Research Project: Alternatives to Antibiotics Strategies to Control Enteric Diseases of Poultry

Location: Animal Biosciences & Biotechnology Laboratory

2022 Annual Report

Objectives
Objective 1: Develop immunological tools to evaluate avian immunity. Sub-objective 1.a. The development of immunological assays and validation of their uses for assessing host immune responses to infectious diseases and vaccination Sub-objective 1.b. Development of multiplex immunoassays for the simultaneous detection of inflammatory cytokines and chemokines as screening tools to evaluate the efficacy of alternatives to antibiotics feed additives Objective 2: Develop alternatives to antibiotics for preventing enteric diseases of poultry Sub-objective 2.a. Discover vaccine platforms that could reduce the use of antibiotics in poultry production and study their modes of action using coccidiosis and necrotic enteritis disease models Sub-objective 2.a.1. Development of Bacillus subtilis spore-vectored recombinant vaccines against necrotic enteritis using B. subtilis spore surface display technology Sub-objective 2.a.2. In ovo delivery of recombinant protein vaccines against necrotic enteritis Sub-objective 2.a.3. Coccidiosis recombinant vaccines delivered with T cell-promoting adjuvants Sub-objective 2.a.4. Coccidiosis recombinant vaccines, cytokine adjuvants, and Bacillus subtilis spores carrying a chicken antimicrobial peptide, cNK2 Sub-objective 2.b. Develop multi-faceted non-antibiotic approaches for preventing priority enteric diseases of poultry and study their modes of action Sub-objective 2.c. Determine intestinal metabolites that enhance host immunity, gut health, and growth performance as feed additives and develop antibiotic-free postbiotic strategy to reduce the need for antibiotics in poultry production.

Approach
Develop novel antibiotic alternative strategies to countermeasures against highly resistance pathogens, Eimeria and Clostridium perfringens, to achieve resiliency in antimicrobial resistance challenges in animal agriculture. Develop critical immune reagents(genes, recombinant cytokines, mAbs)and immunoassays for deciphering host Th1, Th2, Th17 and Treg immune responses to better understand host-pathogen interaction in avian coccidiosis and necrotic enteritis. Develop novel strategies such as vaccines and antibiotic alternatives to beneficially modulate innate host response. Identify potential biomarkers of gut health and assess the levels of gut health biomarkers in vivo.

