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

Location: Animal Biosciences & Biotechnology Laboratory

2020 Annual Report

Objectives
Necrotic enteritis (NE) and coccidiosis are considered the most important enteric diseases impacting poultry production in the U.S. and Europe. With increasing regulation on the use of antibiotics to control infectious diseases and as growth promoters, the incidence of clostridial infections has been rising. This project will focus on developing new poultry immune reagents and immunoassays to promote progress in poultry disease research, and to understand the immunobiology of host-pathogen interactions for developing mitigation strategies for coccidiosis and NE. Specifically, synergistic, non-antibiotic-based strategies will be developed that promote host innate immunity and induce innate effector molecules, thereby decreasing commercial antibiotic usage in the field. In our previous research projects, we developed a unique Clostridium perfringens/Eimeria co-infection model system, and identified a heightened proinflammatory response as a major factor in NE-induced intestinal immunopathology. Furthermore, we identified several plant products and host-derived antimicrobial peptides (AMPs), each of which reduced inflammation-mediated gut damage, activated poultry innate immune responses, and exerted direct cytotoxic activity against C. perfringens and Eimeria. Herein, we propose continued development of critical immune reagents and immunoassays for poultry species and disease research to: use them to better understand the host-pathogen immunobiology of coccidiosis and NE, develop sustainable antibiotic-free alternative strategies to reduce economic losses due to coccidiosis and NE, and enhance the overall gut health of commercial poultry. Objective 1. Develop immunologic tools to evaluate avian immunity including tools to detect host effector molecules associated with immune responses to enteric diseases, and tools to determine the role of host effector molecules in disease resistance to enteric diseases of poultry. [C5, PS5C] We will continue to develop new immunologic tools to evaluate avian immunity, including the next-generation of tools to detect host effector molecules associated with immune responses to enteric diseases, and to determine the role of these effector molecules in avian resistance to enteric diseases. This objective is highly relevant to the current state-of-the-art in poultry research which suffers from a critical shortage of immune reagents and methodologies to evaluate host-pathogen interactions and where traditional vaccines are not effective. Objective 2. Develop alternatives to antibiotics for preventing or treating enteric diseases of poultry including discovering vaccine platforms that could reduce the use of antibiotics in poultry production, and develop non-antibiotic approaches for treating priority enteric diseases of poultry. [C2, PS2B] We will identify additional, non-antibiotic-based immunotherapeutics to 1) reduce the harmful inflammatory response and associated collateral intestinal damage that develop during coccidiosis and NE, 2) activate broad spectrum innate immune responses, and 3) directly target the viability of C. perfringens and Eimeria pathogens.

Approach
Develop immune reagents (genes, recombinant cytokines, mAbs) and immunoassays for Th1, Th2, Th17 and Treg immune responses for the investigation of host-pathogen interaction on the gut mucosa in avian coccidiosis and NE. Develop novel strategies to immunomodulate innate host response. Identify potential biomarkers of gut health and assess the levels of gut health biomarkers in vivo. Develop antibiotic alternative strategies including recombinant vaccines and passive immunization methods.

