Historical overview and current approaches in determining a mycobacterial etiology of Crohn's disease.

Rodrick J. Chiodini

Mycobacteriology unit, Division of Biological and Medical Sciences, Department of Medicine, Brown University and the Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903 U.S.A.

Source: Mulder and Tytgat GNJ (eds): Is Crohn's disease a mycobacterial disease. Dordrecht: Kluwer Academic Publishers, 1992, pp 1-15.

Reproduced with the kind permission of the author

Although the earliest descriptions of Crohn's disease are said to date back to the later 1600's to early 1700's ²⁴, they are difficult to sort from other undefined intestinal disorders of that era. Perhaps more realistically, since Crohn's disease and intestinal tuberculosis were confused for years and such confusion continues to this date, the earliest descriptions probably occurred sometime after the discovery of the tubercle bacillus in 1882 ²⁵. Nevertheless, sometime after that period, an intestinal disease was recognized that was similar to intestinal tuberculosis, but acid fast bacilli could not be visualized in tissues or isolated in culture and the characteristic caseous nodules of tuberculosis were absent. By 1913, cases of intestinal tuberculosis which did not fit the classical pattern were well recognized, but nevertheless, were classified among the tuberculous lesions and the disease was known as hyperplastic tuberculosis ⁴⁴. By the 1920's, these cases of intestinal tuberculosis which did not contain caseous necrosis or acid fast bacilli were reclassified as nonspecific granulomata ^30,44 in an attempt to recognize these cases as distinct from the classical or hypertrophic forms of intestinal tuberculosis. Each description, however, discussed the remarkable resemblance of these cases to intestinal tuberculosis - the only difference being the absence of the pathognomonic lesions of tuberculosis, caseous necrosis and acid fast bacilli.

In 1932, the landmark article of Crohn, Ginsberg, and Oppenheimer ¹⁴ brought recognition of a disease to be known as regional ileitis as an distinct entity and separated these cases from intestinal tuberculosis. Crohn and his colleagues also recognized the "remarkable resemblance" of their regional ileitis and intestinal tuberculosis. For over 50 years, regional ileitis or Crohn's disease as it is now known, has been regarded as a granulomatous ileocolitis of unknown etiology, distinct from that of intestinal tuberculosis and other mycobacterioses. Unfortunately, identification of the etiology and treatment for patients inflicted with this disorder has progressed little over the last 50 years.

As early as 1826, a chronic enteritis was reported in cattle that could not be associated with any currently known cause of diarrhea ⁸. The characteristic gross thickening and corrugation of the intestinal mucosa in these animals was recognized, but it was not until 1895 that the etiologic agent was discovered to be an acid fast bacillus ²³. At that time, the disease was called pseudotuberculous enteritis, and was thought to be caused by the avian tubercle bacillus (Mycobacterium avium) since the morphologic characters of the organism resembled that species more than Mycobacterium tuberculosis. The name pseudotuberculosis was given to this disease in recognition of its remarkable similarity to intestinal tuberculosis, but without caseous necrosis. However, unlike M. avium or M. tuberculosis, this organism resisted cultivation. It was fifteen years later, in 1910, that Twort ³⁸ succeeded in isolating the causative agent of this disorder. The isolation was largely the result of a laboratory mistake and a perceptive eye for fine detail - Twort noticed small satellite colonies arising around colonies of the common hay bacillus (Mycobacterium phlei) in culture tubes which had inadvertently not been cleaned for several months. It is now known that this organism requires an exogenous growth factor, mycobactin, extracted from other species of the genus and requires at least 3-4 months for laboratory growth ⁸. The organism was named, at the time, Mycobacterium enteriditis chronicae pseudtuberculosae bovis Johne³⁹. Between the period of its discovery in 1895 and the isolation of its etiologic agent in 1910, pseudotuberculous enteritis was reported from around the world ⁸. Over the years, the disease became known as paratuberculosis, commonly as Johne's disease, and in 1932, the organism was officially given the name M. paratuberculosis, although synonyms such as M. johnei were used for years after. Since the discovery of paratuberculosis as a disease, little progress has been made in its diagnosis, control, or treatment. For almost 100 years now, paratuberculosis has been spreading through the world's ruminant domestic livestock and wildlife - it remains difficult to diagnose or control, and no animal has ever been cured ⁸.

