Location: Animal Biosciences & Biotechnology Laboratory
2017 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 first 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 also on Problem Statement 5C: Enteric Diseases of Poultry. Under Objective 1, progress has been made in identifying critical poultry immune genes and immune reagents. We have identified 15 candidate poultry genes for use as intracellular and extracellular markers which are associated with chicken immune responses, such as characterizing chicken CD127 biomarker, perforin and granzyme which are involved in effector memory T cells and their anamnestic immune responses. In addition, 15 poultry genes encoding selected cytokine and chemokine markers are also identified for bioengineering in eukaryotic expression cells, and assessment of their functional bioactivities. These new immune reagents will be used to investigate mucosal and systemic immune response to intestinal Eimeria parasites and Clostridium perfringens bacteria, causative agent of Coccidiosis and necrotic enteritis, respectively. They will be very useful in vaccine development in the study of determining the underlying vaccine-induced protection mechanisms against many infectious diseases of poultry. 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. Collaboration will include well-established international network of avian scientists and commercial companies that market veterinary immune reagents worldwide.
In addition, ARS scientists in Beltsville, Maryland, are using the metagenomics and metatranscriptomics approaches to investigate the gut microbiota subpopulation profiles, and to define the differential expression of genes that govern the processes involved in chicken host defense against enteric diseases: Eimeria spp. and Clostridium perfringens.
Under Objective 2, significant progress has been made in isolating, characterizing and toxin genotyping many field isolates of C. perfringens (CP) strains from the necrotic enteritis-afflicted broiler farms. Among them, 5 strains carrying genes of very virulent toxins (netB+tpel+) have been identified using molecular typing method at ABBL. These virulent CP strains will be excellent CP sources that will be used to develop a CP-dependent necrotic enteritis disease model for pathogenesis studies. Establishment of an effective NE disease model would also provide a logical platform to evaluate the efficacies of various alternatives to antibiotic strategies to reduce the use of antibiotics in poultry production. Some of field CP strains have been sequenced, and potential vaccine target genes are being identified, cloned and their recombinant proteins expressed. In addition, significant progresses have been made in development of many effective antibiotic alternatives, including lysin, probiotics, phytochemicals, and hyperimmune egg yolk antibodies that led to commercial feed additives by ARS scientists in Beltsville, Maryland, in partnership with private industries. The efforts towards vaccination for NE is being made since vaccination is often regarded as one of the effective means to control the infectious diseases. Furthermore, ARS scientists are investigating the interaction of gut microflora, diet, host and pathogens (Eimeria spp. and C. perfringens) that could lead to new knowledge on how gut pathogens and diets impact the gut microbial communities and interact with the host immune system in poultry. These results would benefit the poultry production and human health by reducing negative effects of Coccidiosis and necrotic enteritis, and reducing the use of antibiotics in commercial poultry products.
Accomplishments
1. Genomic characterization toxin-producing bacterial field C. perfringens (CP) strains. C. perfringens (CP) can cause enteric bacterial disease in chickens called necrotic enteritis (NE) which has re-emerged as a major problem due to the governmental restrictions on the use of in-feed antibiotic growth promoters. The economic cost of NE, currently estimated to be $6 billion, is likely to increase. Better understanding of pathogen-associated factors will be necessary in order to develop a drug-free strategy to manage NE. ARS scientists in Beltsville, Maryland, screened a variety of CP strains which were isolated from field NE disease outbreaks. Screening of virulent field CP strains identified 5 unique CP isolates which possess a very virulent gene called tpel that has been associated with enhanced disease severity. These virulent strains will be very useful as challenge model strains to induce the NE diseases, thus simplifying the studies of CP pathogenesis in chickens. More importantly, new NE disease challenge model that ARS developed will facilitate the discovery of antibiotic alternatives that will mitigate the use of drugs in poultry production.
