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

Location: Animal Biosciences & Biotechnology Laboratory

2023 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 second 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 and adaptive immune responses 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 sandwich ELISA assays to specifically measure poultry cytokines and chemokines have been developed. Because cytokines and chemokines are major mediators of host innate and adaptive immunities, the availability of these immune reagents and poultry-specific immunoassays will enhance progress in our understanding of poultry immune responses 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 progress has been made in the development of antigen-specific sandwich ELISA assays for the detection of critical antigens of Eimeria parasites and C. perfringens including NetB toxin and collagen adhesion protein (CNA). Significant progress has also been made in the development of several effective antibiotic alternative strategies against coccidiosis and necrotic enteritis, such as Bacillus subtilis-vectored products, and the recombinant chimeric proteins for subunit vaccines in protection against the Eimeria spp. and C. perfringens challenge infections. B. subtilis has been genetically engineered to carry major immunodominant genes of Eimeria spp., and the expression of parasite proteins has been verified. Bacillus amyloliquefaciens, a subvariant of B. subtilis which inhibits the growth of pathogenic C. perfringens, was genetically engineered to carry C. perfringens genes. This progress 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 have been characterized and quantitative antigen capture ELISA assays were used to assess host immune response to coccidiosis and Necrotic enteritis (NE) infections in serum, gut secretions and in other biological fluids. The availability of quantitative immunoassays for the chicken cytokines via commercialization will facilitate further understanding of host-pathogen immunobiology of coccidiosis and NE and allow investigation of feed additives and vaccines that will replace antibiotics. Collectively, novel results from in vitro and in vivo studies will enhance our understanding of coccidiosis and NE pathogenesis and will facilitate the development of antibiotic alternative strategies against coccidiosis and NE in commercial poultry. Progress on the development of diagnostic tests for avian coccidiosis. A major immunodominant antigen of Eimeria apicomplexan parasites, profilin, was produced in E. coli, and several mouse monoclonal antibodies detecting profilin were developed. Using these antibodies to 3-1E, a novel diagnostic antigen-capture immunoassay was developed for large-scale screening, and its specificity was validated using serum samples from coccidia-infected chickens of field poultry farms. A better understanding of the host immune response to 3-1E immunodominant antigen of Eimeria parasites will enhance the development of effective vaccines and antibiotic alternative feed additives that will replace antibiotics. Detection of major antigens associated with the pathogenesis of NE. Two major antigens of C. perfringens which are Immunodominant and are being recognized by NE-afflicted chickens have been identified and mouse monoclonal antibodies have been developed against these antigens. Several mouse monoclonal antibodies which specifically detect NetB and CNA antigens of C. perfringens were cloned, and their proteins were expressed to develop mouse monoclonal antibodies. Antigen-specific immunoassays then validated using serum and fecal samples to quantitate the levels of NetB and CNA antigens and antibodies against them in C. perfringens-infected chickens. 