Duration: 10/01/2013 to 09/30/2018

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

STATEMENT OF THE ISSUE Disease losses represent a significant component in the overall cost of poultry production. These costs not only include the direct losses due to higher morbidity, mortality and condemnations, but also increased production costs caused by suboptimal feed conversion, cost of vaccines and vaccination, biosecurity and eradication programs. Additionally, poultry can carry a number of potentially zoonotic pathogens such as avian influenza viruses and food-borne bacteria like Campylobacter and Salmonella, which may inflict human illness or even death. With continued selection of poultry for production traits, high intensity commercial growing conditions, and the continuous arms race in vaccine development and pathogen resistance, the goal of producing inherently robust, disease resistant poultry with optimal immune system function remains an evolving challenge. To improve poultry health, reduce disease-associated losses and keep poultry health management at a reasonable cost, efforts need to continue to advance understanding of the genetic bases for resistance and immunity to avian diseases. In this context, it is critically important to focus efforts on identification and characterization of genes affecting innate and adaptive immunity, as well as, on understanding the mechanisms underlying optimal immune system development and function and the influence that physiological and environmental factors have on these processes. These efforts also need to go hand-in-hand with technological advances in research approaches and tools and continued development and maintenance of genetic lines with characterized immune system capabilities. JUSTIFICATION Globally, animal protein (meat, milk and eggs) production accounts for about 40% of agricultural production. Currently, U.S. poultry production is estimated to be 19 million metric tons (MMT)/year and is expected to reach 22 MMT by 2020, exceeding production of beef or pigs by more than 50% throughout. U.S. poultry production continues to be highest in the world, contributing 22.4% of global output in 2011. In 2011, U.S. poultry production was valued at $35.9 billion (USDA NASS, 2011). These figures included 8.6 billion broilers and 91.9 billion eggs making the U.S. the worlds largest poultry meat producer and a leading egg producer (US Poultry and Egg Association, 2011). U.S. poultry meat consumption has increased to nearly 52 kg/person/year with little variation over the past 6 years; in South America, poultry consumption has increased since 2005 from roughly 25 kg/person/year to 34 kg/person/year. Similar trends are observed and expected for other developed and developing countries regarding demand for poultry meat and eggs and other poultry species/products. The U.S. has one of the worlds most efficient poultry production systems and is currently an exporter of poultry products, including 3.6 MMT for broiler and 0.32 MMT for turkey to 116 and 91 export markets, respectively (Poultry & Egg Export Market Data, 2011), placing the U.S. second for broiler-export and first for turkey-export in the World. Considering the U.S. and global trends in poultry and egg production, consumption and international export/trade, control of disease and development of healthy animals that are able to thrive under various environmental conditions and rearing practices is of utmost importance. The total economic cost of disease in poultry was estimated by Biggs (1982) to make up about 20% of the gross value of production (GVP), including vaccines and condemnations, and to constitute about three times the cost of losses from mortality. A more recent cost analysis by Bagust, University of Georgia, U.S., reports that costs associated with diseases made up about 8.2% of the 2005 $28.5 GVP by the U.S. Poultry Industry. Efforts of the NE-1034 multistate project and its predecessor five-year projects (NE-1016; NE-60) have rapidly enhanced understanding of the interactions between genetic background and disease resistance and the processes involved in natural defenses. These efforts also led to the development of reagents and tools for use in genetic selection, assessment of immune system development and function, and improvement of poultry health. However, with continued genetic selection of poultry for production traits, ongoing changes in poultry management and husbandry practices, emerging variants of disease-causing agents and higher incidence of non-communicable diseases with complex etiology, there is great demand for continued and renewed research to develop strategies that assure the health and welfare of poultry and the production of poultry products safe for human consumption. To better understand the contributions and goals of this multi-state research effort on the genetic-bases of resistance and immunity to avian diseases, a brief overview of the immune system and its genetic regulation seems appropriate. Organisms undergo continual assault from pathogens. The ability to produce effective immunity against invading bacteria, viruses and parasites determines the survival or demise. Immune responses may be categorized according to their specificity or the functioning agents executing the actions. Using specificity as the criterion, immunity is either innate or acquired (adaptive). Innate immunity consists of physical and chemical barriers that prevent entry of the pathogen into the host. These barriers include most prominently the epithelium of the skin and mucosal membranes as well as a variety of chemicals (acids, defensins, mucus, etc.) that are produced at these surfaces and the presence of commensal (good) bacteria. Knowledge of genetic factors that determine robust defenses at the barrier level is very limited in poultry, yet will have high impact on resistance to disease. Once pathogens have gained entry, there are arrays of soluble factors and cells of innate immune system that are poised to act in the elimination of the pathogen. Knowledge regarding this innate ability to effectively control infection at this stage is rapidly advancing, in both mammalian and avian species, but the full extent of components, recognition and signaling mechanisms as well as effector functions is far from being fully understood. Moreover, tools to examine this important arm of the immune system in poultry lag greatly behind those available for swine, cattle, mice and humans. Innate recognition systems designed to detect pathogens by their pathogen associated molecular patterns (PAMP; e.g. cell wall product of bacteria), as well as, signals arising from the damaged host cells (danger-associated patterns (DAMPs); e.g. heat-shock proteins) appear to be similar in poultry and mammals. PAMPs and DAMPs can be recognized by a variety of soluble receptors present in the blood and tissue fluids, as well as, by receptors present on the membrane of cells or in their intracellular compartments (e.g. Toll-like receptors, NOD receptors, inflammasomes, etc). These receptors are encoded in the germ-line DNA and hence, like the other components of innate immunity, are in place to defend the host against pathogens without the adaptation lag phase required by adaptive immunity. Again, the knowledge regarding the genetic bases of the capabilities of the innate defense system and its regulation is very limited for poultry, yet this system greatly impacts an individuals resistance to disease. Adaptive immunity is the highly specific arm of the immune system that becomes protective several days after a pathogen challenge. The main cellular components of adaptive immunity are T and B lymphocytes that play important roles in cell-mediated and humoral immunity, respectively. There are many subsets of T cells that can be broadly categorized as T helper (Th) cells and cytotoxic T cells (Tc). Th cells are critical in initiating and directing the adaptive immune response to a cell-mediated, inflammatory, or humoral response that can most effectively eliminate an antigen. B lymphocytes produce several types of antibodies that mediate humoral immunity. Together T and B lymphocytes are responsible for the protective effect of vaccination. Genes encoded in the MHC genomic region (B-locus, Y-locus) influence the function of cells of the innate and adaptive immune systems, as well as, encode soluble proteins important in innate defense and inflammation (e.g. complement factor, cytokines). For example, B-F genes (MHC class I) encode MHC class I proteins that are expressed in every cell in the body and serve as recognition molecules to indicate a normal, healthy cellular state or, if associated with foreign antigen peptides, signal intracellular infection of the cell requiring its elimination. B-L (MHC class II) molecules which are also encoded in the MHC are critical in alerting adaptive immunity to infection and play an important role in the activation of an adaptive response. These MHC class II proteins are expressed by antigen-presenting cells (APC), such as dendritic cells, macrophages and B cells, and function in presenting peptides from the foreign invaders to CD4+ Th cells of adaptive immunity. Th cells physically interact with antigen-presenting cells by binding to MHC class II-antigen-peptide complexes on the APC with their T cell antigen-receptor (TCR). The recognition of antigen in this from by the TCR constitutes the first signal for activation of the Th cell. A second signal is provided by the APC which, depending on the nature of the infection, can influence the differentiation of Th cells to become different types of Th effector cells and hence drive the adaptive response into certain directions. Types of Th cells are categorized based on their signature cytokines (soluble signaling molecules) that they produce upon activation as Th1 (interferon-gamma), Th2 (interleukin (IL)-4 and 13), Th17 (IL-17), etc. Th1 cells direct the adaptive response towards a cell-mediated response that is effective against intracellular antigens (e.g. viruses, intracellular bacteria and parasites, tumor antigens) and involves Tc cells, natural killer (NK) cells and macrophages as effector cells. Th2 cells direct the adaptive response to an antibody-mediated response that is effective against extracellular antigens (e.g. extracellular bacteria, parasites). Th2 cells not only help in the activation of antigen-specific B cells to produce antibodies, but also direct B cells to produce the appropriate isotype of antibody that can effectively eliminate a specific antigen. Th17 cells activate a phagocytic response designed to eliminate extracellular antigens, involving both antibody and cell-mediated mechanisms. In addition to Th cell activation, there are many more interactions between and within innate and adaptive immunity designed to activate effective defense systems against an invader. Vaccinations have long been used to protect individuals from pathogens, by making use of the protective nature of adaptive immunity. During an adaptive immune response, memory cells with TCR or BCR (B cell-antigen-receptors) specific for the pathogen are set aside and maintained until the pathogen invades again. Upon a repeat encounter with the pathogen, large numbers of these memory cells are ready to act quickly in the elimination of the pathogen before the infection results in disease. In fact, vaccination is used extensively to protect poultry species from disease. A better understanding of the genetic regulation of the immune response and the qualitative and quantitative nature of a protective response is needed to produce the most effective and appropriate protection. For this, the nature of the host-pathogen interaction also has to be understood and taken into account, as well as, the effects of environment, nutrition