Progress 09/16/04 to 05/30/08
Outputs
Progress Report Objectives (from AD-416) 1. Compare immune responses following Marek's disease virus (MDV) infection between susceptible and resistant lines of chickens. 2. Compare cytotoxic T lymphocyte (CTL) responses between strong and weak responders to avian leucosis virus (ALV) infection. 3. Integrate DNA microarray and protein results with whole genome sequencing. Approach (from AD-416) Three aims are defined. Aim #1 compares the response of chicken cells to MDV infection. We have previously shown that gene expression changes in response to MDV infection in CEFs and between lymphocytes from MD resistant and susceptible chicken lines. We extend these studies by examining the cellular response in MDV-infected cells using laser capture micodissection (LCM) and comparing them to the surrounding uninfected cells. Taking advantage of our characterized inbred lines, we examine how genetic resistance status influences the response. Finally, in addition to profiling gene expression, we will isolate and characterize specific MDV-chicken protein complexes that include host proteins known to be important for the immune response and genetic resistance to MD. Incorporation of defined MDV mutants examines how perturbing key MDV- chicken interactions influence the composition of these functional complexes. Aim #2 is similar to aim #1 in that we will utilize DNA microarrays to profile gene expression changes in the immune response. In this aim, ALV subgroups A and J will be the infectious agents, which are known to produce strong or weak CTL responses depending on the MHC haplotype of the chicken. Revealing specific responses in CD4+ or CD8+ lymphocytes as a function of ALV subgroup-chicken MHC combination will provide basic knowledge on what genes are necessary or sufficient to elicit a strong CTL response against ALV. Aim #3 integrates and extends our results using bioinformatics. Comparisons will be made between gene profiling results and, for Aim #1 only, between proteins and their corresponding RNAs. The chicken genome sequence will be integrated by identifying positional candidate genes that confer genetic resistance to MD, and revealing common regulatory elements for key genes. Significant Activities that Support Special Target Populations This project is also related to 3635-31320-008-03R entitled �High Resolution Microarray and Protein Analysis of Chicken Immune Responses.� To validate that genes were differentially expressed between chicken lines that are resistant or susceptible to Marek�s disease virus, a pathogenic virus that leads to death and lost productivity, a limited number of genes were evaluated using an independent method. The results were confirmed. This project has ended and was being monitored monthly through e-mails between the participating groups as well as telephone calls and, when possible, direct interactions at scientific meetings.
Impacts
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Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) 1. Compare immune responses following Marek's disease virus (MDV) infection between susceptible and resistant lines of chickens. 2. Compare cytotoxic T lymphocyte (CTL) responses between strong and weak responders to avian leucosis virus (ALV) infection. 3. Integrate DNA microarray and protein results with whole genome sequencing. Approach (from AD-416) Three aims are defined. Aim #1 compares the response of chicken cells to MDV infection. We have previously shown that gene expression changes in response to MDV infection in CEFs and between lymphocytes from MD resistant and susceptible chicken lines. We extend these studies by examining the cellular response in MDV-infected cells using laser capture micodissection (LCM) and comparing them to the surrounding uninfected cells. Taking advantage of our characterized inbred lines, we examine how genetic resistance status influences the response. Finally, in addition to profiling gene expression, we will isolate and characterize specific MDV-chicken protein complexes that include host proteins known to be important for the immune response and genetic resistance to MD. Incorporation of defined MDV mutants examines how perturbing key MDV- chicken interactions influence the composition of these functional complexes. Aim #2 is similar to aim #1 in that we will utilize DNA microarrays to profile gene expression changes in the immune response. In this aim, ALV subgroups A and J will be the infectious agents, which are known to produce strong or weak CTL responses depending on the MHC haplotype of the chicken. Revealing specific responses in CD4+ or CD8+ lymphocytes as a function of ALV subgroup-chicken MHC combination will provide basic knowledge on what genes are necessary or sufficient to elicit a strong CTL response against ALV. Aim #3 integrates and extends our results using bioinformatics. Comparisons will be made between gene profiling results and, for Aim #1 only, between proteins and their corresponding RNAs. The chicken genome sequence will be integrated by identifying positional candidate genes that confer genetic resistance to MD, and revealing common regulatory elements for key genes. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between ARS and University of Delaware. Additional details can be found in the report for the parent project 3635-31320-007- 00D, Genomics and Immunogenetics of Economically Important Traits of Poultry. The cDNA microarrays were found to produce reliable, reproducible results only with highly expressed genes. This was due in part to the caveats of cDNA arrays, but also with the repeated amplifications needed to obtain adequate amounts of RNA from laser captured material. Affymetrix GeneChip arrays were made available, and we compared cDNA to Affymetrix arrays and found the Affymetrix produced more reliable, reproducible results and extended the list of differentially expressed genes to include moderately and low abundantly expressed genes. However, we were unable to obtain sufficient laser captured RNA to apply to these arrays. The Affymetrix arrays were used to compare gene expression profiles in spleens from resistant and susceptible birds infected with MDV. The Berkeley RMA Express software was implemented for quality control evaluation and analysis. Differential expression of a subset of genes was confirmed using qRT-PCR. Data are now being annotated and analyzed further. This project is being monitored by weekly e-mails between the participating groups as well as telephone calls and, when possible, direct interactions at scientific meetings.