Progress Report
This is the fourth annual report for the project of 8042-32000-107-00D which started in October, 2016. Progress was made on both objectives and their sub-objectives, all of which fall under National Program 103, Component 2, Antimicrobial Resistance and Component 5, Priority Endemic Diseases. Progress on this project focuses on Problem Statement 2B: Alternatives to Antibiotics and on Problem Statement 5C: Enteric Diseases of Poultry. Under Objective 1, progress has been made in the development of critical poultry immune reagents. Several major chicken cytokine and chemokine genes that mediate host immune response in poultry have been cloned, recombinant proteins have been expressed as immunogens for monoclonal antibody development and these monoclonal antibodies have been characterized. The cytokines play important roles in host cell mediated immune response mediated by T lymphocytes, NK cells and macrophages. Availability of these immune reagents which are specific for poultry species will be critical for investigation of mucosal and systemic immune responses to Eimeria (protozoan parasites) and Clostridium perfringens (anaerobic bacteria), causative agents of coccidiosis and necrotic enteritis, respectively. These immune reagents for poultry species will be commercialized to address the critical immunological reagent gap for basic immunology research and applied research to facilitate the development of novel strategies to reduce antibiotics in poultry production. Under Objective 2, significant progresses have been made in identification of potential vaccine antigens against C. perfringens, understanding of host-Eimeria (parasite) interaction and pathogenicity of bacterial Clostridium perfringens (CP) strains from different genetic backgrounds in inducing necrotic enteritis (NE), investigation of molecular mechanisms of antibiotic growth promoters, development of novel vaccine strategies against coccidiosis and NE. Furthermore, significant progresses have been made in development of several effective commercial feed additives as antibiotic alternative strategies against coccidiosis and NE for commercial poultry in partnership with private industries under several formal agreements, Cooperative Research and Develoment Agreements (CRADA), trust agreements and Memorandum of Understanding (MOU) agreement that reduced the use of antibiotics in poultry production. These results will impact the animal welfare, food safety and food animal production. Some of progresses, such as in investigating the mechanisms of bacterial CP infection and antibiotic growth promoters, and feed supplements (beneficial bacterium Bacillus subtilis as direct-fed microbials and dietary magnolia bark extract), were listed below. Progress 1 - Enhanced understanding of pathogenicity of different strains of Clostridium perfringens (CP) bacterium from different genetic backgrounds: Limited information on the pathogenicity among CP isolates from different sources hinders development of a good dual infection model of NE for efficacy evaluation of various antibiotic alternative approaches. To better understand the nature of CP interactions with Eimeria spp., ARS scientists in Beltsville, Maryland, compared the pathogenicity and genomic sequences for five CP isolates from different sources: LLY_N11 (healthy chicken intestine), Str.13 (from soil), SM101 (from food poisoning case), Del1 (netB+tpeL-) and LLY pel 17 (netB+tpeL+, both from infected farms) in commercial broiler chickens using the E. maxima /CP co-infection NE challenge model. The strains that have an essential NE pathogenesis locus in their chromosome, EM/LLY_N11, EM/Del1, LLY_Tpel17 were found to be more virulent than those without such locus (Str 13 and SM101). The data indicated that LLY_Tpel17 was highly pathogenic to induce severe gut lesions and would be a good CP challenge strain for future studies to investigate NE pathogenesis and to evaluate the protection efficacy for antibiotics alternative approaches. Progress 2 - Much progress was achieved in understanding the molecular mechanisms of antibiotic growth promoters. Regulatory agencies warn that the emergence of multidrug-resistant pathogens could potentially be the greatest threat to human health of our time due to the overuse of antibiotic growth promoters (AGPs) in food animal production. Increased understanding of how AGPs promote animal growth will facilitate the development of alternatives to antibiotics that will replace the need for AGPs. In this study, ARS scientists in Beltsville, Maryland, conducted immunological analysis of chickens treated with AGPs (virginiamycin or bacitracin methylene disalicylate) during infection with enteric pathogens to induce necrotic enteritis (NE) in an experimental model and compared host immunological profiles associated with inflammatory response. As anticipated, increased intestinal levels of inflammatory cytokine transcripts (IL-1ß, IL-2, IL-6, IL-8 and IL-17A) were observed in chickens fed a basal diet and infected with NE compared with uninfected chickens. In contrast, chickens fed an AGP-supplemented diet and challenged with NE disease model showed decreased inflammatory cytokine and antioxidant enzyme transcript levels, compared with pathogen-infected birds fed the unsupplemented diet. The current study suggests that dietary AGPs might increase poultry growth, in part, through down-regulation of pathogen-induced, cytokine-mediated inflammatory responses. These new insights on the mode of AGP action will facilitate the development of alternative feed additives to replace AGPs in commercial poultry production. Progress 3 - There has been increasing understanding of how beneficial Bacillus subtilis bacterium affects poultry growth and immunity. Direct-fed microbials (DFMs) are dietary supplements containing live microorganisms which confer a health benefit to the host, but their mechanisms of action are unclear. Understanding how dietary DFMs enhance host gut health will facilitate the application of this technology to animal husbandry. ARS scientists in Beltsville, Maryland, characterized the metabolic alterations in the chicken gut following dietary supplementation with B. subtilis DFMs with the goal of identifying potential metabolites that might directly contribute to enhanced poultry growth performance. Body weight gains of chickens fed the B. subtilis-supplemented diets were increased, compared with chickens fed the unsupplemented control diet. Compared with unsupplemented control, the changes in the levels of intestinal metabolites provided a distinctive biochemical signature unique to each B. subtilis-supplemented group, characterized by alterations in the levels of certain dipeptides, nucleosides, fatty acids, and carbohydrates (fructose). These results provide the foundation for future studies to identify the nature of small molecular weight biochemicals that might be associated with improving poultry growth performance in the absence of antibiotic growth promoters.

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.