass drug administration programs, has resulted in a rise in drug resistance in a variety of helminth species (Tinkler 2020). This has led to a search for alternative control measures, particularly biological controls. Unlike with anthelmintics, in which the goal is complete elimination of all parasites from the host, the objective of biological control is not to fully eliminate the target organisms, but rather to reduce their negative impacts by using ecological mechanisms to lower parasite populations below harmful levels (Gronvold et al. 1996; Narí et al. 2003; Sagüés et al. 2011; Maqbool et al. 2017).

Diverse potential agents of biological control of parasitic nematodes may be found in natural soils, including bacteria, viruses, mites, and free-living nematodes, but the most promising of these are predatory fungi (Waller and Faedo 1996). Nematophagous fungi are widely distributed and naturally occurring in soils and other substrates. Their ability to survive in a variety of extreme climates and in conditions of low nutrient availability explain their extensive geographic distribution in diverse climates and environments, from high arctic soils to the tropics (Saumell et al. 2008; Hoyos 2011). Nematophagous fungi can capture, parasitize or paralyze nematodes at various life stages (Mendoza de Gives 1999; Hoyos 2011; Sagüés et al. 2011; Abd-Elgawad and Askary 2018; Da Silva et al. 2018). Some fungi, such as those in the genus Arthrobotrys, develop specialized structures out of their hyphae, while others, such as Drechmeria coniospora, are obligatory endoparasites. There are fungi that parasitize nematode eggs, such as Catenaria auxiliaris and Lagenidium spp., and others such as Pleorothus ostreatus that affect nematodes through the production of toxins (López-Llorca and Jansson 2001; Piedra 2008; Hoyos 2011; Henríquez 2015; Najafi et al. 2017). A particularly promising group of nematophagous fungi which occur in the genus Paecilomyces are saprophytes, generally found in soil or air, that parasitize nematode eggs, larvae, and female adults of various species of nematodes, including those of humans, other animals, and plants (Inglis and Tigano 2006; Vargas et al. 2015; Winarto and Liswarni 2018).

Previous research on the biocontrol value of fungi for controlling pathogens of animals and plants has focused on nematophagous properties of fungi; however, a variety of protozoan gastrointestinal parasites of domestic animals and humans are commonly found alongside gastrointestinal nematodes in mixed infections in areas where these parasites are endemic. While antagonistic fungal interactions with parasitic nematodes has been explored for a wide range of fungi and nematodes, to our knowledge, antagonistic fungal interactions against protozoan gastrointestinal parasites has not been documented in the literature (Gronvold et al. 1996). The goal of this study was to explore the parasitic action of naturally occurring soil fungi of the genus Paecilomyces against a diverse set of gastrointestinal parasites, particularly nematodes and protozoans, found in domestic pigs. Specifically, the objectives of this exploratory study were to isolate and cultivate native species of Paecilomyces spp. from natural soils in Panama, and to evaluate isolated strains of the fungi for their capacity to parasitize gastrointestinal nematode and protozoan parasites recovered from the faeces of naturally infected domestic pigs.

Materials and methods

Collection of soil samples for isolation of nematophagous fungi

Soil samples were collected from forest and agricultural areas, as these fungi are known to be found in soils with high organic matter (Peraza et al. 2011). In the district of Chepo (Panama Province), 3 soil samples were collected from rice fields in the villages of Tortí and 1 sample was collected from El Llano; in the district of Nata de los Caballeros (Province of Coclé) 3 samples were collected from pine plantations in the village of Capellanía; and in the district of El Valle de Antón (Province of Coclé) 3 samples were collected from La Cruz and 1 from Chumical (Fig. 1).

Fig. 1.

Fig. 1

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Map of Panama, showing locations of sampling sites for soil for nematophagous fungi, and pig faecal samples for gastrointestinal parasites

Soil samples of approximately 100 g (10 cm by 10 cm by 1 cm depth) were collected, kept at 5 °C, and transported back to the Laboratorio de Investigaciones en Parasitología Ambiental (LIPAAM) of the University of Panama, Panama City campus, for further processing.

Isolation, characterization, identification, and cultivation of nematophagous Fungi

As described by Vanbreuseghem (1952), fungi were isolated by incubating soil samples with keratin (horsehair) traps. Each soil sample was incubated in triplicate in Petri dishes (145 × 20 mm) to which 0.5–1.0 g of finely sieved soil was added. With sterile forceps, 10–15 sterilized horse hairs of 4–5 cm in length were placed on the soil. The Petri dishes were then sealed and incubated for 5–25 days at ambient temperature.

