Enhancing Food Safety Through Control of Food-Borne Disease Agents

NON-TECHNICAL SUMMARY: The types of bacteria that are important for prevention of the establishment of pathogenic bacteria in the chicken cecum are currently not known. The goal of the study is to detect those bacteria that adhere to the inner surface of the cecum and might form barriers for the entry of pathogenic bacteria such as Salmonella into the tissue of the chicken.

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APPROACH: The identification of the cecal bacteria adhering to the cecal wall (mucus layer) will be done by using the fluorescent in-situ hybridization (FISH) technique. This technique is advantageous over other approaches because no culturing of bacteria is necessary and no separation of luminal and adhering bacteria is required. Fourteen oligonucleotide probes complementary to 16S ribosomal RNA sequences for groups of bacteria known to be inhabitants of the chicken cecum have been selected from the literature. A panel of representative species of bacteria has been assembled and will be used to test the specificity of the probes. Hybridization conditions will be varied by the use of formamide at different concentrations. The bacteria will be fixed using methanol/paraformaldehyde and bound to microscope slides. After dehydration with ethanol, hybridization solution containing an oligonucleotide probe will be added and enclosed in a hybridization chamber to prevent evaporation. Hybridization will be done overnight at 50C. The hybridization chambers will be removed and the slides will be washed in buffer to remove unbound probe. An antifade reagent will be spotted onto the slide and covered with a cover slip. The results of the hybridization procedure will be viewed with a confocal microscope. Once appropriate hybridization conditions have been established, the probes will be used to detect bacteria in tissue sections of chicken cecae. The tissue sections will be prepared either from frozen ceca and cryosectioning, or by embedding fixed tissue in plastic resin and sectioning with a microtome. The tissue sections (appr. 20 um thick) will be bound to microscope slides, and hybridization and wash procedures will be done as described above. Confocal microscopy will be used to view the slides and to detect the location and prevalence of bacteria exhibiting fluorescence with a specific probe. We plan on applying FISH to tissue sections from different chicken and from different locations within the cecum. If the hybridization data reveals that certain types of bacteria are preferentially adhering to the cecal wall, culturing of these bacteria can be attempted in future experiments.