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. 2018 Jul 21;11(1):498.
doi: 10.1186/s13104-018-3601-5.

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

Robert J Greenstein  1   2 , Liya Su  3 , Peter S Fam  4 , Judy R Stabel  5 , Sheldon T Brown  6   7
Affiliations

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

Robert J Greenstein et al. BMC Res Notes. .

Abstract

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).

Results: Repetitively, false positive signal was observed in our "no probe" negative control. Attempts to correct this according to the manufacturers suggestions, and with multiple derivative techniques have been unsuccessful. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view® cannot be used to detect MAP in pre-frozen intestine of cattle with Johne's disease.

Keywords: In situ hybridization; Johne disease; Mycobacteria; Mycobacterium avium subspecies paratuberculosis.

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Figures

Fig. 1
Fig. 1
A composite of four images of the same section, of Bovine Johne’s disease tissue. a DAPI; b Texas Red (IS900); c Cy-5 (Bovine β-actin) d composite of a–c. With probes: Note “positive: signal in b–d. Marker bars, in µm, indicate magnification of x 40
Fig. 2
Fig. 2
“No-probe” control for Fig. 1. Processed identically as in Fig. 1, during the same experiment, but no probes were added during the hybridization step. a DAPI; b Texas Red (IS900); c Cy-5 (Bovine β-actin) d composite of a–c. No- probes: Note “positive” signal in the “No-probe” control b–d. Marker bars, in µm, indicate magnification of x 40
Fig. 3
Fig. 3
“No-probe” negative control for Additional file 7. This was a 35-min exposure to 0.2 M HCl. a DAPI, identifying the presence of DNA. Note the positive signal in this “No-probe” control for b–d. Marker bars, in µm, indicate magnification of x 40
See this image and copyright information in PMC

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