Crohns disease and the mycobacterioses
Rodrick J. Chiodini, Ph.D.

Table of contents

Summary.
What is Crohns Disease?
Is Crohns Disease an infectious process?
Mycobacteria and Crohns Disease: A historical perspective.
The search for a mycobacterial etiology.
Cultural Data
Discussion of Cultural Data
Immunological Data
Discussion of Immunological Data
Histochemical Data
Discussion of Histochemical Data
Animal Model Data
Discussion of Animal Model Data
Treatment Data
Discussion of Treatment Data
Similarities between Crohns Disease and other mycobacterial disease.
Pathology
Epidemiology
Immunology
Chemotherapy
Conclusions
Acknowledgements
Abbreviations used
Literature cited
Tables
Table 1. Isolates of mycobacteria from Crohn's disease patients and control populations.
Table 2. Pathogenic characteristics of mycobacteria isolated from Crohn's disease patients and controls.
Table 3. Isolation of pathogenic M. paratuberculosis from patients with Crohn's Disease.
Table 4. Methodologies used for the isolation of mycobacteria from Crohn's disease tissues.
Table 5. Clinical similarities between Crohn's disease and mycobacterioses.
Table 6. Pathologic similarities between Crohn's disease and mycobacterioses
Table 7. Systemic similarities between Crohn's disease and mycobacterioses.
Table 8. Epidemiologic features of Crohn's disease, ileocecal tuberculosis, and paratuberculosis.
Table 9. Time span between investigations seeking a mycobacterial etiology of Crohn's disease.

Information appearing here is a small excerpt from the extensive originating article.....  RM

Summary
Crohn's disease is a chronic granulomatous ileocolitis, of unknown etiology, which generally affects the patient during the prime of life. Medical treatment is supportive at best and patients afflicted with this disorder generally live with chronic pain, in and out of hospitals, throughout their lives.

The disease bears the name of the investigator that convincingly distinguished this disease from intestinal tuberculosis in 1932. This distinction was not universally accepted and the notion of a mycobacterial etiology has never been fully dismissed. Nevertheless, it was 46 years after the distinction of Crohn's disease and intestinal tuberculosis before research attempting to reassociate mycobacteria and Crohn's disease was published.

Recently, there has been a surge of interest in the possible association of mycobacteria and Crohn's disease due largely to the isolation of genetically identical pathogenic M. paratuberculosis from several patients with Crohn's disease in the United States, the Netherlands, Australia, and France. These pathogenic organisms have been isolated from only a few patients and direct evidence for their involvement in the disease process is not clear; however, M. paratuberculosis is an obligate intracellular organism and strict pathogen which strongly suggests some etiologic role.

Immunologic evidence of a mycobacterial etiology, as assessed by humoral immune determinations, have been conflicting, but evaluation of the more relevant cellular immunity have not been performed. Data from histochemical searches for mycobacteria in Crohn's disease tissues have been equally conflicting with acid-fast bacilli detected in 0 to 35% of patients. Animal model studies have demonstrated the pathogenic potential of isolates as well as elucidating the complexity of mycobacteria-intestinal interactions.

Treatment of Crohn's disease patients with anti-mycobacterial agent has not been fully assessed, although case reports suggest efficacy. The similarities in the pathology, epidemiology, and chemotherapy of Crohn's disease and the mycobacterioses are discussed. The issue is froth with controversy and the data generated on the association of mycobacteria and Crohn's disease are in their infantile stages such that a general conclusion of the legitimacy of this association cannot be made.

While no firm evidence clearly implicates mycobacteria as an etiologic agent of Crohn's disease, the notion is supported by suggestive and circumstantial evidence, and a remarkable similarities to other known mycobacterial diseases.

...........