2. Gut microbiome and parasite/bacterial infections. The increasing trends of legislative restrictions and voluntary removal of antibiotic growth promoters worldwide has impacted poultry production and health. The rising incidence of Clostridium infections and development of Clostridium perfringens (CP)-caused necrotic enteritis (NE) in chickens is associated with the withdrawal of antibiotics during production. Normal animal intestinal population, called microbiota, plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. The balance among intestinal microbiota can be disrupted by the use of antibiotics, diets and harmful bacteria that cause infections. ARS scientists in Beltsville, Maryland, used 16S rRNA gene sequencing technology to investigate the presence of specific changes in the gut microbiota following parasite and bacterial infections in chickens. Using this technique, ARS scientists identified unique alterations in gut bacterial population following infections with bacteria Clostridium perfringens, parasites called Eimeria, or antibiotic treatments. Investigation of the effects of various treatments on gut microbiota and host disease response will lead to the development of logical antibiotic-free disease control strategies.
3. 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 a 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 have made great efforts towards developing many critical immune reagents, such as monoclonal antibodies (mAbs), by identifying around 30 major white blood cell subpopulations and their biological markers which are associated with different types of host immune responses. These new immune reagents will allow us to describe the function of important molecules involved in poultry immunity and the assessment of host immune response to microbial pathogens, and will facilitate the development of effective vaccines against many poultry diseases to which there are no effective vaccines at present.
4. Identification and characterization of chicken cytokine Interleukin-8 (IL-8). There is a significant lack of knowledge on poultry immunology due to the lack of poultry-specific immune markers. Nonspecific defense processes that include the natural inherent immunity of the host, come into play immediately or within hours of an invasion of infectious agent in the body. These processes include physical barriers like skin, chemicals in the blood, and immune cells that attack and remove foreign organisms from the host. ARS scientists in Beltsville, Maryland, investigated one of the key molecules which is involved in host immune response in poultry to enhance our understanding of early events in inflammation, a process associated with infectious diseases. New immune reagents that attracts white blood cell called lymphocyte that plays a role in wound healing will now be used to study infection-induced inflammation and disease process in poultry, and they will serve as valuable immune tools for basic and applied research.
5. Important roles of dietary phytochemicals as alternatives to antibiotic growth promoter in poultry. In-feed antibiotic (IFA) use is a well-established practice in the animal industry and has contributed to the intensification of the poultry production. To address increasing concerns on the IFA leading to the development of antimicrobial resistance, ARS scientists in Beltsville, Maryland, collaborated with scientists at the government agricultural agency in Korea to discover new compounds in a plant of Allium hookeri, which provided many beneficial effects that enhanced gut health in commercial broiler chickens. Dietary Allium hookeri clearly enhanced growth performance in chickens, reduced inflammatory immune activities in the intestine, and improved intestinal barrier function during the immunological stress that was induced by injecting bacteria particles called lipopolysaccharides. These findings demonstrate that dietary Allium hookeri, when given to newly hatched chickens, provides many beneficial effects and improves the growth of commercial broilers.
6. Development of novel host-derived antimicrobial peptides as antibiotic alternative. Increasing concerns associated with using antibiotics in poultry production make it necessary to find drug alternatives for treating diseases in poultry. ARS scientists in Beltsville, Maryland used a highly automated process to analyze genetic expression of chicken produced antimicrobials that are toxic against a parasite called Eimeria. The most promising chicken produced antimicrobial was found to be NK-lysin which possesses many beneficial effects to promote host resistance against Eimeria. ARS scientists previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core structure region of cNK-lysin, have antimicrobial activity against parasites such as Eimeria which causes significant economic losses to poultry industry. In addition to the antimicrobial activity of cNK-lysin, ARS scientists further demonstrated that cNK-lysin enhances natural host defense against many pathogens. These new results clearly show that cNK-2 is a potential novel immunity-modulating agent which can be commercially utilized as an antibiotic alternative to reduce the negative effects of enteric infections in young chickens.
