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United States Department of Agriculture

Agricultural Research Service

2010 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.

3.Progress Report
Progress was made on all three objectives and their subobjectives, all of which fall under National Program 103 Action Plan, Component 2, Genetic and Biological Determinants of Disease Susceptibility, Problem Statement 2C, Mucosal Pathogens. Under this project, we addressed three major objectives, Objective 1: Identify host genetic variations associated with coccidiosis susceptibility, Objective 2: Discover biological determinants that drive immune response variations and prevent cross-protective immune responses against Eimeria strains, including mechanisms controlling immune responses to coccidia, necrotic enteritis, and other mucosal pathogens, and Objective 3: Discover genomics-based immune intervention strategies against coccidiosis and necrotic enteritis. Under Objective 1, we made significant progress identifying genetic markers associated with host resistance against intestinal protozoan disease using genome wide association study. Significant progress was made in the identification of genetic polymorphisms associated with various clinical parameters of resistance to coccidiosis. Under Objective 2, we made significant progress identifying transcriptional profiles of infections caused by three different species of Eimeria using a intestine-specific chicken cDNA microarray system and understanding molecular markers associated with intestinal protozoan infections. Progress was also made in identifying common innate intestinal genes which control immediate response to intestinal parasites. This technology was also applied to identify potential host immune-related genes that control innate immune response to avian influenza, Salmonella and clostridium bacteria. Significant progress was also made in developing many immune reagents specific for poultry species that can serve as biological markers of host innate and adaptive immune responses to diseases and stress. These immune reagents are being transferred to a commercial company for distribution to poultry scientists. Under Objective 3, significant progress was made developing drug-free disease control strategies against coccidiosis using probiotics, nutrition and hyperimmune IgY antibodies. We made significant progress in applying these alternative technologies to commercial application and demonstrating potential effectiveness to reduce pathogens in the environment and to enhance gut health without using anti-microbial drugs. Major progress also includes the technology transfer of new knowledge on host-pathogen immunobiology, gut immunity and host genomics to commercial companies to develop practical disease prevention strategies against many economically important infectious diseases via formal agreements.

1. Single nucleotide polymorphisms (SNPs) associated with resistance to avian coccidiosis have been identified. Avian coccidiosis is caused by several distinct Eimeria species, and is an economically important disease of poultry. Current disease control strategy for avian coccidiosis has many drawbacks and there is a timely need for the development of DNA-based strategy against this infection. In this study, the associations between parameters of resistance to coccidiosis and single nucleotide polymorphisms (SNPs) in 6 candidate genes were determined by genotyping 24 F1 generation and 290 F2 generation animals. Single marker association analysis for coccidiosis resistance showed SNPs of TCR-ß and MLF2 were associated with oocyst shedding and body weights. Associations between parameters of resistance to coccidiosis and SNPs in 6 candidate genes located around QTL on chromosome 1 (Zyxin, CD4, TNFRSF1A, TCR-ß, MLF2, and Lymphotactin) were recently determined and the results showed that the SNPs in MLF2 are the most probable locus associated with coccidiosis resistance in chickens. These results suggest that the SNPs in MLF2 or zyxin gene can be used as gene markers to select coccidiosis resistance in commercial meat-type chickens. Identification and validation of gene markers associated with coccidiosis resistance will enable genome-based selection strategy for coccidiosis resistant chickens that will reduce the use of anticoccidial drugs.

2. Immune reagents detecting chicken cytokine IL-18 have been developed and transferred to a commercial company. A major obstacle in basic and applied poultry research is the lack of sufficient immunological reagents. As 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 ( For this reporting period, several mouse monoclonal antibodies (mAbs) which detect chicken Interleukin 18 (IL18) which can be used for basic and applied research in poultry were developed and transferred to a commercial company. IL18 molecule is important molecule which is critically involved in host innate immune response to many intracellular pathogens. Therefore, these mouse mAbs which detect chicken IL18 will be important new immune reagents and useful tools for basic and applied research in poultry.

3. Application of nutrigenomics to study the interplay of nutrition, innate immunity and coccidiosis resistance. In view of rising concerns on antibiotics and emerging diseases, there is an increasing interest for developing an alternative control strategy to enhance animal health and to reduce the use of antimicrobials. One promising new possibility to achieve this goal is the use of natural foods and herbal products to enhance feed efficiency, gut health, and innate immunity. Recently, there has been increasing scientific evidence in clinical medicine to show that dietary factors enhance host innate immunity, especially plant-derived products. Many of these dietary compounds appear to act on multiple target signaling pathways, but there is limited information on the use of phytonutrients in veterinary medicine. To explore how different phytochemicals can be used for enhancing poultry immunity, we used nutrigenomics technology to investigate the molecular and genetic mechanisms of dietary modulation of host innate immunity. Analysis of gene expression profiles of intestinal tissues from phytonutrient-fed birds indicated that dietary supplementation with these phytonutrients significantly protected chickens against live coccidiosis challenge infection based on body weight, parasite fecundity, and gut lesion. These results provide clear evidence to support the idea that plant-derived phytochemicals possess immune-enhancing properties in chickens and these new findings create a new possibility for commercial companies to develop effective drug-free strategies for poultry infectious diseases.

