2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Coccidiosis is a ubiquitous intestinal protozoan infection of poultry which seriously impairs the growth and feed utilization of infected chickens. Avian coccidiosis is caused by several distinct species of obligate intracellular parasites of the genus Eimeria. In general more than one species of Eimeria are involved in field coccidiosis infections. Major clinical sign of coccidiosis involves intestinal pathology which is caused by the rapid replication of the parasite during asexual reproduction in the cells of the gut, resulting in diarrhea, weight loss, and anorexia. Poultry coccidiosis has been particularly problematic where annual losses of over $1.2 million per year have been sustained by the U.S. poultry industry alone. Each of the more than 30 billion chickens raised annually worldwide is at risk for coccidiosis. Continual emergence of drug resistant parasites in the field, and increased feed costs to producers coupled with reduced weight gain indicate that novel approaches are urgently needed to reduce coccidiosis related economic losses. Recently, several problems have been identified which prevent the development of effective treatments for avian coccidiosis:. 1)Drug resistant coccidia strains have been emerging very rapidly worldwide, therefore the effectiveness of existing therapeutics have decreased steadily,. 2)The nature of protective immunity elicited by natural infection is poorly understood. In addition, many host- and parasite- associated factors which include immunology, nutrition, physiology, pathology and genetics need to be better investigated,. 3)The limited number of existing vaccines consist of 4 or more live Eimeria species and do not offer cross-protection against all seven species of Eimeria that can potentially infect chickens, and. 4)Information on the host-parasite interactions involved in host cell invasion and intracellular killing is limited. It is our long-term goal to develop new strategies that will control coccidiosis outbreaks. Since billions of animals will need to be treated, it is necessary that the new control measures be economical and easy to administer. To make these control strategies a reality, there is a critical need for basic research to better understand the mechanisms of host cell invasion, early parasite development and protective immunity. The current research is designed to address all these issues. The project plan has 4 specific objectives: 1). to identify Eimeria genes and gene products which are likely candidates for a subunit, anti-Eimeria vaccine that is broadly effective in poultry using high throughput sequencing of expressed sequence tags (ESTs) from Eimeria cDNA libraries and proteomic analysis of expressed proteins. Develop viral/bacterial vectors or DNA-based delivery systems for a mucosal vaccination strategy,. 2)to investigate immunological and molecular basis of disease resistance and the nature of host innate and adaptive immune responses that lead to protection against coccidiosis using functional genomics. Identify quantitative trait loci (QTL) associated with disease resistance to coccidia and use this information to develop marker-assisted selection strategy for commercial broiler industry,. 3)to improve and develop in vitro culture systems for multiple Eimeria spp. to investigate parasite invasion of host cells and to study metabolic changes associated with early developing stages, and. 4)to investigate the immunobiology of host-parasite interactions in the gut and identify protective intestinal immune mechanisms involved in parasite elimination and characterize the relationship between early acute host response and feed intake. This project is part of the National Programs 103 on Animal Health. It is aimed at reducing infection of poultry with parasitic organisms to improve production efficiency and growth performance. This project relates to Animal Health NP 103, Component 4-Animal Immunology; Component 5- Mechanisms of Disease, and Component 8-Strategies to Control Infectious and Non-Infectious Disease. Future control strategy against avian coccidiosis will have to integrate many different options combining therapies (vaccine as well as chemotherapy) and applying them in chickens that have been genetically selected for improved disease resistance. The advent of new molecular techniques to manipulate the genomes of pathogens and host, and an enhanced understanding of interactions between the parasite and host local immune system will enable the development of new approaches for vaccination against Eimeria to be realized within the near future. Developing a multidisciplinary approach using immunology, cell and molecular biology, proteomics, genomics and functional genomics, as well as bioinformatics, will lead to the development of a logical control strategy against avian coccidiosis. Successful completion of these studies outlined in this CRIS will enhance our understanding of Eimeria biology, the development of host immunity to parasites, innate disease resistance to coccidiosis, and will assist in the development of practical, effective control strategies against Eimeria, including the development of an effective multi-species vaccine. 