and various physiological systems that can impact immune system activities. Considering that the powerful response initiated during innate and adaptive immune system activation that eliminates a pathogen may also cause tissue injury, a multitude of regulatory mechanisms have to be in place to control activation, the extent and the direction of the response, as well as, to reestablish homeostasis and maintain tolerance. Maintenance of tolerance and prevention of attack of self components is critical because the random generation of TCR and BCR can result in self-reactive T and B cells. The immune system has a variety of mechanisms in place to avoid attack of self-components, including selection processes resulting in deletion of self-reactive T and B cells during development (central tolerance) and maintenance of peripheral tolerance through T regulatory cells (Treg). Failure of tolerance and dysregulation of immune responses can result in autoimmune disease and other chronic/progressive inflammatory diseases. In summary, considering the central role of immunity and disease resistance in the health of an individual, research into the genetic bases of disease resistance and immunity to avian diseases requires multiple efforts and multi-faceted approaches. To assess and optimize natural health for poultry, new knowledge is needed to better understand the development and function of the avian immune system in health and disease. Specifically, knowledge regarding the genetic regulation of susceptibility versus resistance to disease in poultry would go a long way towards development of markers that could be used in genetic selection of poultry for both desirable production traits and health. A better understanding of the nature of host-pathogen interactions that influence disease resistance/susceptibility would lead to more effective prophylactic and interventions strategies. Knowledge of the functional consequences of environmental, physiological and nutritional alterations on immune system development and function would provide the basis for management strategies designed to optimize poultry health and improve wellbeing of this important food production animal. a) Need, as indicated by stakeholders Poultry breeders and producers seek to increase production efficiency and lower production cost, while providing consumers with a wholesome product. Poultry disease is still a major issue for the poultry industry. Economic loss due to morbidity, low performance, and mortality is significant and some zoonotic pathogens such as Campylobacter and Salmonella can cause human illness or death. While significant advances have been made in controlling many types of poultry diseases, the impact of diseases is one major impediment for increased productivity. Total losses caused by specific diseases not only include losses due to mortality, decreased meat and egg production, and condemnations, but also costs of vaccinations, chemotherapy and eradication programs. Use of pharmacological agents (e.g., antibiotics) to control and treat disease poses its own risk and challenges to animal production, the environment and the wellbeing of poultry and the consumer. The U.S. Poultry and Egg Association defines 28 critical needs for controlling disease and ensuring food safety in poultry (5) that include prevention of critical diseases while decreasing the use of chemical agents. This project addresses the important issues of genetic bases of resistance and immunity to diseases in poultry as well as the processes involved in natural and adaptive immunity. Primary stakeholders (the most immediate users of the data, reagents and tools generated in this project) are poultry breeding companies and vaccine manufacturers. Their frequent participation in the annual meetings of the Technical Committee and their many collaborative research projects with the members, clearly indicate the importance that stakeholders assign to the research of this project. b) Importance of the work, and what the consequences are if it is not done This work is essential to the continual development of disease prevention and control strategies to ensure the sustainability of the poultry industry and increased production for a growing world population. Genetic variability is inherent within populations of species and is no doubt the result of natural selection. The project addresses the important issues of genetic bases of resistance and immunity to diseases in poultry providing stakeholders with a better understanding of genetic variability within their stocks in order to produce future populations with sustainable, desired traits. Disease resistance as a genetic trait is very complex, often having low heritability, and is difficult to improve by traditional genetic selection methods. To this end, although there is a chicken genome sequence, much remains to be learned about loci that are naturally polymorphic and about the functional outcomes of interactions between polymorphic loci that are increasingly under directed selection. Disease is one of the major limiting factors in poultry production, both in large-scale and small-scale settings. The move to managing birds in less-controlled environments, and reduction of use of antibiotics in animal production will exacerbate disease issues in the future. This project addresses the important issues of environmental and physiologic factors that regulate or affect immune system development, optimal immune function and disease resistance in poultry. If this work is not done, disease will increase, production efficiency will decrease, food safety will be greatly compromised, and export markets will be closed. This project also addresses the need for development of methods, reagents and specialized genetic stocks to be able to assess, monitor and modulate immune system development, patterning and function. In contrast to biomedical model species, such as mice, few of the reagents needed for research in poultry are commercially available. If immune development is not monitored, immune responses to vaccine and disease organisms could deteriorate, thereby increasing morbidity and mortality. Understanding immune system development as affected by genetic and environmental factors will find direct application in the breeding of poultry stocks that have improved health and effective responses to vaccine and in efforts to contain production costs. c) Technical feasibility of the research The NE-1034 Technical Committee collectively possesses the necessary technical expertise to execute the research. The membership possesses a range of technical expertise encompassing immunology, genetics, virology, physiology, nutrition, biochemistry, microbiology, and molecular biology. The work is technically feasible as it is rooted in methodologies used in other species, most notably human medicine, and has already been demonstrated as successful. In the past decade, cutting edge technology, including next generation sequencing and many both simple and highly sophisticated methods are now available for examining the expression and interactions of genes important in disease resistance. These can be readily applied in investigations of disease especially in genetically defined experimental lines. Although the techniques applied are sophisticated, the researchers work on the cutting-edge of science and have demonstrated their expertise and resources to successfully complete the described work, if sufficient financial support is made available. d) Advantages for doing the work as a multistate effort Conducting this work as a multistate project offers the advantage of pooling and sharing resources to address critical scientific questions. The members of the NE-1034 committee are well-established scientists who have supporting research programs in poultry genetics, genomics, virology, immunology, nutrition, physiology, biochemistry and microbiology with combined expertise to examine disease resistance at all levels. In addition, participants have unique skills or specialized resources such as genetic stocks and poultry-specific reagents that are needed to conduct the work. The multistate effort is required for the synergistic and collaborative conduct of research that is based upon the combination of biological materials (including experimental lines of birds, antibodies, cell lines, pathogen stocks), facilities, equipment and expertise from multiple stations. No single station possesses all of these to address the major scientific issues for the project. Conducting this work as a multistate effort allows for the greatest use of resources as it brings to bear 24 independent laboratories from 12 U.S. states [AL (1), AR (2), CA (5), DE (2), IA (1), IN (1), MI (USDA-ADOL, 1), NC (2), NH (1), NY (1), VA (1), WI (1)], and 3 other countries [Canada (2, ON, PEI), the Netherlands (2, NL) and Denmark (1, DK)] in the current NE-1034 group of scientists - each addressing overlapping yet distinct aspects of the problem. The truly essential, cooperative, multidisciplinary nature of the project is illustrated by the many joint-authored publications among participating stations. Over the past 4 years of the NE-1034 project, this group has produced 282 refereed publications, 23 book chapters and 250 abstracts many of which feature joint authorship among multiple stations (see Appendix). The extensive expertise of the NE-1034 Technical Committee members and collaborators is also very clearly illustrated in the contributions by the members to the new book on Avian Immunology (published in 2009, in revision for a 2013 edition) with 7 of the 22 chapters and one of the two appendices contributed by NE-1034 members. The book is co-edited by a NE-1034 member. In addition, efforts of NE-1034 Technical Committee members were recognized during the last 4 years for their contributions with the Poultry Science Association Early Achievement Award for research (2), Hy-line International Poultry Science Research Award (3), Embrex Fundamental Science Award (1), and Induction as Poultry Science Association Fellow (1). e) Likely impacts from successfully completing the work Impacts are expected to include but not be limited to: a better understanding of polymorphic loci and the consequence of selection on poultry health and production; new breeding strategies to produce more robust, disease resistant lines of poultry; improved efficacy of vaccines and other pharmaceutical agents; new vaccine programs for controlling existing as well as emerging diseases; and a better fundamental understanding of how the avian immune system functions, which will aid future scientists in dealing with new problems still not recognized. Improved disease resistance will enhance production efficiency, animal health and hence welfare, and producer acceptance, as well as, reduce the need for antibiotics and improve food safety of products of poultry origin. Much new knowledge in the basic and applied sciences, as well as, reagents and tools generated by this project, will constitute valuable resources to the stakeholders. References Cited: 1. Evans, T. 2010. http://www.themeatsite.com/articles/1021/global-poultry-trends-poultry-meat-consumption-in-the-americas-well-above-world-average 2. http://www.ers. usda.gov./news/broilercoverage.htm 3. http://www.fas. usda.gov./dlp/circular/ 4. http://www.uspoultry.org/economic_data/ 5. http://www.uspoultry.org/research/ 6. Speedy, AW (2003) Global Production and Consumption of Animal Source Foods. J. Nutr. 133: 4048S-4053S 7. Steinfeld, H (2004). The livestock revolution - a global veterinary mission. Veterinary Parasitology 125: 19-41 8. Bagust, T. J. Poultry health and disease control in developing countries. http://www.fao.org/docrep/013/al729e/al729e00.pdf 9. Biggs, P. M. 1982. The world of poultry disease. Avian Pathol. 11:281-300.