Progress Report
This is the first annual report for the project of 8042-32000-115-000D which started in October 2021. Progress was made on both objectives and their sub-objectives, all of which fall under National Program 103, Component 2, Antimicrobial Resistance Problem Statement 2A: Combat the antimicrobial resistance through the development of Alternatives to Antibiotics. Under Objective 1, progress has been made in the development of critical poultry immune reagents that can be used to assess innate immune response of poultry in normal and disease states. 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 antigen capture ELISA assays to specifically measure poultry cytokines and chemokines have been developed. Because cytokines and chemokines are major mediators of host innate and adaptive immunity, the availability of these immune reagents and poultry-specific immunoassays will enhance progress in our understanding of poultry immune response to pathogens including parasites, bacteria, and fungus. Furthermore, these poultry-specific immune reagents developed in this project have been commercialized to address the critical immunological reagent gap for basic and applied immunology research in poultry species to facilitate the development of novel strategies to reduce the use of antibiotics in commercial poultry production. Under Objective 2, significant progresses have been made in the development of antigen-specific sandwich ELISA assays for the detection of critical antigens of C. perfringens including NetB toxin and collagen adhesion protein (CNA). Significant progresses have also been made in the development of several effective antibiotic alternative strategies against coccidiosis and necrotic enteritis, such as Bacillus subtilis-spore surface display, the recombinant chimeric proteins for subunit vaccines and hyperimmune egg yolk antibody production in protection against the pathogenic C. perfringens challenge infection. These progresses may eventually lead to commercializable feed additives that will replace antibiotics to promote food animal welfare and production. Progress on chicken immune reagent development: Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific cytokines including CSF, TNF and IFN-kappa have been developed and quantitative antigen capture enzyme-linked immunosorbent assay (ELISA) assays to measure these cytokines in biological fluids have been validated. Compared to their mammalian counterparts, the immunological properties of chicken CSF, TNF and IFN-kappa have not been clearly defined. Interferon (IFN) is a type I IFN that plays a central role in antiviral host defense response. Tumor necrosis factor-a (TNF-a) is a type II transmembrane protein with either membrane-bound or soluble forms and is a pleiotropic cytokine associated with the regulation of systemic inflammation. Macrophage colony-stimulating factor-1 (M-CSF-1 or CSF-1) is a hematopoietic growth factor that stimulates the survival, proliferation, and differentiation of the mononuclear phagocytes and is involved in inflammatory processes. Availability of quantitative immunoassays for these three cytokines will enhance fundamental and applied studies on innate host immune response in the normal and diseased states in poultry. Using antigen-capture ELISA for chicken TNF-a, in-depth functional characterization and immunomodulatory properties of TNF-a were conducted and the levels of native TNF-a in the circulation of Clostridium perfringens-, Eimeria-, or dual Eimeria/C. perfringens-infected chickens were determined. Collectively, new results of our studies will enhance our understanding of the functional characteristics of CSF, TNF, and IFN-kappa cytokines and new immunoassays will serve as valuable tools to decipher inflammatory responses and disease pathogenesis in poultry. Progress on the development of diagnostic tests for important proteins of C. perfringens: Several mouse monoclonal antibodies which specifically detect two critical antigen epitopes of pathogenic C. perfringens, were developed to construct new sandwich immunoassays to quantitate the levels of NetB and CNA antigens of C. perfringens in infected chickens, specifically in the serum, gut contents, and fecal samples. These assays are the first immunoassays that make it possible to quantitate the two toxin/virulence-associated factors of C. perfringens in biological fluids and will allow an early detection large-scale screening for the NE disease outbreak in broiler farms to minimize economic losses due to NE. Progress on the production and protection using hyperimmune egg yolk IgY antibodies against Eimeria- and C. perfringens-mediated inflammatory enteric infections: Passive immunity from hyperimmune egg yolk IgY is an antibiotic alternative strategy which utilizes pathogen-specific antibodies to reduce pathogen burden on chickens. Antigen-specific hyperimmune IgY antibodies against protective antigens of Eimeria and C. perfringens were developed using whole protein or synthetic protein fragments of these two pathogens, and the effects of orally-fed hyperimmune IgY antibodies were evaluated for their protective effects in coccidiosis- and NE-afflicted broiler chickens. These key antigens chosen were 3-1E, EF1alpha, NetB, and various synthetic protein fragments of NetB toxoid and Alpha toxoid (Plc), Fructose-1,6-bisphosphate aldolase (FBA), a Zinc metalloprotease (Zm), and one pilus subunit (Collagen adhesion protein (CNA). In addition, two recombinant chimeric proteins (NetB-Plc-Cna and FBA-Zm) targeting these toxins and virulence-associated factors were also used to immunize the hens of layer chickens to obtain therapeutic hyperimmune egg yolk IgY antibodies for oral treatment. Preliminary trial results showed significant protection against coccidiosis and NE in chickens fed with various combinations of these IgY antibodies based on body weight gains using two disease models developed at ARS. Furthermore, hyperimmune IgY antibodies against two chimeric C. perfringens proteins conferred protection to the broiler chicks against pathogenic C. perfringens challenges. These results indicate a potential application of these combined IgY antibodies specific for chimeric proteins of Eimeria and C. perfringens to prevent field coccidiosis and NE disease outbreak in commercial poultry farms. Further trials are planned to verify the lab results in the field trials. Hyperimmune egg IgY strategy is a promising non-antibiotic strategy to reduce the clinical impacts of necrotic enteritis in commercial broilers at the early stage. Progress on alternatives to antibiotic strategies using B subtilis spore-vectored vaccines: B. subtilis has been approved as Generally Regards As Safe (GRAS) by FDA and may be used as a probiotic. Bioengineered B subtilis is a practical mucosal vector to deliver key genes encoding beneficial proteins that can reduce pathogenic effects in enteric infections such as coccidiosis and NE. Coccidiosis which is caused by several distinct species of Eimeria parasites and NE that is commonly associated with co-infection with E. maxima and pathogenic strains of C. perfringens, are two leading enteric diseases responsible for significant economic loss of more than $ 20 billion in the global poultry industry. Significant progress was made in constructing transgenic Bacillus strains carrying immunogenic antigens of Eimeria and C. perfringens. Recombinant vaccine candidates using NetB and Plc fragment of Alpha toxin, FBA and Zm genes have been successfully constructed using Bacillus subtilis to be tested against coccidiosis and NE. Developing an effective and practical oral delivery strategy to express vaccine antigens of Eimeria and C. perfringens will effectively reduce the gut damage and reduces economic losses due to these enteric infections.

