|Line, John - Eric|
|Buhr, Richard - Jeff|
|Cox, Nelson - Nac|
Submitted to: International Poultry Scientific Forum
Publication Type: Abstract Only
Publication Acceptance Date: 1/28/2013
Publication Date: 1/28/2013
Citation: Oakley, B., Yoon, S.C., Line, J.E., Berrang, M.E., Buhr, R.J., Cox Jr, N.A. 2013. Fluorescence in-situ hybridization (FISH) as a tool for visualization and enumeration of Campylobacter in broiler ceca. International Poultry Scientific Forum. P.53.
Technical Abstract: Food-borne human pathogens are typically detected and enumerated by either cultural methods or PCR-based approaches. Fluorescence in-situ hybridization (FISH) is a standard microscopy tool for microbial ecology but has not been widely used for food safety applications despite important advantages over existing methods. The main strengths of FISH are the ability to design fluorescent probes to visually detect individual bacterial cells with almost any level of taxonomic resolution, and the ability to rapidly enumerate these cells of interest while maintaining information about their spatial context in the original sample. In this study, we have designed and validated several new FISH probes targeting Campylobacter and have written a new image-processing algorithm to rapidly enumerate cells from acquired images. Validations of the new algorithm show it outperforms existing ‘industry-standard’ software. Newly designed probes successfully targeted either the entire genus or different strains of C. jejuni on the basis of a single base-pair mismatch in the small subunit of the ribosomal RNA. Hybridization conditions were optimized to distinguish among C. jejuni strains ATCC49943, 33250, 81-176, and 11168. Additionally, sample preparation protocols were optimized to perform FISH directly from chicken ceca and are currently being used to investigate the effects of various anti-microbial feed additives on Campylobacter populations in the cecum.