Project : USDA ARS

ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Research Project #432073

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

Location: Animal Biosciences & Biotechnology Laboratory

2019 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 third annual report for Project 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 progress has been made in the understanding of the mode of action of antibiotic growth promoters (AGP) in commercial broiler chickens, identification of potential vaccine antigens of necrotic enteritis (NE), Eimeria interaction with the chicken host, vaccine tests for coccidiosis and NE, and efficacy studies of phytonutrient as alternatives to antibiotics. Virulence factors associated with necrotic enteritis were identified from field isolates of virulent C. perfringens (CP) strains which have been collected by the ARS scientists from field NE disease outbreaks in farms have been sequenced to identify virulence-associated genetic factors. Among them, 5 field CP strains carrying genes of very virulent toxins (netB+tpel+) have been characterized and their pathogenicity were evaluated using the NE disease challenge models. New studies using the pathogenic CP strain provided a consistent virulence platform to identify pathogen-related factors associated with pathogenesis and evaluate the efficacies of various alternatives to antibiotic strategies to reduce the use of antibiotics in poultry production. Adjuvanted proteins were also evaluated as vaccine candidates for coccidiosis and NE. In addition, ARS scientists are applying multi “omics” technologies, including metagenomics, metatranscriptomics, and metabolomics, to investigate the 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, and /or the action mode of AGPs. Furthermore, 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, trust and MOU. These results would benefit the poultry welfare, food security and novel antibiotic-free strategies reducing negative effects of Coccidiosis and necrotic enteritis.

Accomplishments
1. Enhanced understanding of mechanisms of action of antibiotic growth promoters. Although antibiotics growth promoters (AGPs) have been widely used globally to make a significant contribution to the expansion of modern animal agriculture, underlying modes of action of AGPs still remain unknown. Understanding how AGPs work will enable industry to develop logical antibiotic alternatives. Scientists in Beltsville, Maryland, used “omics” technology to identify biochemical pathways which are associated with the effects of AGPs. They collected intestinal contents from broiler chickens fed a diet or a diet supplemented with two kinds of AGPs called virginiamycin or bacitracin methylene disalicylate (BMD) for comparative metabolite analysis. Compared with unsupplemented controls, the levels of 218 biochemicals were altered (156 increased, 62 decreased) in chickens given the virginiamycin-supplemented diet whereas 119 were altered (96 increased, 23 decreased) with the BMD-supplemented diet. The changes in the levels of intestinal small molecular weight metabolites provided distinctive biochemical signatures unique to each antibiotic-supplemented group. These biochemical signatures were characterized by increases in the levels of metabolites of amino acids, fatty acids, nucleosides and vitamins. These results enhance our understanding of the mode of AGP action and provide the framework for future studies to identify natural chemical compounds as antibiotic alternatives to improve poultry growth performance without the use of in-feed antibiotics.

2. Development of species-specific immune tools. The lack of poultry immune reagents and immunoassays for the detection and evaluation of cell-mediated immunity in chickens hinders progress in developing antibiotic alternative strategies including vaccines. To address these technical gaps, scientists in Beltsville, Maryland, have identified and developed new immune reagents that will be critical for measuring host immune response to infectious agents and to evaluate novel antibiotic alternatives to replace antibiotics in poultry. During this reporting period, we have completed 9 target chicken proteins in monoclonal antibody (mAb) development which are critical in measuring host innate immunity in poultry. These new murine hybridoma cell lines which secrete mAbs that specifically detect chicken cytokines and chemokines including IL4, IL10, IL13, CCL4, CCL5, IL7 and granzyme A have been characterized and more importantly, detection assays for many chicken immune proteins in sera have been developed. Availability of these new immune tools would significantly advance avian immunology field and will facilitate the development of vaccines and support the fundamental study of host immune response against various infectious diseases.

3. Understanding poultry innate immunity. Comprehensive understanding of how host cells eliminate pathogens quickly will enable scientists to develop effective alternatives to antibiotics. Scientists in Beltsville, Maryland, investigated how chicken cytokine called interleukin 4 (IL-4) mediates early host defense response using macrophages which are large phagocytic cells at the site of infection. The results of this study showed that the macrophage-mediated immune regulation of innate immunity is mediated by IL-4. To better study the mechanism of action of IL-4 in chickens, several mouse monoclonal antibodies were developed against chicken IL-4. The results showed that IL-4 inhibited nitric oxide (NO) production by LPS-activated macrophage cells and, induced robust expression of genes associated with M2 phenotype than M1 phenotype-related genes. In summary, this study showed that IL-4 may override LPS functionality and regulates alternative activation of macrophages in chickens via increased arginase activity and expression of M2 associated markers. This finding enhanced our understanding of the role of macrophages in host pathogen interaction in chickens.