The first suggested connection between Johne's disease and Crohn's disease occurred in 1913 when Dalziel ¹⁵ described several cases of enteritis in human which, although resembling intestinal tuberculosis, he believed represented a new disorder. Recognizing the incredible similarity between his cases in humans and those of pseudotuberculosis recently described in cattle, he proposed "that the diseases may be the same". He also recognized a major distinction between these two diseases and expressed a view, still largely held to this date, that "In my cases the absence of acid fast bacilli would suggest a clear distinction...". He felt, however, that the histologic lesions were so similar that it justified his proposition even in the absence of demonstrable acid fast bacilli. Dalziel's views were never given much merit and the classification of these human cases as hyperplastic tuberculosis as proposed by Ingram ⁴⁴ prevailed. If Dalziel were alive today, ...

Although Crohn and his colleagues ¹⁴ ended the dispute of a mycobacterial etiology of the disorder in humans for almost 50 years, a few sporadic individuals failed to accept this general dissassociation and attempted to search for mycobacteria in Crohn's disease. Many, however, did their searching quietly, perhaps in fear of waking an old dead horse. In 1952, van Patter ⁴¹, in an effort that remains unpublished except as part of his dissertation, attempted to culture mycobacteria from the intestinal tissues of 43 patients with Crohn's disease. Although reported as a negative study, acid fast organisms which failed to grow on subculture, were isolated from 3 (7%) patients. Further efforts to associate Crohn's disease and mycobacteria took another 25 years to emerge. In 1978, Burnham et al ² reported the isolation of M. kansasii from the lymph node of a patient with Crohn's disease and pleomorphic variable acid fast organisms from 22 of 27 Crohn's disease patients, 7 of 13 ulcerative colitis patients, and 1 of 11 controls. These investigators proposed that Crohn's disease and ulcerative colitis were caused by cell wall deficient forms of M. kansasii. This hypothesis, however, did not receive widespread recognition and the role of cell wall deficient forms of M. kansasii as etiologic agents in inflammatory bowel disease was short lived. A few of the investigators, although remaining largely in the shadows, continued their efforts and search for a connection between their cell wall deficient organisms and Crohn's disease ³⁷.

In recent years, a variety of different approaches have been used to examine the relationship between mycobacteria and Crohn's disease. These have included cultivation, examination of immune reactivity, animal model studies, anti-mycobacterial treatment, and molecular biology.

CULTURAL STUDIES

In 1984, the isolation of an M. paratuberculosis - like organism from 2 patients with Crohn's disease, but not from 3 patients with ulcerative colitis or 3 with other bowel disorders was reported ⁹. These isolations were particularly interesting because the organism resembled that which caused a similar and well recognized granulomatous ileocolitis in ruminants known as paratuberculosis ⁸. It was perhaps this report which initiated the current controversy and which has lead to perhaps the first true concerted effort to determine whether or not mycobacteria are etiologically associated with Crohn's disease. In the last few years, there have been more reports on mycobacteria and Crohn’s disease than in the last 100 years or more. This current worldwide effort and excitement (by at least some) over a mycobacterial etiology of Crohn’s disease was perhaps generated by 2 unrelated circumstances: 1) the isolation of an organism with a reputation for causing a disease very similar to Crohn's disease in animals; and 2) after 50 years of getting no where, with all theories including autoimmune, dietary, viral, etc., loosing ground, and with medical treatment supportive at best, the time was perhaps right to awaken an old dead horse.

Although the status of investigations of the role of mycobacteria and Crohn's disease has recently been reviewed in detail ⁴, there have been many achievements in the last few years. These advances have largely been possible by the discovery of IS900, a species specific insertion sequence of M. paratuberculosis ¹⁸, and the application of the polymerase chain reaction (PCR) and other molecular approaches. Readers are referred elsewhere for detailed discussions of previous findings ⁴.

The original studies suggesting an etiologic role of M. paratuberculosis - like organisms in Crohn's disease were expanded, and it was determined that primary isolation of this organism was in the form of a spheroplast or cell wall deficient form ¹². These spheroplast-like agents were reported to have been isolated from 16 of 26 patients with Crohn's disease (61 %), but not from 13 patients with ulcerative colitis or from 13 patients with other bowel disorders ¹². Of these 16 Crohn's disease - associated spheroplasts, 4 had transformed into classical bacillary forms resembling M. paratuberculosis. One of these cultures required 5 years for the emergence of macroscopically visible colonies. By a variety of molecular techniques, including DNA:DNA hybridization ^28,45, 5s ribosomal DNA analysis ^5,12, and restriction fragment length polymorphism ²⁹, these organisms were definitively identified as M. paratuberculosis. Recently, the remaining 12 unidentified spheroplasts from the Crohn's disease patients and several from controls were examined by PCR for the presence of IS900. An additional 6 cultures from Crohn's disease patients were identified as containing M. paratuberculosis, bringing the total number of isolates from these investigators to 10 out of 26 or 38% of all Crohn’s disease cultures⁴². IS900 was not detected in any culture from non - Crohn's disease sources.

In addition to these initial investigators, others have participated in searching for mycobacteria in Crohn's disease patients. In Australia, Dr. P. Coloe of the Royal Melbourne Institute of Technology isolated M. paratuberculosis from a patient with Crohn's disease, representing the first group to reproduce earlier findings. Other isolations soon came from France, California, Houston, and The Netherlands ⁴. Dr. G. Gitnick at the University of California in Los Angeles, isolated a strain of M. paratuberculosis from 1 of 27 patients with Crohn's disease, but not from 29 patients with ulcerative colitis or 26 with other bowel disorders ¹⁷; although other Mycobacterium spp. were isolated from ulcerative colitis, control, and Crohn's disease cultures. Other investigators have also attempted cultivation, but their efforts are less defined and the reader is referred elsewhere for these discussions⁴.

At present, the 3 most active groups attempting to culture mycobacteria from Crohn's disease patients are Chiodini, who's efforts have largely already been described above, D. H. Graham from the Baylor College of Medicine, and J. Haagsma of the Veterinary Research Institute in The Netherlands.

Graham and coworkers ²⁶ initially reported a vast number of mycobacterial isolates from Crohn's disease patients (5 of 59 or 15%), ulcerative colitis patients (9 of 19 or 47%), and non-IBD controls (18 of 27 or 67%). Most of these isolated were environmental Mycobacterium spp., primarily Mycobacterium avium and Mycobacterium fortuitum. In addition to these bacillary forms, these investigators also isolated spheroplast or assumably cell wall deficient variable acid fast organisms, primarily from Crohn's disease tissues. Examination of these unidentified spheroplast forms revealed the presence of IS900 (M. paratuberculosis) in 6 of 18 (33%) Crohn's disease cultures examined, in none of 6 ulcerative colitis cultures, and in only 1 of 5 non-IBD controls ³¹.

Haagsma et al ¹⁹ cultured tissues from 100 patients with Crohn's disease and 50 from patients with ulcerative colitis or other intestinal disorders. Of the 100 Crohn's disease cultures, approximately 50 have been lost to contamination by other microbes and were considered unsuitable for evaluation. Of the remaining 50 Crohn's disease cultures, they have been successful in isolating M. paratuberculosis from 10 of them, but not from any of their ulcerative colitis or other control tissues ³². In addition, this group has been successful in identifying acid-fast bacilli from an additional 10-15 patients, but these have not been identified as yet.

In summary, of the 3 major groups involved in cultivation of mycobacteria from Crohn's disease, M. paratuberculosis has been isolated or otherwise detected in culture from 38%, 33 %, and 20% out of a total of approximately 135 patients, but from only 1 of over 121 non-Crohn's disease controls. This sort of distribution suggests more than a casual role for this organism in the etiology of Crohn's disease.

