Mycobacterium paratuberculosis DNA in Crohn''s disease tissue.

Crohn's disease has long been suspected of having a mycobacterial cause. Mycobacterium paratuberculosis is a known cause of chronic enteritis in animals, including primates, but may be very difficult to detect by culture. IS900 is a multicopy genomic DNA insertion element highly specific for M paratuberculosis. A polymerase chain reaction (PCR) based on the 5' region of IS900 and capable of the specific detection of a single M paratuberculosis genome was developed. This was applied to DNA extracts of full thickness samples of intestine removed at surgery from 40 patients with Crohn's disease, 23 patients with ulcerative colitis, and 40 control patients without inflammatory bowel disease. Stringent precautions were taken that excluded contamination artefact. M paratuberculosis was identified in 26 of 40 (65%) Crohn's disease, in 1 of 23 (4.3%) ulcerative colitis, and in 5 of 40 (12.5%) control tissues. Positive samples from Crohn's disease were from both the small intestine and colon, those from control tissues were from the colon those from control tissues were from the colon only. All PCR internal control reactions were negative. The presence of M paratuberculosis in a small proportion of apparently normal colonic samples is consistent with a previously unsuspected alimentary prevalence in humans. The presence in two thirds of Crohn's disease tissues but in less than 5% of ulcerative colitis tissues is consistent with an aetiological role for M paratuberculosis in Crohn's disease.     

克隆产现与副结核杆菌

分支杆菌、尤其是副结核杆菌长期被疑为克隆病的致病菌。采用聚合酶链反应技术对手术及内镜活检的７４例石蜡包埋组织中的副结核杆菌ＤＮＡ进行检测。扩增的靶ＤＮＡ为副结核杆菌染色体特异重复插入序列ＩＳ９００１上４００ｂｐ的片段。其产笺特异性通过生物素杆主民的副结核杆菌全染色体探针Ｓｏｕｔｈｅｒｎ杂交证实。     

Mycobacteria in the Intestine of Japanese Patients with Inflammatory Bowel Disease

Objectives : It is still controversial whether or not a mycobacterial infection may be a cause of Crohn's disease. Mycobaclerium paratuberculosis may be very difficult to detect using routine culture techniques. To clarify this, we delected mycobacterial DNA in patients with inflammatory bowel disease. Methods : IS900 sequences highly specific to M. paratuberculosis and groEL gene encoding a conserved mycobacterial antigen were studied in colonic mucosa using polymerase chain reaction (PCR). PCR products were analyzed by Southern blot hybridization. Results : IS900 sequences were detected in all (100%)of 10 patients with Crohan's disease, in 11 (61.1%) of 18 patients with ulcerative colitis, and in 14 (87.5%) of 16 control patients with noninflammatory bowel disease. All IS900 positive samples had groEL PCR products. Conclusions : Our results, on the basis of the prevalence, do not support the hypothesis that M. paratuberculosis is involved in the pathogenesis of Crohn's disease.     

Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn''s disease and control tissues.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.     

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.     

Molecular evidence for Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn''s disease correlates with enhanced TNF-α secretion

BACKGROUND: Support for a role of Mycobacterium avium subspecies paratuberculosis in Crohn's disease is largely based on epidemiological evidence, as no data on mechanisms linking the presence of M. avium subspecies paratuberculosis with gut damage is available. AIMS: To determine whether the presence of M. avium subspecies paratuberculosis contributes to the pathogenesis of Crohn's disease by promoting cytokine secretion within gut mucosa. PATIENTS AND METHODS: A total of 235 subjects were recruited: 63 with Crohn's disease, 53 with ulcerative colitis, 45 with irritable bowel syndrome and 74 normal controls. M. avium subspecies paratuberculosis status was defined by nested PCR using IS900 sequence. Gut mucosal organ cultures were established to detect cytokine secretion patterns. RESULTS: Significantly higher tumour necrosis factor-alpha concentrations were found in culture supernatants for Crohn's disease compared to ulcerative colitis (p<0.05), irritable bowel syndrome (p<0.01) and controls (p<0.0001). When tumour necrosis factor-alpha levels were correlated with the presence of M. avium subspecies paratuberculosis, significantly greater concentrations were only found in M. avium subspecies paratuberculosis-positive Crohn's disease patients (p<0.05). Tumour necrosis factor-alpha levels in M. avium subspecies paratuberculosis-positive Crohn's disease were significantly higher than in M. avium subspecies paratuberculosis-positive ulcerative colitis (p<0.01), M. avium subspecies paratuberculosis-positive irritable bowel syndrome (p<0.05) and M. avium subspecies paratuberculosis-positive controls (p<0.01) and all M. avium subspecies paratuberculosis-negative specimens. CONCLUSIONS: The data link M. avium subspecies paratuberculosis with a pathogenic mechanism in Crohn's disease and is consistent with abnormal macrophage handling of M. avium subspecies paratuberculosis.     