The culture results of Graham and co-workers (106) provide some useful and important information and illustrate the ubiquitousness of some Mycobacterium spp. These workers applied tissue processing techniques of lower stringency than that recommended for the isolation of M. paratuberculosis, and these methods probably account for their results. Although the authors suggest that their method using 0.1% hexadecylpyridinium choride (HPC) is that recommended by the National Animal Disease Center (NADC), this laboratory actually recommends 0.1% benzalkonium chloride (279) or more recently, 0.75% HPC (187). At concentrations less than 0.75% HPC, contaminants, which include environmental mycobacteria, commonly overgrow cultures from clinical specimens. Since organisms of the MAI and M. fortuitum complex are widespread in the environment, processing techniques of low stringency would result in the isolation of these species from a variety of sources. The results of Graham et al (106) are comparable to those obtained from environmental sources. MAI complex can be isolated from 26-63% of soil samples, 50% of tap water samples, 13% of dust samples, and 35% of air samples (27, 95, 122, 241, 289). M. fortuitum can be found in 39% of soil samples, 63% of dust samples and 25% of air samples (137, 241, 284).

An interesting feature of the culture results of Graham et al (106) is the specimen type from which mycobacteria were isolated. Except for a single strain of M. fortuitum complex, mycobacteria could not be isolated from resected tissues; but 35 strains of mycobacteria (predominantly MAI and M. fortuitum) were isolated from biopsy specimens of aphthous ulcers. These ulcers provide a suitable micro-environment for the propagation of such environmental organisms. On the other hand, Graham et al (106) isolated as yet unidentified spheroplasts primarily from resected tissues of Crohn's disease patients rather than aphthous ulcers. Retrospectively, these culture data appear to support, rather than refute, a CWD mycobacterial etiology. Thus, as in all other diseases, the area from which material is obtained for culture is of great importance, as are the techniques applied to tissue processing.

At about the same time, Haga (115) briefly reported that he was unable to isolate mycobacteria from 17 fecal specimens, 5 bowel resection, or 9 biopsy specimens from Crohn's disease patients; although, he did report the isolation of acid-fast coccoid bodies from a Crohn's disease patient which could not be identified or subcultured. S. R. Pattyn, F. Portaels, and Y. Van Maercke presented their culture results at the meeting of the International Working Group on Mycobacterial Taxonomy (IWGMT) held in Bithoven, The Netherlands in September of 1987 (S. R. Pattyn, personal communication) and later published their data in the form of a letter (L. J. Colemont, S. R. Pattyn, P. P. Mitchielsen, J. H. Pen, P. A. Pelckmans, Y. M. Van Maercke, and F. Portaels. Lancet 1:294-295, 1988). These workers examined tissues from 32 patients with Crohn's disease and demonstrated acid fast bacilli in 11 (34%) by acid-fast staining. Cultivation attempts yielded 2 strains of M. chelonei which were said to be mycobactin-dependent; mycobacteria could not be isolated from the remaining 9 cases in which acid-fast bacilli were observed. The authors acknowledged that their processing technique, i.e., 0.15% benzalkonium chloride and 0.5% NaOH, may have been too deleterious for recovery of other acid-fast bacilli; M. paratuberculosis is known not to survive exposure to NaOH decontamination.

.........

Discussion of Cultural Data
It is now clear that a host of different mycobacteria can be isolated from Crohn's disease patients, as well as control populations, and that diseased tissue may be a suitable micro-environment for colonization of some of these species

This controversy is perhaps heightened by the evidence that M. paratuberculosis may be an etiologic agent in Crohn's disease because the organism itself is controversial......... This organism is the slowest growing of the culturable mycobacteria and has a variety of sensitive growth requirements for cultivation (51-54). It generally takes years to become fully proficient at working with this species. Often even the most experienced mycobacteriologists have difficulty growing it because conventional methods are not appropriate.

Immunological Data

....... In conjuction with their isolation of M. kansasii in culture, Burnham et al (33, 34) determined that in skin tests with antigens prepared from M. kansasii a high proportion of Crohn's disease patients showed an increased response as as compared to controls. ........ in 1980, Grange et al (107) reported increased IgA and IgM antibodies to M. tuberculosis in patients with Crohn's disease. They noted that responses of tuberculosis patients were predominantly of the IgG class rather than IgA and IgM as found in Crohn's disease.

