Location: Toxicology & Mycotoxin Research
2024 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 11 counties at the end of the 2022 growing season. Surface sterilized kernels (n=1740) produced 1854 isolates with 50% being species within the mycotoxin-producing genus Fusarium. Another 13% of the isolates were identified as Sarocladium zeae. Interestingly, only 1% of the isolates were Aspergillus flavus, which produces the aflatoxin mycotoxin. Interestingly, preliminary experiments have demonstrated that S. zeae has 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 structural identification. Population analyses are underway using genomic sequencing.
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, continued into FY2024 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. We’ve also recently shown that Trichoderma can reduce the production of N2O by F. verticillioides, thus less N2O may be emitted from fields where Trichoderma is applied as a biocontrol agent. 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. That study is complete, and manuscripts are being prepared for submission.
We have received over 1000 samples from over 10 states and run them on near infrared (NIR) to determine nutritional content. Roughly 500 of those samples (corn, wheat, soybeans, soybean meal, DDGs, different phases of complete poultry feed) have been analyzed for mycotoxins via LCMS. Of the 328 corn samples tested so far, all samples were found to be contaminated with at least one mycotoxin, with fumonisin being the most frequent. Most of the samples were contaminated with two or more mycotoxins, with only 18.29% of the samples containing a single mycotoxin. A correlation was observed between mycotoxin contamination and altered nutrient content in corn. This manuscript is currently undergoing review. We are broadening our data set by focusing on the other feed ingredients we have received. Correlations between contamination and nutritional content will be studied for each. This work will inform the 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. Industry is utilizing our data to adapt their strategies to mitigate mycotoxin issues and to help improve the negative effects of these mycotoxins on poultry health, production, and performance. Our findings are having a significant impact on poultry diet formulation specifically, but they can be applied throughout the feed industry. We have maintained strong relationships with local feed mills and stake holders in several states.
We identified synergy between organic acids and essential oils in combating aflatoxin contamination in feed. Both essential oils and organic acids are already used in the poultry industry to combat the spread of bacterial pathogens and improve poultry gut health. However, we found that combinations of these compounds can be used at concentrations that are less than half of the respective minimal inhibitory concentration of either compound individually. As the ratios can be altered between compounds and still have complete inhibition of A. flavus, this may offer cheaper options for fungal growth control, which should reduce mycotoxin contamination and improve feed quality. Discussions are underway with a company that has developed “softer” versions of these acids and oils so they are less caustic, and these new products will soon be tested in our assays.
Poultry feed ingredients contaminated with multiple mycotoxins at doses below the FDA tolerance levels for individual mycotoxins still worsen intestinal damage, cause intestinal dysbiosis, and increase food-borne pathogen loads in the gut. Dose-dependent effects of mycotoxins on the intestinal tight junction proteins of broiler chickens was shown. Multiple mycotoxins, even well below the recommended level, reduced the expression of genes for intestinal tight junction proteins and caused a significant increase in foodborne pathogen loads. Additionally, mycotoxin doses 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 clinical mycotoxicosis in the field. Further, we identified nine different miRNAs that were expressed differentially in the liver as early as 14 days after mycotoxin exposure. These miRNAs could be potential biomarkers for the early detection of mycotoxicosis, which could help the poultry industry save millions of dollars.
To mitigate the adverse impact of mycotoxins on poultry health, commercially available mycotoxin deactivators and synbiotics were assessed in vivo to improve chicken gut health and production performance. Our findings showed that probiotics metabolized the mycotoxins in vivo and improved chicken health, while mycotoxin deactivators positively impacted the immune response of mycotoxin-exposed birds. Further studies with different concentrations of mycotoxins and deactivators are needed to further evaluate the effects on broiler chicken production performance.
Accomplishments
1. Corn is commonly co-contaminated with various mycotoxins, and the nutrient profile of corn is diminished. Although mycotoxin regulatory guidelines are based on the hazards of individual mycotoxin contamination, feed and feed ingredients may be contaminated with multiple mycotoxins. Such mycotoxin co-contamination and its impact on the nutrient content of corn grain were evaluated. Corn samples (n=328) originating from various regions in the Southeastern United States were quantitatively analyzed by ARS researchers in Athens, Georgia, for fumonisin, deoxynivalenol, aflatoxin, and zearalenone. Nutritional content was analyzed by near-infrared spectroscopy. All samples were found to be contaminated with at least one mycotoxin, with fumonisin being the most frequent. Most of the samples were contaminated with two or more mycotoxins, with only 18.29% of the samples containing a single mycotoxin. A correlation was observed between mycotoxin contamination and altered nutrient content in corn. This study provides further evidence that co-contamination of mycotoxins is the norm in corn, and that mycotoxin contamination likely impacts on the nutrient profile of feed corn. This research provides poultry industry nutritionists information essential to formulating poultry diets for improved performance.
