In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy.

The diversity in the methodology employed to investigate Crohn's disease (CD) etiology has added significantly to the controversy of the mycobacterial role in this chronic inflammatory bowel disease. Mycobacterium avium subsp paratuberculosis (MAP), a proposed and suspected agent in many CD patients, is a fastidious and very slow grower bacillus, which causes Johne's disease (JD) in cattle. The methodology that has been widely and successfully used for isolation and identification of MAP from and in JD animals is not reliable and has proven to be unsuccessful in achieving the same objectives for CD diagnosis. In this study, a Confocal Scanning Laser Microscopy (CSLM) system has been employed in an attempt to detect MAP in CD patient. In situ hybridization was performed on full thickness tissue using rabbit anti-MAP polyclonal antibody that was adsorbed with E. coli protein extracts. Consequently, MAP was detected in the microvilli region in tissue specimens from CD patient and not in the controls. In the same CD tissue specimen, MAP was not detected when isotype normal rabbit sera was employed. The polyclonal antibody marker may be replaced with monoclonal antibodies, if available, or with MAP-specific-DNA or RNA probes. This technique adds an additional approach to investigate MAP role in CD etiology especially when the culture approach is long and inconsistent.

Open clinical trial of rifabutin and clarithromycin therapy in Crohn's disease.

BACKGROUND: Crohn's disease, an inflammatory bowel disease in humans, has a suspected aetiology of Mycobacterium avium subsp. Paratuberculosis. AIMS: To evaluate the role of rifabutin and clarithromycin anti-Mycobacterium avium subsp. Paratuberculosis treatment in Crohn's disease patients using an open clinical trial. METHODS: . A total of 36 patients with acute presentations of Crohn's disease, whose sera tested positive against p35 and p36 antigens (two recombinant proteins of Mycobacterium avium subsp. Paratuberculosis), were selected for treatment with rifabutin and macrolide antibiotic therapy Rifabutin and macrolide antibiotic therapy medications included 250 mg 1 po bid clarithromycin and 150 mg 1 po bid Ri-fabutin accompanied with a probiotic. Crohn's disease patients' response to rifabutin and macrolide antibiotic therapy was monitored over a period ranging from 4 to 17 months. RESULTS: Seven patients (19.4%) withdrew from the study since they were unable to tolerate medications. Of the remaining 29 patients, 21 (58.3%) reached a sustained state of improvement, traditionally defined as a decrease of 70 points between their entrance and exit Crohn's disease activity index scores together with the absence of the need of all other Crohn's medications, such as immunosuppressants and corticosteroids. Three Crohn's disease patients [8. 3%) noticed significant improvements, but required other Crohn's medications, concurrently with rifabutin and macrolide antibiotic therapy, to achieve and sustain improvement. Only 5 Crohn's disease patients (13.8%) were non-responders, noticing no marked improvement while on rifabutin and macrolide antibiotic therapy. CONCLUSION: The data add further evidence to support the role of rifabutin and macrolide antibiotic therapy in the treatment of Crohn's disease specifically in those patients with evidence of Mycobacterium avium subsp. Paratuberculosis infection. A large multi-centre clinical trial is needed to further explore these findings.

Review article: Mycobacterium avium subsp. paratuberculosis as one cause of Crohn's disease.

A number of theories regarding the aetiology of Crohn's disease have been proposed. Diet, infections, other unidentified environmental factors and immune disregulation, all working under the influence of a genetic predisposition, have been viewed with suspicion. Many now believe that Crohn's disease is a syndrome caused by several aetiologies. The two leading theories are the infectious and autoimmune theories. The leading infectious candidate is Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of Johne's disease, an inflammatory bowel disease in a variety of mammals including cattle, sheep, deer, bison, monkeys and chimpanzees. The evidence to support M. paratuberculosis infection as a cause of Crohn's disease is mounting rapidly. Technical advances have allowed the identification and/or isolation of M. paratuberculosis from a significantly higher proportion of Crohn's disease tissues than from controls. These methodologies include: (i) improved culture techniques; (ii) development of M. paratuberculosis-specific polymerase chain reaction assays; (iii) development of a novel in situ hybridization method; (iv) efficacy of macrolide and anti-mycobacterial drug therapies; and (v) discovery of Crohn's disease-specific seroreactivity against two specific M. paratuberculosis recombinant antigens. The causal role for M. paratuberculosis in Crohn's disease and correlation of infection with specific stratification(s) of the disorder need to be investigated. The data implicating Crohn's as an autoimmune disorder may be viewed in a manner that supports the mycobacterial theory. The mycobacterial theory and the autoimmune theory are complementary; the first deals with the aetiology of the disorder, the second deals with its pathogenesis. Combined therapies directed against a mycobacterial aetiology and inflammation may be the optimal treatment of the disease.

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients. Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.

Use of short-term culture for identification of Mycobacterium avium subsp. paratuberculosis in tissue from Crohn's disease patients.

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.