7. Dietary supplementation with the thermally-processed clays, fermentable fiber and organic acid (CFC) as antibiotic alternatives. Necrotic enteritis (NE) is a poultry enteric disease caused by Clostridium perfringens and distinguished by severe intestinal damage. The incidence of avian NE has been progressively increasing following the removal of antibiotics from poultry feed. ARS scientists in Beltsville, Maryland, collaborated with a private company in Chicago, Illinois to evaluate the effect of diets supplemented with CFC mixture on clinical signs, and immune response in broiler chickens using an experimental model of NE. The results showed that chickens fed a diet supplemented with CFC had increased body weight gain, reduced gut lesions, and increased serum antibody levels to two C. perfringens’s toxins (a-toxin and NetB toxin) compared with the control group that did not receive CFC. Intestine and spleen cells of chickens fed CFC-supplemented diets also showed significant levels of substances that are important to immune regulation, and these substances came in the form of messenger RNA. Dietary supplementation of broiler chickens with CFC might be useful to control avian NE in the field as alternatives to antibiotics.
8. Direct-fed microbials as an antibiotic alternative. With an increase in concerns regarding the development of antibiotic resistance and efforts to promote the judicious use of antimicrobials in food-producing animals, there is a timely need for the development of viable alternatives to ensure and maintain optimal animal health and performance. Direct-fed microbials (DFMs), often referred to as probiotics, are a potential non-antibiotic replacement that has been studied extensively and used in commercial applications. DFMs are beneficial bacteria and often used as a feed supplement to promote gut health. To better understand the health benefits of probiotics in enhancing gut health in poultry and the mechanisms used by non-pathogenic probiotic bacteria, Bacillus subtilis, ARS scientists in Beltsville, Maryland, carried out extensive animal studies to show that certain Bacillus strains stimulate host innate immune response, decrease harmful inflammatory response, and promote gut integrity when used as a feed additive in young chickens. These results clearly provided scientific evidence for beneficial effects of probiotic bacteria and the potential use of Bacillus subtilis as a feed additive to promote gut health in commercial poultry production to reduce the use of antibiotics.
Review Publications
Lee, S., Dong, X., Lillehoj, H.S., Lamont, S.J., Sup, X., Kim, D.K., Lee, K., Hong, Y.H. 0216. Comparing the immune responses of two genetically B-complex disparate Fayoumi chicken lines to Eimeria tenella. British Poultry Science. 57(2):165-71.
Del Cacho, E., Lillehoj, H.S., Gallego, M., Quilez, J., Lillehoj, E.P., Sanchez-Acevedo, C. 2016. The induction of protective immunity against experimental Eimeria tenella Infection using serum Exosomes. Veterinary Parasitology. 224:1–6.
Rengaraj, D., Truong, A.D., Lee, S., Lillehoj, H.S., Hong, Y.H. 0215. Expression analysis of DNA-sensing pathway genes in the intestinal mucosal layer of necrotic enteritis-induced chicken. Veterinary Immunology and Immunopathology. 170:1-12.
Gadde, U., Rathinam, T., Lillehoj, H.S. 2015. Passive immunization with hyperimmune egg yolk IgY as prophylaxis and therapy for poultry diseases-A review. Animal Health Research Reviews. 16(2):163-176. doi: 10.1017/S1466252315000195.
Lillehoj, H.S., Lee, S.H., Park, S.S., Jeong, M., Lim, Y., Mathis, G.F., Lumpkin, B., Chi, F., Ching, S., Cravens, R.L. 2016. Calcium Montmorillonite-based dietary supplement attenuates Necrotic Enteritis induced by Eimeria maxima and Clostridium perfringens in broilers. Poultry Science. 53:329-340.
Oh, S., Lillehoj, H.S. 2016. The role of host genetic factors and host immunity in necrotic enteritis. Avian Pathology. 45(3):313-316. doi: 10.1080/03079457.2016.1154503.