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 early detection of NE outbreaks in commercial broiler farms to minimize economic losses due to NE. Using field samples from a commercial company, the specificity, and validity of these immunoassays to quantitate the two toxin/virulence-associated factors of C. perfringens in biological fluids were confirmed. These immunoassays will allow an early detection of large-scale screening for the NE disease outbreak in broiler farms to minimize economic losses due to NE. Progress on the molecular characterization and detection for major proteins of C. perfringens: New sandwich immunoassay (ELISA) was developed using mouse monoclonal antibodies specific for the detection of two critical antigens: necrotic enteritis B-like toxin (NetB) and collagen adhesin protein (CNA) of pathogenic C. perfringens in infected chickens in the jejunal digesta samples from commercial broiler farms in the United States. The CNA and NetB levels showed a positive correlation with each other, and CNA and NetB levels in jejunal digesta were significantly decreased in certified-organics-used farms compared with those from non-antibiotic farms. These new C. perfringens antigen-specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens. Progress on alternatives to antibiotic strategies using novel phytochemical combinations. ARS scientists and a collaborating company have identified a phytochemical combination that mitigates coccidiosis response and reduces gut damage. A beneficial combination of three phytochemicals that enhanced host immunity was identified using in vitro screening system using three different chicken cell lines using unique biomarker combinations associated with different cell functions. Both in vitro and in vivo studies confirmed the beneficial effects of green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In vivo studies confirmed that chickens fed with a diet that is supplemented with this unique phytochemical mixture showed enhanced growth performance and reduced disease severity following coccidiosis. In conclusion, the combination of GT, CO, and PO in the diet of broiler chickens infected with Eimeria maxima induced enhanced the host disease resistance including innate immunity and gut health, which contributed to improved growth and reduced disease responses. These findings will provide the scientific rationale to develop a science-based antibiotic-independent strategy to mitigate coccidiosis response in poultry production to reduce the economic cost associated with commercial broiler production. Progress on alternatives to antibiotic strategies using B subtilis and its subvariant-vectored vaccines: B. subtilis subvariant Bacillus amyloliquefaciens (B. amylo) has been used as a probiotic in the poultry industry. B. amyloliquefaciens was shown to inhibit the growth of pathogenic Listeria monocytogenes and C. perfringens and is a potential candidate as a mucosal vaccine vector to deliver vaccines encoding bacterial virulence factors against enteric infections such as coccidiosis and necrotic enteritis, which are two leading enteric diseases responsible for substantial economic loss of more than $ 20 billion in the global poultry industry. Significant progress was made in bioengineering recombinant B. amylo strain carrying immunogenic antigens of C. perfringens such as the fructose-1,6-bisphosphate aldolase (FBA) gene. 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 resulting from these enteric infections. Development of recombinant vaccine to protect against coccidiosis in commercial broiler chickens. Progress in recombinant vaccination strategy against coccidiosis based on recombinant vaccine mixture composed of the immunodominant antigen of Eimeria, T-cell promoting chicken immune-modulating cytokine and chicken antimicrobial peptide have been formulated. In in vivo trials, recombinant rEF-1a vaccination with rchIL-7 or simultaneous oral administration of cNK-2 induced an enhanced protective immunity against E. maxima infection compared to the rEF-1a vaccination alone. Furthermore, EF-1a vaccination of newly hatched broiler chickens protected them with a significant level of protective immunity against E. maxim and with reduced fecal oocyst output. Furthermore, EF-1a-vaccinated chickens showed mitigated pro-inflammatory cytokine profiles in the gut, where parasites underwent intracellular development following E. maxima challenge infection, compared to non-immunized chickens.