In conjunction with the isolation studies reported by Chiodini et al (48,49), these authors (273) presented data suggesting increased serologic reactivity to M. paratuberculosis antigens in Crohn's disease by the ELISA technique. Patients with Crohn's disease had a statistically significant increase in antibody titer to a protoplasmic antigen of M. paratuberculosis as compared to controls. Examining cross-reactivity between antigens, these authors found 52.5% and 39% cross reactivity of their antigen with M. kansasii and M. tuberculosis, respectively. As a result of this cross-reactivity, a significant proportion of Crohn's disease patients' sera also reacted to M. kansasii antigens.

Jiwa et al (130) described IgG serum antibodies to mycobacterial PPD's in Crohn's disease patients. These investigators examined seroreactivity to PPD's prepared from M. tuberculosis, M. kansasii, M. phlei, M. paratuberculosis, and M. smegmatis and found that Crohn's disease patients have elevated antibody titers to all species examined. Serologic studies conducted with a crude antigen and 3 antigenic fractions of M. paratuberculosis also showed a slight, but insignificant, increased antibody titer in Crohn's disease patients as compared to controls. Such widespread reactivity to PPD, probably based on a ubiquitous cross-reactive antigen, is highly indicative of sensitization by environmental organisms gaining immune access through a defective mucosal barrier.

Das et al (Das, P. K., J. L. G. Blaauwgeers, A. W. Slob, J. Spies, A. Chand, A. Kolk, and H. J. Houthoff. Gastroenterol. 94:A88, 1988) examined the possible relationship of mycobacteria and Crohn's disease by using immunoblot analysis and a lymphoproliferative assay. They found that sera from patients with Crohn's disease reacted with various mycobacterial and gut-associated antigens, and that many sero-reactive epitopes were shared between mycobacteria and human gut tissue. Thus they concluded that the pathogenesis of Crohn's disease could involve either cross-reactive epitopes or idiotypes, without the persistent presence of viable mycobacteria. Their lymphoproliferative assay showed that the lymphocytes from 5 out of 6 patients with Crohn's disease reacted specifically to M. paratuberculosis antigens, whereas those from controls, ulcerative colitis, and bowel cancer patients did not....... these preliminary studies represent the only investigation of CMI responsiveness to mycobacterial antigens in Crohn's disease.

Discussion of Histochemical Data
The inability to detect mycobacteria or their antigens in tissue from patients with Crohn's disease is perhaps the most damaging evidence against their role as etiologic agents; however, if an agent associated with Crohn's disease could be easily demonstrated, it would have been found years ago. It is also unclear why one investigator failed to find any evidence of mycobacteria in 30 patients with Crohn's disease (148), another found evidence in one of 15 patients with Crohn's disease (282), and yet another found evidence in 34% of Crohn's disease specimens (Colemont, L. J. et al. Lancet 1988; 1:294-295). As Rubin and Pinner (237) have said about mycobacteria and sarcoidosis "the failure to find tubercle bacilli in the majority of cases ... is not a convincing argument against tuberculosis as an etiologic factor, nor is the occasional tubercle bacillus which is found positive proof that tuberculosis is the cause".

Animal Model Data
In 1984, using infant goats, Chiodini et al (49) described the first successful production of a granulomatous ileocolitis, or Crohn's disease-like infection, in experimental animals with a putative etiologic agent. Further, more detailed, studies with this goat animal model were later published in 1986 (281). Oral inoculation of goats with their putative agent, later identified as M. paratuberculosis, produced intestinal disease in approximately 5-6 months. The earliest lesions occurred within Peyer's patches of the ileum and consisted of non-caseous granulomatous clusters of epithelioid cells which often occurred in a mantle of lymphocytes between germinal centers and the muscularis mucosae; quite similar to the early lesions of Crohn's disease. Other features of the disease included tuberculoid granulomas without caseation, confluence of granulomas, ulcerations of the mucosa, and lymphocytic lymphangitis. Several animals had no demonstrable acid-fast bacilli although the bacillary organisms were isolated from all except controls. The authors concluded that the lesions produced in these animals were distinctly similar to those occurring in Crohn's disease.