T-cellular immune reactions (in macrophage inhibition factor assay) against Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium avium in patients with chronic inflammatory bowel disease.

A mycobacterial aetiology has been suggested for Crohn's disease. A slow growing mycobacterium, biochemically and genetically identical to M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease), has been isolated from gut specimens of patients affected by Crohn's disease. If M paratuberculosis or other mycobacteria play a role in the pathogenesis of Crohn's disease, then patients may have been sensitised to these mycobacteria or show an anergy immune reaction. We therefore investigated the T-cell mediated immune response to sonicates of M paratuberculosis, M kansasii, M avium, and M tuberculosis in 35 patients with Crohn's disease, 28 with ulcerative colitis, and 25 controls using a macrophage inhibition factor assay on peripheral blood lymphocytes. Two types of reaction patterns were identified--that is, 'responders' (subjects with a macrophage inhibition factor assay in which a dose response relation was present and a percentage of inhibition exceeding 20%), and 'non-responders'. There was no significant difference in the prevalence of responders (59%-80%) and non-responders (20%-41%) to these mycobacteria between the group of Crohn's disease, ulcerative colitis, and control group. We found also that a large proportion of controls showed T-cell immunisation to the mycobacteria which supports the contention that the antigens are practically commensal. Our results do not support the proposed involvement of mycobacteria in the pathogenesis of Crohn's disease.     

Mycobacterium paratuberculosis DNA not detected in Crohn's disease tissue by fluorescent polymerase chain reaction.

The role of mycobacteria in the aetiology of Crohn's disease has been a contentious subject for many years. Mycobacterium paratuberculosis is known to cause a chronic granulomatous enteritis in animals (Johne's disease) and has been implicated as a possible infectious cause of Crohn's disease. However this fastidious organism is only rarely detected by conventional microbiological techniques. This study used oligonucleotide primers to the species-specific M paratuberculosis IS900 DNA insertion element and the polymerase chain reaction to amplify any M paratuberculosis DNA from intestinal tissue DNA extracts. One oligonucleotide primer was fluorochrome-labelled and the presence of fluorescent amplified product was determined using an automated DNA sequencer with a computerised gel-scanning laser. This method was shown capable of detecting 1-2 mycobacterial genomes. Intestinal tissue samples were obtained from 68 patients with histologically confirmed Crohn's disease, 49 patients with histologically confirmed ulcerative colitis, and 26 non-inflammatory bowel disease controls. In no case was M paratuberculosis detected in any of the inflammatory bowel disease tissue samples and only one non-inflammatory bowel disease case was positive. These results do not support the hypothesis that M paratuberculosis has an aetiological role in Crohn's disease.     

Mycobacterium paratuberculosis and Crohn''s disease.

The possible aetiological role of Mycobacterium paratuberculosis in Crohn's disease was investigated. The immunological response was studied using an enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunocytochemistry. The antibody response to two protoplasmic antigen preparations of M paratuberculosis in the sera of patients with inflammatory bowel disease was measured by ELISA. IgG and IgM antibodies to these antigens were measured in serum samples from 52 patients with Crohn's disease, 15 patients with ulcerative colitis, and 41 control patients without inflammatory bowel disease. Although there was wide variation in the concentrations of antibody detected, patients with Crohn's disease had concentrations that were not significantly different from those of the other two groups. In addition, mycobacterial antigens were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the immune response to each antigen was then examined separately and assayed for IgG and IgM in 10 patients from each of the three groups. An indirect peroxidase test was also used to detect M paratuberculosis in sections of tissue from 18 patients with Crohn's disease and 10 with ulcerative colitis. The results were negative in all cases. This study does not support a role for M paratuberculosis in Crohn's disease.     