4. Enhanced understanding of poultry immunology. Following important new discoveries that enhanced fundamental principles of avian immunology were made by scientists in Beltsville, Maryland, in collaboration with Korean University scientists: 1). Interleukin-34 (IL-34) is a newly recognized cytokine which is expressed in macrophages and fibroblast cells where it induces cytokine production. The mechanism of chicken IL-34 (chIL-34) signaling and its function revealed that chicken IL-34 increased the expression of Th1 and Th17 cytokines, and 2). The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. These new findings provide novel insights into the regulation of immunity and immunopathology and will facilitate the development of new immunotherapeutics.

5. Deciphering the effects of dietary indoles on the immune mechanisms of avian coccidiosis. Limited understanding of host-parasite interactions in avian coccidiosis hinders the development of effective disease control strategy against this disease. ARS scientists in Beltsville, Maryland, collaborated with scientists at a Korean university to investigate the immune homeostasis during parasitic infections in chickens. In mammals, indoles, as the ligands of aryl hydrocarbon receptor (AhR), have been shown to possess immune-modulating property in terms of the balancing between regulatory T cells (Treg) and T helper 17 cells (Th17). Using dietary indoles, these scientists showed that indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) induced decreased number of CD4+IL-17A+ (T h17) cells and mRNA levels of Th17-related cytokines, IL-17F, IL-21, and IL-22 while increasing CD4+CD25+ (T reg) cells and mRNA expression of IL-10 in the spleen and intestine; furthermore, when chickens were treated, there was a significant reduction of intestinal lesions in Eimeria tenella-infected ceca although body weight gain and fecal oocyst production were comparable to non-treated control group. These findings indicate that the increase of Treg/Th17 ratio induced by dietary indole treatment likely reduced parasite-induced local inflammatory response or facilitated repairing process of inflamed gut. Furthermore, this study also shows that AhR ligand modulates the T cell immunity through the alteration of Treg/Th17 cells with Treg dominance. These new findings enhance our understanding of importance of immune homeostasis in parasitic infections like coccidiosis and will direct the development of novel disease intervention strategy against avian coccidiosis.

6. New anti-infective chemicals for coccidiosis treatment. Due to the increasing health hazards associated with extensive drug uses in animal agriculture, discovery of new non-antibiotic alternatives is becoming important. ARS scientists in Beltsville, Maryland, in collaboration with scientists at University of Maryland, School of Medicine, Maryland, identified low molecular weight compounds derived from the antimicrobial peptide Human Neutrophil Peptide 1 that bind to Lipid II, an essential precursor of bacterial cell wall biosynthesis. These compounds act as anti-bacterials on multiple biosynthesis pathways with specificity against Gram-positive organisms. These compounds demonstrated killing effects against bacterium Clostridium perfringens and sporozoites of Eimeria tenella, an intracellular protozoan parasite that causes severe intestinal disease in poultry. Mechanism-of-action studies further reveal that some of these compounds affect cell wall biosynthesis, but also inhibits additional biosynthetic pathways. Combined, these results indicate that these new compounds have therapeutic potential to act as an anti-infective against various organisms simultaneously. This compound can be a substitute for currently used antibiotics as a novel alternative that can decrease negative effects of avian coccidiosis.

7. New vaccine candidate antigen for coccidiosis prevention. Limited information on potential vaccine candidates that can be used for coccidiosis prevention hinders the development of effective control measures against avian coccidiosis. ARS scientists in Beltsville, Maryland, showed that a novel coccidia antigen, elongation factor -1a, and chicken IL-7 DNA vaccine formulation confers significant protection against coccidiosis. Coccidiosis, which is caused by several distinct species of the intestinal parasite Eimeria, is one of the most economically important intestinal infectious diseases, responsible for an economic loss of more than $ 3.2 billion worldwide and has been identified as a major risk factor for other enteric diseases 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. ARS scientists in Beltsville, Maryland, showed that chickens immunized with EF-1a and chIL-7 showed overall improvements in clinical disease manifestation, mainly due to gain weight, reducing oocyst shedding, ameliorating lesion score, and up-regulation of pro-inflammatory cytokine gene expression, possibly through the activation of CD8+ cytotoxic effector T-cells. The results of this study demonstrated the beneficial effects of using EF-1a DNA vaccine and/or host cytokine chIL-7 DNA to improve T-cell-mediated effector function in coccidiosis-challenged broiler chickens. 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.