Accomplishments
1. Commercialization of monoclonal antibodies to detect chicken immune molecules. Eleven mouse monoclonal antibodies (mAbs) detecting chicken chemokines (mediators of cellular immunity) have been commercialized by ARS scientists in Beltsville, Maryland. These mAbs detect poultry-specific cytokines which are involved in the early initiation phase of host immune responses to pathogens such as parasites and viruses, and their levels in the blood provide predictive values in determining the quality of host immune responses. These accomplishments address the critical immunological reagent gap for basic immunology research in poultry species, and the availability of these immune reagents will enhance basic and applied research to facilitate our understanding of host immune responses to pathogens and vaccines in poultry that will promote the development of novel immunity-based strategies to reduce antibiotics in commercial poultry production.

2. Development of bioassays to detect poultry immunity. ARS scientists in Beltsville, Maryland, cloned several major chicken cytokine and chemokine genes that mediate poultry immune responses to pathogens, their corresponding recombinant proteins were expressed as immunogens for mAb development, and these mAbs further used to develop sensitive bioassays to measure poultry immunity in chickens after infection or vaccination. The availability of these new immunological tools will be valuable for advancing fundamental and applied studies in avian species to enhance our understanding of host immunity in poultry.

3. Elucidated function of CSF-1 during coccidiosis. Colony-stimulating factor-1 (CSF-1) stimulates the survival, proliferation, and differentiation of macrophages and is involved in bone metabolism, fertility, pregnancy, inflammatory processes, and homeostasis. Five monoclonal antibodies which specifically detect chicken CSF-1 were developed and characterized by ARS scientists in Beltsville, Maryland, and used to develop a bioassay to detect CSF-1 in chickens undergoing infections. Circulating serum CSF-1 was detected in chickens following infection with E. acervulina, E. maxima, and E. tenella, demonstrating an important role of CSF-1 in the immune response to intracellular coccidia parasite infection and suggesting CSF-1 is a biomarker for coccidiosis infection. This mAb-based bioassay will be a valuable tool for the assessment of CSF-1 levels during various poultry infections, and these new antibodies will facilitate fundamental and applied research related to CSF-1 function in normal and disease states in chickens.

4. Biological function of TNF-a during coccidiosis. One of important chicken cytokines called tumor necrosis factor alpha, TNF-a, is well known for its essential and far-reaching regulatory functions in facilitating inflammatory responses in host defense against microbial infections. ARS scientists in Beltsville, Maryland, developed monoclonal antibodies to measure the levels of chicken TNF-a in the circulation of diseases chickens. Augmented serum TNF-a levels were observed at varying time points following Eimeria parasitic infections. Overall, E. tenella-infected chickens showed the highest level of TNF-a suggesting significant damage to the intestinal epithelial cells and systematic inflammatory responses induced by E. tenella infection. The availability of the new TNF-a assay to measure this cytokine in coccidiosis during the early phase of infection will enable the development of counteracting strategies to ameliorate severe gut tissue damage and inflammation in enteric infections.

