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.     

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.     

Possible role of Mycobacteria in inflammatory bowel disease

An unclassified Mycobacterium species has been isolated from two patients with Crohn's disease (CD). Antibodies to the unclassified mycobacteria cross-reacted with Mycobacterium paratuberculosis. Because of this cross-reactivity, an enzyme-linked immunosorbent assay (ELISA) was used to examine the sera of inflammatory bowel disease (IBD) patients, both CD (N = 56), and ulcerative colitis (UC) (N = 34), for antibodies to M. paratuberculosis, Mycobacterium kansasii, and Mycobacterium tuberculosis. Controls consisted of healthy, PPD-negative individuals (N = 67), and from PPD-positive patients (N = 41). Eighteen resected CD patients were also examined. CD patients had a statistically significant increase in antibody titer (P = 0.0003) to M. paratuberculosis compared to healthy controls. Although patients with positive PPD had elevated titers to this organism, the positive response of CD patients was not related to PPD responsiveness, area of involvement in the gut, nor to activity of the disease process.     

Serum Antibodies to Mycobacterium paratuberculosis in Patients with Crohn's Disease

Mycobacterium paratuberculosis has beensuggested as a causative organism of Crohn's disease.Despite a long-term debate to prove this possibility,the role of this bacteria in the pathogenesis of Crohn's disease is still a subject of controversy. Inthe present study, serum antibodies (IgG, IgA, and IgM)to the protoplasmic antigen of M. paratuberculosis werequantified in patients with Crohn's disease and in control subjects by using anenzyme-linked immunosorbent assay whose specificity wasincreased by preabsorbing the sera with cell extracts ofMycobacterium phlei . As compared to normal controls (1/20; 0.062 ± 0.022), a significantdifference was seen in the antibody-positive prevalencerate and mean values of the serum IgG titer in patientswith Crohn's disease (5/13; 0.102 ± 0.039) (P< 0.05), but not in patients with ulcerativecolitis (2/20; 0.065 ± 0.035) and tuberculosis(0/4; 0.053 ± 0.008). No significant differenceswere seen in the antibody-positive prevalence rate andmean values of the serum IgA and IgM titers amongthe four study groups. These results indicate the uniqueimmune response to M. paratuberculosis in patients withCrohn's disease, suggesting that this organism may play some role in the pathogenesis ofCrohn's disease.     

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 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.     

Serum antibodies to mycobacterial antigens in active Crohn's disease

Infection with a species of Mycobacterium has been implicated in the pathogenesis of Crohn's disease. Therefore, we attempted to determine whether a specific serum antibody response to mycobacteria occurs in patients with the disease. We tested sera of patients with active Crohn's disease and several control groups in an enzyme-linked immunosorbent assay for reactivity with two mycobacterial antigens: (a) lipoarabinomannan, a highly immunogenic somatic lipopolysaccharide present in the cell walls of all species of the Mycobacterium genus, and (b) a protoplasmic antigenic preparation from M. sp strain linda, the mycobacterium that has been specifically implicated in Crohn's disease. We found no significant elevation in immunoglobulin A, immunoglobulin G, or immunoglobulin M antibody levels to these two antigen preparations in the Crohn's disease patients. Moreover, no subset of patients (sex, age, Crohn's disease activity index, location of disease, duration of disease, operations, or response to treatment) had elevated antibody levels. As virtually all known chronic infectious diseases have an associated serologic response to the etiologic agent, our findings greatly diminish the likelihood that Crohn's disease is caused by an infection with a mycobacterium.     

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.     

Agglutinins to bacteria in Crohn's disease.

Sera from patients with Crohn's disease were tested for antibodies against organisms which are thought to cause inflammatory bowel disease in animals, or have been implicated in human Crohn's disease. Control sera were collected from healthy individuals and patients with ulcerative colitis. Sera from Crohn's disease and controls failed to agglutinate Clostridium colinum or Campylobacter sputorum subsp. mucosalis and two strains of Mycobacterium paratuberculosis (M26 and M27). Most of the sera agglutinated a Citrobacter freundii variant, Mycobacterium paratuberculosis (M28) and Mycobacterium avium (M41) but Crohn's disease sera did not differ from controls. A complement fixation test against Chlamydia gave more positive reactions in patients with Crohn's disease and colitis than in healthy controls. There was a clear difference between the sera from patients with Crohn's disease and other sera, including ulcerative colitis, in agglutination tests with the commensal coccoid rods of the genera Eubacterium and Peptostreptococcus; in these tests 54% of sera from Crohn's disease were positive compared with 11% in ulcerative colitis and none of the sera from healthy controls. All the results were essentially negative with the exception of those from Eubacterium and Peptostreptococcus and these bacteria merit investigation.     

Effect of intestinal resection on serum antibodies to the mycobacterial 45/48 kilodalton doublet antigen in Crohn's disease.