Some recent animal experiments conducted at the University of Wisconsin in Madison yielded some unexpected results which also have relevance to animal models of Crohn's disease (H. A. Mokresh, S. Hurley, C. Czuprynski, and D. Butler, personal communication) . Newborn rabbits were orally inoculated with M. paratuberculosis and necropsied at various time periods to determine if intestinal lesions could be produced in these animals. Some developed a transient diarrhea and some developed a few intestinal granulomas. Interestingly, culture and prolonged incubation (11 to 15 months) of fecal and ileal tissue homogenates from some rabbits resulted in the growth of very small translucent colonies. The organisms stained poorly, and on electron microscopic examination, were found to be morphologically identical to the mycobacterial spheroplasts described previously in Crohn's disease (55). These CWD forms could not be subcultured. Should these results be repeated, they would represent the first successful in vivo transformation of mycobacteria into CWD forms. They may also represent an animal model for the physiologic and morphologic changes in M. paratuberculosis which may be occurring in the human intestine. These preliminary results have inspired further efforts which are currently in progress.

Discussion of Animal Model Data
Perhaps as a follow-up of the suggestions first made by Dalziel (64) in 1913, and later by Golde and McGill (101) and Patterson and Allen (218), Morgan (194) recently published a theoretical paper comparing Crohn's disease and Johne's disease (paratuberculosis) and suggested that, based on previously reported experimental and epidemiologic data, these two diseases had similar etiologies. He noted the difficulties encountered in experimentally transmitting Johne's disease, not only to other cattle, but also to laboratory animals, as well as a host of other remarkable similarities. This author believed that the similarities, and disease histories, were too remarkable to be coincidental.

It has been disputed that the experimental production of a granulomatous ileocolitis in goats with human isolates of M. paratuberculosis have little meaning and does not support an etiologic role of this agent in Crohn's disease (Graham, D. Y., D. C. Markesich, and H. H. Yoshimura, Letter, Dig. Dis. Sci. 33:251-252, 1988). Since the putative agent of Crohn's disease has been identified as M. paratuberculosis, the experimental infection is not Crohn's disease-like, but expectedly, is only Johne's disease. Such a conclusion, while taxonomically correct, should not be viewed as a simple distinction based on disease classification, and thereby separating two similar diseases. If Crohn's disease is not caused by M. paratuberculosis, then clearly the granulomatous ileocolitis produced in ruminants by human strains of M. paratuberculosis is Johne's disease. But, if Crohn's disease is caused by M. paratuberculosis, then the experimental infection produced in animals does represent Crohn's disease disease, even if the appropriate classification of the infection in animals is Johne's disease.

The isolation of a known animal pathogen from human patients with Crohn's disease has more implications to etiology than the isolation of a new or unknown species with no defined pathogenic characteristics. The fact that M. paratuberculosis is not an environmental organism and cannot replicate in the environment, readily penetrates and has a predilection for the gastrointestinal tract, produces a non-caseating granulomatous intestinal disease in animals, and has been isolated from the diseased tissues of patients with Crohn's disease, must at least raise issues of coincidence and suspicion amongst investigators.

Treatment Data
....... there was no consistent pattern of change in the requirement for steroids in patients receiving anti-mycobacterial drugs. From their experience and data, these authors concluded that rifampin and ethambutol have no place in the treatment of Crohn's disease and that it was unlikely that M. kansasii was etiologically significant (245).

Hampson et al (Hampson, S. J., M. C. Parker, S. H. Saverymuttu, J. J. McFadden, and J. Hermon-Taylor. Gastroenterol. 94:A170, 1988;) treated 17 Crohn's disease patients with quadruple anti-mycobacterial chemotherapy. The antimicrobial agents used in combination were rifampin, ethambutol, isoniazid, and pyrazinamide with clofazimine replacing pyrazinamide in a few cases. Of the 17 patients, 12 (71%) had a statistically significant improvement in their CDAI and 9 of 10 (90%) had been completely withdrawn from steroids. At this date, these investigators have treated 20 patients with quadruple therapy, and after 9-months treatment, 11 of 20 (55%) are considered to be in disease remission. Based on 111 Indium scans, objective evidence of improvement after 1-year of treatment was found in 14 of 20 (70%) of patients (Hampson, S.A., personal communication, 1988).