5. Commercial licensing of hyperimmune egg yolk technology for coccidiosis and necrotic enteritis prevention. In collaboration with a private company, ARS scientists in Beltsville, Maryland, prepared egg yolk antibodies from hens hyper-immunized with Eimeria and C. perfringens antigens and used these antibodies to enhance the immature immune response of newly-hatched chickens and to prevent coccidiosis and necrotic enteritis (NE)(license No. 1916-001 1.2 on hyperimmune eggs and hyperimmune egg supplements to deal with coccidiosis and necrotic enteritis in poultry as encompassed within the claims of the Licensed Patents Serial No. 10,450,364, issued on October 22, 2019). This novel strategy will help to reduce the incidence and economic losses of coccidiosis and NE that are currently estimated to cost around $20 billion annually in global poultry production.

6. Development of bioassays for early detection of necrotic enteritis outbreaks in commercial poultry farms. ARS scientists in Beltsville, Maryland, developed a sensitive bioassay based on the C. perfringens collagen adhesion antigen (CNA) protein and validated its utility using large field screening of necrotic enteritis-afflicted chickens from commercial farms. The results of these screenings indicated that this new assay would serve as a valuable early warning test to allow the accurate measurement of necrotic enteritis outbreak caused by pathogenic C. perfringens strains to monitor flock health status at the farm level or to conduct early detection of necrotic enteritis outbreaks in commercial poultry farms.

7. Dietary sopholipid feed additives as antibiotic alternatives against coccidiosis and necrotic enteritis. Sophorolipids (SPL) are biodegradable, eco-friendly compounds that enhance the growth performance and gastrointestinal functionality in chickens infected with coccidiosis and NE. In collaboration with a private industry research team, ARS scientists in Beltsville, Maryland, developed a novel strategy to use SPL as alternative feed additives to antibiotics to reduce financial losses due to these gut pathogens. The animal studies showed that SPL enhanced growth of chickens infected with these enteric pathogens and decreased gut damage, and suppressed harmful local inflammatory response mediated by cytokine release, therefore enhancing gut integrity. Overall, dietary SPL maintains growth performance, positively modulates gut immunity, and promotes intestinal barrier integrity of young broiler chickens during coccidiosis, demonstrating the potential for using sophorolipids as an alternative to antibiotics for commercial poultry production.

8. Gut-microbiome-derived indole compounds enhance host immunity and increase gut health in broiler chickens. Identification of natural gut compounds that provide beneficial effects to the host may potentially offer a new antibiotic alternative as a feed additive. ARS scientists in Beltsville, Maryland, identified indole compounds as potent immunomodulators which directly contributed to enhanced poultry gut health and growth performance of young chickens afflicted with coccidiosis. Dietary supplementation with indole alleviated intestinal tissue damage of young chickens infected with coccidiosis, reduced inflammation, decreased harmful cytokine expression, and increased expression of genes associated with nutrient transport. These data provide the foundation for future strategies to use natural biochemicals associated with improving poultry growth performance in the absence of antibiotic growth promoters.

9. Development of multivalent recombinant protein vaccines for necrotic enteritis in broiler chickens. ARS scientists in Beltsville, Maryland, developed 3 recombinant C. perfringens proteins and evaluated their protection efficacies with a challenge model of E. maxima/C. perfringens dual infection. Young chickens vaccinated with these C. perfringens proteins demonstrated higher serum antibody levels to the recombinant proteins compared with unvaccinated controls. Following the challenge infection, the pooled antigen-immunized group demonstrated no mortality and the least lesion scores against virulent challenge. These data indicate that the vaccination with these antigens conferred protection against necrotic enteritis challenge in broiler chickens.