One of the major complications to our understanding of the role of these organisms in Crohn's disease is the widespread distribution of mycobacteria in the environment which complicate not only the isolation of particular species, but also our interpretation of their isolation. As we learn more about this particular organism and that of closely-related environmental species, we may begin to understand the role of mycobacteria in Crohn's disease.

GENETIC STUDIES

As discussed earlier, the application of molecular biology to the study of mycobacteria in Crohn's disease and the discovery of IS900 in M. paratuberculosis, has resulted in major advances in our understanding of the distribution of M. paratuberculosis and its possible association with Crohn's disease ²⁰.

Initially, these methods were used to determine the identification of these M. paratuberculosis - like agents from Crohn's disease tissues, and by a variety of methods including DNA:DNA hybridization, analysis of 5s ribosomal DNA genes, and restriction fragment length polymorphisms, these organisms were definitively identified as M. paratuberculosis^5,28,29. Recently, by examination of RFLP of IS900, it was further determined that the human isolates resemble those of cattle strains more than those obtained from ovine or caprine species ^13,43. These findings suggest that the source or reservoir of these organisms in humans may be the domestic cattle population.

The two major advances accomplished by molecular biology has been the use of PCR in combination with IS900 to examine unidentified cultured organisms and the direct examination of tissues for the presence of this sequence.

Many investigators referred to above and many addressed previously⁴, had been successful in isolating unidentified organisms from tissues of both Crohn's disease patients and controls, but the slow growth of these organisms precluded their identification by conventional methods. It has not in the past been possible to intelligently speak of these isolates because their identity was unknown, and even if mycobacterial in origin, the widespread distribution of mycobacteria in the environment preclude any suggestions related to etiologic or pathogenic potentials. The discovery of IS900¹⁸ as a species-specific marker of M. paratuberculosis allowed for the first time the ability to specifically identify this species. Combined with the amplification of PCR, a powerful tool emerged. Examination of unidentified cultures from Crohn's disease and controls revealed IS900 in 38% (10 of 26) of Crohn's disease cultures in one study ⁴² and in 33% (5 of 15) in another ³¹, thereby suggesting that about 1/3 of cultures from Crohn's disease patients have detectable levels of M. paratuberculosis. Based on the hybridization signals, it has been estimated that these cultures contained between 3 and 30 individual mycobacterial cells (genomes) per culture pellet. Such results suggest that these strains either fail to grow in vitro or grow at an extremely slow rate. Using a more ubiquitous probe against the 65 Kd heat shock protein, mycobacterial sequences of unknown species were detected in Crohn's disease as well as control cultures ⁴², again illustrating the ubiquitous nature of mycobacteria in general and the need to define your objectives and organisms at the species level.

Another application of molecular biology has been the direct detection of mycobacterial DNA in tissue specimens. Earlier studies using DNA:DNA hybridization techniques to search for mycobacteria in Crohn's disease patients were complicated by the large number of hybridizations suggesting the presence of mycobacterial DNA in 53% (10 of 19) of Crohn's disease biopsies, 33% (2 of 6) of ulcerative colitis patients, and 17% (1 of 6) of normal controls⁴⁵. These results were comparable to studies on the environmental distribution of mycobacteria and suggested increased colonization of mycobacteria in Crohn's disease. Without the ability to differentiate species, these findings had little etiologic meaning.

With IS900, the ability to specifically search for this species was possible and PCR provided the sensitivity needed to detect an organism that was below the detection level of acid-fast light microscopy or immunohistochemistry. Application of PCR and IS900 to biopsy specimens revealed the presence of M. paratuberculosis in 65% (26 of 40) of Crohn's disease patients, 4.3% (1 of 23) of ulcerative colitis patients, and 12.5% (5 of 40) of non-IBD controls³⁵. These findings have several important implications. The finding of IS900 in controls indicates a previously unrecognized environmental distribution of this organism, suggesting that environmental contact with M. paratuberculosis may be common. The low presence of M. paratuberculosis in ulcerative colitis patients suggest that colonization of the disrupted or abnormal intestinal mucosa by M. paratuberculosis does not occur. Therefore, the high prevalence in Crohn's disease patients would suggest some etiologic role. A casual relationship between IS900 and Crohn's disease is not supported by these studies as a similar distribution should have been observed in ulcerative colitis.

