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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
* Identify host nucleic acid and protein markers and functional genetic variations associated with disease susceptibility and resistance to mucosal pathogens of economic importance. * Discover effective immune interventions strategies to prevent and control mucosal pathogens of poultry. * Determine the host-pathogen interactions that result in immune evasion or protective immunity to avian mucosal pathogens.

1b.Approach (from AD-416)
High throughput genomic approaches will be interfaced with disease modeling studies to decipher genetic and biological determinants of disease susceptibility. This approach will lead to the discovery of innovative tools to prevent and control avian mucosal pathogens such as avian coccidiosis, avian influenza, infectious bronchitis, and other important mucosal pathogens of poultry. BSL-1 Recertified 4/15/11.

3.Progress Report
Under Objective 1, progress was made identifying genetic markers associated with host disease resistance against coccidiosis. Associations between coccidiosis resistance and single nucleotide polymorphisms (SNPs) candidate genes located on chicken chromosome 1 were identified. Following infection of the F2 generation with Eimeria maxima, body weights, fecal oocyst shedding, and biochemical parameters were measured as parameters of coccidiosis resistance. Single marker association tests revealed several new SNPs associated with the disease parameters. These studies provide the identification of the first SNP markers which serve as candidate genes to select coccidiosis resistance in broiler chickens. Under Objective 2, significant progress was made in identifying determinants associated with innate immune response to major Eimeria species. The relative expression levels of immune- and non-immune-related mRNAs in intestinal intraepithelial lymphocytes infected with E. acervulina, E. maxima, or E. tenella were measured. Nearly equal numbers of up-regulated transcripts were detected following E. tenella primary and secondary infections, with a greater number of down-regulated mRNAs following secondary vs. primary infection. Few mRNAs were modulated following primary infection with E. acervulina or E. maxima compared with secondary infection. Sixteen intracellular signaling pathways were identified from the differentially expressed transcripts following Eimeria infection. Progress was also made in identifying host innate immune molecules and antigen-presenting cells which control host innate immune response to pathogens. Isolated antigen-binding cells from Eimeria-infected chickens were, for the first time, were identified as interdigitating dendritic cells (DC) and follicular DC. This ability to isolate antigen-presenting cells from chicken gut makes it possible to investigate their role in initiating an immune response. To facilitate the development of commercially available immune reagents tools through the U.S. Veterinary Immune Reagent Network (VIRN)(, we developed mouse monoclonal antibodies (mAbs) against chicken IL18 that can be used for basic and applied research in poultry. Under Objective 3, we have successfully transferred new technology in the area of drug-free disease control strategies to the poultry industry. The research addresses Agency Performance Measure 3.2.1: Provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health and Performance Measure 3.2.2 and Performance Measure 3.2.3.

1. Development of DNA markers associated with enteric disease resistance polymorphism in broiler chickens. Eimeria causes coccidiosis which inflicts significant economic losses to the poultry industry because of the reduction in production efficiency as a result of mortality, nutrient malabsorption, retarded growth rate, and decreased egg production. Due to the lack of efficient vaccines, the control strategies for coccidiosis have relied on prophylactic chemotherapy. The emergence of drug-resistant parasites and the concern for chemical residues in food hinder the use of coccidiostats. The worldwide current legislation on the use of in-feed antibiotic growth promoters and non-therapeutic antibacterial feed additives results in developments of alternative control strategies for coccidiosis. Recently there has been great interest in an understanding of the complex nature of the interaction among various factors controlling protective immunity to Eimeria in the intestine. Developments in avian functional genomics technologies have resulted in more investigation of host-pathogen interactions during avian coccidiosis. New candidate genes that influence host immune response to Eimeria have been identified using global gene expression analyses of chicken macrophage and intestinal intra-epithelial lymphocyte cDNA microarrays. Global gene expression analysis was used to compare the differences in the global gene transcription following the infection of broiler chickens with three most frequently occurring species, E. tenella, E. acervulina or E. maxima. The information from this work will expand the understanding of host-pathogen immunobiology in avian coccidiosis and enhance the possibility of new control strategies against coccidiosis. Identification and validation of gene markers associated with coccidiosis resistance will enable the development of genome-based selection strategy for coccidiosis resistant chickens that will reduce the use of anticoccidial drugs.

2. Characterization of cell-surface markers to identify antigen-presenting dendritic cells in poultry. A major obstacle in basic and applied poultry research is the lack of sufficient immunological reagents. In an effort to facilitate the progress of veterinary immunology research and to develop commercially available immune reagents tools, the U.S. Veterinary Immune Reagent Network (VIRN) was formed in 2007 ( We developed and characterized mouse monoclonal antibodies (mAbs) against interleukin 18 that can be used for basic and applied research in poultry. Four mouse monoclonal antibodies (mAbs) specific for chicken interleukin 18 were also produced and characterized.

3. Host-pathogen immunobiology in Clostridium infections. Gangrene dermatitis (GD) is an emerging disease of increasing economic importance in chicken and turkey industries that results from infection by Clostridium septicum and C. perfringens. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. We demonstrated that GD-like birds with characteristic clinical signs of Clostridium infections showed altered immune functions compared with GD-free chickens, thereby enhancing our understanding of the Clostridium-chicken interactions that will facilitate the future development of effective prevention strategies against GD. We provide the first evidence of altered systemic and local (skin and intestine) immune responses in GD pathogenesis in chickens and will help poultry industry to develop novel control strategy against Clostridium infection control.