2.List by year the currently approved milestones (indicators of research progress) Allen/Augustine Milestone 1: Optimize conditions for cell invasion by 2 species of avian Eimeria (1 yr). Milestone 2: Develop standard procedures for short-term in vitro cultivation of E. acervulina, E. tenella and E. maxima (1.5 yrs). Milestone 3: Develop assays for antioxidant defense enzymes in E. acervulina, E. tenella and E. maxima as well as for the host (2 yrs). Milestone 4: Develop cDNA probes for genes expressing antioxidant defense enzymes of coccidia and test expressed genes for protective immunity (3 yrs). Milestone 5: Determine the immunological nature of leptin response in primary coccidia infections and its role in infection-induced weight gain reduction and muscle turnover and growth (2 yrs). Fetterer/Jenkins Milestone 1. Isolate E. tenella sporozoites for proteomic analysis including determination of membrane proteins (1 yr). Milestone 2. Characterize E. tenella proteome using antibodies directed against specific parasite structures; characterize and sequence these proteins (1 yr). Milestone 3. Develop methods to selectively radiolabel intracellular stages of E. tenella and characterize the labeled proteins by proteomic analysis; determine the difference in protein expression during intracellular invasion. (2 yrs). Milestone 4. Identify mRNAs associated with E. tenella sporozoites (2 yrs). Milestone 5. Test vaccine efficacy of recombinant EASZ240 and EAMZ250 in various vectors against E. acervulina challenge infection and evaluate CpG adjuvant (3 yrs). Lillehoj, Hyun Milestone 1: Identify candidate gene(s) of Eimeria and host that affect resistance to avian coccidiosis using DNA microarray analysis (3 yrs). Milestone 2: Develop a strategy for effective oral immunization of chickens to induce a protective mucosal immune response against Eimeria. (3 yrs) Milestone 3: Express recombinant cytokines (IFNs, IL-1, IL-2, IL-6, IL-15) and develop monoclonal antibody-based antigen capture ELISA (3 yrs). Milestone 4: Develop strategies to enhance the molecular vaccine efficiency using chicken cytokines and effector molecules (3 yrs). Milestone 5: Develop recombinant chicken antibodies detecting apical complex protein of coccidia and test their effect on the parasite invasion of host cells (2 yrs). Milestone 6: Investigate immunology of intestinal T lymphocytes and NK cells in local defense against coccidia (2 yrs). 4a.List the single most significant research accomplishment during FY 2006. Eimeria biology: A rapid method for isolating and determining the species composition of Eimeria collection has been developed which will allow poultry producers and vaccine companies to select particular live oocyst vaccine to use in any poultry operation. Immunotherapeutics: Novel lymphocyte-derived anti-microbial peptide which kills Eimeria sporozoites has been identified and its gene cloned. Chicken NK-lysin is an anti-microbial and anti-tumor protein expressed by T lymphocytes and is consisted of an 868 bp DNA with a predicted molecular mass of 15.2 kDa. High expression of NK-lysin is detected in the gut of Eimeria-infected chickens indicating an important role of this protein. Furthermore, recombinant chicken NK-lysin killed Eimeria sporozoites. This is the first lymphocyte-derived anti-parasitic peptide and has a great potential as an immunotherapeutic compound with no risk of inducing resistance by Eimeria. Poultry functional genomics: Avian macrophage microarray containing 4,906 unique gene elements obtained from macrophage cDNA library was used to characterize differential gene expression of three most important Eimeria species, E. tenella, E. acervulina, and E. maxima. This technology allowed for the first time to identify the nature of host immune elements which are associated with different phenotypes of clinical pathology caused by three major Eimeria species. This approach will enable poultry industry to identify transcription patterns unique to each individual Eimeria species providing increased insights into the underlying immunopathology associated with Eimeria species. 