After 3 days of incubation, keratin traps were observed daily for up to 25 days, and horse hairs that had fungal growth were isolated on individual Petri dishes of potato dextrose agar (PDA). The PDA dishes were monitored daily for 10 days to determine whether isolated colonies of fungus were growing on the medium.

To characterize the isolated soil fungi, microcultivation techniques, using PDA, water agar (WA), malt extract agar (MEA), and V8 agar (V8A), were followed. The four types of culture media were individually placed on glass slides. Isolated fungi were placed onto each of the four culture types in order to observe the nutritional preference of each isolated fungus. Development of hyphae and conidia were observed. These structures were used to identify the genera of isolated fungi using the taxonomic key developed by Barnett and Hunter (1998).

Once fungi of the genus Paecilomyces were identified, they were cultivated in PDA. Following procedures from Cañedo (2004), conidia from isolated sporulated Paecilomyces were suspended in distilled water with 0.1% Tween 80 to a total volume of 5 mL and homogenized using a vortex. Counts of conidia were made using spectrophotometric comparisons, starting with the 0.5 McFarland standard, and serially diluting 1:10.

Collection, identification, and isolation of gastrointestinal parasites from domestic pigs

Fresh faeces were collected from 10 pigs of 6 months of age on small family-operated pig farms in the village of Pacora in the district of Panama (Province of Panama). Preliminary analysis of samples was done at the collection site through direct observation using light microscopy of 3 replicates of each sample. Samples positive for parasite stages were collected in plastic sample containers and transported at 5 °C to LIPAAM. Coprological diagnostic flotation methods were used on 1 g samples to isolate parasite stages for identification. Parasites were identified using keys from Zajac and Conboy (2012) and Hendrix and Sirois (2007). Spontaneous sedimentation of 150 g of each faecal sample in 1 L of water was done to wash these parasite stages and estimate the quantity present in samples (Chester et al. 1990). Following the washing procedure, 10 replicates were counted to estimate the number of parasite forms present per mL of faeces.

Bioassay to evaluate effect of Paecilomyces on eggs, cysts, and oocysts of pig gastrointestinal parasites

A solution with approximately 100 parasitic forms (Balantidium suis, coccidia, Trichuris suis and hookworm) were inoculated into 24-well microplates, followed by addition of a solution of fungal conidia concentrations of 1.5 × 108, 1.5 × 107 and 1.5 × 106. Two replicates of each conidia concentration were tested, for each of the 4 strains, for a total of 24 wells. Wells were monitored daily for 4 days, during which time parasite stages (eggs, cysts or oocysts) were examined for adhesion and penetration by fungal hyphae. Parasite stages were then stained with lactophenol blue and mounted on slides for examination using light microscopy. Different stages of adhesion by fungal spores and penetration by fungal hyphae were identified and photographed to demonstrate the antagonistic effects of the different isolated Paecilomyces strains on eggs, cysts and oocysts.

Results

Isolation of Paecilomyces sp. from soil

Four strains of the native soil fungus, Paecilomyces spp., were isolated from Capellanía (CPE3), Chumical (CHU3), and Tortí (TA1, TA2; Fig. 1), from soil taken from pig pens, a pine plantation, and a rice field, respectively.

Parasitologial analysis of pig samples

Parasites identified from faeces of naturally infected domestic pigs included Balantidium suis, coccidia, Trichuris suis and hookworms. Balantidium suis was the most common parasite stage recovered, with approximately 2683 cysts per mL of sample and an overall prevalence of 82%. The coccidia group was the second most common type of parasite, with a prevalence of 14% and intensity of 466 oocysts/mL observed. Less-commonly observed parasites were hookworms and Trichuris suis, with prevalences of 3% (30 eggs/mL) and 1% (83 eggs/mL), respectively.

Antagonistic effects of Paecilomyces spp. against Balantidium suis, coccidia, Trichuris suis and hookworms

Of the four fungal strains isolated, three strains from Chumical (CHU3) and Tortí (TA1, TA2) showed antiparasitic activity at all three concentrations tested (1.5 × 108/plate, 1.5 × 107 and 1.5 × 106). All three strains showed the adhesion phase for all four gastrointestinal parasite species tested (Balantidium suis, coccidia, Trichuris suis, and hookworms) whereas the hyphal penetration phase was also observed for all four parasite stages by the strains from Tortí (TA1, TA2). Detailed images of adhesion of spores and penetration of hyphae of the TA1 Paecilomyces sp. strain at concentration 1.5 × 108/plate are shown in Fig. 2.

