Attempts to demonstrate Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease (CD) tissues by many laboratories 1994-1999 using a variety of sample processing and PCR conditions, lead to conflicting results. We have developed reliable procedures for the detection of MAP in fresh human intestinal tissues, incorporating mechanical disruption of the tissue lysate to access MAP DNA and PCR conditions uniquely specific for IS900. In an initial ongoing open-label clinical study, DNA is prepared from fresh ileocolonoscopic mucosal biopsies using SDS proteinase K, mechanical disruption in the Hybaid Ribolyser system, and phenol chloroform extraction. Five µl aliquots of DNA extract are amplified by nested IS900 PCR. A second mucosal biopsy is decontaminated for 20 mins in 500µl BBL Mycoprep, transferred directly to a BBL MGIT culture system containing the antibiotic cocktail PANTA and incubated at 37ºC. The cultures are tested for MAP at intervals. To date MAP has been identified in 17 of 18 (94%) of CD samples and 4 of 16 (25%) samples of uninflamed mucosa (p=0.0004). In a parallel double blinded study, fresh full thickness surgical resection gut samples from CD and control patients are cut with crossed scalpels, digested in trypsin collagenase, and lysed using BBL Mycoprep. The MAP-enriched centrifugal pellet is divided into three portions and tested for MAP using nested IS900 PCR, incubation in the MGIT system, and by feeding to cultures of Acanthamoeba polyphagia maintained for many months and in which MAP can persist and replicate. To date 23 of 44 (52%) of surgical samples are MAP positive and 21 of 44 (48%) are MAP negative. These studies confirm that new and optimized methods can reliably detect MAP in human intestine. The highly significant association of these chronic enteric pathogens with inflamed CD tissues is consistent with a causative role for MAP in CD.