The flu-like syndrome, which may or may not be associated with leukopenia, that is frequently observed in patients with Crohn's disease receiving rifampin or its derivatives is not understood. Other patients receiving rifampin or rifabutin, including patients with tuberculosis, leprosy, atypical mycobacteriosis, or acquired immune deficiency syndrome (AIDS), do not develop these symptoms; the flu-like syndrome appears unique to Crohn's disease patients. Although this syndrome was considered grounds for withdrawal from therapy in some studies, most consider that the severity is not sufficient to warrant drug withdrawal. Patients receiving steroids at the time rifabutin therapy is initiated fail to develop flu symptoms, i.e., steroids prevent the flu-loke syndrome. Thus, this condition is probably either related to drug toxicity or, considering the intervention by steroids, some form of immune phenomenon.

Similarities between Crohns disease and other mycobacterial disease
As has been noted since the first description of Crohn's disease in 1932, the similarities between Crohn's disease and mycobacteriosis are remarkable (59). Since that time, Crohn's disease and mycobacteria have been pushed apart so distantly that many of the common features have become obscure. Many consider that Crohn's disease received a too enthusiastic and uncritical acceptance as a unique disease entitity and that the diagnosis of primary intestinal mycobacteriosis was too lightly discarded. Primary hypertrophic intestinal tuberculosis does occur, and although early investigators thought that this disease was Crohn's disease, the two have been distinguished. In the western world, intestinal tuberculosis is generally misdiagnosed as Crohn's disease, and such cases are properly diagnosed only post-surgically (3, 39). On the other hand, in underdeveloped and those developing countries where tuberculosis is common, cases of granulomatous intestinal disease are generally diagnosed as tuberculosis. The similarities and dissimilarities of Crohn's disease and the mycobacterioses will be difficult to understand fully in the near future. A literature search from 1966 to September 1987 retrieves 7,661 reports on Crohn's disease or IBD and 31,429 on mycobacteria, tuberculosis, or leprosy. Comparisons must be made almost exclusively between Crohn's disease and tuberculosis or leprosy, because relevant data are limited to these human diseases.

Every clinical, radiologic, endoscopic, and pathologic feature of Crohn's disease may occur in primary intestinal tuberculosis or some other mycobacterioses, and they are indistinguishable (Table 5, Table 6, Table 7). Both occur most frequently in the ileocecal region, and both may occur anywhere in the gastrointestinal tract from mouth to anus. In the United States where ileocecal tuberculosis is rare, such cases are generally diagnosed only after surgical resection for Crohn's disease (3). When the features of these two diseases are compared, the only distinguishing criteria are the presence of caseating granulomas and acid-fast bacilli in tuberculosis. Thus, the absence of caseation necrosis and failure to isolate or demonstrate mycobacteria are the chief if not sole criteria for the diagnosis of Crohn's disease. As discussed, these are not reliable criteria. Taylor (271), in his study of intestinal tuberculosis and Crohn's disease, concluded that "it is impossible on the basis of clinical features or morbid anatomy to distinguish between these two conditions". Cattel and Mosely (41) shared this view and stated that ileocecal tuberculosis and Crohn's disease "may be virtually impossible" to distinguish. Even Crohn himself, in a discussion of the paper of Watson et al (287), conceded that "any pathologist would have difficulty in differentiating the pathology of so called pseudotuberculosis, of defining histologically sarcoidosis, ileitis, or ileojejunitis". Without doubt, certain cases of hypertrophic tuberculosis of the intestine are difficult, if not impossible, to differentiate pathologically from Crohn's disease. Some investigators have compared intestinal tuberculosis and Crohn's disease, and have provided detailed pathological descriptions that allow their differentiation (269). These reports, however, did not describe hypertrophic tuberculosis but dealt primarily with secondary intestinal disease or the ulcerohypertrophic variety.

