TO THE EDITOR: Mycobacterium avium subsp paratuberculosis (MAP) is a slow growing, acid fast bacillus that is mycobactin-dependent and an intracellular pathogen of the intestinal tract of animals with Johne's disease. Characteristics seen in Johne's disease include weight loss, diarrhea, granuloma, and lymph node alterations resembling those in humans with Crohn's disease (CD) (1). Consequently, MAP has been sought in intestinal tissue specimens from CD patients with increasingly positive results, leading to speculation that this microorganism is associated with CD. Most recently, MAP has been identified in commercial pasteurized milk, raising speculation that CD may be associated with consumption of contaminated cattle milk (2). To date. many studies have reported the presence of MAP in CD tissue specimens after long-term incubations (3, 4). None of these studies included breast milk specimens from lactating CD mothers. In our laboratory, we recently developed a short-term culture protocol using modified 7H9 broth media with supplements to culture MAP from tissue specimens obtained from CD patients. Using this protocol, MAP was present in more CD tissue specimens than in controls.
In this study, seven breast milk samples (two mothers with CD and five healthy controls who have recently given birth) were investigated for the presence of MAP. Milk samples were centrifuged and each of the cream and centrifugal pellets was transferred to new tubes, decontaminated, and then inoculated into two different 7H9 broth base media. The l2B* Bactec bottles and the Mycobacterial Growth Indicator Tube (MGIT) supplemented with OADC enrichnient, Mycobactin J, and PANTA (Polymyxin B, Amphoterin B, Nalidixic acid, Triniethoprim and Azlocillin) mixture (Becton Dickinson). After 12 wk of incubation at 370C and 5% CO2. acid fast staining, mycobactin dependency, PCR analysis using two 1S900-derived oligonucleotide primers (P90: 5'-TCGGGGCCGTCGCTTAGG-.3' and P91: S'-GAGGTCGATCGCCCACGTGA-3') and hybridization with an internal probe were performed (5). Although microbial growth was observed in some cultures, only MGIT media inoculated with centrifugal pellets from each of the mothers with CD were positive for the presence of MAP (Fig. 1, lanes 4 and 6). MAP was not present in the cream fraction of both CD samples (Fig. 1, lanes 3 and 5). The five control samples were negative for MAP in all fractions using both media. Cultures positive for MAP were subcultured in MGIT media and on 7H10 agar plates supplemented with OADC in the presence and absence of rnycobactin J. The colonies were confirmed to be acid fast bacillus and myeobactin-dependent. As shown in Figure 1. the 400 bp PCR-amplified fragment analyzed on 2.0% agarose gel (A) from CD cultures was also confirmed by hybridization (B). Moreover, the 400 bp fragment was sequenced and the analysis provided 99% homology with the 1S900 sequence.
This report is the first to describe the isolation of MAP from the milk of humans with CD. It also describes a practical protocol for short-term cultivation of MAP from clinical specimens. The finding adds more support to the MAP role in CD pathogenesis. Interestingly, the data further demonstrates the gross similarity between humans diagnosed with CD and Johne's disease mammals infected with MAP.
Our thanks are due to COHPA and CD3 at UCF for financial support, to patients for providing milk samples and to Dr. John Biggerstaff and Chris Piromalli of Florida Hospital and Dr. Najih Naser of NovaSense for their technical advice.
Saleh A. Naser, Ph.D.
Deidre Schwartz, B.S.
Department of Molecular Biology and Microbiology Center for Discovery of Drugs and Diagnostics
University of Central Florida
Orlando, Florida
Ira Shafran, M.D.
Florida Hospital
Orlando, Florida
Reprint requests and correspondence: Saleh A. Naser, Ph.D.. Molecular Biology and Microbiology. University of Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816.
Received Nov. 10, 1999; accepted Nov. 30. 1999
.Figure 1. PCR detection of MAP from cultured milk samples from CD patients. PCR products were analyzed on 2.0% agarose gel (A) and by southern hybridization (B) using a nonradioactive labeled probe derived from the 1S900. Lane I represents DNA from MAP strain 43015, lane 2 has no DNA; lanes 3 and 5 represent DNA from MGIT cultures inoculated with cream fractions of two CD milk samples: and lanes 4 and 6 represent DNA from MGIT cultures inoculated with centrifugal pellets from same samples. M is the molecular weight marker in base pair (bp). The presence of 400-hp amplified fragment is indicated with arrow.