10. Deciphering Clostridium perfringens-induced host-pathogen gene expression profiles in the small intestine of broiler chickens. The molecular mechanisms of C. perfringens-induced pathogenesis in the gut and how C. perfringens interacts with other gut microbes remain to be investigated. ARS scientists in Beltsville, Maryland, compared the gene expression profiles of both the host intestine and gut microbiome in C. perfringens-infected and uninfected chickens using RNA sequencing technology. In total, 13,473 genes were differentially expressed between the infected and uninfected groups, of which a subgroup of 96 genes achieved statistical significance. These latter genes were involved in immunity, energy production, and metabolism. Additionally, the levels of 4 foodborne pathogens (C. jejuni, E coli O157, L. monocytogenes and C. perfringens) were significantly elevated in C. perfringens. This study provides new insights into the mechanism of host-pathogen interactions in necrotic enteritis in broiler chickens.

11. Protein analysis of Eimeria necatrix. Eimeria necatrix is a causative pathogen causing coccidiosis that can reduce the growth performance of poultry. The protein structure of this parasite at the sporozoite stage is unknown, and better understanding of this structure is important for the development of effective biological controls. ARS scientists in Beltsville, Maryland, in collaboration with scientists in China, elucidated the complete protein profile of E. necatrix sporozoites. A total of 680 proteins were identified, of which 98 were found to react with E. necatrix-specific antibodies. These new findings will enhance our understanding of parasite immunogenicity and immune evasion mechanisms of E. necatrix and may facilitate the discovery of highly effective recombinant vaccine candidates.

