Project : USDA ARS

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

Location: Animal Biosciences & Biotechnology Laboratory

2021 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 final 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 (mAbs) 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 have been 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 establishment of mAbs against C. perfringens antigens including NetB toxin and collagen adhesion protein (CNA). In vitro assay for testing collagen binding activity of C. perfringens strains from field bacterial isolates has been developed. Significant progress has been made in the identification of potential vaccine candidate antigens of necrotic enteritis (NE), and efficacy studies of phytonutrient and vaccines as alternatives to antibiotics for coccidiosis and NE. Significant progresses have been made in development of several effective antibiotic alternative strategies against coccidiosis and necrotic enteritis in partnership with private industries under several formal agreements, CRADA, and trust agreements. These results will impact the animal welfare, food safety and food animal production. Progress on immunoassays for important proteins of C. perfringens: Monoclonal antibodies detecting NetB and CNA antigens of C. perfringens were developed and large scale screening immunoassays for NE detection have been established. To better understand the pathogenicity of various C. perfringens, the levels of NetB and CNA in various bacterial cultures and NE challenged samples (sera, intestinal digesta, and fecal samples) were evaluated using antigen capture ELISA assays. NetB- and CNA-specific ELISA assays detected the higher levels of CNA and NetB in the fecal and intestinal digesta samples, but not from serum samples which were obtained from chickens challenged with netB+tpel+ C. perfringens strain. Progress on C. perfringens proteins: Enhanced understanding of collagen binding capability of different strains of Clostridium perfringens (CP) strains has been made and the mAbs detecting collagen adhesion protein (CNA) were developed. Since pathogenicity of CP field strains may be related to the collagen binding capability, the binding capability of various CP isolates to gelatin and 3 types of collagens (type I from rat tail, type II from bovine nasal septum, type III from human placenta) were evaluated by in vitro collagen adhesion assay. CP isolates from healthy birds and food poisoning SM101 cases showed a poor binding to all collagens whereas the isolates from the NE-afflicted chickens showed strong binding to at least one of the collagens or gelatin. For example, the CP19 isolate (netB+tpel+), a pathogenic CP strain bound to all gelatin and all the collagen types. The data indicated a potential role of collagen adherence in the pathogenesis of necrotic enteritis. Progress on NE vaccines: Optimization of vaccination strategies against NE was done in vivo using necrotic enteritis (NE) challenge model using commercial broiler chickens. NE is caused by pathogenic strains of CP and is one of leading enteric infectious diseases responsible for economical loss of more than $ 6 billion in the global poultry industry. There are no effective vaccines against NE which are commercially applicable. To identify potential vaccine candidate antigens, five immunogenic CP proteins were identified, and two hybrid recombinant proteins were constructed. Comparative challenge studies led to the identification of suitable vaccine candidate combinations which will be tested in commercial broiler chickens. Further vaccine trials will identify the hybrid candidate vaccine combination of CP proteins that elicit effective protection against NE. Progress on chicken interferon-kappa: Interferon (IFN)-kappa is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. To better understand the role of chicken IFN-kappa (chIFN-kappa), an antigen-capture ELISA was developed using newly developed mouse monoclonal antibodies (mAbs) which are specific against chIFN-kappa. This ELISA can measure native chIFN-kappa production during the activation of macrophages by polyinosinic: polycytidylic acid (poly I:C). This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-kappa in the normal and diseased states.

Accomplishments
1. Development of poultry immunoassays. The critical lack of immune reagents and the immunoassays to measure host immune mediators and immune response to pathogens in chickens hinders progress in developing novel vaccines and immunotherapeutics against many economically important diseases of poultry. To address these technical gaps, ARS scientists in Beltsville, Maryland, have developed new poultry immune reagents that specifically detect the levels and types of soluble mediators of cellular immunity of chickens to assess a wide spectrum of host immune responses. A total 65 new monoclonal antibodies (mAbs) have been developed and their specificities were validated using several assays including ELISA, immunohistochemistry, Western blot, flow cytometry, qPCR, and cell proliferation. These novel poultry immune reagents have been commercially licensed to U.S. commercial companies for public distribution for use in fundamental and applied research in poultry immunity. These important new immune reagents will facilitate the discovery phase of their roles in host immune response against various infectious diseases which will impact the development of novel vaccines and antibiotic alternative strategies against various infections in poultry.

2. Role of chicken CCL5 chemokine. Lack of scientific information on chemokine mediators of chicken innate immunity has delayed the development of immunotherapeutic agents to treat many poultry infectious diseases. CCL5 belongs to the CC (or ß) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. ARS scientists in Beltsville, Maryland, identified chicken CCL5 gene and showed that CCL5 is mainly produced by T cells, macrophages, epithelial cells, and fibroblasts and it acts as a chemoattractant to recruit effector cells to the inflammation sites during the infection. Furthermore, eight new monoclonal antibodies (mAbs) which specifically detect chicken CCL5 were developed and used to develop a large scale screening immunoassay. Using this new ELISA, CCL5 secretions during coccidiosis and NE infections were measured in commercial broiler chickens. Studies on the molecular characteristics of chicken CCL5 demonstrate the potential application of using anti-CCL5 mAbs and CCL5-specific ELISA for detecting native CCL5 chemokine in biological samples from diseased chickens. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species and enhance our understanding of innate immunity in chickens.