Immunology

Immunologic studies on the reactivity of Crohn's disease patients to mycobacterial antigens have been sparse and inconclusive, and the reader is referred elsewhere for a review of previous efforts⁴. In general, patients with Crohn's disease fail to elicit any consistent humoral or cell mediated response to mycobacterial antigens. Although we and others have observed some responsiveness, the results are generally not reproducible⁴. While superficially these findings would suggest an etiologic dissassociation between Crohn's disease and mycobacteria, the nature of the studies performed do not provide a solid framework on which to reach that conclusion.

The pathologic lesion of Crohn's disease, in general, can be considered a delayed type hypersensitivity type reaction resembling a tuberculoid lesion. By definition, such a lesion is mediated by cellular elements of immunity which may not be accompanied by a humoral response. Therefore, the absence of any demonstrable humoral response to mycobacterial antigens in Crohn's disease would not be unexpected. On the other hand, one would expect a cellular response to mycobacterial antigens in Crohn's disease if these agents were etiologically related. No such response, however, has been consistently demonstrated. There may be several reasons for such, few of which have been adequately examined.

Crohn's disease is a mucosal disease and the sequestered nature of the lesions suggest that effector cells may be compartmentalized within the intestine. Therefore, examination of peripheral reactivity may not be appropriate. There are multiple examples in chronic infectious diseases, including mycobacterial, in which vigorous responses can be demonstrated in infected tissues in the absence of peripheral activity^33,36. Cases with extraintestinal lesions may be the only cases in which humoral reactivity might be expected.

Additionally, and perhaps more importantly, the widespread reactivity of the general population to environmental antigens^1,16 may preclude detection of a specific response. In our studies of peripheral reactivity to mycobacterial antigens in Crohn's disease by a lymphocyte blastogenesis assay (unpublished observation), most Crohn’s disease patients responded well to M. paratuberculosis antigens. However, despite a strong response by the Crohn's disease patient group, it was not significantly different from the control population. The response of the control group was so high that a specific response in Crohn's disease would not have been demonstrated even if such existed. It is not a case that Crohn's disease patients do not respond, but rather that their response cannot be differentiated from normal responses.

Current evidence suggests that if mycobacteria are causative agents in Crohn's disease that they exist in an altered form, probably as a spheroplast or other pleomorphic form without a full cell wall. Under such conditions, the antigens present and expressed may be quite different from those found in the bacillary form since most of the immunogenic antigens are cell wall associated. Therefore, to find a specific response in Crohn's disease patients, it may be necessary to look at reactivity of specific antigens in order to separate specific responses from those obscured by general environmental reactivity. In tuberculoid leprosy, for example, humoral reactivity to M. leprae is generally not demonstrable unless specific antigens, such as PGL- 1, are used and only then can specific antibody responses be detected³⁴. Unfortunately, few specific antigens currently exist for M. paratuberculosis.

In consideration of immunologic reactivity, one must also consider statistical data which in itself may preclude the detection of immune reactivity. There is considerable debate on whether or not Crohn's disease is a single disease entity. Regardless of the position one wishes to hold, most will agree that the characteristics of Crohn's disease are ambiguous enough that it could comprise several etiologies. If we assume cultural and molecular biology data to represent the possible infection rate with M. paratuberculosis, i.e., 65 % or less; recognize the inconsistency in detection of immune reactivity in most chronic infections, e.g., compartmentalization, anergy, etc.; the effects of contact with the agent by nonsusceptible hosts; and then add a 20% diagnosis error as suggested by epidemiologic studies³; a consistent immunologic response in Crohn's disease to its etiologic agent, what ever it might be, would be difficult to detect. This is not to imply that investigations are not warranted or unlikely to show cellular reactivity to mycobacteria, but rather to bring to light that such studies may begin with many lines drawn against success.