Accomplishments
1. Commercialization of mouse monoclonal antibodies detecting chicken immune molecules. Ten hybridoma cell lines that secrete mouse monoclonal antibodies detecting chicken chemokine Tumor Necrosis Factor-alpha (TNF-apha) have been commercialized by ARS scientists in Beltsville, Maryland. These mouse monoclonal antibodies detect poultry-specific TNF-alpha 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 cell lines address the critical immunological reagent gap for basic immunology research in poultry species, and the commercialization of these immune reagents will enhance basic and applied research to facilitate our understanding of host immune responses to pathogens and vaccines in poultry. Furthermore, these immune reagents will facilitate the development of effective immunity-based strategies to reduce antibiotics in commercial poultry production.

2. Development of large-scale immunoassay detection kits for biomarkers of necrotic enteritis infection in commercial broiler chickens. Necrotic enteritis (NE) is an enteric bacterial disease caused by Gram-positive toxicogenic Clostridium perfringens (CP) and inflicts more than $6 billion annual economic cost to global poultry industry. Antibiotic-free production of commercial poultry has been associated with increasing incidence of NE, with many current commercial poultry productions becoming more dependent on using antibiotic-alternative feed additives. ARS scientists in Beltsville, Maryland, and collaborators at the University of Georgia developed ELISAs to detect Collagen Adhesion Antigen and NetB toxin proteins which are two major virulence factors involved in NE pathogenesis for early detection of NE outbreaks in susceptible flocks in commercial broiler farms. Immunoassays were based on two novel mouse monoclonal antibodies which detect specific biomarkers associated with CP. These assays showed that pathogenic strains of CP can be identified in fecal samples from commercial farms within 1-2 weeks post-hatch allowing timely intervention of NE outbreak in the poultry farms. The use of these antigen-capture sandwich ELISAs will facilitate early detection of NE-associated biomarkers in disease susceptible poultry populations in commercial farms enhancing effective management of NE in commercial poultry production.

3. Development of orally deliverable Bacillus subtilis spores carrying chicken NK-2 antimicrobial peptide to protect against coccidiosis and to stabilize gut microbiota in commercial broiler chickens. Avian Coccidiosis, a ubiquitous intestinal disease caused by several distinct Eimeria species, inflicts major economic losses to global poultry industry due to intestinal damages including inflammation, bloody lesions, high morbidity and mortality, and poor nutrition absorption. Due to intensifying public/legislative pressure to reduce the use of antibiotics in poultry production and to develop safe alternatives to reduce the economic losses due to coccidiosis, ARS scientists in Beltsville, Maryland, in collaboration with a biotechnology company, developed a stable Bacillus subtilis spores carrying NK-lysin antimicrobial peptide (B. subtilis-cNK-2) as an effective oral therapeutic against Eimeria parasites in broiler chickens. In vivo trial showed that a higher dose oral B. subtilis-cNK-2 treatment improved disease protection and intestinal health. Therefore B. subtilis-cNK2 spores exerted protective effects against coccidiosis by reducing fecal oocyst shedding, enhancing local protective immunity, and restoring t gut microbiota homeostasis.

4. Dietary phytochemical mitigates coccidiosis infection in commercial broiler chickens. With increasing governmental restriction of growth promoting antibiotics in commercial poultry production, there is a timely need to develop novel feed additives to enhance gut health and to mitigate negative disease impacts of coccidiosis to increase animal resilience on animal agriculture. ARS scientists in Beltsville, Maryland, and a collaborating company have identified a phytochemical combination that mitigates coccidiosis response and reduces gut damage. A beneficial combination of three phytochemicals that enhanced host immunity was identified using in vitro screening system based on three different chicken cell lines using unique biomarker combination associated with different cell functions. Both in vitro and in vivo studies confirmed the beneficial effects of three phytochemicals, green tea extract (GT), cinnamon oil (CO), and pomegranate extract (PO) on avian coccidiosis. In vivo studies confirmed that chickens fed a diet supplemented with this unique phytochemical mixture showed enhanced growth performance and reduced disease response following coccidiosis. These findings provide scientific rationale to develop a science-based antibiotic-independent strategy to mitigate coccidiosis response in poultry production to reduce the economic cost associated with commercial broiler production.

5. Development of a new immunodiagnostic tool for poultry coccidiosis. Avian coccidiosis, an intestinal disease caused by parasites of the genus Eimeria is an economically important disease for the poultry industry worldwide and costs more than $ 13 billion in annual losses. Currently, seven distinct species of Eimeria are known to infect different intestinal sites. There is a timely need for sensitive detection methods for early diagnosis of coccidiosis in commercial poultry production systems for more effective disease management to reduce economic losses associated with coccidiosis. ARS scientists in Beltsville, Maryland, developed a novel antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E, which is present in all Eimeria species. This assay uses a set of 3-1E-specific mouse monoclonal antibodies which were developed by ARS scientists and provides a sensitive diagnostic tool to monitor coccidiosis in commercial poultry farms before clinical symptoms develop. The availability of a sensitive quantitative immunoassay that can specifically detect coccidiosis-infected chickens in large commercial poultry flocks will reduce the economic cost associated with coccidiosis.

