Genetic Association Between Copy Number Variation (CNV) and Resistance to Campylobacter Jejuni Colonization in the Chicken

<p>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.
</p><p>PROGRESS: <br/>2012/01 TO 2013/08 <br/>Target Audience: The target audience for this project is the poultry genetic scientific community and those with an interest in chicken immunology and/or food safety research. Changes/Problems: There were no major problems to report. We did, however, make a change in one approach for our last goal. This goal was to use an array CGH platform to validate some of our findings in a commercial population of broiler chicken. We changed technologies from an array approach to a high-throughput sequencing approach. This approach is more powerful than using an array, as we were able to sequence to a high coverage, while the array was only able to cover a portion of the genome and is based on hybridization dynamics. What opportunities for training and professional development has the project provided? TRAINING: This project involved several
laboratory techniques in which students and visiting scientist were trained. As this project also involved animal trials, training was provided to students and scientists on proper handling and surgical procedures. The PD was further trained by the mentor on array and sequencing technical methods as well as bioinformatics methods. PROFESSIONAL DEVELOPMENT: Professional development was provided for as the PD was afforded the opportunity to present data at major conferences. This also presented networking opportunities for the PD as well as further learning about the USDA grant process at the PD meeting in Washington D.C. How have the results been disseminated to communities of interest? This information has been disseminated as both oral and poster presentations at several major conferences. Information is further disseminated by publication in peer-reviewed journals. Genetic maps, custom
array platforms, array data and sequencing data will be released to the public upon publication and/or submitted to public repositories. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported. <br/>
PROGRESS: <br/>2012/01/01 TO 2013/08/14 <br/>Target Audience: The target audience for this project is the poultry genetic scientific community and those with an interest in chicken immunology and/or food safety research. Changes/Problems: There were no major problems to report. We did, however, make a change in one approach for our last goal. This goal was to use an array CGH platform to validate some of our findings in a commercial population of broiler chicken. We changed technologies from an array approach to a high-throughput sequencing approach. This approach is more powerful than using an array, as we were able to sequence to a high coverage, while the array was only able to cover a portion of the genome and is based on hybridization dynamics. What opportunities for training and professional development has the project provided? TRAINING: This project involved several
laboratory techniques in which students and visiting scientist were trained. As this project also involved animal trials, training was provided to students and scientists on proper handling and surgical procedures. The PD was further trained by the mentor on array and sequencing technical methods as well as bioinformatics methods. PROFESSIONAL DEVELOPMENT: Professional development was provided for as the PD was afforded the opportunity to present data at major conferences. This also presented networking opportunities for the PD as well as further learning about the USDA grant process at the PD meeting in Washington D.C. How have the results been disseminated to communities of interest? This information has been disseminated as both oral and poster presentations at several major conferences. Information is further disseminated by publication in peer-reviewed journals. Genetic maps, custom
array platforms, array data and sequencing data will be released to the public upon publication and/or submitted to public repositories. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported. <br/>
PROGRESS: <br/>2012/01/01 TO 2012/12/31 <br/>OUTPUTS: The following outcomes have been reached since the beginning of this project. ACTIVITIES: A candidate pool of Copy Number Variants (CNVs) in four genetic lines (two inbred and two broiler lines) has been identified through the use of the Agilent chicken 244K array Comparative Genomic Hybridization (aCGH). We took those candidate CNVs and built a custom Agilent aCGH platform to saturate the identified CNVs with probes in order to reduce the spacing, referred to here as fine-mapping, of the chromosomal aberrant regions closer to the Quantitative Trait Loci (QTL) affecting genetic resistance to Campylobacter colonization. The original line A and line B broiler samples were then analyzed by the custom Agilent aCGH. We now have a list of candidate CNVs that have been fine-mapped from the original 244K platform. We have since
aligned this data with existing data (including but not limited to gene expression information, gene ontologies, QTL databases) and created a genetic map in which these regions might be associated with Campylobacter colonization resistance. We have further validated a selected number of these identified CNV regions using alternative experimental approaches including quantitative and digital PCR. These experiments conclude 2 of 3 goals of the project. EVENTS: We have presented this data at the 21st Annual International Plant and Animal Genome Conference in San Diego, California and the 33rd International Society of Animal Genetics Conference in Cairns, Australia. PRODUCTS: We created a custom Agilent 4x44K chicken aCGH array for fine-mapping chromosomal aberrations. We have also created a genetic map with candidate regions that might affect Campylobacter jejuni colonization resistance in
the chicken. This information has been disseminated as both oral and poster presentations at several major conferences. Information will further be disseminated by publication in peer-reviewed journals; the preparation of manuscripts for publication is underway. Genetic maps and custom array platforms will be released to the public upon publication. PARTICIPANTS: INDIVIDUALS: The PD, Jason Abernathy, was the main participant of this project as this was his postdoctoral fellowship. Jason performed the necessary laboratory experiments and data analyses to meet the goals specified in the grant proposal. Throughout the duration of the project, the mentor, Huaijun Zhou, provided guidance where needed and allowed the PD the use of all general laboratory supplies and equipment. PARTNER ORGANIZATIONS: The USDA-ARS in College Station, Texas allowed the use of facilities and equipment as specified
in the proposal. COLLABORATORS: Our collaborator at the USDA-ARS in College Station, Michael Kogut, provided use of the facilities, equipment, and chicken samples to our laboratory. He also provided skilled scientific expertise. TRAINING AND PROFESSIONAL DEVELOPMENT: The PD has been afforded many opportunities for training and professional development after being awarded this postdoctoral fellowship. The PD has presented this work at major scientific conferences and is preparing for publications. As the PD, and with the support of the mentor, Jason has been allowed to lead a team of undergraduate students, graduate students, and a postdoc in the execution of major parts of the project, such as field work with chickens and large-scale genomic data analyses. Jason attended his first PD meeting in Washington D.C., and as such was able to learn more about the USDA grant process and provide
feedback on the fellowships program. Additionally through the use of travel funds from this grant, the PD was able to network with fellow scientists, sharpen presentation skills, and establish potential collaborations. The PD also had informal bioinformatic training from the mentor and other institute members in order to meet the established goals. TARGET AUDIENCES: The target audience is the poultry / genetic scientific community as well as the general public with an interest. EFFORTS: Partial information has been provided at scientific conferences; complete information will be provided at completion of all goals and publication of the work. PROJECT MODIFICATIONS: Not relevant to this project.</p>