Crohn's disease and mycobacterioses not only share the features of primary intestinal disease, but of extra-intestinal manifestations as well. In Crohn's disease, arthritis, iritis, erythema nodosum, and amyloidosis are occasionally encountered and are considered to be important extra-intestinal manifestations (28, 66, 124, 142, 152, 212, 300). Arthritis is a well-known complication of mycobacterial infections (126), and in recent years, it has been shown that arthritis can be produced by mycobacterial antigens alone (127, 280).

Epidemiology
The population epidemiology of Crohn's disease and the mycobacterioses are not readily comparable because of the manner in which these studies have been conducted. Whereas the epidemiology of tuberculosis is well defined and based on total populations studies, that of Crohn's disease is not. There are no methods for population surveillance (such as PPD reactivity), and prevalence/incidence data are determined regionally through hospital records. Additionally, Crohn's disease population epidemiology is hampered by a long lapse between onset of clinical symptoms and diagnosis and an unequal precision in the use of diagnostic criteria in different study centers. In a proportion of studies it has been determined that approximately 20% of the study group are misclassified and do not have Crohn's disease (35). Thus, epidemiology will be addressed breifly.

Crohn's disease occurs with its highest incidence in the United States, the United Kingdom, and Scandinavia. It is less frequent in Central Europe and rarely is reported in Africa, Asia, and South America. The disease is seldom reported in underdeveloped or developing countries (35, 36). In the United States, the incidence is somewhere between 3.1 to 13.5 per 100,000 population, and is between 0.3 to 7.3 in other countries where the disease is reported (35, 36). Reports are conflicting, but the incidence of Crohn's disease in the United States and in other countries has been increasing, particularly in certain regions (35, 36, 91, 208, 262). Generally, the prevalence of disease appears to have stabilized in most countries. In contrast, tuberculosis (and leprosy) occur with highest frequency in those areas where Crohn's disease is rarely seen, and with low frequency where Crohn's disease is most frequent.

Morgan (194) has proposed an .... explanation based on the epidemiology of M. paratuberculosis infection in animals. In this disease, animals are infected with M. paratuberculosis during early childhood (before 30-days of age) but disease becomes manifested later in adult life. An age-dependent resistance develops such that adult animals not exposed to the agent during early life rarely become infected, even experimentally. Thus, Morgan postulated that early exposure to an infectious agent (M. paratuberculosis) would account for the occurrence of Crohn's disease in siblings and mono- and di-zygotic twins, and the rarity of Crohn's disease in half-siblings. He felt that the case for early infection was particularly supported by the disease occurrence in twins separated since childhood. Since paratuberculosis is rarely transmitted to adult animals due to age-dependent resistance, a low incidence in adult married couples would be expected. Morgan proposed a time-space clustering study of Crohn's disease patients during their first 5-years of life to address these issues.

In Crohn's disease, as in tuberculosis and leprosy, steroids are used to provide clinical relief, but neither disease can be cured by such treatment. Although disease remission occasionally occurs in Crohn's disease patients receiving corticosteroids, it is difficult to determine if this remission is due to the steroid therapy, occurs spontaneously (placebo effect), or reflects a masking of the disease as occasionally observed in leprosy (227).

Non-steroidal anti-inflammatory agents are known to activate quiescent Crohn's disease (140) and induce intestinal inflammation in other types of patients as well (24). Therefore, their use is contraindicated. Non-steroidal anti-inflammatory drugs are also known to activate quiescent pulmonary tuberculosis (275) and are also contraindicated in mycobacterioses.

In summary, chemotherapeutic schemes offer several similarities between the mycobacterioses and Crohn's disease: i. anti-mycobacterial agents appear effective in only a portion of Crohn's disease patients as they are in intestinal mycobacterioses; ii. corticosteroids offer clinical improvement in Crohn's disease, leprosy, and some cases of tuberculosis and other mycobacterioses; and iii. non-steroidal anti-inflammatory agents activate quiescent Crohn's disease and tuberculosis. Despite these similarities, the efficacy and appropriateness of antimycobacterial chemotherapy in Crohn's disease remains to be evaluated.

The originating article cites over 300 references
http://alan.kennedy.name/crohns/research/diagnos/chiodini.htm