Blood samples from 58 subjects were blindly coded and investigated by three independent laboratories for the presence of Mycobacterium avium subspecies paratuberculosis (MAP) using MGITpara specialized media and nested PCR. Consequently, viable MAP was detected in 22/40 (55%) IBD (11/20 CD and 11/20 UC) compared to 4/18 (22%) NIBD (P<0.009). At least two centers detected MAP in 41% IBD samples compared to none (0%) in NIBD (P<0.0001). No sample was positive by all three centers. Despite result variability between centers, the study strongly demonstrates that using MGITpara culture media and nested PCR are essential for successful detection of MAP in human blood. Overall, the study supports a mycobacterial role in CD pathogenesis.