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

Authors: GREENSTEIN, ROBERT - James J Peters Vamc; SU, LIYA - James J Peters Vamc; FAM, PETER - James J Peters Vamc; Stabel, Judith; BROWN, SHELDON - James J Peters Vamc

Submitted to: BMC Research Notes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/6/2018
Publication Date: 7/21/2018
Citation: Greenstein, R.J., Su, L., Fam, P.S., Stabel, J.R., Brown, S.T. 2018. Failure to detect M. avium subspecies paratuberculosis in Johne’s disease using a proprietary fluorescent in situ hybridization assay. BMC Research Notes. 11:498. https://doi.org/10.1186/s13104-018-3601-5.
DOI: https://doi.org/10.1186/s13104-018-3601-5

Interpretive Summary: Johne's disease is a chronic, debilitating intestinal disorder in cattle characterized by diarrhea, reduced feed intake, weight loss and death. Cattle usually become infected as young calves by ingesting feces containing the causative bacteria. During the subclinical phase of infection the animal may be shedding the organism in its feces without showing any clinical signs of disease. In addition infected cattle can shed the organism into the milk, thereby presenting a potential mode of infection for humans. Infection with Mycobacterium avium subsp. paratuberculosis is difficult to diagnose and therefore to control. Development of accurate and sensitive diagnostic tests is critical for control of this disease. In the present study, the sensitivity and specificity of a commercial fluorescent RNA probe assay on tissue from infected cattle was evaluated. Results suggest that this test was nonspecific and, therefore, would not be useful for the detection of infection. Although this study reports negative results it is important to make these results known so that other researchers and clinicians do not utilize this assay for their diagnostic work.

Technical Abstract: Background: M. 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. Methods: 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 ß-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.) Principal Findings: 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. Significance: We conclude 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.