Drug resistance to synthetic anthelmintics is a widespread issue, primarily due to excessive use and improper administration, particularly in small ruminants. The nematophagous fungus Duddingtonia flagrans is employed as a biological control agent against gastrointestinal nematodes in ruminants. Its chlamydospores are administered orally and excreted in feces, where the fungus traps nematodes. Mature D. flagrans chlamydospores have thick walls with globular surface protuberances, which help protect the fungus from physical, chemical, and microbiological stress. However, the harsh conditions of the ruminant digestive system can reduce its biocontrol effectiveness. Understanding the impact of each segment of the gastrointestinal tract on chlamydospore survival is essential for developing a delivery system that enhances fungal viability.

The survival of D. flagrans under gastrointestinal conditions has been assessed in vivo, with the final concentration and viability of the fungi in feces shown to correlate with the initial orally administered dose. Some studies have investigated fungal survival by creating fistulas in the animal’s digestive tract, but these methods have drawbacks, including high maintenance costs and the need for specialized personnel. Additionally, from a practical standpoint, these procedures are labor-intensive and time-consuming, limiting the number of samples that can be analyzed. In contrast, in vitro studies offer the advantage of isolating the impact of each digestive segment.

In vitro techniques that simulate gastrointestinal tract conditions provide an efficient way to evaluate the effects fungi are exposed to as they pass through the digestive system. These methodologies are based on a procedure originally developed to assess forage digestion, utilizing discontinuous containers incubated with ruminal fluid and a buffer under anaerobic conditions at 39°C. Previous studies have primarily focused on ruminal and abomasal digestion. This study aimed to expand the evaluation to include four consecutive gastrointestinal segments: the oral cavity, small intestine, rumen, and abomasum. Key response variables related to the fungi’s biocontrol activity, including concentration and nematode predatory ability, were monitored.

Conducting this research in vitro has several drawbacks despite its advantages. One major limitation is the inability to fully replicate the dynamic and complex environment of a living animal's digestive system. Factors such as peristalsis, enzyme fluctuations, immune responses, and microbial interactions are difficult to simulate accurately. Additionally, in vitro models do not account for the influence of the host’s physiology, including mucus secretion and immune modulation, which can impact fungal survival and activity.

Another challenge is the simplified microbial communities present in controlled environments. While ruminal fluid is often used in in vitro digestion models, the microbial diversity and interactions do not fully reflect the complexity of the in vivo gut microbiome, which plays a crucial role in digestion and fungal viability. Furthermore, the time fungi spend in each digestive segment varies in a living animal due to factors like digestion rates, feed composition, and gut motility. In vitro studies typically rely on standardized incubation times that may not accurately represent actual conditions.

In addition, in vitro models do not account for animal-specific variability. Differences in species, breed, diet, and health status can all influence gastrointestinal conditions, potentially affecting the survival and predatory activity of D. flagrans chlamydospores. This controlled environment may lead to either an overestimation or underestimation of the fungus’s biocontrol efficacy compared to real-world applications. Despite these drawbacks, in vitro studies remain valuable for controlled, cost-effective preliminary research, allowing for the isolation of specific digestive effects before conducting more complex and expensive in vivo studies.

Method & Materials

The study was conducted at AGROSAVIA in the Bioproducts Department at the Tibaitatá Research Center in Mosquera, Colombia. The Duddingtonia flagrans strain used in the research was originally isolated from soil in Cota, Cundinamarca, and is preserved at -20°C in a glycerol and peptone solution. Biomass for the study was produced through biphasic fermentation, ensuring a minimum concentration of chlamydospores and high nematode predatory ability.

Ruminal fluid was obtained from a healthy, three-year-old wool sheep with a ruminal fistula. The fluid was collected after fasting, filtered, and used in the assays within an hour to maintain its integrity. The pH of the fluid was recorded at 6.69.