4b.List other significant research accomplishment(s), if any. Immune response of chickens to Eimeria infection: As part of a non-fundable CRADA with BMI Inc. we investigated novel immune refocusing technology to identify potential immunogenic Eimeria epitopes. Two separate time course experiments were conducted to examine the host humoral immune response to low and high doses of E. maxima. These studies led to the identification of Eimeria proteins which are recognized by hyperimmune serum and led to the characterization of initial innate immune responses associated with body weight gains, plasma levels of carotenoids and NO2- + NO3- . Probiotic mediated enhancement of gut immunity: In collaboration with Imagilin Inc., novel probiotic product, Mitogrow was tested in its effect against avian coccidiosis. A Pediococcus-based probiotic supplementation of day-old broiler chickens during early growth period significantly enhanced local protective immunity against coccidiosis lowering fecal oocyst shedding. MitoGrow when supplemented in the standard poultry at the 0.1% level effectively improved weight gain and elicited higher level of Eimeria-specific antibodies following live parasite challenge.These results demonstrated for the first time that MitoGrow (Pediococcus acidilactici-based probiotic) enhances gut natural immunity in birds and protects against the negative growth effect associated with coccidiosis. A novel immunopotentiating effect of a lectin (FFrL) extracted from the mushroom Fomitella fraxinea which enhances poultry cell-mediated immunity and poultry coccidiosis resistance has been identified and U.S. patent has been applied. This study demonstrated the extraction method of FFrL, its in vitro mitogenic activity and in vivo protection against an oral challenge infection with Eimeria acervulina. FFrL showed a potent mitogenic activity on chicken splenic lymphocytes and significantly induced nitric oxide secretion in chicken macrophages and suppressed tumor cell growth. When injected into eighteen-day-old chicken embryos followed by a post-hatch oral E. acervulina challenge infection, FFrL-treatment significantly protected chickens against weight loss associated with coccidiosis. The results of this study demonstrate for the first time that FFrL is an effective growth promoting and immunostimulating natural product in poultry (Patent pending). Collaboration was continued with scientist from Kazakhstan as part of former Soviet Union program (FSU). Studies were completed demonstrating the effectiveness immuno-stimulatory complexes (ISCOMs) in the delivery of Eimeria antigens. Routes of administration of ISCOms was investigated. In ovo immunization with ISCOMs was shown to be effective. The antigen SO7 was expressed as a recombinant and antibody to the protein prepared. Expression of recombinant will be scaled-up to produce adequate protein to prepare ISCOMs and to do immunization trial with ISCOMs containing recombinant antigen. Described for the first time the transcription, expression and localization of the antigen SO7 in Eimiera tenella. The protein was localized to the refractile body and is developmentally regulated being present only in sporulated oocysts and sporozoites with residual amounts of protein in first stage merozoites. The intracellular fate of SO7 was described during in vitro development in cell culture. Anti-coccidial effect of a natural product, xanthohumol (XN) has been demonstrated using in vivo treatment of sporozoites of E. acervulina and E. tenella with XN. In vivo treatment of sporozoites with 20 and 10 ppn XN causes significant morphological alterations in outer and inner sporozoite membranes of E. acervulina, E. tenella, and E. maxima. Completed the sequencing of the gene for acetyl coA carboxylase (ACC1) from E. tenella. Several portions of ACC1 gene have been expressed, and antibodies prepared for use in detecting this protein in parasite stage preparations and in tissue sections from infected chickens. Various methods are being tested to maximize extraction of ACC1 in order to quantitatively compare its levels among developmental stages as well as among species. ACC1 appears to be quite susceptible to degradation in absence of protease and phosphatase inhibitors. Bioinformatics searches have focused on enzymes, cofactors , receptors and other factors