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

Location: Toxicology & Mycotoxin Research

2023 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
Diversity of fungal populations detected in Georgia corn. Field-grown corn ears were collected from 10 counties at the end of the 2022 growing season. Surface sterilized kernels produced nearly 1000 isolates, most of which are putatively identified as Fusarium verticillioides and Sarocladium zeae. A survey of preplant seed for seedborne fungi provided very few isolates, indicating most of the fungi recovered from the harvested kernels were from endemic populations in the agricultural fields and not from vertical transmission of seedborne fungi. This work is being done by a PhD student working with ARS. Interestingly, preliminary experiments have demonstrated that S. zeae does have the potential for vertical transmission from inoculated seed to the subsequent kernels produced on new ears. This supports the concept of treating corn seed with S. zeae as a biological control agent. The diversity and abundance of antifungal secondary metabolites produced by S. zeae is being assessed using liquid chromatography mass spectrometry (LC-MS), including the identification of possibly novel compounds. More refined fractionation will purify one or more candidate compounds for molecular identification. Biological control of Fusarium verticillioides. A collaboration with the Institute of Bioanalytics and Agro-Metabolomics, University of Natural Resources and Life Sciences (BOKU) in Tulln, Austria, was initiated to conduct targeted metabolomic studies on the fumonisin producing fungus F. verticillioides. These studies will provide better understanding of how pyrrocidines inhibit the production of fumonisins. Another project is investigating the potential of Trichoderma species as biocontrol agents with a value-added trait of producing minimal nitrous oxide (N2O) gas, which is a major greenhouse gas. We have shown that a commercialized biological control strain of Trichoderma produces only a fraction of N2O compared to the corn pathogen, F. verticillioides. Thus, if Trichoderma can reduce the abundance of F. verticillioides in agricultural fields, then less N2O should be emitted from those fields. Lastly, another N2O project is the first functional characterization of a fungal p450 nitric oxide reductase (NOR1) that is essential for F. verticillioides to produce N2O. Determine natural contamination of feed ingredients. Corn collections from silos, on the ground piles (flat storage), and other storage forms were analyzed for mycotoxins using LC-MS protocols as well as a VICAM commercial system. We have currently received corn samples from local feed mills and stakeholders in 12 states. In addition to mycotoxins, over 1000 samples of corn, other feed ingredients, and finished feed were analyzed using near infrared (NIR) for their nutritional content. Preliminary data indicate a correlation or trend between mycotoxin contamination levels and some nutritive properties, but more data are needed to strengthen these analyses. The data will help to determine and predict the severity of effects on poultry and other animals that consume contaminated feed. Impact of organic acids and essential oils on fungal growth. Organic acids (OA) are commonly used antimicrobials applied to stored corn and animal feed. Synergies between different OA and between OA and essential oils (EO) were tested in vitro. We found lemongrass oil completely inhibits fungal growth when combined with acetic acid or butyric acid, although the inhibitory concentrations differed. Overall, propionic acid exhibited the best synergy with lemongrass. In contrast, assays on corn kernels showed significant differences, most notably a synergy between the three OA and cinnamon oil. Complete fungal inhibition on the kernels required more OA and EO than found with agar media. Fungal influence on gut microbiome in broilers. Three research trials were conducted to investigate the effects of fumonisin (FUM) and deoxynivalenol (DON) combinations on the gut microbiome of broiler chickens, revealing a decrease in Lactobacillus and Faecalibacterium abundance alongside increased presence of Clostridia. This imbalance resulted in inflammation, intestinal dysbiosis, and subclinical necrotic enteritis. Moreover, mycotoxins had detrimental effects such as reduced expression of genes for intestinal tight junction proteins and a significant increase in cecal Salmonella abundance. The mycotoxins also caused hepatic damage and increased liver enzymes, which can be used as biomarkers for mycotoxin damage. Additionally, the presence of feed mycotoxins adversely impacted broiler production performance, gut health, apparent ileal amino acid digestibility, immune cell response, and intestinal lesion scores.

Accomplishments
1. A fresh look at organic acids and essential oils as antifungal treatments finds synergies. Essential oils and organic acids have been extensively studied for their antimicrobial qualities. A recent study by ARS researchers in Athens, Georgia, had a fresh perspective and identified synergies that lower inhibitory concentrations. Analysis of both organic acid and essential oils demonstrated that prior literature results were inaccurate and that significantly less of the compounds are needed to synergistically inhibit the growth of Aspergillus flavus, the fungus most often associated with aflatoxin contamination of crops. Fungal growth on corn and finished feed is a big concern to the animal feed industry, and the new data from ARS enhances the ability of stakeholders to manage mycotoxin contamination of stored feed and feed ingredients by using improved formulations of organic acids and essential oils.

2. Feed contaminated with multiple mycotoxins, even at low concentrations, increases food borne pathogen loads in the chicken gut. ARS researchers in Athens, Georgia, demonstrated that feed contaminated with multiple mycotoxins at doses below established FDA tolerance levels can synergistically worsen intestinal damage, cause intestinal dysbiosis, and increase food borne pathogen loads in the gut. Thus, even subclinical concentrations of mycotoxins predispose poultry to food borne pathogen outbreaks and exacerbate necrotic enteritis (NE). This valuable data allows poultry producers to effectively manage and hopefully limit mycotoxin exposures by focusing on the presence of multiple mycotoxins in the feed, even when concentrations are low.