Key variables evaluated in the study included chlamydospore concentration and nematode predatory ability. Chlamydospores were counted under a microscope, with any structurally damaged spores recorded separately. Nematode predatory ability was assessed using Panagrellus redivivus larvae, with captured larvae counted to determine effectiveness compared to a control.

The in vitro methodology simulated the ovine gastrointestinal tract, incorporating findings from previous research and digestion studies. Exposure times for each digestive segment were evaluated to account for variations caused by diet, water intake, and health conditions, ensuring a comprehensive analysis of fungal survival and effectiveness.

The proposed methodology consists of four sequential steps:

1. Oral Cavity: Artificial chewing was simulated using a disk mill to mix artificial saliva with grass and biomass. The chlamydospore concentration, nematode predatory ability, and percentage of broken chlamydospores were evaluated.

2. Rumen: Ground samples from the oral cavity step were incubated with ruminal fluid and artificial saliva under controlled pH and temperature conditions. The pH was monitored and adjusted regularly.

3. Abomasum: The mixture from the rumen step was adjusted to a low pH and treated with pepsin to simulate digestion in the abomasum. Conditions were maintained and adjusted throughout incubation.

4. Small Intestine: The abomasum-treated samples were adjusted to an alkaline pH and treated with pancreatin to simulate intestinal digestion. pH adjustments were made periodically.

Control treatments consisted of biomass without mechanical processing, incubated under similar conditions but in sterile water. Samples were taken at each stage for nematode predatory ability assessment.

Results

After passing through the oral cavity, there was no significant difference in chlamydospore concentration between the control and treated groups in the short-exposure assay, but a significant difference was observed in the long-exposure assay. Despite mechanical milling, chlamydospore concentration remained stable, with only a small percentage breaking. The nematode predatory ability remained high in the short-exposure assay but showed significant differences in the long-exposure assay, where it exceeded 97%.

In the gastrointestinal tract, chlamydospore concentration was not significantly affected during short exposure but was impacted during long exposure. The fungus's nematode predatory ability declined progressively through each digestive segment, with significant reductions observed in both assays. The interaction of exposure time and treatment had a notable effect on predatory ability but not on concentration.

The treated group consistently showed higher chlamydospore concentration than the control in all digestive cavities, though both exceeded expected levels. No significant differences were found between cavities in the control group, while differences were observed in the treated group, particularly in the long-exposure assay. Chlamydospore breakage increased through the digestive tract, with the highest percentage in the small intestine.

Nematode predatory ability remained stable in the control group throughout both assays. In the treated group, short exposure led to gradual reductions through the digestive tract, while long exposure resulted in a sharp decline, with the fungus losing all predatory ability in the small intestine. Significant differences were observed between the control and treated groups in the rumen, abomasum, and small intestine, but not in the oral cavity.

Conclusion

The study found that prolonged exposure to the digestive system negatively affected the fungus D. flagrans' ability to trap nematodes but did not significantly reduce its spore concentration. The longer the exposure, the less effective the fungus became, completely losing its nematode predatory ability after 51 hours. Additionally, more spores showed physical damage as they passed through each digestive stage, especially in the long-exposure test.

Researchers

The study (which is available here, if you'd like to read it) was conducted by Elizabeth Céspedes-Gutiérrez, Diana Marcela Aragón, Martha Isabel Gómez-Álvarez, Jaime Andrés Cubides-Cárdenas, and Diego Francisco Cortés-Rojas. Elizabeth Céspedes-Gutiérrez, Martha Isabel Gómez-Álvarez, and Diego Francisco Cortés-Rojas are affiliated with the Corporación Colombiana de Investigación Agropecuaria AGROSAVIA, Sede Central, Mosquera, Colombia. Diana Marcela Aragón is affiliated with the Universidad Nacional de Colombia, Sede Bogotá, Bogotá, Colombia. Jaime Andrés Cubides-Cárdenas is affiliated with the Corporación Colombiana de Investigación Agropecuaria AGROSAVIA, Sede Tibaitatá, Mosquera, Colombia. Diego Francisco Cortés-Rojas is the corresponding author.