Research Project: Non-antibiotic Strategies to Control Enteric Diseases of Poultry

Location: Animal Biosciences & Biotechnology Laboratory

2020 Annual Report

Accomplishments
1. Development of poultry immune reagents. The critical lack of immune reagents and the methods to assess poultry immunity to pathogens hinders progresses in developing novel vaccines and therapeutics against many economically important diseases of poultry. To address these technical gaps, ARS scientists in Beltsville, Maryland, have developed new monoclonal antibodies that detect the levels and types of soluble mediators of cellular immunity of chickens to measure a wide spectrum of host immune responses. Fourteen novel poultry immune reagents (monoclonal antibodies) have been commercially licensed to a U.S. commercial company for public distribution for use in fundamental and applied research in poultry immunity. Availability of these new immune tools will facilitate our understanding of complex host immune system in this economically important food animal for avian community worldwide.

2. Alteration of gut microbiome and body weights following a dual-infection with Eimeria maxima and Clostridium perfringens. With the voluntary and regulatory withdrawal of antibiotics growth promoters from animal feed, it is important to understand how the composition of cecal microbiota changes with the body weights of chickens which are infected with both Eimeria parasites and Clostridium perfringens anaerobic bacteria. ARS scientists in Beltsville, Maryland, investigated the effects of these dual infections on the gut microbial composition and their correlations with body weight gains in broiler chickens using 16S rRNA gene sequencing technology. The parasitic and bacterial coinfection successfully induced necrotic enteritis (NE) with its typical gut lesions, reduction of body weight gains, and changes in the cecal microbial composition. Additionally, significant correlations between the cecal microbiota networks and body weights were identified in the control, Eimeria-infected and Clostridium-infected groups. Understanding of host-microbiota interaction in NE will facilitate the development of antibiotics-independent strategies to reduce the harmful effect of NE on U.S. poultry industry and improve the well-being of commercial poultry.

3. Vaccine efficacy tests for necrotic enteritis (NE) in broiler chickens. NE caused by C. perfringens (CP) bacterium is a prevalent enteric infectious disease costing more than $ 6 billion economical loss in the global poultry industry. No effective vaccines are commercially available to control NE in broiler chickens. ARS scientists in Beltsville, Maryland, identified and expressed 4 recombinant CP fusion proteins as vaccine candidates against NE. These potential vaccine antigens were evaluated for their protection efficacies in young broiler chickens in an ARS-established NE disease challenge model using -Eimeria maxima and CP dual infections. Young chickens which were immunized twice subcutaneously with adjuvanted CP vaccines on days 4 and 15 showed higher serum antibody responses, especially against the 3 CP pilus subunits and enzyme protein vaccines. After virulent NE challenge, the chickens immunized with all pooled antigens demonstrated the best vaccine efficacy with no mortality compared to about 39 percent mortality in the sham control group. These results indicate that immunization of commercial broiler chickens with the pooled CP protein vaccine candidate confers significant protection against virulent NE challenge.

4. Mode of action of poultry defensin molecules. Defensins are antimicrobial peptides that host produce to mediate direct killing of pathogens. Chickens express ß-defensin types that have antimicrobial properties against many pathogenic bacteria and fungi. The mechanism of action of avian ß-defensins is thought to be similar to those of mammalian antimicrobial peptides (AMPs) and involves the disruption of bacterial cell membrane. In this study, ARS scientists in Beltsville, Maryland, collaborated with scientists in South Korea to identify a new chicken defensin called “Avian Beta Defensin 5” (AvBD5) which contains six conserved cysteine forming three disulfide bonds. The collaborative research showed that chicken AvBD5 protein regulated signaling pathways associated with poultry innate immunity that could be used as non-antibiotic immunological therapeutics.

5. Dietary Allium hookeri (garlic chives) changes gut microbiome in chickens. Beneficial effects of dietary A. hookeri is not much known but may enhance gut health by reducing oxidative stress. To better understand the mode of action of dietary A. hookeri, ARS scientists in Beltsville, Maryland, collaborated with scientists in South Korea to investigate the impacts of dietary A. hookeri on the gut microbiome with 16S rRNA sequencing technology using samples obtained from the cecum of chickens fed with A. hookeri leaf. The results demonstrate that the microbiome composition in the groups supplemented with A. hookeri leaf showed higher proportion of beneficial bacteria compared to the unsupplemented control group. Modulation of gut microbiome by the A. hookeri leaf correlated with growth traits including body weight, and bone strength. Therefore, A. hookeri diet is beneficial for gut health of broiler chickens via mediation of gut microbiome.

