Mycobacteria distinct from Mycobacterium avium subsp paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable by IS900 polymerase chain reaction: implications for diagnosis

PCR targeting the 5' end of IS900 has been considered specific for identifi cation of Mycobacterium avium subsp. paratuberculosis and is frequently app lied to confirm the presence of this organism in the diagnosis of Johne's d isease. IS900 PCR has also been applied to studies of the aetiology of Croh n's disease. Mycobacterium spp. isolated from the faeces of 3 clinically no rmal animals in 2 Australian slates appeared not to be M. paratuberculosis but were positive by IS900 PCR. The isolates were characterized using mycob actin dependency, biochemical tests, IS900 and 16 S rRNA sequencing and res triction fragment length polymorphism (RFLP) using IS900 as probe. DNA sequ encing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS900 amplified, were most closely relat ed to Mycobacterium scrofulaceum, and confirmed the usefulness of restricti on enzyme analysis of amplified product to identify the false positive resu lts. RFLP analysis with BstEII detected three to five copies of the IS900-l ike element in the isolates. These were located in molecular weight fragmen ts that were clearly different to IS900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to prov ide differentiation of M. paratuberculosis from these organisms. We recomme nd the adoption of restriction endonuclease analysis of IS900 PCR product a s a routine precaution to prevent the reporting of false positive IS900 PCR results. (C) 1999 Academic Press.