The insertion sequence IS900 has been considered highly specific for Mycobacterium avium subsp. paratuberculosis until the discovery of an IS900-like element in non- Mycobacterium avium subsp. paratuberculosis strains, isolated from ruminant feces. Nevertheless, IS900-PCR still remains widely used as diagnostic PCR because the strains containing the IS900-like element are thought to be exceedingly rare.408 bulk milk samples were analysed in the period August 2004 - February 2005 by traditional cultural methods on Herrold's egg yolk medium with mycobactin; suspected colonies were confirmed by IS900-PCR.We isolated 21 mycobactin-independent Mycobacteria (5.1%), positive for IS900-PCR, which failed to be Mycobacterium avium subsp. paratuberculosis by restriction endonuclease analysis of the amplified products.One strain was subjected to further investigations by 16S rRNA gene sequencing using the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system (Applied Biosystems Division, Foster City, Calif:); sequence analysis by Microseq database system could identify Mycobacterium porcinum with 100% homology.The high number of IS900 false positive results indicates, once again, that PCR results based on IS900 alone should be interpreted with caution. In particular when analysing milk, we strongly recommend to confirm the presence of Mycobacterium avium subsp. paratuberculosis by primary culturing (mycobactin dependency) or molecular methods different from IS900-PCR.

Sponsorship

Attendance to this Congress was sponsored by the EU-funded project SSPE-CT-2004-501903