Interest in the role of mycobacterial infection in Crohn's disease has been revived by the cultural detection of Mycobacterium paratuberculosis in patients with Crohn's disease. This hypothesis was examined serologically using assays with high specificity for Crohn's disease. The effect of intestinal resection on serum antibodies specific for Crohn's disease was investigated with an immunoblot assay and an enzyme linked immunosorbent assay using the 45/48 kilodalton doublet antigen of Mycobacterium tuberculosis. Antibodies were detected in 64.7% of patients with Crohn's disease (n = 17), 10% of patients with ulcerative colitis (n = 10), 5% of patients with carcinoma of the colon (n = 20), and none of 10 healthy subjects with the immunoblot assay. Statistical comparison of the Crohn's disease patients with each control group resulted in p = 0.0000236. Immunoglobulin G was essentially unchanged 75 days (mean) after surgery. After more than 180 days, however, the antibody response was reduced in all of five patients studied, and was no longer demonstrable in two of them (40%). Simultaneously, the Crohn's disease activity index (CDAI) decreased. Both the high specificity of this assay for Crohn's disease and the diminished antibody response after intestinal resection in parallel with decreased CDAI support a mycobacterial aetiology of Crohn's disease.     

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.     

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.     

Immunohistochemical examination for mycobacteria in intestinal tissues from patients with Crohn's disease

We conducted an immunohistochemical search for mycobacteria in the intestinal tissues of patients with Crohn's disease. Tissues obtained by biopsy or surgical resection and fixed by a variety of methods (formalin, periodate-lysine-paraformaldehyde, fresh-frozen) were reacted by an immunoperoxidase method with antibodies to (a) Mycobacterium paratuberculosis strain linda, (b) M. tuberculosis, and (c) the common mycobacterial antigen, lipoarabinomannan. Each of the antibody preparations was shown capable of detecting a variety of typical and atypical mycobacteria (M. tuberculosis, M. kansasii, M. fortuitum, M. chelonei, M. paratuberculosis, and cell wall-defective as well as cell wall-intact forms of M. avium intracellulare) under conditions identical to those used for staining the patients' tissues. We did not detect mycobacteria in any of the 67 specimens from 30 patients examined. These results, in conjunction with those of our previous serologic studies, do not support the hypothesis that infection with a Mycobacterium causes 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.     

Treatment of severe Crohn''s disease using antimycobacterial triple therapy — approaching a cure?

BACKGROUND: Mycobacterium avium subspecies paratuberculosis is probably the best candidate for a microbial cause of Crohn's disease although arguments to the contrary can be equally convincing. Growing evidence suggests that prolonged antimycobacterial combination therapy can improve Crohn's disease in some patients. AIM: To report long-term observations in patients with severe Crohn's disease treated with triple macrolide-based antimycobacterial therapy. PATIENTS: A series of 12 patients (7 male, 5 female; aged 15-42 years) with severe, obstructive or penetrating Crohn's disease were recruited. METHODS: Patients failing maximal therapy were commenced prospectively on a combination of rifabutin (450 mg/d), clarithromycin (750 mg/d) and clofazimine (2 mg/kg/d). Progress was monitored through colonoscopy, histology, clinical response and Harvey-Bradshaw activity index. RESULTS: Follow-up data were available for up to 54 months of therapy Six out of 12 patients experienced a full response to the antiMycobacterium avium subspecies paratuberculosis combination achieving complete clinical, colonoscopic and histologic remission of Crohn's disease. Four of these patients were able to cease treatment after 24-46 months, 3 of whom remained in total remission without treatment for up to 26 months and one patient relapsed after six months off treatment. A partial response to the anti-Mycobacterium avium subspecies paratuberculosis combination was seen in 2 patients showing complete clinical remission with mild histologic inflammation. Return to normal of terminal ileal strictures occurred in 5 patients. Harvey-Bradshaw activity index in patients showing a full or partial response to therapy fell from an initial 13.4 +/- 1. 91 to 0. 5 +/- 0. 47 [n = 8, p < 0. 001) after 52-54 months. CONCLUSIONS: Reversal of severe Crohn's disease has been achieved in 6/12 patients using prolonged combination anti-Mycobacterium avium subspecies paratuberculosis therapy alone. Three patients remain in long-term remission with no detectable Crohn's disease off all therapy These results support a causal role for Mycobacterium avium subspecies paratuberculosis in Crohn's disease while also suggesting that a cure may become possible.     

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。     

Detection and Isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia

OBJECTIVES: Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johne's disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohn's disease were infected with this pathogen. METHODS: Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing. RESULTS: Twenty five patients (83.3%) with Crohn's disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohn's patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens. CONCLUSIONS: Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohn's disease patients. The finding of the organism colonizing a proportion of people without Crohn's disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.     

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.     

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.