High prevalence of Mycobacterium avium subspecies paratuberculosis IS900 DNA in gut tissues from individuals with Crohn's disease

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. METHODS: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. 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.     

Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900.

This paper describes the evaluation of a newly developed DNA probe for Mycobacterium paratuberculosis. DNA probe PCR278 is a 278 bp fragment obtained by polymerase chain reaction (PCR) amplification of the 5'-region of IS900, an insertion element contained in the genome of M paratuberculosis. This DNA probe can specifically distinguish M paratuberculosis from a wide range of other organisms, including members of the M avium-M intracellulare complex. When used in conjunction with the PCR amplification technique DNA probe PCR278 could detect as little as 10 fg (equivalent to two genomes) starting material of M paratuberculosis genomic DNA. Use of PCR amplification assays based on IS900, for the detection of M paratuberculosis, and homologous IS elements found in disease isolates of M avium should greatly help our understanding of the role of these organisms in Crohn's disease and other chronic inflammatory disorders.     

Mycobacterial aetiology of Crohn's disease: serologic study using common mycobacterial antigens and a species-specific glycolipid antigen from Mycobacterium paratuberculosis.

Crohn's disease is a granulomatous form of enteritis superficially similar to Johne's disease (paratuberculosis) of ruminants. Recently, a Mycobacterium sp closely related to Mycobacterium paratuberculosis was cultured from tissues of patients with Crohn's disease suggesting that M paratuberculosis may be the aetiologic agent in some cases. In addition, greater seroreactivity to M paratuberculosis has been reported in patients with Crohn's disease. In the present study, we have evaluated the serum antibody response to disrupted M paratuberculosis using ELISA and serum specimens from 33 people with Crohn's disease, 21 with ulcerative colitis, and 12 non-inflammatory bowel disease controls. We failed to find a consistent IgG, IgM, or IgA antibody response to Mycobacterium paratuberculosis. The results indicate that, as in bovine paratuberculosis, serum seroreactivity is not a reliable tool for examining the relationship between human intestinal disease and mycobacteria.     

Antibodies to Mycobacterium paratuberculosis and nine species of environmental mycobacteria in Crohn's disease and control subjects.

Cultural and serological studies have provided limited, often conflicting, evidence of a role for mycobacteria in the pathogenesis of Crohn's disease. Interest has focussed on Mycobacterium paratuberculosis, previously considered to be common in the environment with no major role as a human pathogen. Whether a specific serum antibody response to mycobacteria occurs in Crohn's disease or ulcerative colitis was investigated. Sera from patients with Crohn's disease (n = 38), ulcerative colitis (n = 15), and a healthy control population (n = 30) were assayed in an enzyme linked immunosorbent assay (ELISA) using eight filtered sonicate mycobacterial preparations and a purified protein derivative made from the bovine tubercle bacillus. In addition, IgG, IgM, and IgA levels to M paratuberculosis were determined in sera from patients with active (n = 24) or inactive (n = 29) Crohn's disease and the control populations. There was strong evidence of contact with environmental mycobacteria in all patients and control populations, with the greatest responses to preparations of M avium, M tuberculosis, and M kansasii. A large proportion of patients with Crohn's disease had antibodies that bound most antigens tested but there were no statistical differences between these values and those of the control population. Similarly, there were no differences in antibody levels to M paratuberculosis in patient and control groups. Although a subset of patients with active Crohn's disease (25%) had IgG concentrations that exceeded the control mean by more than 2 SD, this phenomenon may not be specific to Crohn's disease: 20% of a small group of patients with coeliac disease had similarly raised IgG levels to M paratuberculosis. These findings do not provide serological evidence of a role for this organism in the pathogenesis of Crohn's disease.     

PCR detection of Mycobacterium paratuberculosis in Crohn's disease granulomas isolated by laser capture microdissection

BACKGROUND AND AIMS: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn's disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn's disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). METHODS: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn's granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. RESULTS: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn's granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. CONCLUSIONS: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn's disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.     

Investigation of mycobacteria in Crohn's disease tissue by Southern blotting and DNA hybridisation with cloned mycobacterial genomic DNA probes from a Crohn's disease isolated mycobacteria.

A mycobacterial aetiology for Crohn's disease (CD) has been suggested. Slow growing mycobacteria indistinguishable from M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease) have been isolated from CD tissues. We have used cloned genomic DNA probes derived from a CD isolated mycobacteria strain Ben, to investigate the presence of mycobacterial DNA sequences in CD tissues. DNA was extracted from total tissue from 17 CD and four control gut specimens. DNA was digested with restriction endonucleases, electrophoresed and transferred to nylon membranes by Southern blotting and hybridised to radiolabelled DNA probes. No mycobacterial DNA was detected in any tissue sample studied. Reconstitution experiments with known numbers of in vitro cultured mycobacteria showed sensitive detection of mycobacterial DNA. DNA extracted from mouse liver, infected with M lepraemurium revealed a strong hybridisation signal and showed the applicability of the experimental approach to the detection of mycobacterial DNA in naturally infected tissues. The results do not provide evidence for the involvement of mycobacteria in the pathogenesis of CD but do not exclude the possibility of low levels of infection in subsets of intestinal cells with spheroplast or cell wall deficient forms of mycobacteria.     

Lack of support for a common etiology in Johne's disease of animals and Crohn's disease in humans.

The superficial similarity of Johne's disease to Crohn's disease led to the hypothesis that, like the former. Crohn's disease was caused by Mycobacterium paratuberculosis. Detailed pathologic comparisons, however, reveal little similarity between these two entities, including the lack of important extraintestinal manifestations. Attempts to recover M. paratuberculosis by culture have only rarely succeeded and the significance of spheroplasts that appear more frequently on culture is seriously in question. Five immunocytochemistry studies have failed to find mycobacterial antigens in diseased tissues and the five most recent polymerase chain reaction (PCR) attempts to find genomic evidence of M. paratuberculosis were uniformly negative. Numerous serologic studies failed to demonstrate antibody to M. paratuberculosis and attempts to show cell-mediated immunity were also unrewarding. Inoculation of numerous experimental animals with Crohn's disease tissue has failed to induce Johne's disease, and inoculation of various animal species with M. paratuberculosis has equally failed to result in Crohn's disease. Controlled studies of the treatment of Crohn's disease with antimycobacterial agents have generally resulted in no improvement, and most studies that have shown a positive response are either uncontrolled or include broad-spectrum antibiotics that may be acting on pathogens other than mycobacteria. Finally, although Johne's disease is common in farm animals, and infected animals shed M. paratuberculosis in large numbers, no record of zoonotic transmission has been recorded.     

Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis

This systematic review assesses the evidence for an association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease. We analysed 28 case-control studies comparing MAP in patients with Crohn's disease with individuals free of inflammatory bowel disease (IBD) or patients with ulcerative colitis. Compared with individuals free of IBD, the pooled odds ratio (OR) from studies using PCR in tissue samples was 7.01 (95% CI 3.95-12.4) and was 1.72 (1.02-2.90) in studies using ELISA in serum. ORs were similar for comparisons with ulcerative colitis patients (PCR, 4.13 [1.57-10.9]; ELISA, 1.88 [1.26-2.81]). The association of MAP with Crohn's disease seems to be specific, but its role in the aetiology of Crohn's disease remains to be defined.     

Detection of mycobacteria by polymerase chain reaction]

The polymerase chain reaction (PCR) was used to detect mycobacterial DNA sequences in the cultured or the clinical specimens. Four oligonucleotide primers derived from the sequence of a gene coding 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA samples of all the 11 species of mycobacteria tested. Serial dilution of M. bovis BCG showed that DNA extracted from only 12 bacilli was enough for the detection by PCR method. However, mycobacteria in sputum were detected by PCR when more than 10(3) bacilli were present. The PCR method may become a useful tool for the rapid diagnosis of mycobacterial infections.