that have potential importance in control of Eimerian growth and development. These entities include DAF-12, acyl transferases, ubiquitin ligase, phosphatidyl phosphate kinase, lipoic acid synthase, protein disulfide isomerase, all of which show significant amino acid sequence similarities to various contigs in the Sanger E. tenella data base. 4c.List significant activities that support special target populations. None 4d.Progress report. None 5.Describe the major accomplishments to date and their predicted or actual impact. Development of a sensitive method for recovering Eimeria oocysts directly from poultry litter and speciation of oocysts using polymerase chain reaction will greatly reduced the time required to obtain information on the species profile of Eimeria on a poultry farm and this method will be a useful technique for estimating drug resistance patterns of Eimeria present in the poultry operation. Finding that showed that drug-resistant strains of Eimeria were present in farms using live oocyst vaccines although drug resistance was more widespread in Eimeria isolated from poultry farms utilizing anti-coccidial drugs indicate that we need to pay more attention to biosafety in both traditional and organic poultry farms. Lactobacillus-based probiotic stimulated the local immune system of broiler chickens and improved resistance to E. acervulina]. Moreover, feeding broiler chickens a single species probiotic (MitoGrow®) recently proved effective in protecting the birds against a live E. acervulina challenge. A greater understanding of the mechanisms of probiotic-mediated enhancement of intestinal immunity would improve the effectiveness of its use in the field and reduce drug usage. Demonstrated that short oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are effective immunoprotective agents in chickens by inducing both innate and adaptive immune responses using in vitro and in vivo assay systems. CpG 2006 showed strong stimulatory effects on chicken macrophages as demonstrated by increased IL-6 secretion, enhanced NO release, upregulated cell surface marker expression, and increased intracellular bacterial killing. Furthermore, using avian-stimulating CpG ODNs to activate chicken innate immunity, their ability to enhance protective immune response against Salmonella and coccidia has been demonstrated. Injection of CpG to 18 day-old embryos enhanced resistance against live coccidia challenge given after hatch and significantly reduced oocyst shedding demonstrating that CpG ODNs can amplify protective response when given with live vaccine. Furthermore, when CpG ODN was co-injected with a recombinant microneme protein (MIC2), ODN-injected chickens showed significant reduction of oocyst shedding and improved weight gain. Taken together, these studies showed that CpG ODNs administered in ovo enhance innate immunity following Eimeria infections and the feasibility of using CpG ODNs as adjuvants in recombinant DNA vaccination. Major progress has been made in the identification and characterization of several important Eimeria antigens using functional genomics. Cloning and characterization of Eimeria macrophage inhibitory factor (eMIF), HSP90, proteases, ACC and ABC transporter were accomplished. Understanding the function and molecular structure of these parasite genes will enhance our understanding of parasite biology and will lead to the development novel vaccine against coccidiosis. The underlying mechanisms why eMIF is expressed during intracellular development will lead to host evasion mechanisms that some pathogens use to hide from host immunity. New discovery to show possibility of inducing protective immunity against coccidiosis using a novel vaccine delivery technique by a commercial high speed in ovo injection system and finding that Eimeria recombinant protein or Eimeria gene elicits embryo immune response will facilitate the development of future vaccine against coccidiosis. Understanding the molecular basis of antigen diversity in Eimeria is an important first step toward development of control strategy against coccidiosis. New finding showed that the genetic diversity of ribosomal DNA in E. maxima displays significantly greater amounts of sequence diversity. Based on ITS-1, 5.8S, and ITS-2 sequences collected thus far suggests, it appears that the genetic organization of ribosomal DNA in E. maxima may be substantially different than in other Eimeria species. This data provides the first genetic evidence that E. maxima may be fundamentally different from other Eimeria species so far