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

Location: Toxicology & Mycotoxin Research

2022 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
The unit officially began its new project #6040-42000-046-000D in March 2022 after operating under bridge project #6040-42000-045-000D in FY2021 due to a 1-year postponement of our NP108 review through the Office of Scientific Quality Review. Last year’s annual report provided a five-year summary of the previously expired in-house project #6040-42000-043-000D (2016–2021). The report provided below is based on this year’s progress made to-date on our new objectives from project #6040-42000-046-000D. Objective 1: Our first collection of field-grown corn ears will not happen until the end of this current growing season (approximately August and September). Fields for sampling have been identified through Georgia’s county extension agents, and logistics have been arranged. A survey of preplant seed for seedborne fungi has so far exhibited low infection frequency, suggesting most fungal infections of the corn plant result from endemic populations in the agricultural fields. Yet, preliminary experiments have demonstrated the potential for vertical transmission of the fungus Sarocladium zeae from inoculated seed to the subsequent kernels produced on new ears. Additional experiments are needed to confirm, but this does support the concept of treating seed with S. zeae as a biological control agent. We are also 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 strain of Trichoderma produces only a fraction of N2O compared to the corn pathogen, Fusarium verticillioides. Regarding the monitoring of animal feed and raw feed ingredients for mycotoxin contamination, new instrumentation has been installed that will simultaneously quantify up to six mycotoxins in different types of grain and finished feed. Data will be validated using liquid chromatography mass spectrometry (LC-MS). Objective 2: Protocols for growing Fusarium species to produce ample mycotoxins for the broiler feeding trials required more optimization than originally planned, but parameters were identified that drastically improved the production of mycotoxins on rice culture material. Such hyper-production of the mycotoxins allows for diet formulations that provide the target doses of mycotoxins without adding a lot of culture material, thus not altering nutritional profiles of diets. A broiler feeding trial demonstrated the effects of chronic exposure to subclinical levels of fumonisins and deoxynivalenol in broiler chickens and their role in inducing subclinical necrotic enteritis. The level of these mycotoxins in the experimental diets was much lower than the FDA tolerance levels. Our findings indicated synergistic effects of the mycotoxins and predicted that subclinical doses of combined toxins not only directly affected the production performance but also influenced chicken health by inducing necrotic enteritis and exacerbating its severity. The mycotoxins may also increase susceptibility of the birds to pathogenic bacteria. A second feeding trial was conducted to identify the highest doses of the two mycotoxins combined that does not affect broiler production performance. Data are being gathered and analyzed, but the outcome should provide a foundation to suggest upper limits for mycotoxins in poultry feed.

Accomplishments
1. Fumonisin and deoxynivalenol mycotoxins exacerbate development of necrotic enteritis (NE) in broiler chickens. NE is one of the most economically important diseases of poultry, causing nearly $6 billion in increased annual costs to the poultry industry worldwide due to reduction in body weight and increase in feed conversion ratio compared with healthy birds. ARS researchers in Athens, Georgia, have shown that fumonisin and deoxynivalenol mycotoxins, even when at subclinical concentrations, are predisposing factors causing intestinal damage and act in tandem with coccidial pathogens to exacerbate NE in poultry. The data will aid poultry producers as they manage mycotoxin exposures, particularly when multiple mycotoxins are present in the feed.

2. 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 survey was conducted to assess the possibility of semicarbazide production in poultry processing in the absence of nitrofurazone abuse. The data suggest that some plants are producing appreciable amounts of semicarbazide during processing, but many aren’t. For those plants producing semicarbazide, data clearly implicated the chill tank as the reaction site. ARS researchers in Athens, Georgia, are now assessing processing parameters to provide an understanding of how incidental production of semicarbazide can be avoided, primarily by carefully regulating the pH of the chill tank. These data are critical to the poultry industry to provide a basis to reopen export markets in South Korea.

Review Publications
Yosri, M., Elaasser, M.M., Abdel-Aziz, M.M., Hassan, M.M., Alqhtani, A.H., Al-Gabri, N., Ali, A.B., Pokoo-Aikins, A., Amin, B.H. 2022. Determination of therapeutic and safety effects of zygophyllum coccineum extract in induced inflammation in rats. BioMed Research International. 2022. Article 7513155. https://doi.org/10.1155/2022/7513155.
Yao, L., Beibei, J., Yoon, S.C., Zhuang, H., Ni, X., Guo, B., Gold, S.E., Fountain, J.C., Glenn, A.E., Lawrence, K.C., Zhang, H., Guo, X., Zhang, F., Wang, W. 2022. Spatio-temporal patterns of Aspergillus flavus infection and aflatoxin B1 biosynthesis on maize kernels probed by SWIR hyperspectral imaging and synchrotron FTIR microspectroscopy. Food Chemistry. 382:132340. https://doi.org/10.1016/j.foodchem.2022.132340.
Shanmugasundaram, R., Acevedo-Villanueva, K., Akerele, G., Mortada, M., Selvaraj, R.K., Applegate, T.J., Kogut, M.H. 2021. Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens. PLoS ONE. 16(11). Article e0260280. https://doi.org/10.1371/journal.pone.0260280.
Acevedo-Villanueva, K.A., Akerle, G.O., Al Hakeem, W., Renu, S., Shanmugasundaram, R., Selvaraj, R. 2021. A novel approach against Salmonella: A review of polymeric nanoparticle vaccines for broilers and layers. Vaccines. 9(9):1041. https://doi.org/10.3390/vaccines9091041.
Gao, M., Gold, S.E., Gu, X., Satterlee, T.R., Duke, M.V., Scheffler, B.E., Glenn, A.E. 2022. Transcriptomic Responses of Fusarium verticillioides to Lactam and Lactone Xenobiotics. Frontiers in Fungal Biology. https://doi.org/10.3389/ffunb.2022.923112.
Gregorich, J., Lilburn, M., Shanmugasundaram, R. 2022. Effects of induced moisture loss in chicken embryos at embryonic day 18 and post-hatch immune response during salmonella enteritidis lipopolysaccharide challenge in broilers. Frontiers in Physiology. 13:1-12. https://doi.org/10.3389/fphys.2022.820349.
Livingston, M.L., Pokoo-Aikins, A., Frost, T., Laprade, L., Hoang, V., Nogal, B., Phillips, C., Cowieson, A.A. 2022. Effect of heat stress, dietary electrolytes, and vitamins E and C on blood biochemistry of the modern broiler chicken. Frontiers in Animal Science. 3. Article 807267. https://doi.org/10.3389/fanim.2022.807267.
Shahna, F., Shanmugasundaram, R., Adams, D., Selvaraj, R. 2022. Gastrointestinal microbiota and their manipulation for improved growth and performance in chickens. Foods. 11(10):1401. https://doi.org/10.3390/foods11101401.