Diaz, J.A., Kim, W.H., Fernandez, C.Y., Jeong, J., Afrin, F., Lillehoj, H.S., Kim, S., Kim, S., Dalloul, R.A., Min, W. 2016. Identification and expression analysis of duck interleukin-17D in Riemeralla anatipestifer infection. Developmental and Comparative Immunology. 61:190-197. doi: 10.1016/j.dci.2016.04.002.
Fernandez, C.P., Kim, W.H., Diaz, J.A., Jeong, J., Afrin, F., Kim, S., Jang, H., Lee, B., Lillehoj, H.S., Min, W. 2016. Interleukin-17A critically contributes to susceptibility to Riemerella anatipestifer infection. PLoS Pathogens. 63:36-46. doi: 10.1016/j.dci.2016.05.009.
Truong, A., Ban, J., Park, B., Hong, Y., Lillehoj, H.S. 2016. Characterization and functional analyses of a novel chicken CD8a variant X1 (CD8a1). Veterinary Immunology and Immunopathology. 94(7):2737-2751. doi: 10.2527/jas.2015-0133.
Matsubayashi, M., Minoura, C., Shintauro, K., Hiroyuki, T., Masaru, F., Haruo, M., Hiroshi, N., Lillehoj, H.S., Hatta, T., Tsuji, N., Sasai, K. 2016. Identification of Eimeria acervulina conoid antigen using chicken monoclonal antibody and liquid chromatography coupled to mass spectrometry. Parasitology Research. 115(11):4123-4128.
Truong, A.D., Rengaraj, D., Hong, Y., Hoang, C.T., Lillehoj, H.S., Hong, Y.H. 2017. Analysis of JAK-STAT signaling pathway genes and their microRNA in the intestinal mucosa of genetically disparate chicken lines induced with necrotic enteritis. Veterinary Immunology and Immunopathology. 187:1–9. doi: 10.1016/j.vetimm.2017.03.001.
Kim, W.H., Min, W., Lillehoj, H.S. 2017. Immunomodulatory activity of chicken NK-lysin peptides. Scientific Reports. 7: 45099.
Kim, W.H., Min, W., Sullivan, Y.B., Kakach, L., Labresh, J.W., Lillehoj, H.S. 2017. Development and characterization of mouse monoclonal antibody reactive with chicken IL-8. Developmental and Comparative Immunology. 72:30-36. doi: 10.1016/j.dci.2017.02.008.
Gadde, U., Kim, W., Oh, S., Lillehoj, H.S. 2017. Various alternatives-to-antibiotics for maximizing the efficiency of growth performance and feed efficiency in poultry: a review. Animal Health Research Reviews. 9:1-20. doi: 10.1017/S1466252316000207.
Lillehoj, H.S., Lee, Y., Lee, S.H., Jeong, M.S., Kim, J.B., Jang, H.H., Choe, J.S., Kim, D.W. 2016. Evaluation of the in vitro effects of Allium hookeri on broiler chicken lymphocytes, macrophages and tumor cells. Poultry Science. 54(2):142-148.
Lee, S.H., Lillehoj, H.S., Jang, S.I., Lee, K., Kim, D.K., Hong, Y.H., Kim, J.B., Choe, J., Bravo, D.M. 2016. Comparing immune and anti-oxidant effects of selenium sources by in ovo treatment on post-hatch experimental avian necrotic enteritis. World's Poultry Science Journal. 4(7): 420-427.
Kim, W., Lillehoj, H.S., Gay, C.G. 2016. Genomics for the identification of novel antimicrobials. Scientific and Technical Review. 35(1):95-103. doi: 10.20506/rst.35.1.2420.
Li, C.Z., Lillehoj, H.S., Gadde, U.D., Oh, S., Ritter, D. 2017. Characterization of Clostridium perfringens strains isolated from clinically healthy and necrotic enteritis-afflicted broiler chickens. Avian Diseases. 61:179-186.
Lee, K., Lillehoj, H.S. 2016. An update on direct-fed microbials in broiler chickens in post-antibiotic era (an invited review) . Animal Production Science. doi.org/10.1071/AN15666#sthash.mCFBJd58.dpuf.