8. Deciphering the alteration of mRNA gene profiles in avian coccidiosis. Coccidiosis is one of the leading enteric infection diseases caused by several parasite Eimeria species in poultry industry and is also the major predisposing factor for another major enteric disease-Necrotic enteritis (NE) caused by Clostridium perfringens (CP). These diseases are economically responsible for devastating loss of more than $ 3.2 and 6.0 billion annually worldwide, respectively. The increasing incidences have been associated with the global voluntary removal of antibiotic growth promoters from animal feed in poultry production. There is an urgent need to control such huge loss from coccidiosis. However, the mechanism on host-parasite interaction remains poorly defined. ARS scientists in Beltsville, Maryland, applied RNA-sequencing technology and bioinformatics tool to investigate intestinal gene profiles of chicken in response to Eimeria maxima (EM) infection. The results showed that EM induced significant differences in the cecal mucosal gene expression. Expressions of 332 genes were significantly increased, while the expresses of 363 genes were decreased. The broad gene categories represented by the highly differentiated host genes suggested enrichment in immune responses, and decreased expression in the metabolic pathway, signaling pathway, vascular smooth muscle contraction, and proteins processing after EM infection. These findings provide new insights into the host-parasite interaction to enhance our understanding of the molecular mechanism of avian coccidiosis.

9. Deciphering genomes of pathogenic C. perfringens strains for establishing consistent disease challenge models in inducing necrotic enteritis in meat chickens. C. perfringens (CP)-induced necrotic enteritis (NE) and related subclinical diseases have become financially devastating problems for the global poultry industry with estimated loss of $ 6 billion annually and these problems are mainly associated with the recent increasing government restrictions on the use of in-feed antibiotic growth promoters worldwide. Establishment of successful challenge models will be critical for understanding mechanisms of pathogenesis, development of novel vaccines, and evaluation of efficacy of antibiotic alternatives strategies in disease prevention. ARS scientists in Beltsville, Maryland, sequenced and analyzed the genomes from several important CP strains from field NE disease outbreaks and deposited 4 genomes to public gene banks in the website of National Center for Biotechnology Information. One strain containing genes for large lethal toxin and pore-forming toxin was found to be the most pathogenic and significantly retarded the chicken growth rate following CP infection. This virulent strain was successfully used to develop a NE disease challenge model. Easy public accesses to the genome information of these strains will contribute to the enhanced understanding of the CP evolution and NE pathogenesis in commercial poultry. In addition, availability of the optimal challenge stain and stable NE disease challenge models will significantly contribute to the progress in finding alternatives to antibiotics and help avian researchers to evaluate the efficacies of various antibiotics alternatives approaches in NE prevention.

10. Dietary phytochemical antibiotic alternative that promotes growth and enhance innate immunity. Although in-feed antibiotics have dramatically increased the efficiency of commercial poultry production over the last 50 years, we are now faced with an increasing global crisis concerning the heightened use of antibiotics in animal agriculture and the emergence of multidrug-resistant superbugs that threaten disease management in animals and humans. Therefore, much interest has focused on the development of alternative, antibiotic-free methods of commercial poultry production. In collaboration with scientists at RDA in South Korea, ARS scientists in Beltsville, Maryland, studied beneficial effect of dietary supplementation of Allium hookeri (AH) plant as an antibiotic alternative feed additive for commercial broiler chickens. The results showed this phytochemical supplementation improved body weight gains at day 14 as compared to the control group (p < 0.05) and up-regulated genes that reduced oxidative stress. These results indicated that an optimum level of dietary AH supplementation to young broiler chickens influences growth and improves anti-oxidant activities. The findings suggest using plant product like AH to enhance gut health and to reduce the use of antibiotics in poultry.

U.S. DEPARTMENT OF AGRICULTURE

Animal Biosciences & Biotechnology Laboratory: Beltsville, MD