Impacts
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Publications
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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? Under National Programs 101 (Food Animal Production) and 103 (Animal Health), we address poultry, the third largest agricultural commodity in the U.S. At 41% and growing, poultry is the primary meat consumed in this country and soon the world based on current projections. Primarily due to the utilization of advanced methods of selection in breeding programs, tremendous progress in production traits has been made to meet the growing demands of consumers. As the poultry industry continues to mature, future trends can be readily predicted. First, the industry will continue consolidating into large integrated national and international units. As a result, there will probably be only two major layer (egg) and three major broiler (meat) breeding programs left in the world. Second, it is likely
that the remaining breeders will have similar objectives to target key economic traits and markets. Third, chicken rearing will become more concentrated, which reflects the limited number of poultry companies remaining. And fourth, prophylactic agents will decrease or be abolished due to consumer and regulatory pressures. While these changes allow for more economical meat and egg production, they will exacerbate the accompanying undesirable costs, e.g., reduced genetic diversity. Probably the most important issue is that disease outbreaks are likely to occur more frequently. Recent outbreaks of avian influenza, exotic Newcastle disease and other infectious diseases attest to this statement. Besides being unpredictable, these diseases complicate trade issues with foreign countries and are very costly to producers. Given the importance of disease control and animal health in general for poultry production, a detailed understanding of the immune system is warranted. The immune system is
the first line of defense against infectious agents and a beneficial response is required for vaccines to work. Insufficient immune responses have been proposed as a major factor for higher mortality rates with modern chickens raised under suboptimal nutritional or environmental conditions. An understanding of how genetic and environmental factors influence immune competence would enhance breeding programs, animal husbandry, and vaccine design to name just a few examples. In this proposal, we address and compare the immune response of chickens from characterized lines to infection with Mareks disease virus (MDV) or avian leucosis virus (ALV), which are both pathogenic viruses and economically-important disease agents. 2. List by year the currently approved milestones (indicators of research progress) Significant milestones for this project include: (1) replicate the response of chicken cells from defined chicken lines to MDV infection, (2) compare the response of chickens that respond
strongly or weakly to ALV infection, and (3) integrate and extend our results using computer algorithms. 4a List the single most significant research accomplishment during FY 2006. Identifying genes that confer resistance to Mareks disease is important for the poultry industry to continue improvement in genetic resistance as well as for scientists to understand the biological pathways. Using Affymetrix chicken microarrays, we have identified a set of genes that are differentially regulated between line 6 (disease resistant) and line 7 (disease susceptible) birds, at 4 days post inoculation with the Mareks disease virus. Several cytokines and their receptors (A19, AH221, chemotaxin 2, CD116) are expressed at higher levels in line 7 compared to line 6. However, cathelicidin, an antimicrobial peptide, and CD36 are expressed at higher levels in line 6. This shows there are genes that are associated with resistance and susceptibility to Mareks disease. This information enhances our
understanding of Mareks disease resistance, and will provide a foundation to compare with other diseases. 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Informatics Institute, University of Delaware. Additional details of research can be found in the report for the parent CRIS 3635-31320-007-00D Genomics and Immunogenetics of Economically Important Traits of Poultry, and projects 3635-31320-007-10R and 3635-31320-007-12S entitled High resolution microarray and protein analysis of chicken immune responses. 5. Describe the major accomplishments to date and their predicted or actual impact. Since the short inception of this project in 2004, this project has produced several important outcomes. The main goal of this project is to understand the molecular basis for genetic resistance to avian viruses, which should be useful for the poultry breeding industry and academic scientists. We have demonstrated that
it is possible to obtain and analyze material obtained by laser capture microdissection of MDV- infected tissues. As a result, we are able to get more consistent DNA microarray results. This technique lays the foundation for our future studies that include ALV-infected birds. These results address National Program 101 component "Genetic Improvement" and National Program 103 component "Genetic and Biological Determinants of Disease Susceptibility. "
Impacts
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Publications
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? Poultry is the third largest agricultural commodity in the U.S. At 41% and growing, poultry is the primary meat consumed in this country and soon the world based on current projections. Primarily due to the utilization of advanced methods of selection in breeding programs, tremendous progress in production traits has been made to meet the growing demands of consumers. As the poultry industry continues to mature, future trends can be readily predicted. First, the industry will continue consolidating into large integrated national and international units. As a result, there will probably be only two major layer (egg) and three major broiler (meat) breeding programs left in the world. Second, it is likely that the remaining breeders will have similar objectives to target key economic traits and markets.
