Authors: Romero C, Hamdi A, Valentine JF, Naser SA.

Institution: Department of Molecular Biology and Microbiology and Biomolecular Science Center, The Burnett College of Biomedical Sciences, University of Central Florida, Orlando, Florida 32816, USA.

Summary: Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with tissue granuloma and histopathological alteration that resembles aspects in tuberculosis, leprosy, and paratuberculosis.

In non-IBD subjects, MAP DNA was detected in the tissue of only 1 of 6 patients (17%) by PCR and 0 of 6 patients (0%) by FISH. MAP DNA was identified by PCR in inflamed tissue from 2 of 2 patients with ulcerative colitis. The detection of MAP DNA by either technique in tissue from subjects with CD is significant compared with non-IBD subjects (P < 0.005).

Identification of MAP DNA in both inflamed and noninflamed tissue by both techniques suggests that MAP infection in patients with CD may be systemic. The data add more evidence toward a possible association of MAP in the pathogenesis of CD.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15677904&query_hl=13

Authors: Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, Hermon-Taylor J.

Institution: Department of Surgery, St. George's Hospital Medical School, London SW17 0RE, United Kingdom.

Summary: Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen.

In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens.

…In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12843021&query_hl=13

Authors: Naser SA, Ghobrial G, Romero C, Valentine JF.

Institution: Department of Molecular Biology and Microbiology and Biomolecular Science Center, Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32816, USA. nasers@mail.ucf.edu

Background: Crohn's disease, a form of inflammatory bowel disease, resembles some aspects of tuberculosis, leprosy, and paratuberculosis. The role of Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease is controversial.

Findings: MAP DNA in uncultured buffy coats was identified by PCR in 13 (46%) individuals with Crohn's disease, four (45%) with ulcerative colitis, and three (20%) without inflammatory bowel disease. Viable MAP was cultured from the blood of 14 (50%) patients with Crohn's disease, two (22%) with ulcerative colitis, and none of the individuals without inflammatory bowel disease. Current use of immunosuppressive medication did not correlate with a positive MAP culture. Sequencing of PCR products from MAP cultures confirmed the presence of the MAP-specific IS900 fragment. Among 11 MAP isolates assessed, we identified nine strains that were not identical.

Interpretation: We detected viable MAP in peripheral blood in a higher proportion of individuals with Crohn's disease than in controls. These data contribute to the evidence that MAP might be a cause of Crohn's disease.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15380962&query_hl=9

Authors: Autschbach F, Eisold S, Hinz U, Zinser S, Linnebacher M, Giese T, Loffler T, Buchler MW, Schmidt J.

Institution: Institute of Pathology, University of Heidelberg, Germany.

Background and Aims: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in Crohn's disease (CD) tissues. Therefore, we examined IS900 in a large number of gut samples from patients with CD (n = 100) and ulcerative colitis (UC, n = 100), and in non-inflamed control tissues (nIBD, n = 100). We hypothesised that IS900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD.

Results: IS900 PCR detection rate was significantly higher in CD tissue samples (52%) than in UC (2%) or nIBD (5%) specimens (p<0.0001). In CD patients, IS900 DNA was detected in samples from both diseased small bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS900 detection rates and clinical phenotypic characteristics in CD could be established. However, corticosteroid medication constituted a factor which tended to have a negative influence on IS900 DNA detection rates in CD (p<0.01).

Conclusions: The presence of MAP specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn's disease.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15951539&query_hl=64

Authors: Romero C, Hamdi A, Valentine JF, Naser SA.

Institution: Department of Molecular Biology and Microbiology and Biomolecular Science Center, The Burnett College of Biomedical Sciences, University of Central Florida, Orlando, Florida 32816, USA.

Summary: Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) with tissue granuloma and histopathological alteration that resembles aspects in tuberculosis, leprosy, and paratuberculosis. Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of paratuberculosis, with a suspected role in the etiology of CD.

