Development of an <i>in situ</i> hybridization assay for the diagnosis of <i>Mycobacteriaceae</i> infections of veterinary importance

Mycobacteria (<i>Mycobacteriaceae</i> family) comprise five genera (<i>Mycobacterium</i>, <i>Mycolicibacterium</i>, <i>Mycolicibacter</i>, <i>Mycolicibacillus</i>, and <i>Mycobacteroides</i>), which include relevant animal and human pathogens. Histology is a rapid method for preemptively diagnosing mycobacteriosis, contributing to surveillance, control, and eradication. A constraint on histology is the limited sensitivity and specificity of acid-fast stains, as the number of detectable bacilli in formalin-fixed paraffin-embedded (FFPE) tissue varies and other microorganisms are acid-fast positive. Immunohistochemistry has low specificity and is cross-reactive with other bacteria. We developed an RNAscope probe-based <i>in situ</i> hybridization (ISH) assay, targeting a conserved sequence of <i>16S rRNA</i> gene of <i>Mycobacteriaceae</i>, and tested it on archived FFPE tissues from 22 mammals, birds, amphibians, and fish, collected between 1999 and 2024, infected with 23 species of mycobacteria of veterinary importance, and tissue from 7 animals infected with other bacteria. <i>Mycobacterium</i> spp. (n = 17), <i>Mycobacteroides</i> spp. (n = 2), <i>Mycolicibacter</i> spp. (n = 1), and <i>Mycolicibacterium</i> spp. (n = 3) confirmed infected tissues were tested, and results were compared with 2 acid-fast stains, Ziehl-Neelsen and Fite-Faraco, and <i>Mycobacterium</i> spp. PCR from FFPE tissues. Hybridization signals were detected in all FFPE tissues, archived for up to 25 years, with confirmed <i>Mycobacterium</i> spp. (17/17; 100%), <i>Mycobacteroides</i> spp. (2/2), <i>Mycolicibacter</i> spp. (1/1), and <i>Mycolicibacterium</i> spp. (3/3), including cases with few or no acid-fast bacilli. Available FFPE tissues were positive by PCR (15/15, 100%). Hybridization signal was not identified in other bacterial infections. This ISH assay is a rapid screening and specific diagnostic tool for mycobacteria in FFPE tissues.