Related, Current and Previous Work

The NE-1034 multistate research project scientists design, create, maintain, and study unique poultry genetic lines. Some members carry out all of these functions and others a subset as an integral part of our research. These efforts have been our contribution and our responsibility to achieve the project objectives of understanding the genetic bases for immunity to avian diseases. Special genetic lines, established over the last 80 years, are at risk at many research stations. If lost, these unique avian genetic resources (e.g., congenic, recombinant, and inbred lines) are unlikely to be recreated. Since member scientists share these genetic resources in collaborative research, their elimination will impact the project, collaborators and the avian research community at large. The Technical Committee recognizes the imperative to conserve the resources currently available. Several Technical Committee members served on the Avian Genetic Resources Task Force and are now part of the National Animal Germplasm Program, Poultry Committee. The members are committed to the establishment of a national system of networked researchers and a site for orphaned stock conservation to support our objectives of understanding and improving resistance to diseases in poultry. Innovative technologies such as candidate gene identification, applications of recombinant DNA, monoclonal antibodies, DNA probes, QTL analysis, global and targeted transcriptome analysis, gene sequencing and other novel molecular approaches have been effectively used to identify and characterize many facets of disease resistance or immune function. These techniques expand upon the pioneering work conducted by NE-1034 members throughout the project history. Project results continue to be important and readily applicable in both research and industry. Commercial poultry breeders lead other animal breeders in terms of improvement of a variety of economic traits, including genetic resistance to disease. Further research on new methods to select for disease resistance in poultry must, and will, continue in the proposed renewal project for NE-1034. Recent scientific advances in understanding the immune system and enhanced knowledge about poultry pathogens promise imminent and significant improvements in poultry health, production efficiency, food safety and animal wellbeing through genetic selection. Extensive publication searches indexed in the comprehensive databases (Agricola, Biosis, CAB, CRIS, Health Index, and Medline) for the last five year period revealed substantial scientific contributions that NE-1034 members have made in genetics of disease resistance and immune response in poultry. Five of the current Technical Committee members are also part of the NC-1170 multi-state project on advanced technologies for the genetic improvement of poultry. Through interactions with NC-1170, which focuses their research efforts more on genomics and system biology of poultry and elucidation of genetic mechanism that underlie economic traits, duplication can be avoided. The two groups overlap in the creation and sharing of poultry research populations and research tools, including gene transfer technologies, hence maximizing resources. Compared to other multi-state projects that focus on specific diseases [NC-1202; Enteric diseases of food animals; enhanced prevention, control and food safety] or animal welfare beyond animal health [NE-1042; Optimizing poultry welfare], the uniqueness of the NE-1034 group lies in examining genetic bases of resistance to diseases in avian species in the context of all levels of immune system development and function. Working relationships, either formal or informal, exist between NE-1034 stations and the international laboratories conducting similar research. This, too, assures coordination of efforts and avoidance of unnecessary research duplication. Most of the members also participate in meetings of the Avian Immunology Research Group which is an international conference that brings together researchers in avian immunology from all over the world to share advances in this field, build collaborations and avoid duplicate efforts. Participation of international contributors to NE-1034 from institutions in Canada (ON, PEI), the Netherlands (NL) and Denmark (DK) demonstrates the stature of the project. To highlight accomplishments achieved by the NE-1034 investigators during the past 4 years of this project, a summary of major contributions under each Objective and the need for continuation are presented below. Objective 1. Efforts by CA-D, CA-COH, NL yielded a more extensive description of the organization and sequences of the B- and Y-MHC regions and MHC-B and MHC-Y haplotypes (CA-D, CA-COH, NL). Within this effort, the correct order of MHC genes on gga-16 was further defined revealing the presence of a here-to-fore unknown gene segment within the MHC. The nucleolus organizer region (NOR) was found to be tightly linked to the MHC-Y and separated from the telomeric MHC-B by a GC-rich region. Hence, the lack of linkage between the MHC-Y and -B is not the result of being separated by the NOR. This work debunked the untested hypothesis of the NOR being responsible for the lack of linkage of Y from B and opened up an entirely new avenue of research to explore the content of the GC-rich region, which likely includes repeat elements and other genes having important physiologic function relevant to the immune response and resistance or susceptibility to disease. Extensive sequence data for 14 MHC-B haplotypes, as well as, detailed definitions of the binding motifs for two MHC-B class I (BF) antigen-presentation molecules and candidate viral peptides, opened new venue for the study of target cell recognition by Tc. This research also led to the discovery that only few of the MHC-YF class I proteins are expressed in chickens and that at least one of these may represent a new type of antigen presentation molecule with a hydrophobic binding groove able to present non-peptide molecules. Molecular definition of MHC haplotypes was extended beyond the use of the LEI0258 microsatellite marker based on finding micro-variation in the BF1 or BLb2 gene exons. For reliable molecular rather than serological MHC-typing, further characterization of gene exon variation is required in addition to the molecular MHC typing using the LEI0258 microsatellite marker. AR, CA-D, CA-COH, CA-WU, DK, IA, ON, and VA investigators studied the sequences, expression and function of the products of other genes playing a role in immune function. Molecular characterization of chicken natural killer (NK) cells, heterophils and macrophages, as well as, cloning and characterization of avian cytokines and receptors (e.g. IL-19, IL-22, IL22BP, MIF, Nod1/2, TLRs, scavenger receptors) greatly increased our understanding of genes and their products involved in avian immune system development and function. The effort initiated and led by VA to sequence, annotate and analyze the turkey genome and immune related genes provides the much needed basic knowledge and tools for the study of disease resistance and immunity in turkeys. Further, part of the mannose-binding-lectin (MBL) promoter was cloned and sequenced to identify polymorphism in two inbred and various commercial and experimental lines. In total, 14 SNPs were identified which resulted in identification of six different promoter alleles. The allele A1 was found to be associated with low MBL in serum and it was found in inbred lines as well as in commercial lines. The phenotypic consequences of MHC-haplotypes in terms of immune system function and disease resistance were examined through live-bird challenges, quantitative trait locus mapping, single nucleotide polymorphisms (SNP) analyses, establishment of linkage maps based on microsatellite markers and SNPs, global microarray and qPCR assessment of gene expression (CA-D, CA-WU, DK, IA, USDA-ADOL, NH). Using these approaches, many genes, genomic regions and signaling pathways associated with the host response to infection with pathogens such as E. coli, Salmonella, and Campylobacter in the chicken have been identified providing important direction for further study into these economically important bacterial infections. Similarly, microRNAs and signaling pathways related to avian influenza virus infection in chickens have been identified using high-throughput technology including microarray and next-generation sequencing. Other major contributions to our understanding of viral infections in poultry include NHs observation that Mareks disease incidence was affected by a single locus BG1 3-untranslated region difference identified in congenic lines 003.R2 and 003.R4 with serologically identical MHC recombinant haplotypes. DK established that different MHC haplotypes were associated with different amounts of antibodies to infectious bursal disease virus (IBDV) and lower disease severity after experimental infection of chickens. A similar association of MHC-haplotype with antibody levels and pathology was also found in Newcastle disease virus (NDV) infected chickens and for parasite egg burden after a challenge with Ascaridia galli. Greater resistance to clinical illness and better viral clearance in infectious bronchitis virus (IBV) infected chicks with the B2/B2 compared to the B19/B19 MHC-haplotype were also reported by CA-WU and collaborators. These studies offer new opportunity for characterization of genetic regulation of resistance and immunity to pathogens in poultry. Objective 2. The group has also made significant accomplishments regarding basic characterization of innate and adaptive immune functions and examination of the influence of genetic, environmental, nutritional and physiological factors on these processes. For these studies, availability of genetic lines with optimal and suboptimal immune responses to an experimental antigen has been very helpful. Lines selected for high or low antibody responses to SRBC were shown by NH to exhibit differential bursal gene-expression profiles revealing biomarkers unique for high and low SRBC-Ab responders as early as day 15 of incubation. Embryonic testosterone propionate exposure, which results in bursal ablation, influenced distinct pathways in birds from the high and low SRBC-Ab responder lines. USDA-ADOL compared MDV-susceptible (7-2) and resistant (6-3) lines based on expression analysis with a panel of immune-related genes revealing a much more vigorous, especially T cell-mediated, immune response in line 6-3 than line 7-2. Additionally, transient paralysis could be observed in both lines with high pathogenic strains of MDV. Using MHC-defined lines of chickens, known to respond differently to infection with pathogens or to have different innate immune activity to PAMPs or other immunostimulants, several members (CA-COH, CA-WU, CA-D) were able to better define the nature of an effective or ineffective immune response. CA-COH and CA-WU in collaboration with others have helped to elucidate mechanisms underlying the activation of natural killer cells, T lymphocyte responses and macrophages. Evaluation of chicken monocytes as a factor in disease resistance showed that B2/B2 monocytes differentiated more readily into macrophages, were stimulated to significantly greater levels with either poly I:C or IFN-gamma, and exhibited differential upregulation of at least 9 pathways compared to B19 stimulated macrophages. Genetic lines prone to non-communicable diseases with immune system involvement (e.g. autoimmune disease, ascites, skin disorders, lameness, etc.) were used by AR, USDA-ADOL and NH for comparison of aberrant versus normal immune activities. AR identified IL-21, IL-10 and IFN-gamma as the signature cytokine profile associated with autoimmune vitiligo onset and progression in susceptible Smyth Line (SL) chickens. Global gene-expression analysis of the target tissue (feather) before and throughout SL vitiligo development established a role of innate and adaptive immunity, as well as, cellular stress. Independent of serotype, MDV infection administered at hatch was reliably associated with vitiligo expression in susceptible SL chicks. Based on HVT administration, the ability of MDV to trigger SL vitiligo is limited to infection during the first 6 weeks of life. Susceptibility to autoimmune SL vitiligo appears to be manifested in part in target cell (melanocyte) defects. Studies by NH using an atherosclerosis model also reports differentially-expressed genes and soluble proteins found in aortic smooth muscle cells in atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons. Immune activity and pathogenic mechanisms initiated as a result of PAMP administration, viral, bacterial, or parasitic infections were also investigated in selected lines of chickens, commercial layers, broilers and turkeys by members and collaborators of NE-1034 (AR, CA, DE, DK, NC, NL, NY, ON, PEI and VA). These studies yielded critical new knowledge regarding impact of physiological factors on aspects of disease susceptibility, disease progression, activities and interrelationships of innate and adaptive immune systems, virulence of pathogens, immunodominant epitopes, nature of effective or ineffective host responses and approaches for disease intervention and prevention. An eight year longitudinal survey of SPF flocks at NY infected with immunosuppressive chicken infectious anemia virus (CIAV) showed that antibody development to CIAV started in general on or after sexual maturity with significant differences in levels of seroconversion during this period. These findings suggest that the infection may remain latent and that reactivation is linked in part to sexual maturity. Studies on avian influenza (AI) by NY using the highly pathogenic H5N1 (VN1203/04) isolate showed that thrombocytes play an important role in the pathogenesis in chickens but not in ducks. ON conducted extensive study on gene-expression profiles during infection with different serotypes of Mareks disease viruses in a variety of tissues known to play a key role in infection, latency and transmission of these MDV. DE studied the effects of innate immune stimulants on the replication of MDV vaccine strains and overall vaccine efficacy. Inclusion of some select innate inducers (e.g. PAMPs) did not interfere with vaccine virus replication, despite potent induction of innate responses but did not have appreciable effects on cell-mediated immune function and MD protection. Gene expression profiling by USDA-ADOL between rMd5 and rMd5Dmeq infected chickens revealed that Meq functions as an immunosuppressive oncogene that results in down-regulation of many immune-related genes and may be controlling the expression levels of p53 involved in regulating the cell cycle and tumor development. NC worked to characterize numerous circulating strains of the type-2 turkey astrovirus isolated from commercial turkeys across the U.S. to identify potential virulence markers. In the infected host, astroviruses induced a reduction in the apical expression of sodium/hydrogen exchanger-3 which contributes to mal-absorption and diarrhea. The infected epithelial cells responded to infection by expressing inducible nitric oxide synthase (iNOS), which likely plays a key role in eliminating the virus in the immunologically immature host. VA assessed the differential genetic resistance to clostridial toxins in select chicken lines and conducted serum protein profiling and identification of potential blood markers in Eimeria-infected chickens from commercial genetic