Fig. 2.

Fig. 2

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Antagonistic effects of Paecilomyces strain TA1 on parasites, showing fungal spores (S) and hyphae (H): a, b hookworm eggs showing adhesion phase; c hookworm egg in early penetration phase with spore adhesion and hyphae beginning to extend into the egg; d hookworm egg in late penetration phase with hyphae observed inside the egg; e Trichuris egg in adhesion phase; f Trichuris in early penetration phase with hyphal growth on the egg surface; g, h coccidia oocysts showing adhesion phase and fungal spores; i Balantidium showing adhesion and penetration phases; j Balantidium in early penetration phase with hyphae growing into the cyst; k, l Balantidium cysts in advanced penetration phase with extensive hyphae growth observed inside and around the cysts and advanced digestion of the cysts

Discussion

In this exploratory study, four strains of the predatory fungus Paecilomyces were isolated from native soils in Panama, three of which were observed to successfully adhere to and penetrate the eggs of soil-transmitted helminths (STH) Trichuris suis and hookworms, cysts of Balantidium suis, and oocysts belonging to the coccidia group. To our knowledge, this is the first report of Paecilomyces successfully infecting cysts and oocysts of protozoan parasites of mammals. The discovery of multiple predatory strains of Paecilomyces from soil samples in Panama demonstrates possibilities for augmentative biological control, in which populations of naturally occurring biological control agents are increased to enhance their actions, while at the same time avoiding potential catastrophes associated with introduced species (Waller and Faedo 1996). Further, discovery of native strains of Paecilomyces that can infect multiple species of helminth and protozoan parasites suggests effectiveness of single strains in controlling multiple infections, as are very often the case in endemic areas such as rural Panama.

On coprological examination of naturally infected domestic pigs, multiple infections were observed which are consistent with findings of others such as Zumbado et al. (2009), who demonstrated that, in addition to these parasites, examination of faeces of domestic pigs also often show evidence of infection with Ascaris suum, Strongyloides ransomi, Oesophagostomun spp., Hyostrongylus rubidus, Eimeria spp., and Isospora suis. Balantidium suis was highly prevalent, which is consistent with a study by Guzmán et al. (2013), in which the prevalence of Balantidium suis was between 33 and 95%, likely because pigs are the natural host of this protozoan parasite (Borchert 1981; Martínez et al. 1999; Vásquez and Vidal 1999; Aguilar 2010). The high number of coccidian oocysts observed may be due to the fact that these parasite stages are resistant to extreme environmental conditions, coupled with the fact that the pig holding facilities from which they were recovered had favourable environmental conditions for rapid sporulation and transmission of coccidia infection. Recovery of these protozoans as well as eggs from STHs are consistent with conditions such as poor housing conditions of the animals, low hygiene, and sanitation standards with presence of faeces on the floors of pens for prolonged periods of time, as well as other variables such as geographic location of farms (related to climate), lack of deworming protocols for pigs, and age of pigs, which are similarly reported by Freyre (1990), Pinilla et al. (2005), Valle Peguero et al. (2006), and Zumbado et al. (2009).

The locations from which Paecilomyces spp. were recovered are consistent with other reports. According to Carranza (2014), Paecilomyces can be found in a variety of habitats, including cultivated soils, forests, meadows, deserts, sediments, and sewage sludge. In our assays, three of the four isolated strains of Paecilomyces demonstrated a capacity to adhere to all forms of helminth and protozoan parasites tested, but only strains isolated from one location (Tortí, TA1 and TA2) demonstrated the capacity to invade parasite cysts, oocysts, and eggs. From these results, it can be concluded that of the four strains isolated, the strains from Tortí showed the most antagonistic effects, and thus have the greatest promise for use as an antiparasitic fungus.

A number of previously published works on members of the genus Paecilomyces have demonstrated nematophagous activity by this group of fungi on both eggs and free-living larvae (Dávila and Climaco 2005; Degenkolb and Vilcinskas 2016; Hoyos 2011; Peraza et al. 2011, 2014). The antagonistic actions of the isolated fungi we observed on hookworm eggs were consistent with results of Hoyos (2011) and Peraza et al. (2011, 2014), who demonstrated antagonistic effects of Paecilomyces sp. on the eggs of phytoparasitic nematodes such as Meloidogyne sp. and Heterodera sp. This activity may be related to the ability of members of this genus to produce chitinolytic enzymes (Peraza et al. 2014). Chitin is a structural polymer that forms part of the wall of the shell of nematode eggs, and penetration by nematophagous fungi is facilitated by the secretion of chitinases (Henriquez 2015). In addition to chitinolytic enzymes, Degenkolb and Vilcinskas (2016) demonstrated that this genus of fungus also produces a diverse array of other enzymes and metabolites that can help in the destruction of parasite stages, including leucinotoxins, proteases, acetic acid, and paecilocin.

