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Despite an increased understanding of how virulence factors enable avian pathogenic Escherichia coli (APEC) to develop infection and cause disease, practical application of this information to prevent significant economic losses in the poultry industry have not been forthcoming. Most of this research has focused on elucidating the mechanism(s) by which APECs enter the bloodstream, and yet still remain unknown. Consequently, prevention of APEC infection and disease relies on environmental control measures, antibiotherapy, and more recently, vaccines which are effective only against homologous strains. This proposal describes a different investigative approach that focuses on the most direct method of APEC control: reduction [elimination] and control of gastrointestinal APEC contamination.
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We propose a series of microarray and possibly RNA-Seq experiments designed to first measure responses of the entire transcriptome in both the host chicken and APEC simultaneously under controlled notobiotic (anexic) conditions. We will: <OL> <LI> Evaluate and cross-validate APEC-O1 and Gallus gallus microarray designs;<LI> Determine limits of temporal lethality in RPRL-71 chicks orally challenged with APEC-O1; <LI>Establish baseline transcriptome profile in APEC-O1 and RPRL-71 chicks; <LI>Perform time-course oral challenge experiments on day-old RPRL-71 chicks; <LI>Evaluate experimental transcriptome profiles in both APEC-O1 and RPRL-71 chicks.
</ol> Comparative genomic techniques will allow us to interrogate not only the adhesive elements of the APEC in the gastrointestinal tract but also the manner and degree to which the host responds to its presence. Ultimately, we foresee the engineering of persistent probiotics containing the APEC genetic elements required for in vivo adhesion, but instead competitively exclude APEC. Advantageously, the generality of our approach provides an analytical framework applicable to study any host:pathogen interaction.
NON-TECHNICAL SUMMARY: Despite implementation of various therapies, bacterial infections of poultry continue to persist and circulate in the environment posing serious concerns from both agricultural and human health perspectives. Because certain bacterial species can cross species barriers and are known to lethally infect humans, significant concern has increased regarding mechanisms of host:pathogen relationships. The implications of zoonosis have prompted researchers whether it might be possible to develop poultry flocks resistant to certain bacterial strains. Our research will determine how a specfific pathogenic bacterium (E. coli O2; APEC) persists in the gastrointestinal tract of the chicken. The generality of our approach will provide a model of analysis suitable for use in any host:pathogen relationship. Thus, it provides a means to further interrogate many other aspects of such relationships not intrinsic to this specific proposal. Successful completion of this reserach will provde a means to reduce the threat of APEC to the poultry industry thus protecting a multi-billion dollar worldwide enterprise. It will also reduce significantly the potential for poultry operations to act as reservoirs or bridging hosts of zoonotic bacterial strains and contribute to overall pre- and post-harvest food safety.
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APPROACH: Aim(1): We will initiate control tests to evaluate the performance of each microarray design. This will be accomplished by first hybridizing genomic DNA of each organism to its respective microarray. Those targets failing to provide sufficient signal under the defined hybridization conditions will be pruned from the total set of targets. This information allows us to determine unambiguously whether or not a specific probe sequence occurs in the transcriptome in a given sample, at least to detectable limits. Aim(2): White Leghorn chicks will be challenged orally with defined doses of APEC-O1 and observed for lethal effects. For each of three dose experiments (1x105, 1x106, and 1x107 cfus), we will begin with 10 chicks and monitor their responses over a period of 96 hours. Chicks will be necropsied and the parabronchi, air sacs, and gastrointestinal tract will be examined intensively for any pathology consistent with APEC infection. If a positive result is obtained, PCR will be used to confirm genetically APEC-O1 presence with our APEC-O1 specific primer sets. Aims(3/4): For APEC-O1, we will evaluate the transcriptome under two conditions: (1) minimal (MOPS) and (2) rich defined media. Transcriptome evaluation for notobiotic RPRL-71 chicks will be calibrated to the time course corresponding to APEC oral challenge. We will analyze the ileum as it appears to be the primary reservoir of APEC. To increase the quality of Gallus total RNA, poly-A subtraction will be performed prior to cDNA synthesis. Quality control of the RNA samples will be assessed with an Agilent 2100 Bioanalyzer located at the UW GEC. Aim 5: The first phase of data analysis will be to evaluate the reliability of the microarrays. The next stage is to identify differences in transcript abundance for controls in both the APEC-O1 cultures [minimal and rich defined media] and non-challenged, notobiotic RPRL-71 chicks [ileum at 6 and 24 hrs]. The most revealing analyses will be made by comparing changes in the transcript abundance of APEC-O1 and Gallus during the time-course challenge.