Project : USDA ARS

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Research Project: Strategies to Reduce Mycotoxin Contamination in Animal Feed and its Effect in Poultry Production Systems

Location: Toxicology & Mycotoxin Research

2025 Annual Report

Objectives
1. Monitor and mitigate mycotoxins in the poultry feed chain for improved safety and performance. 1.A. Develop and evaluate novel preharvest strategies to reduce mycotoxin contamination in corn and improve sustainability using biological control fungi, Sarocladium zeae (Sz) and Trichoderma harzianum (Th). 1.B. Employ improved mycotoxin detection to identify opportunities for reducing mycotoxin contamination in feed manufacturing practices including storage and management at the feed mill and farm. 2. Determine the impact of chronic mycotoxin exposure on common food safety bacteria, gut health, immunity, and the pathophysiology of poultry. 2.A. Describe the impact of chronic ingestion of combined mycotoxins on intestinal morphology, microbiome, and immune response in poultry, and identify biomarkers of mycotoxin exposure. 2.B. Evaluate the role of FUM and DON on foodborne pathogen loads in NE-induced broilers. 2.C. Investigate the effects of co-contamination of mycotoxins on poultry and identify strategies including the use of feed additives to reduce the harmful effects.

Approach
1. Control and management of mycotoxins in corn, other feed ingredients, and finished feed. Hundreds of fungal isolates will be collected in Georgia to characterize the antagonism and biocontrol potential of Sarocladium zeae (Sz) against Fusarium verticillioides (Fv), Aspergillus flavus (Af) and other mycotoxigenic fungi. This will include assessment of Sz chemotype variation and population structure, including identification of pyrrocidine super-producer strains capable of seed-to-seed vertical transmission. Pyrrocidine produced by Sz suppresses Fv fumonisin production, and the involvement of the gene FvZBD1 will be elucidated. Agricultural nitrous oxide emissions will be reduced along with mycotoxins by use of a non-emitting Trichoderma biocontrol agent. Further, new technology for the rapid detection and quantification of multiple mycotoxins will be deployed for testing the poultry feed chain. Lastly, organic acids and essential oils will be evaluated for inhibition of fungal colonization and postharvest mycotoxin contamination. 2. Impacts of subclinical and chronic doses of fumonisin and deoxynivalenol mycotoxins on poultry gut health, microbiome, immune parameters, and intestinal morphology. Identify miRNA biomarkers to detect subclinical mycotoxicosis in poultry. Also evaluate the role of fumonisin and deoxynivalenol on foodborne pathogen loads in broilers with necrotic enteritis. Evaluate microbial in vivo degradation of mycotoxins in poultry by supplementing feed with deactivators and synbiotics.

