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<OL> <LI> Develop fluorescent hybridization probes for identification of non-culturable Listeria monocytogenes (Lm) biofilm growth from turkey synovial tissues by fluorescense in situ hybridization (FISH) and develop an FISH detection assay for Lm biofilm growth in macrophage cell culture on glass coverslips.<LI> Develop a real-time quantatative PCR assay (TaqMan TM) for detecting nonculturable Lm in mixed culture biofilms isolated from synovial tissues of Lm challenged turkeys.
APPROACH: Initially, we will develop fluorescent hybridization probes for identification of the available sequences for established Lm virulence genes, including Iap (Invasion Associated Protein), Hly (Hemolysin), and Rib (Ribosomal Operon) genes using PCR. Procedures for probe labeling will be obtained from Current Protocols in Cytometry. These probes will be utilized to screen turkey synovial biofilm infected macrophage cell cultures and cell cultures infected with Lm strains Scott A and V7 using FISH methodology as detailed in Current Protocols in Cytometry. We will develop a real-time quantatative PCR (Taqman TM) assay using an ABI Prism 7700 Sequence Detector to detect the presence of Lm in synovial biofilm cultures using methods developed at the NADC. Biofilm material from turkey synovial joints that has been shown to invade avian macrophages will be tested for cytotoxicity in an established in vitro virulence assay utilizing a Mouse (Ped-2E9) hybridoma cell line. A previously described in vitro virulence assay for L. moncytogenes will be used with some modification.