stocks. Influence of environmental factors on mucosal and systemic immunity has been a focus of research by AR, NC, NL, ON, and VA. AR and collaborators observed different vasoactive and cytokine responses to local pulmonary inflammatory activities induced by PAMPs or vascular occlusion in ascites-susceptible or -resistant broiler lines. ON developed a probiotic formulation that possesses immune stimulatory activities, which they plan to test in commercial settings in the near future. VA is evaluating the effects of antibiotic alternatives in commercial chickens (e.g. beta-glucans, probiotics) using various delivery routes and disease models. Immunomodulatory effects of concurrent administration of model-antigens and PAMPs typically present in the air of poultry houses were observed by NL particularly in young broiler and layer chickens. Adaptive systemic immune responses were also shown to be affected by the absence of microflora in the gut following antibiotic treatment as well as by administration of probiotics. Through these studies, the period of 3-6 weeks of age was identified as a critical time in the development of mucosal immunity. Investigations into the role of nutrition in the immune responses of poultry by CA-D established that nutrients that have primarily regulatory functions (vitamins A and D, and essential fatty acids) had greater effects on development of the immune system than nutrients that serve as anabolic precursors (amino acids, energy, minerals). Vitamin A deficiency during development diminished B lymphocyte maturation and the breadth of the antibody repertoire. The entire cost of the adaptive immune response (specific antibodies and lymphocytes) was easily fueled by the decay of the acute phase proteins produced during the innate response, suggesting that adaptive immunity has no net nutritional cost. NC demonstrated that changes in the intestinal microflora of poultry alter the amount of energy consumed by the immune response. PEI characterized local and systemic innate immunity in poultry during nutritional intervention using yeast derivate carbohydrates (YDC). In several comparative studies using broiler chickens fed conventional diets including an anticoccidial (Monensin), a growth promoter (BMD), and an anticoccidial plus YDC they found that the inclusion of YDC (23% mannans) affected several immunological parameters, including regulation of intestinal microflora, intestinal architecture, cytokine expression and enhanced neutrophil activity. DK conducted studies to characterize and examine the function of mannose-binding-lectin (MBL, innate immunity) in susceptibility of poultry to different pathogens. Using chickens selectively bred for a high or a low serum concentration of MBL, low circulating levels of MBL were associated with reduced ability of poultry to respond to pathogens such as IBV, E. coli, Pasteurella muliocida, and Ascaridia galli. These results confirm that MBL, as shown in mammals, plays a major role in the outcome of various infections in chickens and may emerge as a biomarker for disease susceptibility. Similarly, investigations into natural antibodies (NAb) and health in poultry by NL showed that high levels of NAb, especially of the IgM isotype, correlate with lower mortality during a lay period. NAb levels were found to be very heritable (0.4), related to SNP in immune response-, behavioral-, and unknown-gene regions. Whether NAb originate from introduction of the intestinal microbiota or reflect homeostatic auto-antibodies is subject of future studies. Their studies also showed that NAb binding to protein extracts from chicken organs may provide a fingerprint for measuring the health status of individuals. Objective 3. Efforts by the group resulted in development of a variety of tools and basic data for continued research on the genetic bases of resistance and immunity to avian diseases. A number of stations (AR, CA, DK, IA, NC, NL, USDA-ADOL) developed, maintained, characterized, and made available unique genetic resources to the NE-1034 members and the entirety of the poultry research community. These included genetic lines that are highly inbred and contain MHC-congenic sets, are MHC-defined, and/or exhibit defined disease susceptibility/resistance characteristics. DK breeds of chicken were found to have five B21-like haplotypes including B131 (broiler origin) and BW1 (Red Jungle Fowl, Gallus gallus gallus origin). AL generated a better understanding of the mucosal immune system in chickens induced by IBV vaccines or Ad5 vaccine vectors expressing the avian influenza HA gene. AL found the head-associated lymphoid system, i.e., conjunctiva-associated lymphoid tissues (CALT) and Harderian glands (HDGL), to generate immunity to avian pathogens after ocular or in ovo immunization. CALT generated more of a cell-mediated immune response and after priming seemed to contain cytotoxic effector memory T cells, while the HDGL generated more of a B cell response. The spleen played a minor role after mucosal IBV priming, but generated the highest IFN-gamma response after boosting, indicating induction of a central memory T cell response. IgA dominated the primary response, while IgG dominated the memory response to IBV. Advances made by the group to facilitate the study of immunity include development of chicken whole genome 44K gene expression array (CA-D) that has been widely used in the poultry community; increased availability of genetic information to conduct targeted qPCR expression analyses of cytokine and other genes; and, B-cell spectratyping including IgA, IgG and IgM isotypes. Western blotting was optimized to measure post translational polymorphism of NAb and auto-antibody fingerprints. A minimally invasive procedure to monitor cellular immune responses in vivo using the growing feather as an in vivo test-tube was developed by AR. Based on analyses of antibody responses using a peptide array, AL reported that mutations in the S1 protein of IBV contributed to immune escape. VA identified innate immune markers correlating with disease resistance to coccidiosis. NC developed reagents for use in the study of turkey immunity, including polyclonal antibodies to the turkey iNOS protein. NY developed an antigen-antibody complex vaccine that does not cause damage in chickens lacking maternal antibodies to CIAV and protected against replication of a challenge virus. Over the last four years, DEs research on the evolving MDV resulted in identification and development of various MDV mutants for research. Included are mutations in the main oncogene (Meq) of MDV, Meq splice variants observed during MDV pathogenesis, and a glycoprotein L mutation common to high virulence MDVs. Studies with these MDV mutations focused on their effects on transactivation, target gene expression, cell shape and mobility, immune evasion and pathogenesis and the immune response. Use of these mutated MDV viruses has already provided insight into the impact of viral genes on tumor composition, mechanisms by which MDV regulates immune-associated genes, virulence, viral T cell epitopes presented, and effects on the early patterning of immune responses. While transgenic approaches to study the function of genes is not yet readily available for avian species, virus vectors approaches have been employed by members of the group. ON developed a prototype virus-vectored system for down-regulation the expression of IFN-gamma, a system that can be modified for down-regulation the expression of other cytokines in the future. WI has created a generation 3 self-inactivating lentiviral expression vectors for in ovo administration in preparation for expression of the Mx transcript from the highly pathogenic AI virus resistant Blue-winged Teal in chickens under control of an inducible promoter.