3. Molecular characterization of chicken IL-7. Limited information on the nature of immune-enhancing cytokines in chickens hinders progress in developing therapeutic strategies based on immune modulation. IL-7 is a hematopoietic growth factor required for development and maintenance of lymphocytes including T cells, B cells, and NK cells. Although IL-7 plays an important role in host immune protection against infectious diseases, reliable bioassays are not available to detect and quantify bioactive IL-7 in avian species due to the lack of specific antibodies. ARS scientists in Beltsville, Maryland, identified chicken IL-7 (chIL-7) and investigated its role in host response to coccidiosis. To better study IL-7, chicken gene encoding IL-7 was cloned and recombinant protein expressed and five mAbs detecting chicken IL-7 were produced. Considering the critical role of IL-7 in T cell survival, a sensitive sandwich ELISA to monitor circulating chIL-7 levels in the serum samples of coccidiosis-infected chickens. The mAb and immunoassay to measure chIL-7 will be valuable reagents for fundamental and applied immunological studies in poultry to enhance our understanding of the role of chIL7 on lymphocyte development and maintaining homeostasis of immune system in chickens.

4. Immunoassays for a large scale screening of necrotic enteritis. Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type A/G which impacts global poultry industry by compromising performance, health, and welfare of the chickens. NE has been a common disease afflicted in intensive broiler production globally due to the voluntary or regulatory ban on in-feed antibiotics use including anticoccidials. Ability to detect the NE early during broiler production will have a significant impact on the economic cost of commercial poultry production. ARS scientists in Beltsville, Maryland, developed several mAbs detecting the NetB toxin of C. perfringens, a major toxin involved in pathogenesis of NE, and used them to develop a sensitive ELISA that allows a large field screening of NE in commercial farms. The usefulness of this new ELISA was validated on fecal samples collected from a large field testing of commercial chickens undergoing NE infection. Results of field screening indicate that this new immunoassay can detect native NetB protein that will allow the accurate measurement of C. perfringens virulence, monitor flock health status at farm level or to conduct early detection of NE outbreak in NE-susceptible farms.

5. New oral solution to combat avian coccidiosis. Coccidiosis is a major infection of the chicken intestinal tract caused by the protozoan, Eimeria, with global economic losses exceeding $3.2 billion. Antibiotic overuse and abuse on a global scale to treat coccidiosis has led to the emergence of multi-drug resistant “superbugs” in food animals. The United States FDA has requested that agriculture producers discontinue sub-therapeutic dosing of antibiotics into animal feed and there is an urgent need to identify new antibiotic-free growth promotion strategies. ARS scientists in Beltsville, Maryland, in partnership with a U.S. company, developed an oral delivery strategy for chicken anti-Eimeria peptide (NKlysin) that kills the invasive stage of Eimeria parasites using Bacillus subtilis spores to improve poultry growth performance without antibiotics. NKlysin is a natural lytic peptide discovered by ARS scientists in Beltsville, Maryland, with cytotoxic activity against multiple parasites including Eimeria. This novel ARS-patented technology (#63/040718) using Bacillus subtilis-NKlysin (B. subtilis-NK2) to protect poultry against avian coccidiosis was licensed to a commercial partner as an oral antibiotic alternative that will enable technology in the fight against poultry coccidiosis. This genetically engineered probiotic is administered orally in a probiotic powder that can be mixed into current feed processes and be fed to the birds across their lifetime without requiring additional steps or new ingredients. This oral method will help greatly reduce the clinical impact of coccidiosis, lessen spread of the disease, and improve gut health in poultry.

6. Discovery of maltol as a novel antibiotic alternative. Increasing implementation of antibiotic-free poultry production system in the U.S. is making the control of some enteric pathogens such as coccidiosis-causing Eimeria species and necrotic enteritis-inducing Clostridium perfringens strains challenging. Development of antibiotic alternatives is a priority for the animal agriculture industry to maintain the growth performance and health of food-producing animals without losing productivity in the post-antibiotic era. ARS scientists in Beltsville, Maryland, conducted global metagenomics to analyze the entire metabolites of gut microbiota after feeding Bacillus subtilis probiotics. Several important small molecular weight metabolites were identified which are beneficial for gut health and maltol was chosen for further study as a novel antibiotic alternative that enhance host immunity, gut health, and growth performance of broiler chickens challenged with coccidiosis. This is the first report on using maltol as an antibiotic alternative postbiotic to improve the growth and immunity of chickens presenting with enteric diseases.

U.S. DEPARTMENT OF AGRICULTURE

Animal Biosciences & Biotechnology Laboratory: Beltsville, MD