2. Impacts of mycotoxin contaminated corn on poultry can be monitored and potentially mitigated by utilizing novel biomarkers. 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). Additionally, hepatic enzymes and miRNAs may have utility as biomarkers to identify mycotoxicosis as early as 14 days after exposure to mycotoxins. These data will allow poultry producers to monitor and effectively mitigate mycotoxin contamination in the feed.
Review Publications
Yadav, S., Singh, A.K., Selvaraj, R., Applegate, T., Bhattacharya, P., Shinall, S.B., Fenn, L., Shanmugasundaram, R., Kim, W. 2023. Effect of dietary xylo-oligosaccharide on growth performance, intestinal histomorphology, and specific cecal bacteria in broiler chickens. Poultry Science. 103(1), 103189.. https://doi.org/10.1016/j.psj.2023.103189.
Satterlee, T.R., Mcdonough, C.M., Gold, S.E., Chen, C., Glenn, A.E., Pokoo-Aikins, A. 2023. Synergistic effects of essential oils and organic acids against aspergillus flavus contamination in poultry feed. Toxins. 15(11), p.635. https://doi.org/10.3390/toxins15110635.
Helmy, E.A., Amin, B.H., Alqhtani, A.H., Pokoo-Aikins, A., Yosri, M. 2024. Estimation of the antibacterial and anti-tumor impacts of soy milk and ecofriendly myco-manufactured zinc oxide nanomaterials. Polish Journal of Environmental Studies. 33(3):2093-2102. https://doi.org/10.15244/pjoes/174792.
Fathima, S., Hakeem, W., Selvaraj, R., Shanmugasundaram, R. 2024. Beyond protein synthesis: The emerging role of arginine in poultry nutrition and host-microbe interactions. Frontiers in Physiology. Volume 14(2024),1326809. https://doi.org/10.3389/fphys.2023.1326809.
Syamily, S., Selvaraj, R., Shanmugasundaram, R. 2023. Salmonella infection in poultry: A review on the pathogen and control strategies. Microorganisms. 11(11), p. 2814. https://doi.org/10.3390/microorganisms11112814.
Taylor, J., Mercier, Y., Olukosi, O., Kim, W.K., Selvaraj, R., Applegate, T., Shanmugasundaram, R., Ball, E., Kyriazakis, I. 2024. Supplementing low protein diets with methionine or threonine during mixed Eimeria challenge. Poultry Science. 103: (6); 103714. https://doi.org/10.1016/j.psj.2024.103714.
Hakeem, W., Cason, E.E., Villanueva, K.A., Shanmugasundaram, R., Lourenco, J., Selvaraj, R. 2024. The effect of Campylobacter jejuni challenge on the ileal microbiota and short-chain fatty acids at 28 and 35 days of age. Italian Journal of Animal Science. VOL. 23, NO. 1, 299–312. https://doi.org/10.1080/1828051X.2024.2310588.
Hakeem, W.G., Cason, E.E., Adams, D., Fathima, S., Shanmugasundaram, R., Lourenco, J., Selvaraj, R.K. 2024. Characterizing the effect of Campylobacter jejuni challenge on growth performance, cecal microbiota, and cecal short-chain fatty acid concentrations in broilers. Animals. 14(3),473. https://doi.org/10.3390/ani14030473.
Fathima, S., Hakeem, W., Shanmugasundaram, R., Periyannan, V., Varadhan, R., Selvaraj, R. 2024. Effect of 125% and 135% arginine on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge. Poultry Science. 103(7):103815. https://doi.org/10.1016/j.psj.2024.103826.
Fathima, S., Hakeem, W., Shanmugasundaram, R., Selvaraj, R. 2024. Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.. Poultry Science. 103: 7, 103815. https://doi.org/10.1016/j.psj.2024.103815.
Kappari, L., Joseph, D.R., Applegate, T., Selvaraj, R., Shanmugasundaram, R. 2024. MicroRNAs: Exploring their role in farm animal disease and mycotoxin challenges. Frontiers in Veterinary Science. 11: 1372961. https://doi.org/10.3389/fvets.2024.1372961.
Khan, I., Khan, S., Shuaib, M., Hassan, U., Alqhtani, A.H., Pokoo-Aikins, A., Qureshi, A., Ali, M., Alam, W. 2024. Comparison of floored and cage housed broiler breeder farms for energy and economic efficiency in Pakistan: A case study of broiler breeder's farm in district Punjab. Pakistan Journal of Zoology. pp.1-11. https://doi.org/10.17582/journal.pjz/20230810114347.