Objectives

1. Identify and characterize genes that regulate or affect innate and adaptive immunity and determine their relationships to disease resistance in poultry.
   
2. Identify, characterize, and modulate environmental and physiologic factors that regulate or affect immune system development, optimal immune function and disease resistance in poultry.
   
3. Increase poultry production efficiency and disease resistance by developing and evaluating methods and reagents, including specialized genetic stocks, to assess or modulate immune system development, patterning and function.
   

Methods

Over the past 30-40 years, the field of immunology and immunogenetics continuously has seen extensive advances, particularly with regard to immune system function and development in mammals. The increased understanding of immune system development and function stems primarily from studies on the mouse model, where manipulation of gene-expression seems to have unlimited potential in dissecting mechanisms of cell development and function. With the availability of the chicken genome sequence since 2004, holistic approaches such as genome-wide sequencing and whole transcriptome and proteome analyses have been carried out providing new insights into unique features of the avian immune system, disease resistance and susceptibility as well as the mechanistic aspects of immune system development and function in chickens. As additional avian resources are becoming available particularly through contributions of member stations, such as the recently published turkey genome and much improved chicken genome, poultry scientists are well positioned for further discoveries pertaining to avian immunity and their inherent responses to disease challenges. Since the complexity of the immune systems molecular and cellular components, genetic regulation underlying the functional responses of the immune system, and the interplay between genetic, immune system, environmental, physiological and nutritional factors have become apparent, such discoveries will significantly better our knowledge in this arena and further enhance the U.S. global competitiveness. OBJECTIVE 1) Identify and characterize genes that regulate or affect innate and adaptive immunity and determine their relationships to disease resistance in poultry. NE-1034 members will continue to investigate genetic resistance of birds to a myriad of avian diseases using various approaches that employ the latest genomic resources and technologies. Research by member stations will encompass fundamental immunology, developmental immunology in defined lines, and responses to specific pathogens (viruses, bacteria, and parasites) in poultry stocks. CA stations (CA-COH and CA-D) will focus on MHC research using genomics, FISH, molecular and immunological methods to identify and characterize the activities of genes that regulate avian immunity in the MHC-Y region to which the nucleolus organizer region (NOR) is tightly linked but separated from the telomeric MHC-B by a GC-region. Planned research will study this GC-rich region which likely includes repeat elements and other genes having important physiologic function. Within the disease context, the stations will pursue the post-transcriptional regulation of BG1, a polymorphic gene that affects the incidence of Mareks disease and the function of MHC-Y class I molecules in disease resistance. VA will research basic functions of avian pathogen recognition receptors, cytokines and their receptors (e.g. NODs, IL-22, macrophage migration inhibitory factor), particularly in response to mucosal infections and their potential regulatory functions in disease resistance. Additional projects at VA will focus on providing genomic resources (e.g. turkey genome) and extensive tissue-specific transcriptomes coupled with detailed annotations. AR will conduct studies to determine the basis for the genetic susceptibility of Smyth line chickens to develop autoimmune vitiligo by next-generation sequencing in conjunction with quantitative trait analyses. Further, NH will employ the inbred lines 63 (regressor) and 72 (progressor) that differ in v-src DNA tumor growth and metastasis, in addition to the 19 Recombinant Congenic Strains from these two lines, in order to identify the genetic basis for differential tumor growth. In collaboration with VA and NL, NH will use microarray and whole transcriptome sequencing to measure gene expression in embryonic lymphoid tissues from two sets of lines selected for high or low antibody to SRBC. Comparisons among the four lines will be tested at multiple time points before and after SRBC injection based on selection methods. Another focus of the NL station will be on the divergent breeding and selection of layers for high or lower natural antibody levels. SNP typing will be performed to identify genomic regions that are related with the level of natural antibodies and their isotypes. Natural antibodies levels will be related with disease resistance and further refining of the MHC region typing will be performed. In commercial and experimental chicken lines, several SNPs have been identified in the promoter region of the Mannose-Binding Lectin (MBL) gene. DK will use six identified alleles to evaluate the effect of MBL as a genetic marker in relation to susceptibility to bacteria, viruses, and parasites, which can be implemented in breeding strategies. Further, the NC station will identify differences in expression and function of innate antiviral mediators by virally infected cells from pedigreed lines of chickens and determine how allelic variations in these mediators influence the overall resistance of birds to different viral infections. CA-D, IA, and VA will use chicken and turkey genetic lines to determine the host-pathogen interactions during infections with avian pathogenic E. coli (APEC), Campylobacter or Salmonella by examining transcriptomic changes in inflicted lymphoid tissues using RNA-Seq, microarray, qPCR and bioinformatic analyses. USDA-ADOL will continue research on the identification and characterization of chicken genes that mediate production of cell-free infectious Mareks disease virus (MDV) particles in the feather follicle epithelium (FFE). The station will employ the Agilent 44K chip that includes all the known chicken and MDV transcripts and 150 chicken microRNAs to conduct a comprehensive comparative gene expression analysis between infected and control FFE cells and T cell subsets isolated from the skin and splenocytes of MDV-infected chickens. Additionally, the role of NK cells in vaccine-induced immunity against MDV will be investigated by comparative analyses of the functional capacity of NK cells between MDV-susceptible and resistant chickens lines vaccinated with commercial and recombinant formulations. The analyses will include a number of pore forming proteins, effector cytokines, degranulation capacity and NK-induced apoptosis of target cells. Further, the ON station (Guelph) will conduct a systematic analysis of immune responses to MD and avian influenza viruses (AIV) in various host tissues, with the intention of identifying correlates of immunity against these two pathogens. Also, they will study Toll-like receptor (TLR) ligands for their ability to confer protection against Mareks disease and influenza. Using selected haplotypes with resistance and susceptibility to avian coronavirus, CA-WU will study pathogenesis of low path AIV. Adaptive immune responses to AIV and avian coronavirus will be compared in the B2, B8 and B19 homozygous birds. T cell responses will be determined through adoptive transfer of T lymphocytes from infected birds and through stimulation with matched or mismatched antigen presenting cells. Next generation mRNA sequencing and KEGG analysis for several time points will be performed to further investigate affected immunological pathways and differences in activation of the innate immune system between haplotypes. Moreover at the WI station, Mx alleles from avian species sensitive and resistant to AIV will be sequenced and analyzed for positive site-to-site selective pressures to identify putative antiviral regions within the Mx protein, which is known to possess antiviral activity against RNA viruses. Further, the role of mucosal immunity in head-associated lymphoid tissues (HALT) to infectious bronchitis virus (IBV) will be measured and analyzed to better understand resistance to disease and the role of the quickly mutating S1 sequence of the spike protein on protection versus immune escape of the virus. These studies will be conducted at AL station and analyses will be performed at both the cellular level (B and T cell ELISPOT assays, FACS analyses, qRT-PCR) and the molecular level (B and T cell epitope mapping) involving IBV-resistant and -susceptible chicken lines. OBJECTIVE 2) Identify, characterize, and modulate environmental and physiologic factors that regulate or affect immune system development, optimal immune function and disease resistance in poultry. Improved disease resistance depends on understanding how husbandry and management modulate immunity. Nine institutions will examine how probiotic and commensal organisms, nutrition, environment, physiological inducers or inhibitors, and pathogens impact the development and response of the immune system, as well as, molecular details of immunomodulation. Three institutions will examine how beneficial microbes modulate immunity. ON will focus on development of probiotic formulations with the ability to enhance immune responses in chickens. NL will follow the effects probiotics on the development of the immune repertoire (including auto-immune-profiles) in relation with other physiological traits (egg lay and mal-behavior). NC has demonstrated that changes to the intestinal microbiota can affect the level of energy consumed by and activation state of the mucosal and systemic immune system and will focus on the mechanism by which changes in the host microflora is communicated across the gut epithelium and then on to the systemic immune system Nutritional approaches to facilitate and improve immunity through effects on development and modulation of responses will be examined at three institutions. VA will continue investigating the impact of in ovo delivered supplements on gastrointestinal development, microbial populations, and immune responses of poultry post-hatch, in the context of enteric disease challenges. RNA-Seq, qPCR, and flow cytometry will be the main tools used and results