We investigated the presence of MAP DNA in 31 surgical tissue samples from 20 subjects using fluorescence in situ hybridization (FISH) with the aid of confocal scanning laser microscopy and nested polymerase chain reaction (PCR) using the IS900 sequence unique to MAP. MAP DNA was detected by PCR in tissue from 10 of 12 (83%) patients with CD: 7/12 (58%) in inflamed, 6/11 (55%) in noninflamed and in 10 (83%) of either tissue and by FISH in 8 of 12 (67%) patients with CD: 7 of 12 (58%) in inflamed, 4 of 11 (36%) in noninflamed, and in 8(67%) of either tissue.

In non-IBD subjects, MAP DNA was detected in the tissue of only 1 of 6 patients (17%) by PCR and 0 of 6 patients (0%) by FISH. MAP DNA was identified by PCR in inflamed tissue from 2 of 2 patients with ulcerative colitis. The detection of MAP DNA by either technique in tissue from subjects with CD is significant compared with non-IBD subjects (P < 0.005).

Identification of MAP DNA in both inflamed and noninflamed tissue by both techniques suggests that MAP infection in patients with CD may be systemic. The data add more evidence toward a possible association of MAP in the pathogenesis of CD.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15677904&query_hl=64

Authors: Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, Hermon-Taylor J.

Institution: Department of Surgery, St. George's Hospital Medical School, London SW17 0RE, United Kingdom.

Summary: Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere.

Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis.

The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900.

Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis.

In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12843021&query_hl=64

Authors: Schwartz D, Shafran I, Romero C, Piromalli C, Biggerstaff J, Naser N, Chamberlin W, Naser SA.

Institution: Department of Molecular Biology and Microbiology, Center for Discovery of Drugs and Diagnostics, University of Central Florida, Orlando, Florida 32816, USA.

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.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11168138&dopt=Abstract

Authors: Sechi LA, Mura M, Tanda E, Lissia A, Fadda G, Zanetti S.

Institution: Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, Universita degli Studi, Sassari, Italy.

Summary: Crohn's disease is a non-specific chronic transmural inflammatory disease. The disease was associated with a frameshit mutation in the NOD2 gene. Nevertheless, other researchers associated the presence of M. paratuberculosis within the intestinal tissues of patients with the disease.

An adapted "in situ hybridization" technique was used to detect IS900 M. paratuberculosis DNA in paraffin embedded tissue from Crohns tissue disease samples. We were able to identify M. paratuberculosis DNA in around 69% of the paraffine embedded intestinal samples of Crohn's disease patients analysed. The presence of M. paratuberculosis DNA in the intestinal samples analysed does not necessarily mean that M. paratuberculosis is responsible for Crohn's disease.

Our results support the hypothesis that infection may be caused by cell wall defective M. paratuberculosis since no bacteria were detected by Ziehl Neelsen stain.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14964409&query_hl=43

Authors: Hulten K, El-Zimaity HM, Karttunen TJ, Almashhrawi A, Schwartz MR, Graham DY, El-Zaatari FA.

Institution: Veterans Affairs Medical Center, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.

Objectives: Reports about the association between Crohn's disease (CD) and cell wall-deficient (CWD) forms of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) are controversial. This may be due to the heterogeneous nature of CD where only about 50% of the patients show granulomatous inflammation. Detection of CWD forms of M. paratuberculosis in tissues from patients with CD would support its association with the disease. To help identify these forms in inflamed tissues, a previously developed and optimized nonradioactive in situ hybridization method was applied on well-defined tissue materials obtained from patients with CD, ulcerative colitis (UC), and controls.

Results: Six of 15 (40%) patients with CD and granulomas showed positive signals in myofibroblasts and macrophages. Interestingly, no positive signals were observed within granulomas. Only 4.5% of 22 CD samples from patients with nongranulomatous disease, 9.5% of 21 UC, and remarkably, none of the 22 non-IBD patients were M. paratuberculosis positive.

Conclusion: The demonstration of DNA from CWD forms of M. paratuberculosis in this limited number of CD tissues further supports and confirms previous reports of its association with the granulomatous type of the disease.

Study link: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11374694&query_hl=41