Paecilomyces has most commonly been described as a nematophagous fungi with particular activity as an endoparasite of nematode eggs, but the various antagonistic chemical effects observed against nematode ova could also explain its activity against protozoan parasites. For example, chitin is present in the walls of cysts of Balantidium and oocysts of coccidia, and the antagonistic effects of Paecilomyces on these parasites may be due to its capacity to degrade the walls of these parasites through the presence of chitinases. The chemical content of the double coating of the cystic wall of Balantidium remains poorly understood; however, other protozoa, such as Entamoeba and Giardia, possess lectins such as chitin in the walls of their cysts, with proteins rich in leucine, which may be similar to Balantidium, and which may explain the mode of action of the isolated strains of Paecilomyces on Balantidium cysts in our study (Gallego-Berenger 1998; Samuelson et al. 2013). As for coccidia, Samuelson et al. (2013) analyzed the chemical structure of the walls of oocysts, which they found to possess fatty acids similar to those of alcohol-resistant acid bacteria, and porous glycocalyx fibers. On the other hand, Borchert (1981) described oocysts as very resistant to external environmental factors because of the three layers of wall that cover them: their external wall is gelatinous and mucoid, which covers a thick keratinoid layer, with an innermost third layer containing cholesterol and other lipid components. Therefore, isolated fungi in our study must have enzymes that can break down all three walls, such as the paraherquamide alkaloids, chitinases, proteases and many other enzymes that work to destroy parasitic forms (Degenkolb and Vilcinskas 2016).

The study has some limitations. As it was exploratory and observational in nature, quantitative comparisons of the effectiveness of various strains of fungus against the different parasite stages or between inoculates with different concentrations of conidia could not be made. Further, this study was limited to a small number of possible soils (for isolation of Paecilomyces) and possible parasites of domestic pigs (and humans). However, as a proof-of-concept that native strains of soil fungi could be used against freeliving stages of a variety of endemic gastrointestinal parasites, this study demonstrates great promise for future development of native nematophagous fungi as a biocontrol for a variety of endemic parasites. In considering the use of native isolated Paecilomyces strains for biological control of environmentally-transmitted gastrointestinal parasites, it is important to note potential negative health impacts such as renal toxicity and mutagenicity, as reported by Che et al. (2014). The positive health impacts of decreasing environmental transmission of the parasites will need to be considered against these potential health hazards in any future use of native strains of Paecilomyces for biological control, and human contact should be avoided, for example in irrigation of crops for human consumption or in chicken manure used as fertilizer for crops.

Conclusions

Strains of the fungus Paecilomyces sp. were isolated from native soils in Panama and shown to have antagonistic activity against endemic gastrointestinal protozoan and nematode parasites of domestic pigs in rural Panama. Our results verify that fungi isolated from Panamanian farms have a high potential as biocontrol agents against Balantidium suis, coccidia, Trichuris suis, and hookworms in soils contaminated with faeces from naturally infected domestic pigs. Importantly, some of these fungal strains can infect multiple types of parasitic forms, both protozoa and nematodes, which is important for biocontrol of natural infections, particularly in mixed infections that pose risks not only for domestic pig producers, but for humans also.

Acknowledgements

Samples from Coclé were collected with the help of Mario Urriola and Mario Arosemena. The authors are grateful to the farmers and landowners who allowed collection of samples from their properties. The authors gratefully acknowledge the support from anonymous donors whose financial contributions made this work possible.

Authors’ contributions

NS designed and supervised the study. GdlC, LdL and NS collected the soil and pig faecal samples. GdlC and LdL prepared the samples and completed the lab work. GdlC and LdL analyzed the data, with the help of AB in identifying and analyzing the fungal strains, NS in identifying the pig parasites, and RK in the final interpretation of the data. GdlC was the primary writer of the manuscript, with help from RK, including help in translating the manuscript from Spanish into English.

Funding

The funds for this project came from anonymous donations from students and local businesses.

Availability of data and material

Not applicable.

Compliance with ethical standards

Conflict of interest

Not applicable.

Ethics approval

Not applicable.

Code availability

Not applicable.

Consent for publication

Not applicable.

Consent to participate

Not applicable.

Footnotes

Publisher's Note

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