will be compared to similar studies conducted with post-hatch supplementation and antibiotic applications. NL will determine dietary impacts on the development of the immune repertoire in relation to productive and behavioral traits. CA will establish the nutritional cost of maintaining and using the immune system by determining the mass and nutrient content of the mucosal immune system and quantify the increase nutritional needs for a robust enteric response to pathogens. Two institutions will investigate environmental factors. NL will focus on husbandry conditions that modulate immunity and its relationship to egg production and mal-behaviors. AR will examine the role of environmental factors in triggering expression of multifactorial disease in susceptible individuals. AR will determine immune system dysregulation leading to immunopathology in multifactorial non-contagious diseases (autoimmune vitiligo, uveitis, ascites) in chickens. Methods will include a range from whole animal studies to histological, cellular and molecular examinations, including gene-expression at the transcriptome and protein level. The immunomodulatory role of physiological inducers or inhibitors will be investigated by three institutions. DE will continue working with Industrial partners to test innate immune inducers that increase the resistance of poultry to various microbial agents. DE will use transcriptomic analysis of the effects of these inducers on innate signaling and ultimate patterning of acquired immune responses. AR-USDA will study the modulating effects of the acute phase protein ovotransferrin on HTC macrophages after stimulation with LPS, peptidoglycan, and phorbol ester, which induce discrete effects on phagocytosis, matrix metalloproteinase production, respiratory burst and chemotaxis. AR will determine local innate- and adaptive-cellular immune activities in tissues in response to a first and second exposure to antigen. DK will focus on the interaction between innate and adaptive immunity as an avenue to improved vaccination efficiency and discovery of new adjuvants for the vaccine industry. MBL is involved in the regulation of the adaptive immune response to IBV. DK will therefore try to develop vaccination strategies where the contribution from the innate part of the immune system temporarily will be blocked in order to allow enhanced development of the adaptive immune response. Pathogen induced changes in immunity will be investigated at four institutions. Genes, signaling pathways, copy number variation (CNV) and microRNAs associated with Campylobacter infection and avian influenza virus infection in the chicken will be investigated at CA-D. Microarray and next generation sequencing and computational biology approaches will be used to address this issue. In vitro knock-down assay will be applied to elucidate the function of genes. DK will develop biochemical, molecular and immunological assays for measuring the host-pathogen interactions, including real-time PCR assays, ELISAs, and flow cytometric assays. IA will determine the effects of LPS-induced sickness syndrome combined with heat stress on the transcriptomic changes in immune-related tissues by use of live-animal challenge trials, RNA-Seq and bioinformatics analyses. AR-USDA will study the effect of LPS on circulating peptides that potentially function as hormones, antimicrobial, growth, and signaling factors. Direct effects of LPS on HTC macrophages will also be examined. HPLC, electrospray ionization ad MALDI-TOF mass spectrometry will be used to resolve and identify changes in peptides. CA-COH will pursue the fundamental role of miRNAs in modulating the regulation and development of the immune system in poultry. PEI will focus on studying the modulating effects diets containing different forms of vitamin D and different Ca and P levels on immune parameters in broiler chickens. This novel approach is based on the presence of vitamin D receptors on leukocytes, including activated T and B cells, macrophages and dendritic cells, and the important role of Ca in intracellular cell signaling in lymphocyte proliferation, differentiation, effector functions, and memory development, OH,a new member of the group, will examine the function of regulatory T cells (Tregs) and their role in facilitating survival of commensal bacteria at the gut level. Aspects examined will include quantification of the time it takes for Treg numbers and suppressive properties to peak, nadir, and return to baseline. Additionally, since pathogens such as Salmonella enterica use Tregs to avoid efficient immune responses, OH and collaborators will quantify the effects of a S. enterica infection on Treg properties in chickens. The effects of S. enterica infections on Tregs and the mechanisms through which Tregs become super-suppressive following S. enterica infections will be examined by determining Treg percentages in different organs using flow cytometry, Treg suppressive properties using the suppression of T cell proliferation assay, and Treg cytokine expression (IL-10, TGF-beta, and CTLA-4) by qRT-PCR will be measured. Another new investigator at CA-D (Gallardo) will continue to conduct research on the characterization of the immune response, pathogenicity, and immunosuppression in chickens challenged with very virulent infectious bursal disease virus (vvIBDV). Reassortants observed in unique subtypes of vvIBDV were first seen after the first known case of vvIBDV in North America in 2008 which occurred in California. These previously unseen strains have shown to be reassortants of different serotype 1 and even serotype 2 IBDV segments A and B shown by RT-PCR and sequencing of the vvIBDV genome. CA-D will define the pathogenicity and immune response against these new vvIBDV subtypes to understand the significance of these subtypes with respect to morbidity and mortality and plan preventative strategies to control pathologic events. OBJECTIVE 3) Increase poultry production efficiency and disease resistance by developing and evaluating methods and reagents, including specialized genetic stocks, to assess or modulate immune system development, patterning and function. One of the hallmarks for the NE-1034 stations has been the development of genetically-selected chicken strains that have facilitated evaluation of resistance and/or susceptibility to disease. Special B congenic chicken strains that differ only at the B complex have been produced by repeated backcrossing of certain B haplotypes to an inbred line [CA, IA, NC, USDA-ADOL]. Highly inbred lines and experimental chicken lines selected for unique immune functions are available from several stations [AR, CA-D, IA, NC, VA, IL-NIU, NY, USDA-ADOL, NL]. These populations remain valuable genetic resources for research conducted by collaborating NE-1034 members and other research institutions. The planning, cooperation and sharing of resources or expertise which takes place through participation in this project enhances discovery. CA-D will maintain and make available unique genetic resources to the community of NE-1034 members and will continue to do so. They will incorporate resistant and susceptible lines in studies examining the status of MD virus-host genome interactions in specialized immune and feather follicle cells over time post infection. The studies suggest that certain factors of resistance to respiratory viruses may be predicted by responses of macrophages to IFN-gamma and poly I:C. An ideal mechanism for controlling disease in poultry is to breed birds with natural resistance. Using IBV as a model respiratory pathogen, haplotypes have been identified that may provide candidate genomes for resistance. CA-D is identifying mechanisms for this resistance, and with the forthcoming characterization of RNA expression profiles in the more resistant and more susceptible birds, should be able to identify genes and pathways critical for establishing such resistance. IA will develop, maintain, characterize and share with project collaborators several chicken genetic lines that are highly inbred and contain MHC-congenic sets. VA will further evaluate the genetic resistance to Clostridium sp. in poultry and Salmonella Heidelberg in turkey breeders using RNA-Seq, SNP genotyping, and pathological methods. ON (Guelph) has developed a system using RNA interference to knockdown expression of IFN-gamma in chicken cells. This system will be further optimized for in vivo use. In addition to targeted transcription analysis for innate immune function, DE plans to use macrophage-based cell lines for developing cell-based reporter systems for induced innate signaling. DE continues to work on Mareks disease virus (MDV) with targeted goals of understanding immune patterning during pathogenesis and vaccine responses, the contribution of specific gene products to immunosuppression, and the development and testing of vaccines. Compared to selective breeding and vaccination, genetic modification of poultry is a viable solution to create influenza-resistant poultry in a limited timeframe. WI will characterize avian influenza-resistance conferring Mx alleles, and transduce it into isogenic, AI-sensitive White Leghorns. Germline transductants will be exposed to various strains of low and high pathogenicity AI viruses and scored for survival. DK has a Taqman assay that can distinguish between some of the MBL alleles. In collaboration with INRA in France, DK will develop a new genotyping method (KASPar) that will be able to distinguish between all the known MBL alleles, and search for new genetic disease markers in the group of collectins (Ficolin). AR will develop key indices of cellular and humoral immune response activities by monitoring response to antigen in blood and tissues. NC will continue to develop and characterize reagents to specifically assay for changes in the expression of cytokines and immune mediators in chickens and turkeys. GA, a new member, will develop additional genetic and molecular tools for researcher to better address immunological questions that are difficult to study without transgenic technology. New technology will include generation of transgenic birds using the piggyBac transposon system and in vivo transfection reagents, sperm mediated transfection to directly target the egg and thus transfect all cells in the developing embryo, and establish PGC lines that will be used for manipulation of the chicken genome. These tools will allow researchers to establish and test new immunological models in the chicken.