studied, and may lead to the understanding of the mechanisms behind the antigenic diversity present in this species. Developed the first chicken intestinal cDNA microarray to investigate host immunity against coccidia and other mucosal pathogen. Chicken intestinal cDNA library using pooled intestinal tissues from coccidia-infected chickens was used to develop a intestinal chip containing 10,000 EST elements. Using BLAST searches against the GenBank nucleic acid sequence database, we analyzed the 18 cluster contigs containing 15 or more ESTs, which could be regarded as abundant transcripts and therefore were most likely to match previously described genes. Ten sequences were highly homologous to previously described chicken genes. They included NK-lysin, apolipoprotein AIV, fatty acid binding protein, acid ribosomal phosphoprotein, tubulin, GAPDH, and ferritin heavy chain. Comparison of our intestine cDNA sequence data with chicken DNA sequences in GenBank identified 125 clones which encoded novel genes of which 110 genes were unknown and the remaining genes showed weak homologies to identified molecules like CC chemokine receptor type 8, death-associated protein kinase-2 and others. These EST sequences from Eimeria-stimulated intestinal IEL transcripts is being used to study global gene expression profiling and to identify novel immune-related genes during avian coccidiosis, necrotic enteritis, salmonellosis and avian influenza. Recent progress in Eimeria genome sequencing and rapidly developing functional genomics technology will facilitate the identification and characterization of parasite genes which are involved in immunoprotection and immunopathology in avian coccidiosis. Development of recombinant chicken antibodies which detect apical complex proteins and block host call invasion of Eimeria sporozoites will lead to a new immunoprophylactic strategy against coccidiosis. Since antibodies are naturally produced by chicken lymphocytes Eimeria parasites do not develop resistance against them. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? U.S.Patent has been applied that disclosed a novel extraction method for an immunostimulatory lectin from the mushroom, Fomitella fraxinea (FFrl). Furthermore, this study discovered that the FFrl lectin has immunopotentiating effects that enhanced gut health and promoted protective immunity against avian coccidiosis. Since FFrl showed an immunostimulating composition and stimulated non-specific cell mediated immunity in poultry, this is a potentially safe and natural product which can be fed into broilers to reduce economic losses due to disease. Since lectins are carbohydrate-binding proteins or glycoproteins of non-immune origins which have the ability to exert various important biological activities including immunomodulatory activities, this discovery will offer new ways of controlling nutriton based strategy against many poultry pathogens. Potential delivery strategy that activate innate immunity of developing embryos is also disclosed. Demonstrated the effectiveness of immunization of Eimeria with antigens delivered by ISCOMs. These results will be used to develop practical immunization methods using ISCOMs and previously developed recombinant antigens. It is anticipated that with 2-3 years this technology will be available for transfer to producers or industry for large scale trials. Collaborative studies done as a CRADA with Imagilin Inc.(TEDCO-funded project) demonstrated the protective effects of two new commercial probiotics, MitoGrow and MitoMax in Eimeria-infected chickens as measured by performance parameters. Collectively, these studies have shown improved weight gains and reduced oocyst shedding in birds fed the probiotics MitoGrow and MitoMax following infection with Eimeria. The results of these studies have enhanced our undestaning of the immune mechanisms by which immunoenhancing probiotics stimulate local immunity and protect against enteric infections in poultry. This information concerning how the chicken’s immune system can be modulated by these agents to impact the gut immune responses under commercial conditions could lead to more effective means of controlling many poultry pathogens with less dependence on growth promoters and drugs. CRADA was established with Diversa Biotechnology company to apply various strategies to enhance cell-mediated immunity against avian coccidiosis using a proprietary T-cell epitope vector. The results of this collaboration led to better understanding of the importance of T-cell epitopes in the development