ANIMAL MODEL STUDIES

There have been several attempts by various investigators to develop mycobacterial models of Crohn's disease, but in this authors opinion, none have been particularly successful. Our initial studies in goats⁴⁰ do not provide an appropriate model in many respects because, although a natural host of M. paratuberculosis, the disease is of the lepromatous form rather than a tuberculoid disease as seen in humans. Even in the disease observed in primates²⁷, the lesions and corresponding immune reactivity is lepromatous. Since Crohn's disease, regardless of its etiology, clearly has a major immunologic component, comparison between tuberculoid and lepromatous disease is not entirely appropriate since different immunologic mechanisms exist. To compare tuberculoid to lepromatous leprosy is like comparing apples and oranges.

This putative agent, M. paratuberculosis, does not readily infect species other than ruminants⁸. To accomplish such generally requires modulation of the immune response which may destroy the animal as an appropriate model. Can a disease which contains a major immunologic component as the disease causing factor itself be successfully studied in an immunodeficient or immunocompromised host? While meaningful data can be derived from such studies, they do not represent realistic models in which to study pathogenesis.

For years investigators have sought animal models of paratuberculosis but none has been found to be appropriate⁸. The same seems likely with Crohn's disease. It may be necessary to explore other animals but their cost may not offer the advantage generally dictating animal models. For instance, the disease observed in subhuman primates (stumptail macaques) was of the lepromatous form, but we were unsuccessful in infecting other subhuman primates (rhesus). Other primates, however, may be susceptible and provide a tuberculoid-type disease, but their cost and per diem charges would likely preclude their use over human clinical studies.

If mycobacteria are ultimately found to be involved in the pathogenesis (or perhaps more appropriately immunopathogenesis) of Crohn's disease, animal models are likely to play a major role in determining certain aspects of the disease process.

TREATMENT

There have been numerous studies on the efficacy of treatment of Crohn's disease with various antimicrobial (anti-mycobacterial) agents, but few have been scientifically valid enough to draw any conclusive conclusions (see 4 for review). There are a large number of case reports suggesting efficacy of anti-mycobacterial chemotherapy in Crohn’s disease, but to date, no controlled study has shown any conclusive (objective) benefit of such treatment. The reasons for such failures may be based on the manner in which these studies were conducted, and unfortunately, continue to be conducted. No animal with M. paratuberculosis infection has ever been cured.

We have learned over the years that one cannot extrapolate data from the treatment of tuberculosis to leprosy, or visa versa, or to other mycobacterial infections. Even within the Enterobacteriacae, it is known that effective therapy with one species does not necessarily provide efficacy with infection by another. Therefore, the choice of treatment regimes based on tuberculosis may be inappropriate. In recent years we have gained a great deal of knowledge in the treatment of M. avium infections, in both compromised and non-compromised patients, and such data would perhaps be more appropriate to base treatment regimes. Alternately, or concurrently, susceptibility patterns for M. paratuberculosis strains isolated from Crohn's disease patients have been determined for single drugs and multiple combinations ^6,7,11 and should be considered. It must, however, be appreciated that in vitro effects and therapeutic efficacy may not always agree, particularly with regard to intracellular organisms.

Current advances in the treatment of M. avium pulmonary infections in non-immunocompromised patients suggests that at least 3 or more drugs need to be used simultaneously and continued for 3.6+-0.5 years in order to achieve clinical improvement ²¹. In light of these findings, the use of single or double anti-mycobacterial drugs in the treatment of Crohn's disease for a 6-month period would seem rather inadequate. It must be appreciated that all antimicrobial agents work only during the replicative phase of bacterial growth - these agents have no effect during stationary or other nonproliferative state. According to all data accumulated to date, if mycobacteria, particularly M. paratuberculosis, are involved in the pathogenesis of Crohn's disease, they exist within the intestine in very small numbers and in a form which rarely replicates. Under such a situation, the 3.6 years required for M. avium infections may not be long enough to eliminate M. paratuberculosis and its antigens should they exist in Crohn's disease.