Review Publications
Lu, M., Yuan, B., Yan, X., Sun, Z., Lillehoj, H.S., Lee, Y., Li, C.Z. 2021. Clostridium perfringens-Induced Host-Pathogen Transcriptional Changes in the Small Intestine of Broiler Chickens. Pathogens. 10(12):1607. https://doi.org/10.3390/pathogens10121607.
Lee, K., Lillehoj, H.S. 2020. Role of Clostridium perfringens necrotic enteritis (NE) B-like toxin in NE pathogenesis. Avian Pathology. https://doi.org/10.1637/0005-2086-64.4.490.
Teng, P., Choi, J., Tompkin, Y.H., Lillehoj, H.S., Kim, W. 2021. Impacts of increasing challenge of E. maxima on gut integrity and gene expression of nutrient transporters in broiler chickens. International Journal of Parasitology. https://doi.org/10.1186/s13567-021-00949-3.
Hong, Y., Lee, J., Vu, T.H., Lillehoj, H.S., Hong, Y.H. 2021. Immunomodulatory effects of poly (I:C)-stimulated exosomes derived from chicken macrophages. International Journal of Molecular Sciences. https://doi.org/10.1016/j.psj.2021.101247.
Lee, Y., Lu, M., Lillehoj, H.S. 2021. Development of antigen-capture ELISA to analyze the immunological functions of chicken interferon-kappa using specific monoclonal antibodies. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2021.104204.
Vu, T.H., Hong, Y., Truong, A.D., Lee, J., Lee, S., Song, K., Cha, J., Dang, V.D., Tran, H., Lillehoj, H.S., Hong, Y.H. 2022. Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus-infected lungs of genetically disparate Ri chicken lines. Journal of Animal Bioscience. https://doi.org/10.5713/ab.21.0420.
Qu, G., Xu, Z., Tuo, W., Li, C.Z., Wan, G., Lillehoj, H.S., Gong, H., Huang, J., Tian, G., Li, S., Liu, Y., Liu, L. 2021. Clostridium perfringens-induced host-pathogen transcriptional changes in the small intestine of broiler chickens. Frontiers in Veterinary Science. https://doi.org/10.1016/j.vetpar.2021.109642.
Hong, Y., Lee, J., Vu, T.H., Lee, S., Heo, J., Truong, A.D., Lillehoj, H.S., Hong, Y.H. 2022. Influenza a pathway analysis of highly pathogenic avian influenza virus (H5N1) infection in genetically disparate Ri chicken lines. Veterinary Immunology and Immunopathology. 246:110404. https://doi.org/10.1016/j.vetimm.2022.110404.
Panebra, A., Lillehoj, H.S. 2022. Development of a sandwich ELISA for the detection of chicken colony stimulating factor 1a. Poultry Science. https://doi.org/10.1016/j.psj.2022.101924.
Wickramasuriya, S., Park, I., Lee, K., Lee, Y., Kim, W., Nam, H., Lillehoj, H.S. 2022. Role of physiology, immunity, microbiota and enteric diseases in gut health in poultry. Vaccines. https://doi.org/10.3390/vaccines10020172.
Lee, Y., Lu, M., Lillehoj, H.S. 2022. Coccidiosis: Recent progress in host immunity and alternatives to antibiotic strategies. Vaccine. 10:215. https://doi.org/10.3390/vaccines10020215.
Kim, W., Min, W., Park, K.I., Lillehoj, H.S., Fernandez-Colorado, C.P., Flores, R.A., Cammayo, P.L., Nguyen, B.T. 2022. Expression of chicken NK-Lysin and its role in chicken Coccidiosis induced by Eimeria necatrix. Korean Journal of Parasitology. 59:439. https://doi.org/10.3347/kjp.2021.59.5.439.
Park, I., Oh, S., Goo, D., Celi, P., Lillehoj, H.S. 2022. Effect of dietary sophorolipids on growth performance and gastrointestinal functionality of chickens infected with Eimeria maxima. Animal Feed Science and Technology. https://doi.org/10.1016/j.psj.2022.101944.
Park, I., Oh, S., Nam, H., Celi, P., Lillehoj, H.S. 2022. Antimicrobial activity of sophorolipids against Eimeria maxima and Clostridium perfringens, and their effect on growth performance and gut health in necrotic enteritis. Poultry Science. 101:101731. https://doi.org/10.1016/j.psj.2022.101731.
Lu, M., Lee, Y., Li, C.Z., Lillehoj, H.S. 2022. Immunological characterization of chicken tumor necrosis factor-a (TNF-a) using new sets of monoclonal antibodies specific for poultry TNF. Developmental and Comparative Immunology. https://doi.org/10.1016/j.dci.2022.104374.
Hong, Y., Truong, A.D., Vu, T.H., Lee, S., Heo, J., Kang, S., Lillehoj, H.S., Hong, Y.H. 2022. Exosomes from H5N1 avian influenza virus-infected chickens regulate antiviral immune responses of chicken immune cells. Veterinary Research. 130:104368. https://doi.org/10.1016/j.dci.2022.104368.
Lee, K., Kim, W., Li, C.Z., Lillehoj, H.S. 2020. Detection of Necrotic Enteritis B-like toxin secreted by Clostridium perfringens using capture Enzyme-Linked Immunosorbent Assay. Avian Diseases. 64(4):490-495. https://doi.org/10.1637/0005-2086-64.4.490.
Park, I., Nam, H., Goo, D., Wickramasuriya, S.S., Zimmerman, N., Smith, A.H., Rehberger, T.G., Lillehoj, H.S. 2022. Gut microbiota-derived indole-3-carboxylate influences mucosal integrity and immunity through the activation of the aryl hydrocarbon receptors and nutrient transporters in broiler chickens challenged with Eimeria maxima. Frontiers in Immunology. 13:867754. https://doi.org/10.3389/fimmu.2022.867754.
Vu, T.H., Hong, Y., Truong, A.D., Lee, J., Lee, S., Song, K., Cha, J., Dang, H.V., Tran, H.T., Lillehoj, H.S., Hong, Y.H. 2021. Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus-infected lungs of genetically disparate Ri chicken lines. Journal of Animal Bioscience. 35 (3):367-376. https://doi.org/10.5713/ab.21.0163.