4. Development of antibiotics-free alternative strategies to mitigate the gut damages caused by avian intestinal pathogens. Although live vaccination strategy and conventional drug therapy have been widely used to control avian diseases including coccidiosis, there are major limitations such as inability of live vaccines to induce cross protection against multiple species of Eimeria and the occurrence of drug resistance. Therefore, there is timely need to find alternative disease control strategies for those infections where effective methods are not available. In this reporting period, we have investigated two different alternative strategies to control coccidiosis: recombinant vaccines and phytonutrient-based immune enhancement. Studies using recombinant antigens common to multiple coccidia species to stimulate broad-spectrum immunity have shown limited success, mainly because of their low antigenicity, inadequate stimulation of protective host immunity, and/or restricted expression during the parasite life-cycle. Therefore, interest has been focused on using immunostimulators such as vaccine adjuvants to enhance the immunogenicity of recombinant subunit vaccines. Our work documents the immunologically-based enhancement of innate immunity in chickens by extracts of plants and mushrooms with known medicinal properties, specifically that vaccination with the E. acervulina profilin subunit vaccine in combination with MontanideTM adjuvants enhances protective immunity against avian coccidiosis. This research relates to delivering improved control methods and interventions against mucosal diseases.

5. Immune mechanisms underlying probiotics-mediated immunomodulation of poultry innate immunity. Direct-fed microbials (DFM) are live microorganisms that could confer a health benefit on the host, particularly in the area of host immunity. The role of Bacillus subtilis-based DFMs on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis, two most important innate immune functions of macrophages was investigated. This study indicated that the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens was augmented (NO production increased and increased level of phagocytosis), thus providing a rational scientific basis for future studies to investigate DFMs as immunopotentiating agents to enhance host protective immunity against enteric pathogens in broilers chickens.

Review Publications
Kim, D., Kim, C., Lillehoj, H.S., Lamont, S. 2009. Gene expression profiles of two B-complex disparate, genetically inbed Fayoumi chicken lines that differ in suceptibility to Eimeria maxima. Poultry Science. 88:1565-1579.

Lee, S.H., Lillehoj, H.S., Hong, Y., Cho, S., Park, D., Park, H., Chun, H. 2009. Protective effects of dietary Safflower (Carthamus tinctorius) on experimental coccidiosis. Journal of Poultry Science. 46:155-162.

Lee, S.H., Lillehoj, H.S., Lillehoj, E.P., Chun, H., Park, H., Cho, S. 2009. In vitro Treatment Effect of the Methanol Extracts of Korean Medicinal Fruits (Persimmon, Raspberry, Tomato) on Chicken Lymphocytes, Macrophages and Tumor Cells. Poultry Science 46:149-154.

Lee, K., Lillehoj, H.S., Siragusa, G.R. 2010. Direct-Fed Microbials and Their Impact on the Intestinal Microflora and Immune System of Chickens. International Journal of Poultry Science. 47:106-114.

Lee, S.H., Lillehoj, H.S., Park, J.W., Jang, S.I., Morales, A., Garcia, D., Lucio, E., Larios, R., Victoria, G., Marrufo, D. 2009. Protective effect of hyperimmune egg yolk Ig Y antibodies against Eimeria tenella and Eimeria maxima infections. Veterinary Parasitology. 163:123-126.

Lee, S.H., Lillehoj, H.S., Park, D.W., Jang, S.I., Morales, A., Garcia, D., Lucio, E., Larios, R., Victoria, G., Marrufo, D. 2010. Induction of passive immunity in broiler chickens against Eimeria acervulina by hyperimmune egg yolk IgY. Poultry Science. 88(3):562-566.

Hong, Y., Lillehoj, H.S., Lee, S.H., Park, S., Min, W., Labresh, J., Tompkins, D., Baldwin, C. 2011. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18. Veterinary Immunology and Immunopathology. 138(1-2):144-148.

Lee, S.H., Lillehoj, H.S., Jang, S., Hong, Y., Min, W., Lillehoj, E., Yancy, R.J., Dobinowski, P. 2011. Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection. Vaccine. 28(49):7774-7778.

Lee, S.H., Lillehoj, H.S., Jang, S., Lee, K., Yancy, R.J., Dominowski, P. 2011. Effects of novel adjuvant complex/Eimeria profilin vaccine on intestinal host immune responses against live E. acervulina challenge infection. Vaccine. 28(39):6498-6504.

Lee, S.H., Lillehoj, H.S., Park, M., Baldwin, C., Tompkins, D., Wagner, B., Babu, U., Del Cacho, E., Min, W. 2011. Development and characterization of mouse monoclonal antibodies reactive with chicken CD80. Comparative Immunology Microbiology and Infectious Diseases. 34(3):273-279.

Del Cacho, E., Gallego, M., Lee, S.H., Lillehoj, H.S., Quilez, J., Sanchez-Acedo, C. 2011. Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells (DC) and DC-derived exosomes. Vaccine. 29(21):3818-3825.

Last Modified: 4/23/2014
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