Measurement of Progress and Results

Outputs

• Continue to maintain unique genetic resources including selected lines, inbred, congenic and recombinant congenic lines as well as experimental lines. Develop additional genetic material (e.g. line crosses) as needs arise.
• Generate atlases of transcriptional responses under normal and disease conditions including those to viruses (e.g. MDV, AI, AIV, and IBDV) and bacteria (e.g. Salmonella, Campylobacter, and Clostridium) using RNA-Seq, microarrays, real-time PCR or other methods.
• Identify individual genes or quantitative trait loci (QTL) associated with disease resistance or immune response via next generation sequencing, microarray technology or emerging high throughput genotyping methods.
• Uncover and develop new single nucleotide polymorphisms (SNP) markers, microarrays, peptides, antisera, primer sets, and serum chemistry analyses to categorize immune responses in normal and disease states. Use genetic, environmental, dietary and immunostimulation methods to enhance protective immunity.
• Continue to identify new and characterize recently described immune response elements (e.g. cytokines, receptors, MHC molecules) and their involvement in resistance to disease.
• Use refereed publications, symposia, invited lectures and informal discussions at regional, national and international workshops and meetings to disseminate information to stakeholders and public

Outcomes or Projected Impacts

• Identified individual causative genes or quantitative trait loci will improve poultry health and animal agriculture in general through marker-assisted selection and breeding or technological applications.
• Knowledge of basic mechanisms of immunity and disease resistance will increase through project efforts. This knowledge will allow development of new technologies to assess or improve the immune response.
• Appropriate immune responses and improved disease resistance through immune modulators will augment production efficiency.

Milestones

(2014): Identify new candidate genes or quantitative trait loci (QTL) associated with disease resistance or immune function through next-generation sequencing, microarray technology or high-throughput single nucleotide polymorphisms (SNP) panels.

(2015): Generate comprehensive expression data of tissue transcriptomes during various developmental stages and challenge conditions.

(2016): Generate advanced line crosses, new breeding stock and other resource populations, to produce experimental progeny for the proposed studies.

(2017): Advance knowledge regarding basic aspects of innate and adaptive immune system development and function. Classify specific dietary ingredients that increase, decrease, or differentially modulate immune function for use as tools to optimize immunity for specific disease challenges.

(2018): Improve transgenic technology to more effectively elucidate biological functions of known and unknown genes in the immune system.

Projected Participation

View Appendix E: Participation

Outreach Plan

Industry stakeholders are invited to and frequently attend the annual project meetings. Their attendance provides an opportunity for information exchange. For example, representatives of breeder organizations can learn of the latest genetic advances in disease resistance from the project scientists. Technical Committee members gain knowledge of emerging field problems that the project can address through experiments. The combined efforts of the NE-1034 stations will generate new scientific data. Refereed publications, online data bases of genetic lines and genome/transcriptome information, symposia, invited lectures and informal discussions are some methods used to disseminate information. Project investigators have made significant scientific contributions to the improvement of poultry immune responses as well as the genetics of disease resistance. Cooperation among project members and with other researchers will remain a hallmark of NE-1034. This cooperative effort will include sharing scientific expertise and genetic resources held at numerous project stations. The addition of several international members has expanded the research scope and global dissemination of research findings.

Organization/Governance

The planning and supervision of the Multistate Research project shall be the responsibility of the Multistate Technical Committee. The membership of this committee shall consist of an Administrative Advisor, a technical representative of each participating agency or experiment station, and a representative of the USDA National Institute of Food and Agriculture (NIFA). The voting membership shall consist of the Technical Committee Representatives. The Technical Committee shall be responsible for review and acceptance of contributing projects, preparation of reviews, modification of the multistate project proposal, and preparation of an annual report for transmittal by the Administrative Advisor upon approval to NIFA. Annual written reports will be prepared by each technical committee member and distributed at the annual meeting. A limited number of the compiled annual reports will be available upon request from the Administrative Advisor. The Technical Committee will meet yearly and elect a secretary, who will serve the year after election and as the chairperson the following year. An Executive Committee will be formed to conduct all business of the Technical Committee between annual meetings. The Executive Committee shall consist of the current Technical Committee Chairperson, the Secretary, and the two immediate Past Chairpersons. The chairperson may name other subcommittees as needed to perform specific assignments. They may include subcommittees to develop procedures, manuals, and phases of the multistate project, to review work assignments; to develop research methods, to prepare publications, and to write proposals. Other agencies and institutions may participate and vote at the invitation of the Administrative Advisor. Minimum expectations for Technical Committee members are submission of a written annual report every year, and attendance at an annual meeting including presentation of research results at least one year out of two. Collaborators may include emeritus members with an interest in attending annual meetings, scientists who wish to contribute by virtue of having special expertise or interest, and those who engage in research interactions with an individual Technical Committee member. Collaborators should submit a written report every year, and present their progress when attending the annual meeting. Guests who attend an annual meeting through special connection to the Technical Committee (i.e. host institution) are invited to make a brief presentation of their interests and ongoing research.

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