Progress Report
Under Objective 1 Sarocladium zeae, a potential biocontrol fungus, has a unique population in Georgia. Using whole genome sequencing of nearly 200 isolates of Sarocladium from corn kernels harvested from 11 Georgia counties at the end of the 2022 growing season, two distinct populations of S. zeae were identified. One of the populations consists of a dozen isolates from a single field, and sequencing data indicate it is likely a clonal population. It may represent an introduced population. Importantly, this small population produces much more of the pyrrocidine metabolites compared to the larger, more broadly distributed and genetically variable S. zeae population. Pyrrocidines are key metabolites for biocontrol efficacy of S. zeae. ARS researchers at Athens, Georgia have previously shown that pyrrocidines shut off fumonisin mycotoxin production by Fusarium verticillioides. The diversity and abundance of antifungal secondary metabolites produced by S. zeae is also being assessed. A field trial in summer 2025 will assess biocontrol efficacy of S. zeae isolates from the unique, high-pyrrocidine producing population. There is much greater genomic variability in mycotoxin-producing Fusarium species than originally believed. In addition to the evaluation of S. zeae population genetics, project scientists are actively conducting population diversity studies using whole genome sequencing on the fumonisin producing species F. verticillioides, F. proliferatum, and F. fujikuroi. The diversity within F. fujikuroi has been evaluated the most, and there is solid evidence to support two main populations within this species, namely the high fumonisin-producing genotypes (that produce less gibberellic acid) and the low fumonisin-producing genotypes (that produce more gibberellic acid). This information is important for risk assessment of fumonisin exposure since this species infects various crops from rice to wine grapes. ARS researchers at Athens, Georgia assessed >1500 samples of processed animal feed or raw feed ingredients (e.g., corn, soybeans, etc.) from more than 15 states using near infrared (NIR) instrumentation to determine nutritional content of animal feed products. Roughly 650 of those samples (corn, wheat, soybeans, soybean meal, broiler and layer feeds, etc.) were also analyzed for mycotoxins. All samples were contaminated with at least one mycotoxin, with fumonisin being the most frequent. Most of the samples were contaminated with two or more mycotoxins, with 35% containing 2 mycotoxins and 29% containing 3 mycotoxins in a single sample. Conversely, 52% of soybean meal samples tested had no mycotoxin contamination whereas 48% were contaminated with fumonisin. Of the finished feed samples analyzed, 22% were contaminated with one mycotoxin, 21% with 2 mycotoxins, and 54% were contaminated with 3 mycotoxins. A correlation was observed between mycotoxin contamination and altered protein, starch, and fat content in corn. Thus, mycotoxin contamination in corn can result in reduced value of feed both monetarily and nutritionally. A manuscript on this project has been published. Corn samples contaminated with aflatoxin showed a lower fat content and appeared darker in color. Deoxynevalenol was positively correlated with starch. Fumonisin was positively correlated with protein and moisture and negatively correlated with starch. Zearalenone was negatively correlated with starch. This work is informing the poultry feed industry by helping predict possible ingredient degradation based on which mycotoxin(s) they are contaminated with, allowing for improved formulation prior to animal consumption. The poultry industry is utilizing data from this project to adapt their strategies to mitigate mycotoxin issues and to help improve the negative effects of mycotoxins on poultry health, production, and performance. The research findings are impacting poultry diet formulation specifically but can be applied throughout the animal feed industry. Project scientists have maintained strong relationships with local feed mills and stakeholders in several states. Under Objective 2 over the past three years, project scientists have demonstrated that poultry feed ingredients contaminated with multiple mycotoxins, even at doses below the FDA tolerance levels for individual mycotoxins, still cause significant intestinal damage, intestinal dysbiosis, and increase food-borne pathogen loads in the broiler gut. Findings established dose-dependent effects of mycotoxins on intestinal tight junction proteins of broiler chickens. Multiple mycotoxins, even when well below the recommended level, reduced the expression of genes responsible for maintaining the gut barrier integrity and caused a significant increase in foodborne pathogen loads, particularly Salmonella and C. perfringens, in broiler chickens. Further, dietary mycotoxin levels above 1 mg/kg in the poultry diet caused hepatic damage and increased liver enzymes. These altered hepatic enzymes can be used as potential biomarkers for detecting early clinical mycotoxicosis in poultry flocks. Project scientists identified nine miRNAs that were expressed differentially in the broiler liver as early as 14 days post-mycotoxin exposure. These miRNAs represent novel and sensitive biomarkers for the early diagnosis of mycotoxin induced liver damage, offering potential for significant economic savings in the poultry industry through timely intervention. To address these challenges, scientists evaluated commercially available mycotoxin deactivators and synbiotics in vivo to mitigate mycotoxin-induced decreases in health and production performance. Results showed that probiotics metabolized the mycotoxins within the bird and improved overall chicken gut health, while mycotoxin deactivators positively impacted the immune response of mycotoxin-exposed birds. These findings suggest combined approaches may offer effective protection against multiple mycotoxins in the poultry diet. These data advanced the understanding of subclinical mycotoxin exposure in poultry and provided practical insights to improve feed safety, bird health, and production performance. Project scientists continue to collaborate closely with poultry producers across the US to ensure research translates into real-world improvements in poultry food safety, nutrition, welfare, and profitability.

Accomplishments
1. Improved feed safety and poultry disease prevention can lead to better production performance and reduce economic losses. By understanding how subclinical concentrations of multiple mycotoxins increase food borne pathogen loads in the chicken, producers can take proactive measures to improve overall flock health. ARS researchers in Athens, Georgia demonstrated that feed contaminated with multiple mycotoxins at concentrations below established FDA tolerance levels can synergistically worsen intestinal damage, cause intestinal dysbiosis, and increase food borne pathogen loads in the gut. They also found the key amino acids methionine, aspartate, and serine are significantly reduced when combined mycotoxin levels are above 1mg/kg diet, emphasizing a link between mycotoxins and feed nutritional profiles. Additionally, researchers identified key hepatic enzymes and miRNAs biomarkers to identify mycotoxicosis as early as 14 days after exposure to mycotoxins, which may provide valuable tools for poultry producers to monitor and effectively mitigate mycotoxin exposure.

2. Mycotoxin regulatory guidelines are based on the hazards of individual mycotoxins contaminating feed and feed ingredients, but multiple mycotoxins contribute to total contamination levels. ARS researchers in Athens, Georgia quantitatively analyzed fumonisins, deoxynivalenol, aflatoxin, and zearalenone in corn samples originating from various regions in the Southeastern U.S. All samples were contaminated with at least one mycotoxin, with fumonisins being the most frequent. Most of the samples were contaminated with two or more mycotoxins, with only 18% of the samples containing a single mycotoxin. As mycotoxin contamination can vary by location, this study indicates region specific management strategies may be needed to mitigate mycotoxins in feed ingredients. Further, a correlation was observed between mycotoxin contamination and altered nutrient content in corn. This ongoing research provides further evidence that co-contamination of mycotoxins is common in corn, potentially meaning severe economic losses to poultry producers due to poor-quality feed impacting food safety as well as animal health and productivity.

U.S. DEPARTMENT OF AGRICULTURE

Toxicology & Mycotoxin Research: Athens, GA