title: Use of short-term culture for identification of Mycobacterium avium subsp. paratuberculosis in tissue from Crohn's disease patients.
abstract: OBJECTIVE
  To investigate the role of Mycobacterium avium subsp. paratuberculosis (MAP) in Crohn's disease (CD), using short-term mycobacterial culture media.
  
  
  METHODS
  Sixty-three tissue specimens from 27 CD patients and 36 controls were processed and inoculated into a modified 7H9 broth base medium and incubated at 37 degrees C and 5% CO2 for up to 1 year. Acid-fast staining, determination of mycobactin dependency, PCR analysis using two IS900-derived oligonucleotides and hybridization with an internal probe were performed.
  
  
  RESULTS
  MAP was present in six of seven (86%) surgically resected tissue samples and in four of 20 (20%) biopsies, with an overall 37% from CD patients, as compared to two of 36 (5.6%) of control specimens. The presence of MAP in Mycobacterial Growth Indicator Tube (MGIT) cultures was detected within 10-12 weeks for surgically resected tissue and after 40 weeks for biopsy specimens, with no MAP growth detected in 12B* Bactec cultures.
  
  
  CONCLUSIONS
  Because MAP was present in 86% of resected tissue compared to 20% of biopsy specimens from CD patients, we speculate that MAP resides in the submucosal layer closer to the active part of the ulcer rather than on the surface of the mucosal cells. Thus, surgically resected tissue cultured in MGIT medium is a favorable protocol for rapid cultivation of MAP and for investigating its role in CD pathogenesis. The data support the mycobacterial role in CD pathogenesis.
authors.affiliation: Department of Molecular Biology and Microbiology, Center for Discovery of Drugs and Diagnostics, University of Central Florida, Orlando, Florida 32816, USA.
authors.forename: D
authors.initials: D
authors.lastname: Schwartz
authors.forename: I
authors.initials: I
authors.lastname: Shafran
authors.forename: C
authors.initials: C
authors.lastname: Romero
authors.forename: C
authors.initials: C
authors.lastname: Piromalli
authors.forename: J
authors.initials: J
authors.lastname: Biggerstaff
authors.forename: N
authors.initials: N
authors.lastname: Naser
authors.forename: W
authors.initials: W
authors.lastname: Chamberlin
authors.forename: S A
authors.initials: SA
authors.lastname: Naser
mesh_terms: D003424:Crohn Disease / Q000382:microbiology* / Q000473:pathology; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D009162:Mycobacterium avium / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial