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.     

克隆病与副结核杆菌

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（ＰＣＲ）技术对手术及内镜活检的７４例石蜡包埋组织（３６例克隆病、１８例溃疡性结肠炎和２０例非炎性肠病对照组织）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ＩＳ９００上４００ｂｐ的片段。其产物特异性通过生物素标记的副结核杆菌全染色体探针Ｓｏｕｔｈｅｒｎ杂交证实。结果显示：４７．２％的克隆病、１１．１％的溃疡性结肠炎和１５．０％的非炎性肠病对照组织中检出了副结核杆菌ＤＮＡ，并发现克隆病组织中副结核杆菌的检出率与其病变部位和是否存在肉芽肿病变无关。提示部分克隆病组织中确有副结核杆菌存在，且后者与克隆病间可能存在特殊相关性。     

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。     

Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn's disease.

The role of mycobacteria, specifically Mycobacterium paratuberculosis, in Crohn's disease has aroused considerable controversy for many years. Using the ultra sensitive polymerase chain reaction some studies have reported detection of M paratuberculosis DNA in as many as 65% of Crohn's disease patients but also in patients without disease. Other studies have been negative for both groups. We therefore designed a double blind control trial to investigate the presence of mycobacterial DNA in age, sex, and tissue matched paraffin wax embedded tissues from 31 Crohn's disease tissues, 20 diseased gut control tissues, and 10 ulcerative colitis tissues. The specimens were coded and analysed blind with three separate polymerase chain reactions (PCR) based on DNA sequences specific for M paratuberculosis (IS900), M avium (RFLP type A/1) (IS901), and the Mycobacterium genus (65 kDa gene, TB600). The number of granulomata and presence of acid fast bacilli in each Crohn's disease tissue was also investigated. The sensitivity of the system was determined using similarly prepared gut tissue from an animal infected with M paratuberculosis. Four of 31 Crohn's disease tissues and none of the 30 control and ulcerative colitis derived tissues amplified M paratuberculosis DNA. Crohn's disease tissues containing granulomata were significantly more likely to amplify M paratuberculosis specific DNA on PCR than the non-Crohn's disease tissues (p = 0.02). All the positive Crohn's disease tissues contained granulomata, and none contained acid fast bacilli. Equivalent numbers of Crohn's and non-Crohn's disease tissues amplified the region of the 65 kD gene on PCR for non-specific mycobacterial DNA (11/31 and 9/30 respectively). No sections produced an amplified product with the IS901 PCR. These results suggest that few Crohn's disease gut biopsy sections contain M paratuberculosis DNA in association with granulomata. The absence of such DNA in any control and ulcerative colitic tissue strengthens the case for it having a specific association, which may be pathogenic, with Crohn's disease in this minority of patients.     

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.     

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.     

Search for Mycobacterium paratuberculosis DNA in orofacial granulomatosis and oral Crohn's disease tissue by polymerase chain reaction

Background—Although intestinalCrohn's disease has long been suspected to have amycobacterial cause, possible mycobacterial involvement in orofacialgranulomatosis (OFG) and oral lesions of Crohn's disease has not yetbeen investigated.
Aims—As the slow growingMycobacterium paratuberculosis has been implicated in theaetiology of intestinal Crohn's disease, the potential involvement ofthis mycobacterial species in OFG and oral lesions of Crohn's diseasewas investigated.
Patients—To attempt detectionof the organism in OFG and oral Crohn's disease tissue samples, apolymerase chain reaction (PCR) assay was used on archival formalinfixed, paraffin wax embedded oral tissue sections from 30 patients withOFG, seven with Crohn's disease, and 12 normal controls.
Methods—The PCR assay used wasbased on primers targeting the 5' region of the multicopy IS900 DNAinsertion element of the M paratuberculosis genome. Inorder to achieve maximum sensitivity, two rounds of PCR were carriedout and amplicons confirmed by Southern blot hybridisation to adigoxigenin labelled IS900 DNA probe.
Results—None of the OFG andoral lesions of Crohn's disease samples were positive forM paratuberculosis and all normal controls were also negative.
Conclusions—These results suggestthat M paratuberculosis may not be a major aetiologicalagent in OFG or oral Crohn's disease lesions, although the use ofparaffin wax embedded tissue as opposed to fresh tissue as a samplesource could underestimate the true prevalence of the organism.

Keywords:oral Crohn's disease; Mycobacteriumparatuberculosis; orofacial granulomatosis; polymerase chainreaction

     

The evidence for Mycobacterium paratuberculosis in Crohn's disease.

PURPOSE OF REVIEW: Though long hypothesized, the putative link between Mycobacterium avium paratuberculosis and Crohn's disease remains neither confirmed nor refuted. This article reviews published contributions that directly or indirectly address this question. RECENT FINDINGS: Epidemiologic studies, looking for M. avium paratuberculosis DNA in Crohn's tissue, show a strong association between the agent and this disease. Supporting data, however, are presently inconclusive on a causal role. Genetic studies provide indirect support for a role of mycobacteria in Crohn's disease, by identifying susceptibility genes that encode proteins implicated in innate immunity to intracellular bacteria. Clinical trial data support at least a short-term benefit for antimycobacterial therapy in Crohn's disease, but the microbial specificity of this response is presently unknown. SUMMARY: There appears to be a strong association between M. avium paratuberculosis and Crohn's disease, but the causality of this association is unknown. Consequently, the therapeutic implications of this association require further study. A number of critical questions about the biology of M. avium paratuberculosis remain unanswered. Data from studies of this organism, and its interaction with the immune system, can help address proposed reasons for or against a role of M. avium paratuberculosis in the etiology of Crohn's disease.     

Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.     

Absence of Mycobacterium paratuberculosis DNA in intestinal tissues from Crohn's disease by nested polymerase chain reaction

A role of Mycobacterium paratuberculosis in Crohn's disease has been suggested for many years. To determine whether Mycobacterium paratuberculosis has the potential to cause Crohn's disease, we investigated Crohn's disease tissues for the presence of Mycobacterium paratuberculosis, using highly sensitive nested polymerase chain reaction (PCR). DNA was extracted from samples of intestine obtained from 13 patients with Crohn's disease, 14 patients with ulcerative colitis, and 13 control patients without inflammatory bowel disease. Nested PCR was performed using primer pairs complementary to sequences in the insertion element IS900 gene of Mycobacterium paratuberculosis. No Mycobacterium paratuberculosis-specific PCR product was detected in any of these specimens, although PCR products of the appropriate size were consistently obtained using control DNA template from Mycobacterium paratuberculosis. The results did not suggest that Mycobacterium paratuberculosis is involved in the pathogenesis of Crohn's disease. (Received: May 6, 1998; accepted: Oct. 23, 1998)     

Infective endocarditis in Crohn''s disease.

Infective endocarditis developed in a patient with Crohn's disease. This case highlights a hazard of immunosuppressive treatment in inflammatory bowel disease.