3. Semicarbazide can be produced incidentally in poultry processing facilities. Semicarbazide is a regulatory marker for the use of nitrofurazone, an antibiotic banned from use in animals intended for human consumption. A previous survey by ARS researchers in Athens, Georgia, found that some plants are producing appreciable amounts of semicarbazide during processing while other plants are not. Data clearly implicated the chill tank as the reaction site for those plants producing semicarbazide. Further research is assessing chill tank parameters necessary to minimize the chemical production of semicarbazide during processing, primarily by pH regulation. These data are critical to the U.S. poultry industry to provide a basis to reopen export markets.

Review Publications
Karagianni, E., Kontomina, E., Lowe, E., Athanasopoulos, K., Papanikolaou, G., Garefalaki, V., Kotseli, V., Zaliou, S., Grimaud, T., Arvaniti, K., Tsatiri, M., Fakis, G., Glenn, A.E., Roversi, P., Abuhammad, A., Ryan, A., Sim, R., Sim, E., Boukouvala, S. 2022. Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally, and phylogenetically distinct from its N-acetyltransferase (NAT) homologues. FEBS Journal. 290:2412-2436. https://doi.org/10.1111/febs.16642.
Cason, E.E., Al Hakeem, W., Adams, D., Shanmugasundaram, R., Selvaraj, R. 2022. Effects of synbiotic supplementation as an antibiotic growth promoter replacement on cecal Campylobacter jejuni load in broilers challenged with C. jejuni. Journal of Applied Poultry Research. 32(2):100315. https://doi.org/10.1016/j.japr.2022.100315.
Al-Garadi, A., Quid-Mohammed, M., Alqhtani-Abdulmohsen, H., Pokoo-Aikins, A., Al-Mufarrej-Saud, I. 2022. In vitro antibacterial and antifungal efficacy assessment of ethanolic and aqueous extracts of Rumex nervosus leaves against selected bacteria and fungi. Veterinary world. 15(11):2725-2737. https://doi.org/10.14202/vetworld.2022.2725-2737.
Hakeem, A., Fathima, S., Selvaraj, R., Shanmugasundaram, R. 2022. Campylobacter jejuni in poultry: pathogenesis and control strategies. Microorganisms. 10(11):2134. https://doi.org/10.3390/microorganisms10112134.
Shanmugasundaram, R., Lourenco, J., Hakeem, W.A., Dycus, M., Applegate, T. 2023. Subclinical doses of dietary fumonisins and deoxynivalenol causes cecal microbiota dysbiosis in broiler chickens challenged with clostridium perfringens. Frontiers in Microbiology. 14:1-14. https://doi.org/10.3389/fmicb.2023.1106604.
Fathima, S., Shanmugasundaram, R., Sifri, M., Selvaraj, R. 2023. Yeasts and yeast-based products in poultry nutrition. Journal of Applied Poultry Research. 32(2):100345. https://doi.org/10.1016/j.japr.2023.100345.
Liu, J., Shanmugasundaram, R., Doupovec, B., Dian, S., Ganapathi, R., Applegate, T. 2023. Short-term exposure to fumonisins and deoxynivalenol, on broiler growth performance and cecal Salmonella load during experimental Salmonella enteritidis infection. Poultry Science. 102(6):102677. https://doi.org/10.1016/j.psj.2023.102677.
Avila, L.P., Leiva, S.F., Abascal-Ponciano, G.A., Flees, J.J., Sweeney, K.M., Wilson, J.L., Turner, B., Litta, G.A., Waguespack-Levy, A.M., Pokoo-Aikins, A., Starkey, G.W. 2022. Combining maternal and post-hatch dietary 25-hydroxycholecalciferol supplementation on broiler chicken growth performance and carcass characteristics. Poultry. 1(2):111–124. https://doi.org/10.3390/poultry1020010.
Pokoo-Aikins, A., Timmons, J.R., Min, B.R., Lee, W.R., Mwangi, S.N., Mcdonough, C.M., Chen, C. 2022. Effects of varying levels of dietary DL-methionine supplementation on breast meat quality of male and female broilers. Poultry. 1(1):40-53. https://doi.org/10.3390/poultry1010005.
Chen, C., Pokoo-Aikins, A., Huang, S., Regmi, P., Rehman, M. 2023. Editorial: Perspectives in avian skeletal systems and skeletal abnormalities. Frontiers in Physiology. 14:1229943. https://doi.org/10.3389/fphys.2023.1229943.
Mitchell, T.R., Glenn, A.E., Gold, S.E., Lawrence, K.C., Berrang, M.E., Gamble, G.R., Feldner, P.W., Hawkins, J.A., Miller, C.E., Olson, D.E., Chatterjee, D., Mcdonough, C.M., Pokoo-Aikins, A. 2022. Survey of meat collected from commercial broiler processing plants suggests low levels of semicarbazide can be created during immersion chilling. Journal of Food Protection. 85(5):798-802. https://doi.org/10.4315/JFP-22-012.
Fathima, S., Hakeem, A., Shanmugasundaram, R., Selvaraj, R. 2022. Necrotic enteritis in broiler chickens: A review on the pathogen, pathogenesis, and prevention. Microorganisms. 10(10):1958. https://doi.org/10.3390/microorganisms10101958.
Liu, G., Ajao, A.M., Shanmugasundaram, R., Taylor, J., Ball, E., Applegate, T.J., Selvaraj, R., Kyriazakis, I., Olukosi, O.A., Kim, W.K. 2023. The effects of arginine and branched-chain amino acid supplementation to reduced-protein diet on intestinal health, cecal short-chain fatty acid profiles, and immune response in broiler chickens challenged with Eimeria spp.. Poultry Science. 102 (7): 1-17. https://doi.org/10.1016/j.psj.2023.102773.