Project : USDA ARS

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

Location: Animal Biosciences & Biotechnology Laboratory

2018 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 second annual report for the project of 8042-32000-107-00D which started in Oct, 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 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 and their biological functions have been elucidated. Furthermore, three poultry genes which encode cell surface markers of chicken lymphocytes have been cloned, expressed and their uses in various immunoassays, including intracellular and extracellular staining as markers of host innate and adaptive immunity, have been validated. These include chicken CD127 biomarker, perforin and granzyme which are mostly involved in effector memory T cells and anamnestic immune response in vaccination process. In addition, three genes encoding selected cytokine and chemokine markers were also expressed in Escherichia coli and/or eukaryotic expression cells, and the monoclonal antibodies specific for chicken CD127 were characterized by ELISA and FACS. These new immune reagents will be used to investigate mucosal and systemic immune response to intestinal Eimeria parasites and Clostridium perfringens bacteria, causative agents of coccidiosis and necrotic enteritis, respectively. In addition, the multi “omics” technologies including metagenomics, metatranscriptomics, and metabolomics, were employed to investigate the gut microbiota subpopulation profiles, molecular mechanism of host-pathogen interaction and to identify molecular and metabolite signals that mediate beneficial host response to the causative agents of coccidiosis and necrotic enteritis. Under Objective 2, significant progress has been made in understanding virulence related factors associated with pathogens associated with necrotic enteritis. Fourteen field isolates of virulent C. perfringens (CP), collected since 2011 from field disease outbreaks in the Eastern Shore, have been sequenced to identify virulence-associated genetic factors. Among them, five field CP strains carrying genes of virulent toxins (netB+tpel+) were identified and their pathogenicity were evaluated using an ARS disease challenge model developed in 2011. One CP strain (tpel 17) was found to be the most virulent in inducing necrotic enteritis when birds were challenged with this strain alone. This virulent CP strain is an excellent CP source as a challenge strain could be used to develop a CP-dependent necrotic enteritis disease model for pathogenesis studies and vaccine development. New studies using these virulent CP strains would provide a logical platform virulence and to identify pathogen-related factors associated with evaluate the efficacies of various alternatives to antibiotic strategies to reduce the use of antibiotics in poultry production. In addition, significant progress has been made in development of three effective antibiotic alternative strategies in partnership with private industries under several formal agreements, CRADA, trust and MOU. Furthermore, ARS scientists are investigating the interaction between host and pathogens (Eimeria spp. and C. perfringens) using global gene expression technology to identify host and pathogen genes which are mediating protective innate immune responses.

Accomplishments
1. Development of critical poultry immune reagents. Currently, one of the major challenges for poultry immunology and disease research is the lack of sufficient immunological reagents, and the lack of sensitive detection methods to assess poultry immune responses to environmental stressors, such as infectious agents, in commercial chickens. ARS scientists in Beltsville, Maryland, developed critical immune reagents that will be applied for the detection of several critical molecules of host immune cells in poultry species. These small molecules are proteins which are secreted by chicken immune cells and they mediate various functions which are important in intercellular communications to initiate protective immune response toward pathogens. These new immune reagents will allow scientists to study the function of important chicken molecules involved in host immune response in many disease conditions, and will enable scientists to develop effective vaccines against many poultry diseases.

2. Characterization of a virulent toxin-producing field strains of bacterial Clostridium perfringens (CP). Clostridium perfringens causes an intestinal bacterial disease in chickens known as necrotic enteritis (NE) and this disease has re-emerged as a major problem due to the governmental restrictions on the use of in-feed antibiotic growth promoters worldwide. The economic cost of NE, currently estimated to be $6 billion, is likely to increase. An effective NE model will be necessary for developing a drug-free strategy to combat NE disease. ARS scientists in Beltsville, Maryland, tested a variety of CP strains that were collected from field NE disease outbreaks. Using genomic approaches, genes associated with high infectivity (virulent) were identified for the first time in these field strains and the NE disease was reproduced with typical intestinal lesions using selective field CP strains that possess virulent genes associated with enhanced disease severity. These virulent CP strains will be very useful as challenge model strain for vaccine and CP pathogenesis study in chickens and will greatly facilitate the discovery of antibiotic alternatives to replace drugs in poultry production.

3. Development of antibiotic alternatives. There are heightened concerns globally on emerging drug-resistant superbugs and the lack of new antibiotics for treating human and animal diseases. For the agricultural industry, there is an urgent need to develop strategies to replace antibiotics for food-producing animals, especially poultry. ARS scientists in Beltsville, Maryland, studied the mode of action for novel antibiotic alternatives in collaboration with a private industry and discovered that a combination of two plant-based phytochemical mixture from pepper (Capsicum) and turmeric (Curcuma) promoted gut health and enhanced host immune response against parasitic and bacterial infections in commercial broilers. Furthermore, young chickens which were fed with these plant extracts showed enhanced body weight gains and produced higher levels beneficial immune molecules when they were challenged with parasites and bacteria that commonly cause illness in commercial poultry. Collectively, these studies showed clear beneficial effects of certain dietary plant extracts to promote poultry growth and health. This accomplishment will help scientists from industry, academia and government research institutes to develop other antibiotic-alternative feed additives that can promote a sustainable animal production system in the absence of antibiotics.

4. Beneficial bacterial strains as growth-promoting antibiotic alternatives. The increasing occurrence of antibiotic-resistant bacteria combined with regulatory pressure and consumer demands for foods produced without antibiotics has caused the agricultural industry to restrict its practice of using antibiotic growth promoters (AGP) in feed animals. To develop natural alternatives to AGP to improve the growth performance of commercial poultry, ARS scientists in Beltsville, Maryland, collaborated with scientists at a private industry to screen multiple bacterial isolates for growth promoting property. These scientists discovered that a specific strain of bacteria called Bacillus subtilus (BS) can be used as an AGP alternative because of their growth promoting benefits and their ability to modulate protective host immune response. Furthermore, these bacteria strains interacted directly with the host to produce beneficial metabolites in the gut and the resulting immunological changes were critical to promote poultry growth.

5. Description of the gene expression patterns associated with avian coccidiosis infections. Coccidiosis, which is caused by several distinct species of the intestinal parasite Eimeria, is one of the most economically important intestinal infectious diseases and it is responsible for an economic loss of more than $3.2 billion worldwide and has been identified as a major risk factor for other enteric disease such as salmonellosis and necrotic enteritis. Increasing trends of legislative restrictions and voluntary removal of antibiotic growth promoters from animal feed worldwide has impacted poultry production and health; therefore, controlling coccidiosis is a high priority for poultry industry since it poses as a major risk factor for other enteric diseases that are potentially hazardous for human health. ARS scientists in Beltsville, Maryland, used sequencing technology to investigate underlying molecular changes in the pattern of gene expression in the intestine of chickens after infecting them with one of several Eimeria species that cause coccidiosis in commercial poultry. Using this approach, ARS scientists identified unique alterations in chicken genes which promote parasite-killing and host-protecting gene expression in the intestine. Understanding the molecular mechanisms associated with host protective immunity in this important enteric poultry disease will facilitate the development of logical antibiotic-free disease control approaches against coccidiosis.

U.S. DEPARTMENT OF AGRICULTURE

Animal Biosciences & Biotechnology Laboratory: Beltsville, MD