6. Development of orally deliverable recombinant vaccine formula to protect chickens against coccidiosis. Avian Coccidiosis, a ubiquitous intestinal disease caused by several distinct Eimeria species (spp.), is one of the costliest diseases in commercial poultry production worldwide. Several distinct species of Eimeria infect young chickens resulting in more than $ 13 billion in annual economic losses due to intestinal damages including inflammation, high morbidity and mortality, and poor nutrition absorption. Although antibiotics are effective in controlling coccidiosis, intensifying public/legislative pressure reduced the use of antibiotics in poultry production, and there is a timely need to develop safe alternatives to control coccidiosis. ARS scientists in Beltsville, Maryland, developed a recombinant vaccination strategy against coccidiosis using a novel recombinant vaccine mixture composed of immunodominant antigen of Eimeria, T-cell promoting chicken cytokine and chicken antimicrobial peptide which targets Eimeria parasites. In vivo, chicken trial showed that rEF-1a vaccine formulated with chicken IL-7 or simultaneous oral administration of chicken NK-2 induced an enhanced protective immunity against E. maxima infection compared to the rEF-1a vaccination alone. Furthermore, EF-1a-vaccinated chickens showed mitigated pro-inflammatory cytokine profiles in the gut, where parasites underwent intracellular development following E. maxima challenge infection, compared to non-immunized chickens. More importantly, co-administration of EF-1a vaccine with chIL-7 enhanced protection against coccidiosis and mitigated negative gut response.

7. Dietary organic selenium improves growth performance, gut health, and disease response in broiler chickens. During the early stage of the newly hatched chickens, before they develop full adaptive immunity, the occurrence of enteric diseases such as coccidiosis and necrotic enteritis contributes to high levels of oxidative stress leading to lower antioxidant gene expression in broiler chickens. As an essential element for the antioxidant enzyme system, selenium (SE) is vital for detoxifying lipid peroxide and reactive oxygen species which are generated from oxidative stress response. Coccidiosis is a major broiler disease condition caused by Eimeria protozoan parasites resulting in poor nutrient absorption and hindering growth performance via gut epithelial damage. To further understand the impact and interactions of dietary SE and the Eimeria infection on broiler gut health, ARS scientists in Beltsville, Maryland, assessed many biomarkers associated with gut health, and investigate the effect of dietary organic SE on growth performance in coccidiosis-infected broiler chickens. The results of this study showed improved body weight and confirmed the beneficial effect of dietary SE and the efficiency of organic SE compared with inorganic SE for growth improvement. Specifically, the addition of organic selinized yeast improved growth performance and enhanced SE accumulation in the tissue, and therefore, organic SE supplementation is beneficial to produce SE -enriched organic broiler chickens, which may help provide guidance for poultry nutrition.

8. Characterization of collagen binding activity of Clostridium perfringens strains isolated from commercial broiler chickens. Necrotic enteritis (NE) caused by Clostridium perfringens type A/G is one of the leading enteric diseases which negatively impact the global poultry industry by affecting the growth performance, production cost, animal health, and welfare of poultry. Collagen adhesion antigen (CNA) protein is a major virulence factor involved in the pathogenesis of NE that plays an important role in early bacteria colonization. ARS scientists in Beltsville, Maryland, compared the abilities of collagen binding of several C. perfringens field isolates and showed that the cna gene was detected in high copies in pathogenic isolates carrying netB gene, and the most pathogenic strains exhibited potent binding activities to Collagen Type III. This study showed that the detection of cna gene and its protein product could be valuable factors to determine the virulence and pathogenicity of C. perfringens isolates from poultry farms.