4. Probiotics modulate gut immunity and enhance natural resistance against avian coccidiosis. Direct-fed microbials (DFMs) are live microorganisms which confers a health benefit on the host by balancing its intestinal microbes. Recently, much attention has been paid to the role of DFMs on immune system (i.e., immunomodulation) and their effects on the interaction between gut microflora and host immune system development. In order to develop a novel control strategy for poultry diseases and to reduce antibiotics uses, we investigated the immune mechanisms and possibility of immune enhancement using various DFM products. Feeding dietary DFMs significantly improved intestinal structure and enhanced gut health as revealed by increased villus height and crypt depth compared with normal controls. These studies provided a rational scientific basis for future studies to investigate DFMs as immunopotentiating agents to enhance host protective immunity against enteric pathogens in broilers chickens.

5. Immune mechanisms underlying passive protective immunity against coccidiosis mediated by hyperimmune egg yolk antibodies. One of the current efforts to reduce the use of drugs in poultry production involves the induction of passive protective immunity against coccidiosis using using hyperimmune, pathogen-specific, parasite-neutralizing antibodies. Chicken egg yolk IgY antibodies offer a practicable alternative to mammalian serum antibodies because of their feasibility for large-scale commercial production and the relative non-invasive methods used for their preparation. Based on our earlier research findings that showed passive immunity protection against avian coccidiosis using IgY fraction derived from egg yolk of hens hyperimmunized with Eimeria oocysts, we investigated protective immune mechanisms underlying IgY-mediated passive protective immunity. The results showed that hyperimmune IgY antibodies can directly damage parasites and inhibited invasion of parasites into host cells. These findings provide clear evidence that passive immunization of chickens with hyperimmune IgY antibodies protects against coccidiosis via an immune-based mechanism. Commercial products based on this research is being marketed in the Latin American countries.

6. Gametocyte-specific coccidia antigen provides protection against coccidiosis. Avian coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria which invade discrete regions of the intestinal epithelium causing reduced feed conversion efficiency leading to decreased body weight gain. Furthermore, Eimeria parasites have complex life cycle stages which involve sexual and asexual stages. Prophylactic feeding of coccidiostat drugs is the major disease control method used in commercial settings. However, with increasing consumer concerns over the use of antibiotics in poultry production, the search for alternative strategies against avian coccidiosis have intensified. Several proteins associated with the sexual stage of E. maxima antigens have been identified previously as potential vaccine targets for inducing transmission-blocking immunity. In this reporting period, we investigated the vaccine potential of Gam82 which is involved in the oocyst wall formation. Repeated trials demonstrated that broiler birds vaccinated with Gam82 are protected against live parasite challenge infection. The results of these trials clearly demonstrate feasibility of using a gametocyte protein as a vaccine against avian coccidiosis.

Review Publications
Jang, S.I., Lillehoj, H.S., Lee, S.H., Lee, K.W., Park, M.S., Cha, S.R., Lillehoj, E.P., Subramanian, B.M., Sriraman, R., Srinivasan, V.A. 2010. Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity. Vaccine. 28(17):2980-2985.

Lillehoj, H.S., Li, G., Zeng, Y., Ren, X. 2010. Cloning, Expression and Biological Analysis of Recombinant Chicken IFN-gamma Expressed in Escherichia coli. Hybridoma. 29(1):1-6.

Lillehoj, H.S., Lee, S.H., Park, D., Jang, S., Lillehoj, E., 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.

Kim, C., Lillehoj, H.S., Hong, Y., Lillehoj, E., Keeler, C.L. 2010. Comparison of Global Transcriptional Responses of Chicken Following Primary and Secondary Eimeria acervulina Infections. Developmental and Comparative Immunology. 34:344-351.

Kim, E., Hong, Y., Lillehoj, H.S. 2010. Genetic effects analysis of MLF2 and TCR-B on resistance to coccidiosis in chickens. Poultry Science. 89:20-27.

Lee, K.W., Lee, S.H., Lillehoj, H.S., Li, G.X., Jang, S.I., Park, M.S., Kim, D.K., Lillehoj, E.P., Neumann, A.P., Rehberger, T.G., Siragusa, G.R., Babu, U.S. 2010. Effects of Direct-Fed Microbials on Growth Performance, Gut Morphometry, and Immune Characteristics in Broiler Chickens. Poultry Science. 89:203-216.

Drechsler, Y., Bohls, R.L., Smith, R., Silvy, N., Lillehoj, H.S., Collisson, E. 2009. An avian, oncogenic retrovirus replicates in vivo in more than 50% of CD4+ and CD8+ T lymphocytes from an endangered grouse. Virology. (83)380-386.

Lillehoj, H.S., Li, S., Zhang, Z., Yan, L., Pace, L., Zhang, S. 2010. Induction of CXC chemokine mRNA expression in chicken oviduct epithelial cells by Salmonella enterica serovar Enteritidis via the type three secretion system-1. Avian Diseases. 53(3):396-404.

Lee, S.H., Lillehoj, H.S., Hong, Y.H., Jang, S.I., Lillehoj, E.P., Ionescu, C., Mazuranok, L., Bravo, D. 2010. In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages. British Poultry Science. 51(2):213-221.

Lee, K., Lillehoj, H.S., Siragusa, G. 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., Jang, S.I., Kim, D., Ionescu, C., Bravo, D., Lillehoj, H.S. 2009. Synergistic Effect of Dietary Curcuma, Capsicum, and Lentinus on enhancing local immunity against Eimeria acervulina infection. Poultry Science. 47(1):89-95.

Yoo, J., Jang, S.I., Kim, S., Cho, J., Lee, H., Rhee, M.H., Lillehoj, H.S., Min, W. 2009. Molecular cloning and characterization of duck interleukin-17 . Veterinary Immunology and Immunopathology. 132:318-322.

Last Modified: 4/19/2014
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