Third, chicken rearing will become more concentrated, which reflects the limited number of poultry companies remaining. And fourth, prophylactic agents will decrease or be abolished due to consumer and regulatory pressures. While these changes allow for more economical meat and egg production, they will exacerbate the accompanying undesirable costs, e.g., reduced genetic diversity. Probably the most important issue is that disease outbreaks are likely to occur more frequently. Recent outbreaks of avian influenza, exotic Newcastle disease and other infectious diseases attest to this statement. Besides being unpredictable, these diseases complicate trade issues with foreign countries and are very costly to producers. Given the importance of disease control and animal health in general for poultry production, a detailed understanding of the immune system is warranted. The immune system is the first line of defense against infectious agents and a beneficial response is required for
vaccines to work. Insufficient immune responses have been proposed as a major factor for higher mortality rates with modern chickens raised under suboptimal nutritional or environmental conditions. An understanding of how genetic and environmental factors influence immune competence would enhance breeding programs, animal husbandry, and vaccine design to name just a few examples. In this proposal, we address and compare the immune response of chickens from characterized lines to infection with Mareks disease virus (MDV) or avian leucosis virus (ALV), which are both pathogenic viruses and economically-important disease agents. 2. List the milestones (indicators of progress) from your Project Plan. Significant milestones for this project include: (1) compare the response of chicken cells from defined chicken lines to MDV infection, (2) compare the response of chickens that respond strongly or weakly to ALV infection, and (3) integrate and extend our results using computer algorithms. 3a
List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Compare the response of chicken cells from defined chicken lines to MDV infection. Milestone Fully Met 2. Initiate a pilot study to compare the response of chickens that respond strongly or weakly to ALV infection. Milestone Fully Met 3. Develop computer algorithms to integrate the data. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? During FY 2006, we hope to address the following: (1) continue the DNA microarray work as well as initiate our MDV-chicken protein interactions, which should allow us to identify relevant genes and pathways that are associated with MD resistance and susceptibility, (2) initiate DNA microarrays for ALV-infected chickens,
and (3) begin to compare and contrast the initial results. During FY 2007, we hope to address the following: (1) finish all the DNA microarray work and protein interaction studies, (2) complete the DNA microarray work for ALV-infected chickens, and (3) focus on the computational analyses and integration of the results. The project is schedule to end on May 31, 2007, thus, there are no more research goals list for this project beyond that date. 4a What was the single most significant accomplishment this past year? Identifying genes that confer resistance to Mareks disease is important for the poultry industry to continue improvement in genetic resistance as well as for scientists to understand the biological pathways. A main limitation in progress using DNA microarrays has been the lack of material and the heterogeneity of infected tissues. In FY 2005, we were able to reproducibly demonstrate that we could isolate material using LCM, and obtain quality results with our DNA microarrays.
This result alleviates our concerns over limited material to work with. And it shows there are genes that are associated with resistance and susceptibility to Mareks disease. This information enhances our understanding of Mareks disease resistance, and will provide a foundation to compare with other diseases. 4b List other significant accomplishments, if any. Identifying genes that confer resistance to Mareks disease is important for the poultry industry to continue improvement in genetic resistance as well as for scientists to understand the biological pathways. A main problem with the DNA microarray technology has been to interpret the vast amount of information. One specific aim of this project is to use the DNA microarray technology to assay chicken immune responses to various viral infections. For various technical reasons, microarray data often contains missing values that hamper the subsequent data analysis. Dr. Ming Ouyang of the Informatics Institute of UMDNJ studied
computational methods that estimate the missing values. After the missing values are estimated, it is demonstrated that more meaningful analysis results can be obtained than analysis without estimation. These new findings extend our abilities to extract meaningful results from our datasets. 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Informatics Institute, University of Delaware. Additional details of research can be found in the report for the parent CRIS 3635-31320-007-00D Genomics and Immunogenetics of Economically Important Traits of Poultry, and projects 3635-31320-007-10R and 3635-31320-007-12S entitled High resolution microarray and protein analysis of chicken immune responses. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Since the short inception of this project in 2004, this project has produced several important outcomes. The
main goal of this project is to understand the molecular basis for genetic resistance to avian viruses, which should be useful for the poultry breeding industry and academic scientists. We have demonstrated that it is possible to obtain and analyze material obtained by laser capture microdissection of MDV- infected tissues. As a result, we are able to get more consistent DNA microarray results. This technique lays the foundation for our future studies that include ALV-infected birds. 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? As this project was just initiated, we not yet had the chance to transfer any information or technologies.
Impacts
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