of recombinant vaccines against avian coccidiosis. Trust agreement with the Rural Development Administration (RDA: ARS equivalent) of South Korean government was established to apply various immunological assay technology to evaluate plant-derived phytonutrients using chicken coccidiosis disease model. With increasing regulations on the use of antibiotics in the production of livestock and poultry, new dietary strategies to improve disease resistance capability of poultry against infectious diseases are urgently needed. In this collaboration, RDA provided several phytonutrients from fruits and vegetables and ARS scientists demonstrated the immunomodulatory activities of several of these natural products in enhancing gut health of poultry. Furthermore, appropriate animal disease model has been developed to evaluate natural products using avian coccidiosis. Further understanding of phytonutrient-mediated enhancement of innate immunity in avian disease model will provide important information for understanding the beneficial effect of phytonutrients on human and poultry health. Developed a CRADA with Zeon company and various in ovo vaccination strategies were investigated. Laboratory vaccination trials using recombinant vectors demonstrated possibility of using few coccidial recombinant proteins as a vaccine for embryo vaccination. Novel delivery technology was developed to induce protection against avian coccidiosis using a recombinant DNA vaccine encoding a microneme protein of Eimeria tenella. ETMIC2 represents a microneme antigen of apical complexan parasites and is involved in host cell invasion by apicaomplexan parasites. In this collaboration, Zeon molecular biologists cloned ETMIC2 gene and our investigator demonstrated vaccine potential of this gene in embryo vaccination model that she developed. In two trials of vaccine testing, EtMIC2 gene and the encoded protein induced significant antibodies and antigen specific cell-mediated immune response against live parasite challenge infection with two different Eimeria species. In ovo vaccination with the EtMIC2 gene increased anti-EtMIC2 antibody titers at days 11 and 17 days post-hatching. In addition, vaccinated animals developed protective immunity against infection by both E. tenella and E. acervulina as assessed by significantly increased body weight gain and decreased fecal oocyst shedding compared with non-vaccinated controls. These results indicate potential of using ETMIC2 vaccine to induce cross protection against many Eimeria species. Further studies will be conducted to evaluate potential of this vaccine to induce protection against several other field strains of Eimeria. A trust agreement has been established to evaluate recombinant chicken antibodies developed in this project with scientists at the Investigacion Aplicada, S. A. de C.V (IASA) in Mexico. The main objective of this cooperative research project is to evaluate the use of polyclonal chicken immunoglobulins and recombinant chicken antibodies produced against coccidia antigens as a preventive and therapeutic agent against coccidiosis in commercial broilers. In addition, a novel immunization method will be developed to hyperimmunize hens using an immunogenic recombinant coccidia protein and poultry-specific adjuvants in order to increase the efficacy of IASA’s hyperimmune serum product. Continued collaborative research as identified in the CRADA with the Novus International Inc. company to evaluate cell-mediated immunity against commercial coccidia vaccines. Real time RT-PCR method was developed to measure chicken interferon-gamma as a useful assay for predicting the efficacy of live vaccines against avian coccidiosis field infection. Molecular biology technology to develop a construct to produce potentially therapeutic recombinant chicken antibody fragment, single chain fragment of variable chain (ScFv), was approved for U.S. and Korean patents. This technology will allow the development of novel antibody-mediated immunointervention strategy against avian coccidiosis. Continued CRADAs with Townsends, Inc. to evaluate field strains of avian coccidia and Provided timely information to Townsends, Inc. on the species composition and drug resistance profiles of Eimeria isolated from company poultry farms. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Allen, P.C., Miska, K.B., Jenkins, M.C., and Fetterer, R.F. 2005. Acetyl coA carboxylase in avian Eimeria. IX International Coccidiosis Conference. p. 164. Dalloul, R. and Lillehoj, H.S. 2005. In ovo and dietary modulation of host intestinal immunity and its subsequent effects on coccidiosis. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 139. Dalloul, R. A., Lillehoj, H.S., Klinman, D.M., Ding, X.C., Min, W.G., Heckert, R. and Lillehoj, E. 2005. CpG oligodeoxynucleotides co-administered with the microneme protein MIC2 protect against Eimeria infections. The Eighth Annual Conference on Vaccine Research. Baltimore, MD. May 9-11. p. 64. Hong, Y., Lillehoj, H.S. Dalloul, R.A., Miska, K.B., Tuo, W. and Lee, S.H. 2005. Molecular cloning and expression profiles of chicken NK-lysin during Eimeria infections. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 153. Hong, Y. H., Lillehoj, H.S., Lee, S.H., Park, D.W., and Lillehoj, H.S. 2006. Cloning and characterization of chicken lipopolysaccharide-induced TNF-alpha factor. Proc. American Association of Immunologists. p. 43. Fetterer, R.H., Miska, K.B., Allen, P.C., Lillehoj, H.S. and Barfield, R.C. 2005. Characterization of Serine Proteases in Developmental Stages of Eimeria tenella. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 165. Fetterer, R.H., Jenkins, M.C., Miska, K.B. The Characterization and Localization of the Protective Antigen SO7 in Developmental Stages of Eimeria tenella. Annual Meeting of the American Association of Avian Pathologists, July 15–19, 2006, Honolulu, Hawaii. p. 159. Jenkins, M.C., Wilkins, G., Klopp, S., Miska, K.B. 2006. Application of Polymerase Chain Reaction and Drug-sensitivity Testing to Compare Species Composition and anti-coccidial Drug Resistance in Eimeria Isolated from Vaccine- and Coccidiostat-utilizing Poultry Facilities. Annual Meeting of the American Association of Avian Pathologists, July 15–19, 2006, Honolulu, Hawaii. p. 52. Jenkins, M.C., Miska, K.B., Klopp, S. Molecular Analysis of the Content and Diversity of Eimeria Species Present in Litter of Local Poultry Facilities Using Ribosomal DNA Sequencing. Annual Meeting of the American Association of Veterinary Parasitologists, July 15–18, 2006, Honolulu, Hawaii, p. 26. Lillehoj, H.S. 2005. Host immune response to coccidia. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 63-83. Lillehoj, H.S., Sher, A. and Yarovinsky, F. 2005. Eimeria profilin protein 3-1E is the ligand for toll-like receptor 11 and regulates host innate immune response in coccidiosis. Proceedings of International Cytokine Society Conference, Seoul, Korea. p. 50. Lillehoj, H.S., Hong, Y.H., Dalloul, R.A., Miska, K.B. , Tuo, W., Lee, S. H., Han, J. Y. and Lillehoj, E.P. 2006. NK-lysin: High level of local gene expression in coccidiosis. Proc. American Asso. Immunologists, Boston, MA, p. 43. Lillehoj, H. 2006. Application of macrophage and intestinal cDNA microarrays to study innate mucosal immunity in avian coccidiosis. Proceedings of the annual meeting of the Poultry Science Association, July 16-19, Edmonton, Canada. p. 2. Lillehoj H.S., Dalloul R.A., Hong Y.H., Bliss T.W., Keeler C.L., and Han J.Y. 2005. Application of functional genomics, immunology and molecular biology tools to explore mucosal immune responses against Eimeria. Conference of Research Workers in Animal Diseases, St. Louis, MO, USA. pp. 67-84. Lillehoj, H.S., Dalloul, R.A., Hong, Y.H., Bliss, T.W., Keeler, C.L., Chouikha, I., Park, D.W., and Han, J.Y. 2005. Application of functional genomics, immunology and molecular biology tools to explore host immune response to Eimeria. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 155. Miska, K.B., Lillehoj, H.S., Allen, P.A., Fetterer, R.H., and Jenkins, M.C. 2005. The Macrophage Inhibitory Factor of Eimeria species. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 163. Miska, K.B., Lillehoj, H.S., Fetterer, R.H. 2005. The developmental expression of Eimeria heat shock protein 90. Proceedings of IX International Coccidiosis Conference, Iguasu Falls, Brazil, p. 163. Miska, K.B., Jenkins, M.C., Klopp, S. Molecular Analysis of the Content and Diversity of Eimeria Species Present in Litter of Local Poultry Facilities Using Ribosomal DNA Sequencing. Annual Meeting of the American Association of Avian Pathologists, July 15–19, 2006, Honolulu, Hawaii, p. 162. Review Publications Babu, U., Dalloul, R.A., Okamura, M., Lillehoj, H.S., Xie, H., Raybourne, R.B., Gaines, D., Heckert, R.A. 2005. Salmonella enteritidis clearance and immune responses in chickens following salmonella vaccination and challenge.Veterinary Immunology and Immunopathology. 101:251-257. Jenkins, M.C., Miska, K.B., Klopp, S. 2006. Application of polymerase chain reaction based on its1 rdna to speciate eimeria. Avian Diseases. 50:110-114.