Review Publications
Kim, W., Chaudhari, A., Lillehoj, H.S. 2019. "Involvement of T cell immunity in Avian Coccidiosis". Frontiers in Immunology. https://doi.org/10.3389/fimmu.2019.02732.
Oh, S., Lillehoj, H.S., Lee, Y., Bravo, D., Lillehoj, E.P. 2019. Dietary antibiotic growth promoters down-regulate inflammatory cytokine expression in chickens challenged with LPS or co-infected with Eimeria maxima and Clostridium perfringens. PLoS One. https://doi.org/10.3389/fvets.2019.00420.
Chaudhari, A., Lee, Y., Lillehoj, H.S. 2020. Beneficial effects of dietary supplementation of Bacillus strains on growth performance and gut health in chicken with mixed coccidiosis infection. Probiotics and Antimicrobial Proteins. https://doi.org/10.1016/j.psj.2019.10.023.
Chaudharia, A., Kim, W., Lillehoj, H.S. 2020. Development and characterization of monoclonal antibodies for chicken interleukin-13 and their neutralizing effect in chicken primary monocytes. Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.psj.2019.10.023.
Park, I., Lee, Y., Goo, D., Zimmerman, N., Smith, A., Rehberger, T., Lillehoj, H.S. 2020. The effects of dietary Baccillus subtilis supplementation, as an alternative to antibiotics, on growth performance, intestinal immunity, and epithelial barrier integrity in broiler chickens infected with Eimeria maxima. Poultry Science. https://doi.org/10.1016/j.psj.2019.12.002.
Truong, A., Hong, Y., Tran, H., Dang, H., Nguyen, V., Pham, T., Lillehoj, H.S., Hong, Y. 2019. Characterization and functional analysis of novel chicken leukocyte immunoglobulin-like receptor subfamily B member 4 and 5. Poultry Science. https://doi.org/10.3382/ps/pez442.
Park, I., Zimmerman, N.P., Smith, A.H., Rehberger, T.G., Lillehoj, E.P., Lillehoj, H.S. 2020. Dietary supplementation with Bacillus subtilis direct-fed microbials alters chicken intestinal metabolite levels. Scientific Reports. https://doi.org/10.3389/fvets.2020.00123.
Kurt, T., Wong, N., Fowler, N., Gay, C.G., Lillehoj, H.S., Plummer, P., Scott, M., Hoelzer, K. 2019. Strategic priorities for research on antibiotic alternatives in animal agriculture-Results from an expert workshop. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2019.00429.
Park, I., Lillehoj, E., Lillehoj, H.S. 2020. Dietary supplementation with magnolia bark extract alters chicken intestinal metabolite levels. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2020.00157.
Li, C.Z., Lu, M. 2020. Putting antimicrobial resistance in the corner. Nature Food. https://doi.org/10.1038/s43016-020-0034-9.
Tian, X., Lu, M., Jia, C., Bu, Y., Aimulajiang, K., Zhang, Y., Li, C.Z., Yan, R., Xu, L., Li, X. 2020. Haemonchus contortus transthyretin domain - containing protein (HcTTR): A promising vaccine candidate against Haemonchus contortus infection. Veterinary Parasitology. https://doi.org/10.1016/j.vetpar.2020.109045.
Li, C.Z., Yan, X., Lillehoj, H.S., Gu, C., Sun, Z., Oh, S., Lee, Y., Xianyu, Z., Zhao, H., Liu, L. 2019. Eimeria maxima-induced transcriptional changes in the cecal mucosa of broiler chickens. Parasites & Vectors. https://doi.org/10.1186/s13071-019-3534-4.
Hong, Y., Truong, A., Lee, J., Lee, K., Kim, G., Heo, K., Lillehoj, H.S., Hong, Y. 2018. Identification of duck liver-expressed antimicrobial peptide 2 (LEAP-2) and characterization of its bactericidal activity. Asian-Australasian Journal of Animal Sciences. https://doi.org/10.5713/ajas.18.0571.
Pirgozliev, V., Mansbridge, S., Rose, S., Lillehoj, H.S., Bravo, D. 2019. Growth performance, nutrient availability and immunity responses in broiler chickens fed a commercial blend of phytogenic feed additives. Poultry Science. https://doi.org/10.3382/ps/pey472.
Park, I., Lee, Y., Zimmerman, N., Smith, A., Rehberger, T., Lillehoj, H.S. 2020. The effects of dietary Bacillus subtilis supplementation, as an alternative to antibiotics, on growth performance, intestinal immunity, and epithelial barrier integrity in broiler chickens infected with Eimeria maxima. Poultry Science. https://doi.org/10.1016/j.psj.2019.12.002.
Lu, M., Li, R., Zhao, H., Yan, X., Lillehoj, H.S., Sun, Z., Oh, S., Wang, Y., Li, C.Z. 2020. Effects of Eimeria maxima and Clostridium perfringens infections on cecal microbiome components and correlation with body weight in broiler chickens. Research in Veterinary Science. https://doi.org/10.1016/j.rvsc.2020.05.013.