Assessment of an in situ hybridization test for the diagnosis of paratuberculosis (Mycobacterium avium subsp. paratuberculosis infection) in goats.

Paratuberculosis (PTB) is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis (MAP). The gold standard test for detection of MAP is culture, which requires 8-16 wk. We reviewed archived intestinal cases of goat PTB and performed RNA scope-based chromogenic pan-mycobacterial in situ hybridization (ISH) on formalin-fixed paraffin-embedded (FFPE) tissue in 20 cases and 5 negative controls. Cases included 11 multibacillary and 9 paucibacillary infections, 2 of which had postmortem autolysis. At the time of diagnosis, we compared the results with ancillary testing (MAP culture and/or PCR testing of fresh tissues), Ziehl-Neelsen (ZN) staining, and pan-Mycobacterium PCR testing of FFPE tissues. MAP had been confirmed in 9 of 10 cultured cases. MAP had been detected by PCR in fresh tissue in 7 of 7 cases. We detected Mycobacterium spp. by PCR in FFPE tissue in 18 of 20 cases; the 2 negative cases had been confirmed at the time of diagnosis. We detected hybridization signal in 19 of 20 cases, including 8 of 9 paucibacillary cases, in 6 cases of which we did not detect bacilli by ZN; the case lacking hybridization signal had severe autolysis. We did not see hybridization signal in the negative controls. We found that ISH and PCR tests of FFPE tissues are more sensitive than ZN, can reduce turnaround time, and contribute to the diagnosis of PTB, especially when fresh tissue is unavailable for culture and PCR. Incorporation of ISH into an integrated diagnostic framework could aid in the confirmation of MAP infection.