9. Development and characterization of monoclonal antibodies specific for chicken interleukin-7 receptor a (CD127). Chicken cytokine and chemokine genes such as CD127 that mediate host immune responses in poultry have been cloned, recombinant proteins have been expressed as immunogens for monoclonal antibody development, and these monoclonal antibodies have been further used to develop sensitive immunoassays to characterize host immune responses in poultry. Several mouse hybridomas secreting monoclonal antibodies specifically detecting chicken-specific cytokines including CD127 have been developed and characterized by ARS scientists Beltsville, Maryland, to establish antigen capture ELISA assays to specifically measure them. CD127 plays a critical role in the differentiation and activation of T lymphocytes. Availability of these new sets of chCD127- specific monoclonal antibodies will facilitate the immunological studies on CD127 in poultry, especially in understanding effector and memory T immune cell responses in normal and diseased states.

Review Publications
Lee, Y., Park, I., Wickramasuriya, S., Ben Arous, J., Koziol, M., Lillehoj, H.S. 2022. Co-administration of chicken IL-7 or NK-lysin peptide 2 enhances the efficacy of Eimeria elongation factor-1a vaccination against Eimeria maxima infection in broiler chickens. Poultry Science. 101:102013. https://doi.org/10.1016/j.psj.2022.102013.
Sun, Z., Lu, M., Lillehoj, H.S., Lee, Y., Doo, D., Yuan, B., Yan, X., Li, C.Z. 2023. Characterization of collagen binding capability of clostridium perfringens isolate collections from broiler chickens . Pathogens. 12:778. https://doi.org/10.3390/pathogens12060778.
Li, C.Z., Wang, L., Zhang, S. 2023. Editorial; immunosuppressive diseases in poultry. Frontiers in Immunology. 14:1215513. https://doi.org/10.3389/fimmu.2023.1215513.
Goo, D., Park, I., Nam, H., Lee, Y., Sawall, J., Smith, X., Li, C.Z., Lillehoj, H.S. 2023. Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens. Poultry Science. https://doi.org/10.1016/j.psj.2023.102647.
Lee, Y., Park, I.N., Wickramasuriya, S.S., Lillehoj, H.S. 2023. Short communication: Bacillus subtilis expressing chicken NK-2 peptide enhances the efficacy of EF-1a vaccination in Eimeria maxima-challenged broiler chickens. Animals. https://doi.org/10.3390/ani13081383.
Truong, A., Tran, H., Nguyen, H., Chu, N., Hong, Y., Lillehoj, H.S., Dang, H., Song, K. 2023. Molecular characterization and functional analysis of chicken interleukin 1 receptor 2 (chIL-1R2). Poultry Science. https://doi.org/10.1016/j.psj.2022.102399.
Lee, Y., Lillehoj, H.S. 2023. Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria. Poultry Science. https://doi.org/10.1016/j.psj.2023.102790.
Wickramasuriya, S.S., Park, I., Lee, Y., Lillehoj, H.S. 2023. Effect of dietary organic selenium on growth performance, gut health, and coccidiosis response in broiler chickens. Animals. https://doi.org/10.3390/ani13091560.
Wickramasuriya, S.S., Park, I., Lee, Y., Richer, L.R., Przybyszewski, C., Gay, C.G., Van Oosterwijk, J.G., Lillehoj, H.S. 2023. Orally delivered bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens. Poultry Science. https://doi.org/10.1016/j.psj.2023.102590.
Park, I., Nam, H., Wickramasuria, S.S., Lee, Y., Wall, E.H., Sripathy, R., Lillehoj, H.S. 2023. Beneficial effects of tea extracts, cinnamon oil, and pomegranate on avian coccidiosis. Frontiers in Immunology. https://doi.org/10.3389/fimmu.2023.1145367.
Lee, Y., Park, I., Lillehoj, H.S. 2023. Oral administration of chicken NK-lysin or recombinant chicken IL-7 improves vaccine efficacy of Eimeria tenella Elongation Factor-1a (EF-1a) against coccidiosis in commercial broiler chickens. Poultry Science. 102:102611. https://doi.org/10.1016/j.psj.2023.102611.