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GOALS: Control of Campylobacter jejuni in poultry farms will significantly reduce the risk of human exposure to the bacteria and have a significant impact on food safety and public health. Genetic selection of breeding stocks for genetic resistance to bacterial colonization is an effective means of control in many agricultural environments, yet the specific genes, genomic variations, and mechanisms that underlie differences in resistance or susceptibility to C. jejuni colonization in various chicken lines have yet to be elucidated. The long term goal of this project is to assist in the development of genetic markers that can be used in the selection of chicken breeding stocks with natural resistance to C. jejuni. Understanding the genetic basis of differences in C. jejuni colonization in the chicken gut between genetic lines and individuals with different susceptibilities is the major goal of this project. <P>
OBJECTIVES: The focus of study will be to analyze genetic variation between chicken lines, specifically copy number variation. Copy number variants (CNVs) can be responsible for altered gene dosage, disrupting of genes, or varying gene regulation. CNVs are detected through the use of array comparative genomic hybridization (CGH). A fine-mapping high-resolution CGH array will be developed based on genomic regions previously identified as significant between lines. Through the use of the CGH data, along with microarray gene expression data and single nucleotide polymorphism (SNP) data currently available, the combined data will be used to identify putative regions along the chicken chromosome with association to resistance or susceptibility to C. jejuni. An association test will then be performed to validate the CNVs. A commercial broiler population will be challenged with C. jejuni. The two extremes broilers (most resistant and most susceptible to C. jejuni colonization) will be collected and CGH performed. <P> EXPECTED OUTPUTS: We expect to confirm a majority of copy number variants associated with susceptibilities to C. jejuni colonization identified in the previous studies, but with significantly narrow regions of CNVs. These results will be used in creation of integrated genomic maps using all available gene expression microarray and SNP data. We expect to identify regions of the chromosomes based on the array CGH that overlap with gene expression microarray and SNP data. This information will allow the identification of several unique chromosome regions with at least one genetic marker. Through association analysis, we expect to identify significant CNVs that are associated with C. jejuni colonization in broilers. With the high-density of CGH array, along with correlation to the microarray and SNP data, we further expect to be able to identify genomic regions close to the quantitative trait loci region underlying C. jejuni resistance or susceptibility for future functional studies.
NON-TECHNICAL SUMMARY: <BR> The bacterium Campylobacter jejuni is one of the most commonly reported cause of foodborne infection in the United States. The major source of C. jejuni contamination in the U.S. is chickens. Therefore, control of C. jejuni in poultry farms would significantly reduce human exposure. One major strategy in protecting the food supply from C. jejuni is to decrease the bacteria that colonize the host through genetic selection. Currently, two chicken broiler lines have been identified with genetic differences in their susceptibility to C. jejuni colonization; however, the genes, genomic variations, and mechanisms that underlie these differences are unknown. The major goal of this project is aimed at elucidating the genetic variation that exists among broilers associated with resistance to C. jejuni colonization. Preliminary data available from these lines suggests copy number variation (CNV) could play a role in the differences. The objectives are to generate a high-resolution CNV chicken array and use these genetic lines to pinpoint the genomic regions that differ between birds susceptible and resistant to C. jejuni colonization. The identified CNVs will be validated in a commercial broiler population challenged with C. jejuni. By integrating this CNV experimental data with other chicken genetic information available for these lines, such as gene expression and Single Nucleotide Polymorphism (SNP) data, a high-resolution genomic road map will be constructed. The expected outcomes are to complete this combined genomic marker map and to identify regions along the chicken chromosomes associated with C. jejuni resistance. The results generated will impact the safety of the U.S. food supply by leading to the development of markers that can be used in the genetic selection of breeding stocks with resistance to the bacteria.
<P> APPROACH: <BR> The broiler chickens used will be two distinct genetic broiler lines (one line is resistant to C. jejuni colonization, while the other one is susceptible to C. jejuni colonization). A number of CNVs that have significant aberrations between these two lines have previously been identified. Based on these regions identified, a high-density array CGH will be produced for fine-mapping of the genomic aberrations. The fine-mapping CGH array will be designed using the Agilent eArray software by uploading the genomic intervals found to be significantly different between C. jejuni resistant and susceptible broiler lines in the 244K array. Genomic DNA will be used to hybridize to the high-density fine-mapping array CGH. Sample processing from DNA labeling to scanning will be performed according to Agilent CGH protocols. Significant CNVs will be called using Agilent's Genomic Workbench software. A subset of significant data will be randomly validated by quantitative PCR. The data extracted from the fine-mapping CGH array will be integrated with the data from previous microarray and SNP data based on challenges of chicken broilers with C. jejuni. This data will produce an integrated genomic map of genomic regions between populations of C jejuni susceptibility or resistance. Data will be aligned manually using the JMP genomic software and Microsoft applications including Excel spreadsheets and Access. The array CGH data will be correlated with gene expression microarray and SNPs at various stringency conditions. For association study, chicken broilers will be purchased from commercial breeding sources. A total of 200 birds will be used for the challenge experiment. A total of 20 birds will be used as a preliminary screen for C.jejuni contamination. One hundred eighty 1-day old chickens will be orally inoculated with C. jejuni. At 7 d post-inoculation, whole blood will be collected; then, the birds will be sacrificed by carbon dioxide asphyxiation and the cecum removed from each bird. The cecal contents will be filtered plated on selective agar. After incubation, the number of colonies will be counted. After the plate counts, samples will be divided into two groups based on the number of colonies: the highest and lowest number of bacterial colonies observed. Of the 180 samples, the 18 most resistant and the 18 most susceptible individual samples, based on the number of bacterial colonies, will be selected for genomic DNA isolation. Array CGH will be performed on these individuals as previously completed. The results will be used to validate those genomic regions with significant association in the population to C. jejuni colonization. The project will be successful as measured by the following. First, the research should meet the two-year timeline as proposed. Second, at least two publications are expected from the results of these data. Third, the data from this project will ultimately be available to the public so that future functional studies may be initiated.