Furthermore, there exists the possibility that a 6-month limited drug trial could make the patient transiently worse rather than better. As an immunologically based disease (like most mycobacterial infections) the killing of organisms with their subsequent release of antigens could conceivably cause a worsening of symptoms, perhaps similar to the reversal reaction in leprosy²². It must also be appreciated that intestinal tuberculosis cannot be cured by chemotherapy alone but requires surgical intervention.

In summary, to conclusively evaluate the efficacy of anti-mycobacterial chemotherapy in Crohn's disease, the use of multiple drug combinations for extended periods need to be evaluated. The statistical matters discussed under immunology will also need to be considered in patient selection and analysis of data.

Conclusions.

Progress over the last few years regarding the role of mycobacteria in Crohn's disease have not progressed as fast as many may have anticipated or wished. There are various reasons for the slow progress. If Crohn's disease is caused by M. paratuberculosis or another Mycobacterium spp., it is not the classical picture of tuberculosis or leprosy, but some unique unusual manifestation which is not fully understood. The classical mycobacterioses have puzzled scientists for well over 100 years and something outside this picture is going to be even moredifficult to understand. Pathogenic mycobacteria elicit one of the most complex, and in many respects most poorly understood, immunological events known. After all, disease by these organisms is caused by the host's response to them rather than any effect they cause on the host. Understanding the classical diseases are difficult enough; novel approaches will be required to understand a non-classical disease. There are also a limited number of investigators actively studying this particular subject and funding is in general sparse. As a result, progress has been, and will likely continue to be, slow. Increased efforts are likely to be limited until the controversy surrounding this issue subsides and mycobacteria become convincingly established as etiologic agents in at least some cases of Crohn's disease.

Although there are various aspects of Crohn's disease as related to mycobacteria that are perhaps needed to be understood, the primary objective must be in establishing a definitive role for these agents in Crohn's disease. Aspects unrelated to this primary objective will have little meaning unless an etiologic role is firmly established. Unfortunately, differing views exist, largely because there are no clear cut answers and a variety of approaches will be required to collectively provide a determination. At present, the greatest hope lies in molecular biology and the demonstration of etiologic agents directly in tissues. These techniques are most likely to provide the strongest evidence of an association and these studies are currently in progress. The other major area, since Crohn's disease contains a major immunologic component, is identification of the antigenic determinant(s) and immunologic responses that are giving rise to the chronic inflammation. These may be unique antigens or antigens cross-reactive with mucosal tissue. Reactivity may also be associated only within the intestinal lymphoid cells. There is general agreement that if mycobacteria are etiologically related to Crohn's disease, a cellular response must be present and that response will need to be demonstrated in addition to the demonstration of mycobacteria in tissues.

In summary, there has been a great deal of effort since 1984 investigating the role of mycobacteria, specifically M. paratuberculosis, as etiologic agents in Crohn's disease. The data suggesting a role is largely based on cultural isolation or other methods of detection of a well known animal pathogen from the tissues of Crohn’s disease patients, but interpretation of the data has been complicated by the isolation of closely related environmental mycobacteria from both Crohn's disease patients and controls. Nevertheless, it is now conclusively and irrefutably shown that M. paratuberculosis can be found within the tissues of a major proportion of Crohn's disease patients. With current studies and efforts continuing, this association is likely to become more and more established and the etiologic role of M. paratuberculosis in Crohn’s disease will likely gain momentum.

As methods are improved and an understanding of the environmental distribution of mycobacteria is achieved, the significance of these pathogenic strains will become more evident. Although it has been a slow path down the road, recent advances suggest an answer to this intriguing and controversial subject may be approaching in the near future.

The old horse isn't dead yet and current evidence may even suggest that it is not only being revived, but may be nursed into good health.

REFERENCES