Failure to detect M. avium subspecies paratuberculosis in Johne’s disease using a proprietary fluorescent in situ hybridization assay

Objectives Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in ruminants. The “gold standard” of MAP detection is by culture, DNA sequencing possibly supplemented by identification of Ziehl–Neelsen positive mycobacteria. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view®) fluorescent in situ hybridization (FISH) assay for MAP RNA. Intestine from a steer with documented Johne’s disease was assayed according to the manufacturer’s instructions. Probes were custom designed for MAP and bovine β-actin (as the eukaryotic housekeeping gene) from published genomes. We attempt to prevent false positive signal in the “no-probe” control, by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and “Hope” for Cy-5).